(Medicina Veterinaria) Animalario-Zoonosis Animales Experimentación Parte2

Clin Lab Med 22 (2002) xi–xiii
Preface
Clinical HIV-1 virology
R.J. Pomerantz, MD, FACP

Guest Editor
It has been 8 years since the last issue on HIV-1 and AIDS was published
in Clinics in Laboratory Medicine. Although it has been 8 years in time, it
has been a quantum leap forward in both the basic and clinical science
knowledge regarding HIV-1 infection. Over this time period, data on the
viral life cycle and pathogenesis of disease states has increased remarkably.
We now have a very good handle on viral replication on a molecular level in
a variety of primary cell types, which is of importance in understanding
AIDS pathogenesis. This interpreted into a revolution in antiretroviral ther-
apy for HIV-1 infection. The development of combination chemotherapy
(highly active antiretroviral therapy [HAART]) has led to a true paradigm
shift in this disease. It is analogous to the change in diabetes care before and
after the discovery of insulin. In the developed world, at least, much of HIV-1
infection has become a disease of chronicity. Many hospitals have remark-
ably fewer patients with HIV-1 infection admitted for care, and HIV-1 infec-
tion has become an outpatient disease in many cities in North America and
Western Europe. This is based on the rational design of new drugs to inhibit
HIV-1 in various portions of the viral life cycle, both preintegration and
postintegration of the HIV-1 provirus. Unfortunately, these changes have
not occurred in the developing world, which accounts for over 90% of the
AIDS pandemic worldwide.
This issue of Clinics in Laboratory Medicine is extraordinarily timely and
overdue. I have asked a variety of experts in critical fields in HIV-1 patho-
genesis and clinical care to contribute important articles, which are designed
to give a broad but clear overview of the developments in HIV-1 biology
0272-2712/02/$ - see front matter Ó 2002, Elsevier Science (USA). All rights reserved.
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xii R.J. Pomerantz / Clin Lab Med 22 (2002) xi–xiii
and treatment over the past 8 years. The first article by Dr. Joseph DeSi-
mone and myself demonstrates the new methods for detection of HIV-1,
highlighting the increase in rapid and non–blood sample testing. The second
article by Drs. Mark Holodniy and Karen Relucio discusses the importance
of plasma HIV-1 RNA levels in understanding treatment effects and disease
prognostication. This has been arguably one of the most important labora-
tory advances over the past eight years in HIV-1 clinical care. The next
article by Dr. William O’Brien and colleagues takes on the Herculean task
of reviewing the HIV-1 replication cycle. The amount of detailed under-
standing of HIV-1 replication and its interaction with host cell cofactors has
been truly impressive over the last 8 years. As such, this is an important
article for review by all physicians and scientists who approach HIV-1 either
in the clinic or in the laboratory. Dr. George Hanna writes a very important
and detailed review on HIV-1 genotype and phenotype resistance testing.
Clearly, both secondary and the newly described primary resistance patterns
found in HIV-1 viruses, caused by noneffective or semi-effective treatment,
are two of the major problems that HIV-1 clinicians have had to face over
the last several years.
I contributed the next article on HIV-1 reservoirs. This is a critical area in
which HIV-1 latent proviruses, and cryptically replicating virus, in patients
on virally suppressive HAART occurs throughout the epidemic. HIV-1 res-
ervoirs are clearly the major reason that this lentivirus cannot be eradicated
in patients on any known HAART regimen. The next article by Dr. Ron
Collman and Dr. Linda Starr-Spires also is critical because it demonstrates
the rational design of entry inhibitors, which are some of the newest ther-
apeutic agents and which will likely obtain Food and Drug Administration
approval in the next few years. Understanding the complexity of HIV-1
entry, which has had significant advances over the last few years, is key in
the studies of therapeutic drug design.
Dr. Dennis Kolson contributes a section on HIV-1 neuropathogenesis.
As the era of HAART has advanced, AIDS dementia complex has thank-
fully decreased in frequency. Nevertheless, this remains an enigmatic disease
state in which neuronal drop out leads to a dementia complex, and is only
now being understood on the molecular level. Immune reconstitution, in an
article contributed by Drs. Drew Weissman and Luis Montaner, is also now
of importance in the era of virally suppressive HAART. Many HIV-1–
infected patients are left with a very low CD4+ T-lymphocyte count, even
when the virus is decreased to undetectable levels in the peripheral blood.
Clearly, immune reconstitution is of immense importance for long-term care
in these patients. The next article, and arguably one of the hardest to syn-
thesize based on its voluminous amount of material, is written by Dr. Ian
Frank on antiretrovirals and HIV-1. With the explosion in antiretroviral
agents over the last 8 years, this is a critical article for all physicians and sci-
entists interested in HIV-1, who must be well-versed in this rapidly changing
and expanding field. As a complementary article to the previous one,
R.J. Pomerantz / Clin Lab Med 22 (2002) xi–xiii xiii
Dr. Paul Palumbo describes both HIV-1 infection and treatment modalities
in pediatric patients. Vertical transmission of HIV-1 is still present in the
United States, although decreasing, and the pathogenesis and treatment of
this disease in children is profoundly different from that in adults.
The next two articles are linked. The first by Dr. Charles Rinaldo and
colleagues outlines the importance of CD8+ T lymphocytes and cytotoxic
T lymphocytes in immunity against HIV-1 infection and inhibition of viral
replication after infection. Clearly, for any immune-based therapy or vac-
cine design, understanding cytotoxic T lymphocytes and their interactions
with HIV-1 is of critical importance. Dr. Matthias Schnell and colleagues
write the final article in this issue, on HIV-1 vaccines. He and his group enti-
tled this ‘‘The search continues.’’ I think that this is an appropriate title
because we still, 21 years after the first AIDS cases were reported in the
United States, are searching for an efficacious prophylactic or therapeutic
vaccine.
I believe this issue is an extremely useful addition to the libraries of
internists, infectious disease physicians, pathologists, and other medical sci-
entists interested in HIV-1, and synthesizes the dramatic increase in knowl-
edge regarding this pathogenic human retroviral infection obtained over the
last 8 years. I look forward optimistically to the next several years, because
I predict that there will be a continued dramatic increase in knowledge
regarding the pathogenetic processes and treatment opportunities to combat
HIV-1. It will certainly be interesting to see how the field changes over the
next 8 years.
Roger J. Pomerantz, MD, FACP

The Dorrance H. Hamilton Laboratory
Center for Human Virology
Thomas Jefferson University
1020 Locust Street, Suite 329
Philadelphia, PA 19107
Clin Lab Med 22 (2002) 573–592
New methods for the detection of HIV

Joseph A. DeSimone, MD,
Roger J. Pomerantz, MD, FACP*
Division of Infectious Diseases, Department of Medicine, Jefferson Medical College,
Thomas Jefferson University, Philadelphia, PA 19107, USA
Several strides have recently been made in the ability to detect the pres-
ence of both HIV-1 and HIV-2. Serologic testing has been greatly refined,
vastly improving the sensitivity and specificity of such tests. More rapid
serologic methods now exist, as do kits for performance of testing at home.
HIV-1 antibody tests of nonserum samples, such as urine and oral fluids,
have recently been approved by the Food and Drug Administration (FDA).
HIV-1 antigen testing and HIV-1 culture techniques have become useful in
certain clinical situations. Finally, nucleic acid–based tests, which can allow
direct detection of both HIV-1 RNA and complementary DNA (cDNA),
have played an important role in the detection of acute HIV-1 infection in
adults and in the postpartum period of infants born to HIV-1–infected
mothers. These tests and their clinical uses are discussed in this article.
ELISA and Western blot testing

Serologic tests for the presence of HIV-1 and HIV-2 using the enzyme-
linked immunosorbent assay (ELISA) and Western blot methods remain the
primary methods for the detection of HIV infection. Both the sensitivity and
specificity for most recent HIV ELISA tests are reported to be greater than
99% [1,2]. Detection of antibody is usually possible within 6 to 8 weeks after
infection, and nearly all tests are positive within 6 months, although longer
seroconversion periods have been reported [1,3]. Because of the window
period in antibody development immediately after infection, serologic test-
ing performed during this time may lead to a false-negative result. Also,
because of passive transfer of maternal antibody, serologic diagnosis of an
* Corresponding author. 1020 Locust Street, Suite 329, Philadelphia, PA 19107–6799.

E-mail address: roger.j.pomerantz@mail.tju.edu (R.J. Pomerantz).
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574 J.A. DeSimone, R.J. Pomerantz / Clin Lab Med 22 (2002) 573–592
infant born to an HIV-infected mother is often not helpful. Detection of

HIV infection in these situations is discussed in detail later.
The ELISA is the usual serologic test used initially for screening and
diagnosis of both HIV-1 and HIV-2. Recent improvements in ELISA meth-
odologies, such as the use of recombinant antigens instead of whole viral
lysate, and the use of double-antigen sandwich assays (frequently referred
to as third-generation assays), have improved both the sensitivity and specif-
icity of the HIV ELISA [4,5]. Similarly, difficulties with detection of HIV-2
by ELISA testing have been resolved. Although mostly limited to western
Africa and South America, HIV-2 has been identified in some patients in
North America. Because of gene heterogeneity, HIV-2 is not always
detected in HIV-1 ELISA tests, although cross-reactivity can occur [6,7].
Combined HIV-1–HIV-2 ELISA tests have helped resolve this problem in
many instances. A testing algorithm, using an HIV-2 Western blot confirma-
tory test, has been recommended by the Centers for Disease Control and
Prevention (CDC) and FDA for use with the HIV-1–HIV-2 combination
ELISA [8]. Finally, the addition of HIV-1 subtype O antigens to the ELISA
has allowed improved detection of this uncommon subtype of HIV-1 [9].
There are a number of reasons for false-positive and false-negative
ELISA results. False-positive ELISA results are often weakly or only mod-
erately reactive, are frequently transient, and are usually nonreactive by
Western blot testing [2]. Causes of false-positive HIV ELISA results include
human and technical error, cross-reacting antibodies, and numerous medi-
cal conditions [2]. Many of the problems with cross-reacting antibodies have
been eliminated since ELISA tests began using synthetic or recombinant
HIV peptides, although cross-reaction may still occur. False-positive ELISA
results may also occur in individuals participating in HIV vaccine trials [10].
False-negative HIV ELISA results can occur for a variety of reasons [2].
Importantly, a nonreactive HIV ELISA result in a person at high risk for
HIV infection should always prompt consideration of the window period
before seroconversion. Such patients should have repeat serologic testing
several weeks later in this clinical situation.
Repeatedly reactive HIV ELISAs require confirmatory testing, and the
Western blot technique is the most widely used test in this setting. In this
assay, individual HIV proteins are separated according to size by gel electro-
phoresis and transferred (blotted) onto nitrocellulose paper. After addition of
the patient’s serum, the reactivity of antibodies to specific viral proteins can
be determined. Interpretation of the Western blot is based on the spectrum
of bands that is visualized. It should be noted, however, that different consen-
sus groups have proposed alternative criteria for interpretation of the HIV
Western blot. The Association of State and Territorial Public Health Labo-
ratory Directors and CDC have defined a positive HIV-1 Western blot as the
presence of any two of the following bands: p24, gp41, or gp120–gp160 [11].
These criteria for a positive Western blot have been accepted by most labora-
tories performing this test [2]. If no bands are present, the test is considered
J.A. DeSimone, R.J. Pomerantz / Clin Lab Med 22 (2002) 573–592 575
negative. If a single band is present, or a combination of bands is present that

do not meet criteria for a positive result, the test is termed indeterminate.
The sensitivity and specificity of the HIV-1 Western blot assay are both
excellent, ranging between 96% and 100% in most cases [12]. Nevertheless,
false-negative and false-positive results can occur. For example, because
these assays do not contain certain proteins from HIV-1 serogroup O, con-
ventional Western blot results can be falsely negative when this relatively
rare serogroup is present [13]. False-positive HIV-1 Western blots have been
reported in patients with hyperbilirubinemia; HLA antibodies; other human
retroviruses (including HIV-2); connective tissue disorders; and polyclonal
gammopathies [1].
The indeterminate HIV-1 Western blot result remains a source of confu-
sion and anxiety for both physicians and patients alike. As many as 10% to
20% of Western blot results performed on sera that are repeatedly reactive
by HIV-1 ELISA are interpreted as indeterminate [14]. This result may be
caused by false positivity, as mentioned previously, or may represent true
HIV-1 infection before complete seroconversion. Assessing the patient’s risk
factors for HIV acquisition, and repeat Western blot testing in several
weeks, are both necessary to determine the significance of an indeterminate
HIV-1 Western blot. Because this result may represent recent HIV-1 infec-
tion before complete seroconversion, it is recommended that repeat Western
blot testing occur over the next 6 months. If an evolution of reactive bands
does not occur over time, such patients are usually not infected. Guidelines
from the CDC state that ‘‘a person whose Western blot test results continue
to be consistently indeterminate for at least 6 months—in the absence of any
known risk factors, clinical symptoms, or other findings—may be consid-
ered to be negative for antibodies to HIV-1’’ [11]. If recent HIV-1 infection
is suspected based on risk factors or clinical presentation, additional tests,
such as those used in the diagnosis of acute HIV-1 infection (discussed
later), also may be useful.
Rapid HIV-1 antibody tests

One of the drawbacks of the previously mentioned routine antibody tests
is that several days may elapse before the results are known or reported to
the ordering physician. For this reason, a number of rapid HIV-1 antibody
tests have been developed, in which results can be determined within a mat-
ter of minutes. Clinical settings in which rapid results may be important
include public clinics and emergency rooms, where patients infrequently
return for their HIV-1 antibody test results [15]. By determining HIV-1 sta-
tus quickly, these patients can then be counseled and advised immediately
regarding further testing and treatment. In addition, these rapid tests have
been frequently used in situations that require immediate results, such as
in the case of occupational exposure.
Currently, only one FDA-approved rapid antibody test is commercially

available in the United States (the Single Use Diagnostic System, Abbott
Laboratories, Abbott Murex, Norcross, GA), although other rapid test kits
are being evaluated for approval. These assays, performed on plasma sam-
ples, used rapid ELISA or latex agglutination techniques. Results are
usually available within 10 minutes. In one large trial, the sensitivity of
the Single Use Diagnostic System assay was 100%, and the specificity was
99.5% [16]. This resulted in a positive predictive value of 88% and a negative
predictive value of 100%. Other studies of this assay and other commercially
available rapid assays have found similar results [17,18]. Importantly, it has
been noted that specificity can be lowered with improper specimen handling
or if the test is performed on patients who have indeterminate Western blots,
such as those patients who are acutely infected [16,19]. Because of the rela-
tively low specificity, it is recommended that all positive results be confirmed
with standard antibody testing. The low specificity and positive predictive
value also imply that this test should not be used as a screening test of the
general population.
Home sample collection kits

Home sample collection kits were designed to encourage patients to
undergo HIV-1 testing in a more confidential and convenient manner. Cur-
rently there is a single home sample collection kit which is FDA-approved:
Home Access (Home Access Health Corporation, Hoffman Estates, IL) [20].
This is an over-the-counter kit that also is available by mail order. The
patient collects a sample of blood with a provided lancet, places the blood
on a test card, and mails the card to the company. The Home Access test
utilizes an ELISA and immunofluorescent assay (IFA) on the dried blood
sample. The patient can call a toll-free number for the results, which are
kept anonymous by means of a code number. If the test result is either pos-
itive or indeterminate, the caller is transferred to a counselor regarding the
results. Sensitivity and specificity of this assay approaches 100% [20,21].
It should be noted that this kit differs from home-use HIV-1 test kits,
which allow consumers to interpret their own HIV-1 test results at home,
and which have not been approved by the FDA due to inaccurate results [22].
Non–serum-based HIV-1 antibody tests

To make HIV-1 testing simpler and safer, several non–serum-based anti-
body tests have been developed. There are several advantages to testing for
HIV-1 antibodies in samples, such as oral fluids, urine, and vaginal secre-
tions. For example, specimen collection and handling are easier and safer
for the health care provider. Also, patients who are averse to venipuncture,
or who simply have poor venous access, may be more willing to undergo
HIV-1 testing if venipuncture can be avoided. Finally, such tests may allow
wider HIV-1 testing in developing countries, where laboratory support may

be minimal.
Orasure (Epitope, Inc., Beaverton, OR) is the only FDA-approved HIV-
1 antibody test that relies on oral fluid specimens. Specimens are collected
by placing a cotton pad between the cheek and gum for 2 to 5 minutes. This
pad contains a hypertonic solution to encourage transudation of oral
mucosal transudate, a serous fluid that contains high amounts of HIV-1 IgG
[23]. A health care provider then places the pad in a preservative for trans-
port to an Orasure laboratory. An Orasure ELISA and Western blot can
then be performed on the oral mucosal transudate. In several trials, when
compared with standard testing, Orasure had a sensitivity of 98% to 100%
and specificity of 99% to 100%. This corresponded to a positive predictive
value of 100% and a negative predictive value of 99.97% [23–25]. Impor-
tantly, it has been shown that oral pathology does not affect the results of
oral mucosal transudate testing [26].
The FDA also has recently approved an HIV-1 antibody test for urine
specimens. The Sentinel HIV-1 Urine Enzyme Immunoassay (Calpyte Bio-
medical Corporation, Alameda, CA) is a rapid IgG capture enzyme immu-
noassay with a reported sensitivity of 98.73% [27]. A urine specimen HIV-1
Western blot is now available for confirmation of positive urine enzyme
immunoassay results [27,28]. Further confirmation by blood sample is rec-
ommended for positive urine results because of the lower specificity of the
urine Western blot as compared with the serum-based Western blot [22].
An ELISA for detection of HIV-1 and HIV-2 IgG in vaginal secretions,
although not FDA-approved, may be useful in certain clinical situations.
Because HIV-1 and HIV-2 IgG can be detected in seminal fluid, one potential
use for vaginal secretion testing may be in a rape victim, where serostatus can
guide the need for postexposure prophylaxis [29].
p24 antigen detection

p24 is an HIV-1 core protein encoded by the gag gene. It is expressed
shortly after acquisition of HIV-1, and antibody to p24 forms shortly there-
after [30,31]. p24 antigen can be detected in serum, plasma, and cerebro-
spinal fluid [32]. Assays to detect p24 antigen use an antigen capture
with ELISA technique. The patient’s serum is added to a plate or beads
coated with anti-p24 antibody, allowing the p24 antigen to be captured. An
enzyme-linked anti–HIV-1 IgG is then bound to the complex, and is subse-
quently measured colorimetrically. Because antigen-antibody complexes can
limit the level of detection of p24 antigen, a dissociation step to separate
these immune complexes has been added to the assay. This dissociation step
has increased the sensitivity of the assay significantly [33,34]. Although
uncommon, false-positive p24 antigen results, presumably caused by cross-
reacting proteins, have occurred in uninfected patients [35].
Before the advent of plasma HIV-1 viral RNA measurement, the p24
antigen assay was used as a prognostic tool. Levels of p24 antigen were
found to reappear or increase in HIV-1–infected patients shortly before or
during the development of AIDS [36–38]. Similarly, this assay was useful
in measuring the antiretroviral effect of medical therapy [33,39,40]. Use of
the p24 antigen assay in these roles has been mostly supplanted by quanti-
tation of plasma HIV-1 RNA assays. Although not FDA-approved for use
as a diagnostic test, p24 antigen testing has played a role in the diagnosis of
acute HIV infection and in infants born to mothers who are HIV-infected
(discussed later).
Cultures of HIV-1
The HIV-1 can be cultured from plasma, serum, peripheral blood mono-
nuclear cells (PBMCs), cerebrospinal fluid, saliva, semen, cervical speci-
mens, and breast milk [1]. Standardized culture methods usually use the
patient’s plasma or PBMCs, which are incubated with uninfected donor
PBMCs. Interleukin-2 is present to activate and stimulate cell growth. The
culture supernatant, which then contains progeny HIV-1 virions, can be
tested qualitatively or quantitatively for the presence of HIV-1 with assays
for reverse transcriptase (RT) or p24 antigen. Most cultures from HIV-1–
infected patients, excluding those on virally suppressive highly active antire-
troviral therapy, become positive within 21 days [41]. Sensitivity of these
culture methods in patients who are HIV-1 seropositive has been reported
at greater than 97%, with a specificity of 100% [42,43].
In the age of plasma viral RNA testing, the use of HIV-1 culture in clin-
ical management is minimal. The in vitro rate of replication when culturing
HIV-1 has been shown to correlate with the patient’s clinical status and may
serve as a means of measuring response to antiretroviral therapy [44–47].
When compared with plasma viral RNA testing, however, culture methods
for HIV-1 are far more laborious, time-consuming, and less sensitive. Sim-
ilarly, although culture may be useful in diagnosing infants born to women
who are HIV-1–infected, the sensitivity of this test is far lower when com-
pared with a diagnostic method, such as proviral DNA polymerase chain
reaction (PCR) testing [48,49]. For these reasons, culture testing of HIV-1
has mainly been relegated to the research and clinical trial realm.
Nucleic acid–based tests

On entering human cells, HIV-1 RNA is converted into a complementary
strand of cDNA by RT. These linear DNA molecules then integrate into the
host genome, becoming the proviral form of HIV-1. After translation and
the production of new virions, release of HIV-1 RNA into the plasma
occurs. Molecular tests are now available for the detection of cDNA and
plasma viral RNA. Although primarily used as prognostic and therapeutic
markers, these tests also have been used as diagnostics.
The presence of proviral DNA can be detected by using the PCR. In the
HIV-1 DNA PCR assay, PCR using oligonucleotide primers amplifies a seg-
ment of the highly conserved HIV-1 gag gene. This is followed by hybridiza-
tion of an identifying DNA probe, with a subsequent qualitative enzymatic
colorimetric assay. The sensitivity of this technique has been reported as
greater than 95%, with a specificity of greater than 98% [50,51].
The DNA-PCR method for detecting the presence of HIV-1 is highly reli-
able when testing for HIV-1 subtype B, the most common subtype in North
America. This method, however, is less reliable when infection with non–
subtype B strains of HIV-1 is present [52–54]. Because of the somewhat high
inaccuracy rate, this test has not been recommended as a routine screening
measure [55]. Use of this method in diagnosing infants is discussed later.
Although less commonly used as a diagnostic test, the quantitation of
circulating virion-associated HIV-1 RNA in plasma, commonly referred
to as the plasma viral load, has had an enormous impact on the management
of HIV-1 infection. This measurement has allowed greater understanding of
HIV-1 viral dynamics, and the continuous, high-level rate at which viral
replication occurs [56,57]. The advent of the plasma viral load allowed inves-
tigators to understand that a basal level of high viremia is continuously
present, regardless of the patient’s clinical stage [58,59]. Furthermore,
knowledge of the extraordinary rate at which HIV-1 replication occurs has
helped explain the process and development of antiretroviral-resistant qua-
sispecies, and the reasons behind antiretroviral failure [57].
Perhaps most importantly, the advent of assays to quantitate plasma
HIV-1 RNA in the early 1990s gave clinicians a powerful new prognostic
tool. Several studies performed during the mid-1990s consistently confirmed
that the risk of progression to AIDS and death from HIV-1 infection was
directly related to the plasma viral load [60–63]. In addition, the plasma viral
load was found also strongly to predict the decline of CD4þ T lymphocytes
[64,65]. Subsequent studies revealed the superiority of the plasma HIV-1
RNA level over the CD4þ T-lymphocyte count in predicting disease pro-
gression, but importantly the combined measurement of both plasma
HIV-1 RNA and the CD4þ T-lymphocyte count was found to be a better
prognosticator of disease progression than any single test alone [64,66,67].
New, simpler, and more rapid techniques of quantitating plasma HIV-1
RNA have allowed the plasma viral load to become the most clinically use-
ful and meaningful prognostic tool.
Of equal importance has been the impact of plasma viral load on the use
and understanding of antiretroviral therapy. With the advent of the plasma
viral RNA assays, investigators had a reliable means of measuring both the
short- and long-term impact of antiretroviral therapy [63,68–72]. Baseline
plasma HIV-1 RNA levels are an integral component in determining when
and if a patient should receive antiretroviral therapy. Also, viral load reduc-
tion has now become the standard measure for determining response to
antiretroviral therapy when comparing efficacy of different antiretroviral
medications in clinical trials. Indeed, it was the response in plasma viral load
that allowed investigators to propose that dual or triple antiretroviral ther-
apy is of greater benefit than monotherapy [73]. Finally, the plasma viral
load has proved useful in predicting disease progression in the setting of
antiretroviral therapeutic response, and the progressive change in this level
has been shown to be even more predictive of disease progression than the
pretherapy level [66,74].
The three methods of quantitating plasma HIV-1 RNA, which are com-
mercially available, include the RT-PCR assay, the branched DNA (bDNA)
assay, and the nucleic acid sequence-based amplification (NASBA) tech-
nique [75–81]. In the RT-PCR assay, HIV-1 RNA from the patient is con-
verted to cDNA by adding RT. A well-preserved portion of the gag gene is
amplified by PCR and hybridized to an enzyme-linked DNA probe. Simul-
taneously, a competitive RNA template, with a known standard copy num-
ber, is used in competitive titration. The ratio of detected signal is compared
with the signal of the known standard, determining the amount of HIV-1
RNA in the patient’s plasma.
The bDNA technique for measuring viral RNA differs in concept from
the RT-PCR method. In this assay, plasma HIV-1 RNA is captured by
probes on to a microplate. Multiple DNA probes are hybridized to specific
pol (or gag) gene segments of the bound RNA, thereby amplifying the sig-
nal. Alkaline phosphatase is then added in the presence of a substrate to
generate a chemiluminescent reaction. The chemical light units are then
compared with a standard to determine the amount of RNA in the sample.
The bDNA method is based on signal amplification rather than target
amplification.
The NASBA method of quantifying HIV-1 RNA, like the RT-PCR
method, involves repetitive rounds of target amplification [75]. In this assay,
RNA transcriptases are used to amplify specific HIV-1 RNA targets.
Although similar to the RT-PCR method in principle, this method differs
in that NASBA amplifies viral RNA as opposed to cDNA. Also, with the
addition of a unique RNA extraction step, NASBA can measure HIV-1
RNA in samples other than plasma.
Most commercially available viral load tests are effective for measuring
HIV-1 subtype B only. HIV-2 RNA viral load tests are not commercially
available. Similarly, these tests are frequently ineffective in measuring the
HIV-1 non–subtype B strains (eg, A, C, D, or E), which are commonly
found outside of North America [82]. Because multiple DNA probes are
used in the bDNA method, this may be the best available method for mea-
surement of non–subtype B strains of HIV-1 RNA [83]. Nevertheless, there
is currently no FDA-approved viral load assay for non–subtype B HIV-1
RNA.
These three assay methods seem to be equivalent in sensitivity and specif-

icity, particularly when measuring high or moderately high levels of viremia
[84,85]. Variability among the assays, however, may be more pronounced
when evaluating low or undetectable viral loads [86]. Currently, only the
RT-PCR viral load test has been approved by the FDA for determining
prognosis and for monitoring the response to therapy. Also, absolute levels
of nucleic acid can vary among the assays. For example, the levels of viremia
when determined by RT-PCR are typically two times higher than when
measured by bDNA [64]. For these reasons, direct comparisons of viral load
should occur only if the same methodology is used, and the same assay
should be used when serially testing viral loads.
The issue regarding HIV-1 viral load differences in men compared with
women is not yet resolved. Some studies have noted that women tend to
have lower HIV-1 RNA levels than men at seroconversion and early in
infection, although other studies revealed no such differences [87–91]. Even
when viral load was found to be lower in women than men at seroconver-
sion, the plasma HIV-1 RNA levels seemed to equalize with time [92]. More
recent data suggest that because viral loads are lower at seroconversion in
women, and because the rate of progression to AIDS is similar in both sexes,
guidelines recommending when to initiate therapy should be different for
men and women [93]. Until the issue of gender differences on plasma
HIV-1 viral load is clarified, it is unlikely that different treatment recommen-
dations for men and women will be made.
A number of factors can lead to variation in viral load for an individ-
ual patient. This variation can be related to assay methodology or can be
biologic in nature. For example, use of heparin as an anticoagulant can
result in lower quantities of HIV-1 RNA than with use of ethylenediami-
netetraacetic acid as the anticoagulant [64,94]. Similarly, time to process-
ing of the specimen can alter levels of RNA [95]. In addition, plasma
viral load can be transiently affected by a number of host or biologic fac-
tors. Vaccination against tetanus, the pneumococcus, and influenza have
all been shown to cause a moderate, but transient, increase in plasma
HIV-1 RNA levels [96–99]. The cellular activation that results from vac-
cination may explain the increased HIV-1 viral expression and replication.
Several infectious disorders, such as tuberculosis, herpes simplex virus,
and bacterial pneumonia, have also been shown to cause a transient
increase in HIV-1 viral load [100–102]. In fact, a transient increase in
viral load has been associated with the development of any of several
opportunistic infections, such as Candida esophagitis or Pneumocystis car-
inii pneumonia [103]. Again, this may be related to cellular activation in
response to infection.
Transient changes in plasma viral load may occur for less obvious rea-
sons. A decrease in plasma viral load had been demonstrated in ovulating
women, particularly during the early follicular phase to the mid-luteal
phase, perhaps as a result of hormonal regulation of lymphocytes or
cytokines [104]. Also, missing even a few doses of antiretroviral therapy

before measurement of viral load can result in an increased HIV-1 RNA
level [67]. Given the variability in viral load, it is strongly recommended that
decisions regarding therapy be based on at least two plasma viral RNA
determinations separated over time [105,106].
Recommendations regarding use of plasma HIV-1 RNA level in guiding
therapeutic decisions have been made by the Department of Health and
Human Services and the International AIDS Society [107,108]. For patients
who are naive to antiretroviral therapy, determination of baseline plasma
viral load, in addition to the CD4þ T-lymphocyte count, is an essential
component to these guidelines. The plasma HIV-1 RNA threshold values
used in determining when to initiate therapy differ slightly among the two
sets of recommendations. The guidelines by the Department of Health and
Human Services also consider different threshold values based on which
assay method for plasma viral load is used.
Both sets of guidelines state that if combination antiretroviral therapy is
instituted, the goal of therapy is to obtain and maintain a plasma viral load
below the level of detection. The level of detection, however, depends on the
sensitivity of the assay. Both the RT-PCR and bDNA methods for plasma
HIV-1 RNA can detect viremia as low as 50 copies/mL. Currently, the
guidelines suggest that a viral load less than 50 copies/mL is optimal, and
there are some data to suggest that a more durable virologic response is
obtained if such a viral load can be achieved [109–111].
It is expected that a 1- to 2-log reduction of plasma HIV-1 RNA should
occur within 4 to 8 weeks after starting therapy, and that the plasma viral
load should be undetectable within 16 to 24 weeks. One factor that can affect
the time it takes for the viral load to become undetectable after beginning
therapy is the level of viremia at baseline. A viral load that is greater than
100,000 copies/mL has been associated with a poorer chance of attaining
an undetectable viral load in the setting of highly active antiretroviral ther-
apy [107,112,113]. It has also been demonstrated that the less time it takes to
obtain an undetectable viral load after initiating therapy, the more likely the
viral load remains undetectable while on therapy [113]. The lower the nadir,
and the less time it takes to achieve an undetectable viral load, the greater
the chance of successful therapy.
The optimal frequency for performing plasma viral load once therapy has
begun is not clear. Current guidelines recommend performing plasma viral
load within 1 month after initiating or changing therapy, monthly until the
goal of therapy is reached, and every 2 to 3 months thereafter [107]. The
guidelines also make recommendations regarding a change in antiretroviral
therapy if viral rebound or failure to attain an undetectable viral load
occurs. This issue is somewhat less clear, because the exact plasma viral load
threshold for changing therapy is unknown, and other factors, such as
immune status and adherence and side effects of medications, should be con-
sidered before altering therapy.
The diagnosis of acute HIV-1 infection

Because the development of antibodies to HIV-1 may not occur until
weeks after exposure to the virus, routine serologic testing for HIV-1 using
ELISA and Western blot assays may initially be negative or indeterminate,
particularly during the first few days to weeks after infection. Diagnosis and
treatment of HIV-1 infection shortly after exposure may be beneficial.
Potential benefits of identifying and treating HIV-1 during the acute phase
include preserving immune function, reducing the risk of transmission, and
potentially altering the natural history and progression of disease [114]. Ini-
tial viremia is thought to occur approximately 4 to 11 days after mucosal
exposure [115]. Quantitation of HIV-1 and p24 antigen during this period
has revealed extraordinarily high levels of HIV-1 in the plasma [32,
59,116,117]. Control of viremia with antiretroviral therapy during acute
infection may limit viral replication, enhance the cytotoxic T-lymphocyte
response and T-helper lymphocyte response, and improve overall prognosis
[118–123]. Making the diagnosis of acute HIV-1 infection, combined with
early antiretroviral therapy, may be very beneficial.
Establishing this diagnosis, however, may be difficult. Although a well-
described mononucleosis-like syndrome occurs in at least 50% of patients with
acute HIV-1 infection, these findings are diverse and nonspecific, and easily can
be attributed to other viral illnesses. In fact, less than 10% of acute HIV-1 infec-
tion is diagnosed by clinicians [124]. These symptoms usually occur 2 to 6 weeks
after exposure and last for approximately 1 to 2 weeks [125]. Because HIV-1
serologic tests become positive an average of at least 22 days after exposure,
other methods of detection of HIV-1 infection are necessary [126].
Measurement of serum p24 antigen, HIV-1 DNA in plasma or PBMCs, or
plasma HIV-1 RNA may be useful during this serologic window period. As
mentioned previously, p24 antigen levels are often initially high, but may
wane after 1 to 2 weeks [116,127]. One recent trial determined that the sensi-
tivity of the p24 antigen assay during acute HIV-1 infection is approximately
89%, with a specificity of 100% [128]. A more sensitive method of diagnosing
acute HIV-1 infection may be the plasma HIV-1 RNA level. Studies evaluat-
ing the use of this marker in diagnosing acute HIV-1 infection have found
that the plasma viral load is nearly always greater than 50,000 copies/mL,
and often greater than 100,000 copies/mL [119,128]. The sensitivity of this
assay during acute HIV-1 infection was 100% in a recent study, with a spe-
cificity of 97% [128]. It should be noted, however, that false-positive plasma
viral loads (usually with less than 5000 copies/mL) have occurred and can
lead to misdiagnosis [68,129]. Although these tests are performed more
easily, a third option for diagnosis of acute HIV-1 infection is the PCR for
HIV-1 DNA in plasma or PBMCs. The sensitivity of this assay in this situa-
tion is not known. Finally, if a diagnosis of acute HIV-1 infection is made
based on these tests, standard serologic testing should still be performed at
a later date to confirm the diagnosis.
Detection of HIV-1 infection in infants

The diagnosis of HIV-1 infection in infants, particularly in neonates, can
be challenging. Standard serologic tests, such as ELISA and Western blot,
are not useful in early infancy because of the presence of transplacentally
derived maternal antibody. This maternal antibody may be present in the
child until the second year of life [130]. For this reason, the diagnosis of
an HIV-1–infected infant usually relies on demonstrating the presence of
viral antigens, viral RNA or DNA, or by culturing the virus itself. Early and
accurate diagnosis of an infant born to an HIV-1–infected mother is impor-
tant for a number of reasons, most important of which is deciding on the
need for antiretroviral therapy and opportunistic infection prophylaxis for
the infant.
Measurement of p24 antigen in the blood of such infants has been eval-
uated as a diagnostic method. Sensitivity in this setting varies greatly with
age, and is rather poor (20%) if the infant is younger than 1 month old
[49,131,132]. Although p24 antigenemia has a relatively high specificity in
this setting, false-positive results, particularly in neonates, have been
reported when testing infants [133]. The rather poor sensitivity of p24 anti-
genemia testing has virtually eliminated its diagnostic role in infants born to
HIV-1–infected mothers. The presence of p24 antigenemia at birth, how-
ever, has been associated with the development of early and severe HIV-
1–related disease [49].
The current recommendations for testing in this situation include either
PCR testing for HIV-1 proviral DNA, or culture of the virus itself
[134,135]. Testing is recommended at birth, at 1 to 2 months of age, and
again at 3 to 6 months of age. Umbilical cord blood should not be used,
to avoid maternal contamination. Any positive test result should be con-
firmed with repeat testing from a separate blood sample.
Although considered the gold standard for diagnosing HIV-1 infection in
infants, HIV-1 culture testing is problematic in that it is labor-intensive,
requires a specialized laboratory, and may require 2 to 4 weeks to obtain
a result. Furthermore, the sensitivity of this test varies with the age of the
infant. For example, two studies using HIV-1 culture methods detected less
than 50% of the truly HIV-1–infected infants when the test was performed at
birth [49,136]. The sensitivity of this test improves greatly by 6 months of
age, and the specificity of this test also is very high. Nevertheless, the poor
sensitivity of HIV-1 culture during the neonatal period, and several techni-
cal issues, has resulted in relatively low use of this test during infancy.
Currently, the most commonly used test in diagnosing infants born to
HIV-1–infected mothers is PCR testing for proviral DNA. When compared
with HIV-1 culture in detecting HIV-1 infection in infants, PCR testing for
HIV-1 DNA is superior [48]. The timing of testing, however, can affect
DNA PCR results in infants. The sensitivity of DNA PCR below the age
of 3 months is not clear at this time. Several trials report a sensitivity of
90% for DNA PCR after the age of 3 months, but as low as 50% at 1 month
of age [48]. A recent meta-analysis evaluated 271 HIV-1–infected infants
who were evaluated prospectively to determine age-specific estimates of the
sensitivity of DNA PCR [137]. This analysis found a sensitivity of 38% if
DNA PCR was performed on the day of birth or the day after birth, but
an unexpectedly large increase in sensitivity to 93% by the second week of
life. Although further trials may clarify the true accuracy of DNA PCR test-
ing of the neonate, this test remains the current diagnostic method of choice
in this setting.
Measurement of HIV-1 plasma RNA in infants born to mothers who are
HIV-1–infected may be superior to any of these tests. At least two trials
evaluating plasma RNA levels have been conducted in this setting [138,
139]. These trials used the NASBA technique to measure plasma HIV-1
RNA, and compared this assay with HIV-1 DNA PCR testing. Both found
that plasma RNA testing had an equal or better sensitivity than DNA PCR
testing in infants. Importantly, the plasma RNA test had a much higher
sensitivity than the DNA PCR test when comparing tests in infants less than
1 month of age [139]. It should be noted, however, that false-positive plasma
RNA results, although uncommon, were reported in these studies. Although
plasma RNA testing is not yet FDA-approved as a diagnostic test, its use
in the setting of neonatal testing certainly holds promise.
Numerous advances have been made recently in the ability to detect the
presence of HIV-1 and HIV-2 infection. These assays have enabled quicker
and more efficient diagnosis in the clinical setting, and have had an impact
on therapy and survival. Nevertheless, some uncommon subtypes of HIV-1,
and certain clinical scenarios, continue to be problematic from a detection
standpoint. The diagnosis of HIV infection in these situations requires fur-
ther study.
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Clin Lab Med 22 (2002) 593–610
HIV-1 RNA and viral load

Karen Relucio, MDa, Mark Holodniy, MD, FACPa,b,*
a
Division of Infectious Diseases and Geographic Medicine,
Stanford University Medical Center, 300 Pasteur Drive S-156, Stanford, CA 94304, USA
b
AIDS Research Center, VA Palo Alto Health Care System,
3801 Miranda Avenue (132), Palo Alto, CA 94304, USA
The use of reverse transcription (RT) followed by polymerase chain reac-

tion (PCR) to detect HIV-1 RNA in serum was first described in 1988 [1].
Using plasma-associated HIV-1 RNA (viral load) as a surrogate marker
in clinical practice was first introduced in the mid-1990s, with multiple
reports describing plasma viral load quantification and the relationship of
copy number to stage of HIV disease and response to antiretroviral therapy.
Concurrently, three different methods to quantify viral load were being
developed, resulting in the first United States Food and Drug Administra-
tion (FDA)–approved RT-PCR–based assay for determining prognosis and
monitoring antiretroviral therapy in 1997, and the approval of an ultrasen-
sitive version in 1999. More recently, in November 2001, the United States
FDA approved a nucleic acid sequence-based amplification (NASBA) test
for quantifying HIV-1 in human plasma.
The current use of highly active antiretroviral treatment (HAART) has
resulted in a dramatic reduction in viral replication (viral load) with concom-
itant reductions in AIDS-defining conditions and death. The degree of viral
load reduction has made it possible to distinguish differences in potency
between antiretroviral agents and regimens and in the durability of treatment
responses. Quantification of HIV RNA in plasma is an important surrogate
marker in assessing the risk of disease progression and monitoring antiretro-
viral therapy in routine HIV clinical practice and is now a required surrogate
marker in antiretroviral clinical efficacy studies. Guidelines for intended use
and interpretation of viral load results have been recently published (see later)
[2,3]. This article reviews current methodologies, factors that affect perfor-
mance and interpretation, and intended use of HIV-1 viral load testing.
* AIDS Research Center, VA Palo Alto Health Care System, 3801 Miranda Avenue (132),
Palo Alto, CA 94304.
E-mail address: mark.Holodniy@med.va.gov (M. Holodniy).
0272-2712/02/$ - see front matter 2002, Elsevier Science (USA). All rights reserved.
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 0 8 - 2
594 K. Relucio, M. Holodniy / Clin Lab Med 22 (2002) 593–610
Methodologies
Commercially available assays
There are three commercially available assay kits to detect and quantify
plasma HIV RNA [4–6]. An RT-PCR (Standard and Ultra sensitive Ampli-
cor HIV-1 Monitor 1.0, Roche Diagnostics, NJ) and NASBA (NucliSens
HIV-1 QT, OrganonTeknika/BioMerieux, Marcy-l’Etoile, France) assays
are United States FDA approved for assessment of prognosis and antiretro-
viral response, but not for HIV-1 diagnosis (see later). The third assay uses
branched DNA ([bDNA] Versant HIV-1 RNA 3.0 Assay, Bayer Diagnos-
tics, Tarrytown, NY) and is currently available in the United States for
research-use-only applications.
In the RT-PCR assay, an RNA control of known copy number is
added to the plasma sample. HIV RNA is manually extracted from plasma
and an HIV-1 gag gene sequence is then reverse-transcribed and amplified
in a thermocycler in a single reaction tube. The resulting amplicon is seri-
ally diluted in the presence of the known copy number standard. An
enzyme-linked DNA probe is then hybridized to the amplified product and
a subsequent colorimetric reaction is performed in an automated plate
reader. The measured optical density ratio is directly proportional to the
input copy number. The current dynamic range for the standard HIV-1
Monitor assay is 400 to 750,000 copies/mL and for the ultrasensitive assay
50 to 75,000 copies/mL. Samples not in the dynamic range of either assay
either need to be diluted or need to be reflexed to the alternate assay if
quantitation is required. Recent modifications to this assay include use
of the MagNA Pure LC and AmpliPrep automated nucleic acid purifica-
tion systems and the COBAS platform, which uses robotics to facilitate
automation of RT-PCR and quantitation. The HIV-1 Monitor version
1.5, which incorporates the COBAS system and expands HIV-1 subtype
detection and quantitation, is currently under review by the United States
FDA.
In the NASBA assay, three internal standards or calibrators are added to
the patient plasma sample. NASBA uses the Boom method (guanidine thi-
ocyanate and RNA binding to silicon dioxide particles) for RNA extraction.
Extraction is accomplished manually or can be automated using the NucliS-
ens Extractor. The NASBA assay uses a three-enzyme system (AMV-RT,
RNase H, and T7RNA polymerase) to facilitate repetitive rounds of RNA
template amplification under isothermal conditions (41C). After RNA
extraction and amplification, a probe hybridization reaction with the
unknown patient RNA and the three known internal standards generate
electrochemiluminescent signals proportional to input copy number. Fur-
ther modification of the NASBA assay (NucliSens EasyQ) will include
real-time detection with molecular beacon technology. The current dynamic
range is 40 to 5,000,000 copies/mL.
K. Relucio, M. Holodniy / Clin Lab Med 22 (2002) 593–610 595
The bDNA assay does not require nucleic acid extraction and purifica-
tion. Pelleted viral particles from plasma are lysed, and then HIV-1 RNA
is captured in a microplate well. Multiple DNA probes are hybridized to
specific gene segments, namely the pol gene sequence. Alkaline phosphatase
is added, which attaches to the DNA probes, and in the presence of a sub-
strate subsequently generates a chemiluminescent reaction. The relative light
units produced are compared with an external HIV-1 RNA standard curve
and are directly proportional to the amount of RNA in the sample. The
Bayer System 340 automates the entire process after RNA capture. The
current dynamic range is from 50 to 500,000 copies/mL. In contrast to
the RT-PCR and NASBA reactions, which are template (plasma RNA) ampli-
fication assays, the bDNA assay amplifies the signal from the RNA-DNA
hybridization reaction.
Although all the commercially available assays described previously
measure the same HIV RNA template, the copy numbers derived from
each of these assays are not equivalent because of the differences in assay
methodologies and inefficiencies of sample preparation [7]. Laboratorians
should be advised that they should not interchange assays when monitoring
patients.
Other technologies
A new assay, not yet widely available, is the LCx HIV RNA Quantitative
Assay (Abbott Laboratories, Abbott Park, IL). This assay is a competitive
RT-PCR assay that uses microparticle fluorescent immunoassay detection
on an LCx analyzer. Twenty-one patient samples and controls can be anal-
yzed in an assay run. An external standard curve is used to generate patient
sample copy number. The dynamic range is 50 to 1,000,000 copies/mL for a
0.2-mL sample and 178 to 5,000,000 copies/mL if a 1-mL sample is used
[8]. A transcription-mediated amplification (Gen-Probe, San Diego, CA)
assay is in clinical development and being used for blood donor screening
(see later) [9].
Assay performance issues and problems in interpretation

Although commercial kits are widely available, several laboratories con-
tinue to use home brew RT-PCR assays for HIV-1 RNA detection. All of
the reagents necessary for RT-PCR can be purchased separately. These
reagents can be assembled into viable assays that produce a quantitative
result. Reagent quality control and assay performance are difficult to con-
trol, however, in this format. Proficiency programs have been established
for HIV research laboratories in the Adult and Pediatric AIDS Clinical
Trials Groups through the Virology Quality Assurance program and for
clinical molecular pathology laboratories through the College of American
Pathology. Most laboratories participating in these programs are using

commercially available kits for HIV RNA quantitation.
First-generation versus later-generation assays

Early commercial assays had lower limits of detection in the 400 to 500
copies/mL range. Second- or third-generation assays, which have a lower
limit of detection of around 50 copies/mL, were developed as a result of
modifications in sample preparation and altered chemistry. This enhanced
sensitivity has lowered the upper quantitation limit to 75,000 copies/mL for
HIV-1 Monitor Ultra/Direct and 500,000 copies/mL for Versant 3.0. Some
research-associated assays have been further modified to have sensitivities
below 10 copies/mL [10]. There is increased variability between results once
viral loads reach levels of less than 200 copies/mL, however, and certainly
with less than 50 copies/mL, because of the lack of template for efficient
amplification reactions.
Variability between HIV groups and subtypes

The HIV-1 is categorized into major (M), outlier (O), and N groups.
Group M is further divided into subtypes (clades) A to J [11]. Subtypes are
distinguished by sequence analysis of the HIV-1 envelope, pol, and gag
genes. These subtypes have wide geographic diversity. Subtype B is most
common in North America and Western Europe, whereas subtypes A and
C are found predominantly in Africa. Subtype E occurs in Thailand. Other
M clades are found primarily in Africa and Asia.
Group O HIV-1 strains have been detected primarily in western African
countries. Case reports have reported group O strains occurring in Europe
and more recently in the United States. The first Group N HIV-1 strain was
found in Cameroon in 1998. Viruses from this particular group are exceed-
ingly rare [12].
Viral load assays were developed using a group M, subtype B strain as
the prototype. The HIV-1 Monitor version 1.0 assay has had difficulty
amplifying or quantifying subtype A and E virus and group O. The NASBA
assay also had problems quantitating subtype G and group O [13,14]. The
bDNA assay can reliably quantitate the M clades, but has difficulty with
group O. Although not United States FDA approved, the new HIV-1 Mon-
itor version 1.5 detects all M subtypes effectively, but not group O [15]. The
LCx HIV RNA assay reliably quantitates subtypes A to G and group O [9].
There may be clinical situations in which viral load results are not consistent
with CD4 count or the clinical impression, or are falsely negative. The
laboratorian may need to consider that the assay they currently use may not
detect or reliably quantitate a non-B subtype, which could account for such
discordant results.
Sample handling and precision issues

Important aspects of sample handling for viral load quantification have
been described in several studies. Small but sometimes significant differences
in viral load levels are seen depending on method of viral load assay; type of
blood component (plasma or serum); and type of anticoagulant used. Plas-
ma usually yields a higher copy number compared with serum. Heparin can
inhibit the RT-PCR reaction. Quantitation can be achieved with NASBA
and bDNA assays using samples collected in the presence of heparin. Blood
for viral load testing should be consistently collected in ethylenediaminete-
traacetic acid tubes, because it has been shown that plasma viral load sam-
ples stored in ethylenediaminetetraacetic acid have the highest copy
numbers per milliliter and viral load stability [16,17]. Finally, some viral
load decay has been found in whole blood samples that have not had plasma
separated and stored at 20C or 80C within 8 hours of collection. Plas-
ma samples seem to have stable viral loads after one or two freeze thaw
cycles and can be stored for up to 6 months at 80C [18]. Most package
inserts for commercially available kits require plasma separation from whole
blood within 6 to 8 hours of collection and freezing at a minimum of 20C.
Most studies have found that the intra-assay variability for a particular
sample is less than 0.2 log10 copy/mL [19]. The coefficient of variation
increases for interassay variability and may be kit lot, assay type, and oper-
ator dependent. Variability in viral load is also greater with lower copy num-
ber and with greater times in between measurements [20]. Average variance
in pooled studies was less than 0.5 log10/mL after either multiple time points
over 48 hours or two time points over 2 weeks [21,22]. There seems to be no
diurnal and little intrapatient variation over time in those patients who are
clinically stable. Finally, high concentrations of interfering substances (ie,
antiretroviral drugs, hemoglobin, triglycerides, etc) do not seem to affect
viral load quantification (bDNA) [23].
Issues in clinical interpretation

Gender and racial differences
Several studies have found statistically significant differences (0.1 to 0.25
log10/mL) in baseline viral load between men and women, after controlling
for CD4 count and other variables [24]. The studies are conflicting as to
whether this difference is significant in women based on risk factor. The viral
load results between these studies may have varied because of the medium
used (serum versus plasma); assay used; or length of sample storage before
analysis. Study results are also conflicting as to whether the risk of disease
progression to AIDS is higher in women when viral load and CD4 are con-
trolled. There are no significant differences in long-term virologic responses
to HAART between men and women, although women may achieve
undetectable viral loads more quickly [25]. In addition, the few studies that
have attempted to address viral load variability between races or ethnicities
have found no significant differences [26].
Neonatal and pediatric viral load

Neonatal viral load levels may vary, depending on whether HIV infection
occurred in utero or perinatally. Babies infected in utero were found to have
a significantly higher median viral load at birth and 1 month after birth,
compared with babies infected during the peripartum period [27–29]. In gen-
eral, infants’ viral load increases rapidly over the first 1 to 2 months, fol-
lowed by a slow decline over the next 2 years, and viral loads of greater
than 100,000 copies/mL are maintained during the first year of life [94]. In
contrast, adults experience a reduction in viral load concomitant with sero-
conversion and development of a cellular immune response.
Despite the differences in viral load decline seen in infants after primary
infection, the prognostic value of viral load in infants and children is similar
to that seen in adults. In the absence of therapy, a high viral load level at 4
weeks of age that remains high at 6 months is highly predictive of disease
progression within the first 2 years. Viral loads of greater than 105 copies/
mL correlate with presence of syncytial-inducing strains of virus, growth
retardation, encephalopathy, development of opportunistic infections, and
increased risk of mortality [29,30]. Children started on antiretroviral therapy
or changed to a new regimen tend to have the same magnitude of viral load
reduction when compared with adults [31].
Viral load and risk of transmission

Heterosexual transmission is highly correlated with the level of viral load
in the HIV-infected partner, with significantly higher rates of transmission
occurring for every log10 per milliliter increase in viral load. Female-to-male
and male-to-male transmission rates are similar when viral load levels are
controlled for. Finally, the risk of sexual transmission is unlikely when the
viral load is less than 1500 copies/mL [32,33]. The risk of perinatal transmis-
sion is also highly correlated with viral load levels. In the absence of treat-
ment, the risk of transmission is significantly greater for every log10 per
milliliter increase in maternal viral load [34]. Transmission is also associated
with breast-feeding and mastitis, and maternal cervical HIV DNA load and
genital ulcer disease, independent of maternal plasma viral load [35]. Perina-
tal transmission is extremely unlikely when pregnant women have undetect-
able plasma viral loads while receiving HAART. The thresholds at which
transmission does or does not occur have not been conclusively established.
Viral load in primary HIV-1 infection

The HIV RNA is detectable in primary HIV-1 infection well before sero-
conversion, and has been detected within 2 weeks of infection [36]. Viral
load levels at that time are extremely high, ranging from 105 to more than
2 · 107/mL. During seroconversion, viral load drops precipitously within
the first 4 to 8 weeks after infection, achieving levels at a range of less than
1000 to more than 100,000/mL (a 2 to 4 log10, or 100- to 10,000-fold, reduc-
tion/mL) during early chronic infection. In the Multicenter AIDS Cohort
Study (MACS), less than 2% of patients within 6 months of seroconversion
had viral load levels less than 500 copies/mL [37]. There is no significant dif-
ference in plasma viral load levels during seroconversion between those
patients who are symptomatic or asymptomatic [38]. Patients who have
symptoms during the time of seroconversion, however, have significantly
higher viral loads 6 to 12 months after seroconversion [39]. Viral load levels
remain relatively stable during the period of clinical latency in clinically sta-
ble patients, but increase gradually over the course of disease. Plasma viral
load levels are detectable throughout the course of infection in the absence
of effective antiretroviral therapy. The level of viral load after seroconver-
sion has been defined as the ‘‘set point,’’ or virologic equilibrium between
viral replication and immunologic containment of viral replication. This
steady-state level is highly variable among patients [40]. Some authors
believe that there is no true viral set point [41].
Plasma HIV viral load testing has been used with increasing frequency in
the diagnosis of HIV infection. This is particularly true in patients with
known risk factors, who have negative serum p24 antigen, negative or inde-
terminant HIV serology, or inconsistent serologic results [42]. It is important
to note that HIV viral load testing is not US FDA approved for this indica-
tion. In addition, some false-positives have been described with viral load
testing in patients who manifest acute viral symptoms [43]. In most of the
adult acute infection studies to date, however, plasma viral load was detect-
able by 4 weeks in all patients who were truly infected. During early chronic
infection (after 3 to 6 months of infection) and thereafter, it can be expected
that HIV serology is positive. The need to use plasma viral load as a diag-
nostic test is unnecessary. Diagnosis of HIV infection in newborn infants
can be problematic. In the absence of HIV infection, HIV antibody tests can
be positive for up to 12 months after birth, because of maternal antibody
transfer. In neonatal cases, either plasma viral load or peripheral blood
mononuclear cell DNA PCR has been used with greater frequency in the
diagnosis of HIV infection. Plasma viral load seems to be more sensitive
compared with DNA PCR. Plasma viral load may be positive at birth after
in utero infection, but the sensitivity after peripartum infection is low. Plasma
viral load is almost always positive, however, 4 weeks after birth [44].
Other compartments
Viral load has been found in almost all body fluids and compartments. In
general, viral load in plasma is usually higher when compared with levels
in these other fluids [45]. The full relationship between blood and other
compartment viral loads has yet to be elucidated. The lack of detection

within a compartment or discrepancies between compartments may be the
result of an inability to detect viral load because of inhibitory factors
found in these body fluids that result in an inability successfully to extract
RNA or amplify nucleic acid.
Viral load in semen correlates with blood plasma viral load, but not CD4
counts. In a small number of patients analyzed with serial semen samples,
seminal fluid viral load also increased over time in those patients who pro-
gressed to AIDS [28]. Reductions in seminal viral load are also seen follow-
ing antiretroviral therapy. The magnitude of reduction seems dependent on
the potency of the regimen [46]. Although many men achieve undetectable
viral load in semen after initiation of HAART, many men still maintain sig-
nificant levels of viral load in seminal plasma [47]. Viral load is also detected
in cervicovaginal lavage fluid and within cervical tissue at lower levels than
in blood plasma, and detection is correlated with higher plasma viral loads
[48]. Although blood viral load is not affected by menstrual cycle phase, cer-
vicovaginal lavage viral load level seems to be lower in the follicular phase
and highest during the menstrual phase [49,50]. Viral load in cervicovaginal
lavage has also been shown to decline significantly with initiation or change
in antiretroviral therapy [51]. Co-infection with other sexually transmitted
diseases results in higher semen and cervicovaginal lavage HIV viral load
levels [52]. HIV-1 viral load has been detected in breast milk in a significant
number of infected women, and did not seem to change over time. Breast
milk viral load is significantly higher in women with HIV-infected infants
than in those with uninfected infants and is significantly correlated with
mastitis [53]. Women with postpartum mastitis are probably more likely
to transmit HIV to uninfected newborns than women without mastitis [54].
There is a complex relationship between plasma and cerebrospinal fluid
(CSF) viral load levels. Plasma levels tend to be more than 1 log10/mL greater
than CSF levels, but it remains controversial whether CSF viral load re-
flects only diffused plasma viral load, or is indicative of brain or central ner-
vous system production. There was a correlation between an increase in
CSF neopterin levels and time-dependent increase in CSF viral load levels
(0.5 log10/mL over 3 years), which suggested that increasing levels of
immune activation were occurring to account for endogenous central nerv-
ous system viral production. There is conflicting evidence on whether higher
CSF viral load levels are correlated with the presence and severity of AIDS
dementia complex, AIDS diagnosis, CD4 count less than 200/mm3, lym-
phocyte pleocytosis, and presence of central nervous system opportunistic
infections. Following initiation of HAART therapy, there are profound
reductions of CSF viral load, which parallel those reductions seen in plasma
in terms of magnitude [55].
Lastly, viral load has rarely been detected in saliva and stool, and has not
been reported in sweat or tears [56]. Higher saliva levels, compared with
plasma, have been reported in some patients and may be the result of oral
inflammatory processes or significant lymphocyte counts in saliva [57]. The

clinical significance of this for patients or exposed health care workers is
unclear.
Covariables
Numerous small studies have attempted to evaluate the impact of comor-
bidities, procedures, vaccination, and acute infections regarding their impact
on HIV viral load. Hemophiliacs are more likely to have higher viral loads
and clinical progression to AIDS than individuals without hemophilia [58].
Viral load was found to increase by 0.5 log10/mL following tuberculin skin
testing in drug-naive patients with a positive test [59]. Several studies inves-
tigating effects of vaccination, primarily in patients who were untreated or
had suboptimal treatment with antiretrovirals, have reported that immuni-
zation with influenza and other vaccines or treatment with exogenous inter-
leukin-2 produce transient increases (>0.5 log10/mL) in viral load [60,61].
More recent studies in adults and children have indicated, however, that
vaccination with influenza or diphtheria-pertussis-tetanus vaccines or
administration of interleukin-2, in the presence of HAART therapy, did not
produce significant increases in viral load [62–64].
Several co-infections have also been studied for their impact or associa-
tion with viral load. Co-infection with oncogenic strains of cervical human
papillomavirus, oral Candida, and Kaposi’s sarcoma–associated herpes
virus DNA in peripheral blood mononuclear cells of patients with or with-
out Kaposi’s sarcoma correlate with higher plasma HIV viral loads [65–68].
Acute, significant increases in HIV viral load have been demonstrated with
acute co-infections from Pneumocystis carinii pneumonia, cytomegalovirus,
Mycobacterium avium complex (MAC), and plasmodium falciparum, which
subsequently returned to baseline levels on initiation of effective therapy for
the acute co-infection [69–71]. This phenomenon seems to be consistent in
both adults and children with HIV infection. The interrelationship between
hepatitis C virus and HIV viral load is less clear. Hepatitis C virus viral load
is higher in HIV co-infected patients compared with those patients with
only hepatitis C virus infection. It is not clear whether the converse is true.
Hepatitis C virus clearly affects HIV pathogenesis and progression of HIV
disease [72]. HAART does not affect hepatitis C virus viral load [73].
Viral load in relation to pathogenesis and antiviral efficacy studies

Several studies have attempted to analyze whether there are correlations
between viral load and immunologic markers. Viral load levels seem to be
inversely correlated with CD4 count and cytotoxic (CD8) T-cell responses
[74]. In one study, the number of activated T cells (CD38þ) was found to
be a stronger predictor of disease progression than viral load level [75]. Viral
load levels do not correlate with regulated on activation normal T-cell
expressed and secreted (RANTES) or macrophage inflammatory protein

1-alpha (MIP-1a) levels, but correlate with other cytokines, such as tumor
necrosis factor levels [76,77].
Many studies have demonstrated the rapid and sustained declines in viral
load after initiation of HAART. In almost all treatment-naive patients,
adherence to a regimen of HAART results in a 2 to 3 log10/mL reduction
in viral load and a viral load of less than 500 copies/mL, and is usually
achieved within 4 to 8 weeks of initiating therapy. This may be contingent
on the baseline viral load level, the potency and number agents used in the
regimen, and treatment experience of the patients. Viral load generally con-
tinues to decline to undetectable levels by 16 to 20 weeks, defined as less
than 50 copies/mL. Reaching a viral load of less than 50 copies/mL, how-
ever, may take as long as 6 months. Antiviral efficacy data, which is based
on several clinical trials, strongly suggest that lowering plasma HIV RNA to
undetectable levels (<50 copies/mL) is associated with a more complete and
durable viral suppression, compared with reducing HIV RNA to levels
between 50 and 500 copies/mL [3]. The nadir level of viral load has been
found to correlate with durability of treatment response [78]. Those patients
who did not achieve a viral load of less than 50/mL were more likely to dem-
onstrate virologic failure within the first year than those who achieved unde-
tectable levels [79].
Several studies are currently investigating whether treatment interruption
is a clinically useful strategy. It is known that discontinuation of HAART in
those patients with undetectable viral load can result in the return of viral
load to pretreatment levels or can result in viral loads that overshoot the
baseline level by as much as 10-fold [80,81]. Reinitiation of therapy after
acute discontinuation results in a virologic decline back to undetectable lev-
els in most patients [82]. Recent studies suggest that intermittent treatment
strategies in such patients may improve immunologic recognition of HIV
infection [83]. Other reasons for virologic rebound include development of
drug resistance, poor adherence, and drug interactions that reduce the effec-
tiveness of HAART. Treatment interruption in those patients with multi-
drug-resistant virus can result in further increases in viral load, with
reversion of resistant virus to wild-type (drug susceptible and hence more
cytopathic) strains, resulting in significant declines in CD4 count [84].
Finally, viral load may also not decline in a predictable fashion in some
patients. This may be related to transmission of drug-resistant strains in
recently infected patients [85]. The slope of viral load decline after initiation
or change in HAART regimen is an important measure to follow. In choosing
the next appropriate regimen, the use of resistance testing, instead of
clinical impression, may offer a virologic advantage in previously treated
patients.
There is controversy surrounding transient increases in viral load from
undetectable levels or fluctuations while on antiretroviral therapy, called
‘‘blips,’’ which represent evolution of resistant strains or release of virus from
blood and lymphoid tissue reservoirs. Recent data suggest that these viral
load blips do not result in short-term (1 year) virologic failure [86]. Addition-
ally, patients who have only modest reductions in viral load (1 to 2 log10/mL)
after 8 weeks of therapy, and who have persistently detectable viremia, can
still have improved clinical outcomes. There may be clinical benefits that are
still achievable despite continued detectable viral load [87]. Other discordant
responses have been described. In some patients, CD4 counts have increased
in the presence of continued viremia [88]. In other cases, CD4 counts have
continued to decline despite undetectable plasma viral loads. Further studies
are required to explain these discordant responses.
Finally, an undetectable plasma viral load does not necessarily indicate
that viral replication in lymphoid tissue or other compartments is nonexis-
tent. Recent pathogenesis-based studies have described the presence of
infected, replication-competent cells in blood or lymph nodes despite unde-
tectable plasma viral load and the same level of viral replication in lymphoid
tissues regardless of plasma viral load level [89,90].
HIV treatment guidelines and viral load

The 2001 Department of Health and Human Services (DHHS) and Inter-
national AIDS Society-USA HIV Treatment Guidelines, among other inter-
national guidelines, strongly recommend monitoring of HIV-1 viral load,
CD4þ cell count, and clinical condition of the patient to make decisions
in initiating or changing antiretroviral therapy [2,3]. Viral load and CD4þ
count provide the physician important information on the patient’s risk of
clinical progression, based primarily on data obtained from the MACS
[37]. In general, the MACS study, among others, has found that the viral
load level after seroconversion is highly predictive of disease progression
to AIDS and death. Those patients with viral load levels above 50,000/mL
are at greatest risk of clinical progression and death. Patients with viral
loads of less than 500 copies/mL had little risk of progression, even after 10
years of infection. Some of these patients have been found to be long-term
nonprogressors with slowly replicating or defective virus, efficient immuno-
logic containment, and little or no CD4 cell decline over time.
The DHHS guidelines (www.hivatis.org.) recommend measurement of
plasma HIV RNA levels in treatment-naive patients at the time of diagnosis
and every 3 to 4 months thereafter. They further recommend initiation of
HAART when the CD4 count is less than 350/mm3 or the viral load is
greater than 30 to 55,000 copies/mL. In patients anticipated to receive anti-
retroviral medications, a pretreatment baseline viral load measurement is
recommended before starting therapy and again at 2 to 8 weeks after, which
allows the clinician to determine whether the chosen antiretroviral regimen
is effective. It has also been recommended that viral load testing be repeated
every 3 to 4 months thereafter to evaluate the continuing effectiveness of
therapy. If HIV RNA remains detectable in plasma after 16 to 20 weeks of

therapy, or if viremia is detected after suppression to undetectable levels, the
DHHS guidelines suggest repeating the HIV-1 viral load test to confirm the
result and to consider a change in therapy.
The first set of published DHHS guidelines used the branched chain
DNA assay to determine which viral load levels serve as a threshold for
initiating or changing therapy, based on the MACS study. Subsequent
guideline editions have incorporated the RT-PCR assay, based on later
studies. As a result, a viral load threshold range (RT-PCR values are
2- to 2.5-fold greater than bDNA values, as indicated previously) is recom-
mended based on interassay variability. Because of these differences, it is
recommended that confirmatory plasma HIV RNA levels be measured using
the same technique, to ensure consistent results. In general, viral load and
trends in viral load are believed to be more informative for guiding decisions
regarding antiretroviral therapy than are CD4þ T-cell counts.
Use of HIV-1 viral load testing in blood donor screening

Historically, only serologic methods to detect HIV-1 antibodies were
used to detect HIV-1 infection during blood donor screening. Testing was
expanded a few years ago to include supplemental HIV-1 p24 antigen detec-
tion. The p24 antigen neutralization assay, however, detected fewer than
expected HIV-1 antigen–positive units since the test was introduced [91].
Under an investigational new drug (IND) exemption granted by the United
States FDA, viral load testing for HIV using high-sensitivity, qualitative
nucleic acid amplification testing (NAT) was introduced for blood donor
confirmatory screening in 1999. NAT uses RT-PCR and transcription-
mediated amplification technologies, and can detect HIV-1–containing blood
units that are missed by traditional serologic assays [92]. Compared with
the risk of HIV transmission estimated at the peak of the transfusion-
AIDS epidemic between 1982 and 1984, the addition of NAT into testing
algorithms reduces the risk of HIV transmission significantly. NAT assays
also reduce the HIV window period by 10 to 15 days, compared with HIV
antibody testing alone.
To reduce cost and increase efficiency, NAT is performed in the donor
screening setting on ‘‘mini pools’’ of 16 to 24 donated samples. A pooled
sample with a positive reaction in an assay is resolved by performing the
assay on individual specimens, and then the positive specimen is further
retested by single virus discriminatory tests for final resolution and confir-
mation. The current approach of testing pooled blood donor samples is not
optimal, however, because sample dilution reduces assay sensitivity and pos-
itive tests in a pool may be difficult to identify. It is expected that upgrading
to single donation testing with NAT will be phased in as soon as technically
and financially feasible.
In the blood and plasma donor settings, NAT can also resolve false-
positive Western blot and p24 antigen neutralization results, and discrepant
or indeterminate antibody or p24 antigen test results. Most HIV antibody,
enzyme immunoassay–reactive donated units are either negative or indeter-
minate on supplemental Western blot testing, and are very rarely attribut-
able to primary HIV infection. In a recent study, NAT confirmed HIV-1
infection in all Western blot–positive donors, and ruled out infection in all
donors with indeterminate Western blot results [93]. Using NAT could allow
reinstatement of donors who would otherwise be permanently deferred, and
reassure those individuals of a negative result.
Summary
Viral load monitoring has become the standard of care in clinical practice
to assess risk for disease progression and to monitor treatment response.
Furthermore, viral load monitoring has contributed greatly to the under-
standing of HIV disease pathogenesis and response to various antiretroviral
regimens, and has broadened its applications to include blood bank screen-
ing. The assays that are currently available are more sensitive, precise, and
robust. There is now a better understanding of their limitations and the clin-
ical scenarios and assay performance issues that result in variations of viral
load results.
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Clin Lab Med 22 (2002) 611–635
HIV-1 replication cycle

Monique R. Ferguson, PhDa,*, Daniel R. Rojo, PhDa,
Jana J. von Lindern, BSb,
William A. O’Brien, MD, MSa,b
a
Department of Internal Medicine, Division of Infectious Diseases, University of Texas
Medical Branch, 301 University Boulevard, Galveston, TX 77555–0435, USA
b
Department of Microbiology and Immunology, University of Texas Medical Branch,
301 University Boulevard, Galveston, TX 77555–0435, USA
Acquired immunodeficiency disease is caused by HIV. HIV types 1 and 2

are members of the Lentivirus genus of the Retroviridae family. HIV-1 is
found throughout the world, whereas HIV-2 is still found predominately
in West Africa. Although these viruses have replication processes character-
istic of the Retroviridae family, marked by single-stranded RNA genome,
and replication through reverse transcription and integration, HIV is a new
pathogen that seems to have emerged in the twentieth century, likely from
cross-species infection from chimpanzees in Africa [1,2]. In contrast to other
retroviruses, however, HIV has evolved a variety of accessory genes that can
modulate HIV replication. Some of these genes seem to confer abilities to
establish persistent infection and to control exuberant replication that may
more rapidly cause disease and death in the host.
Epidemic HIV infection exploded worldwide in the late 1970s and 1980s.
AIDS was described in 1981 [3,4], and HIV was identified and cloned in
1983 [5,6]. Much of the information about the molecular biology of HIV
that rapidly accumulated after the cloning of HIV was based on studies with
laboratory-derived HIV clones adapted for efficient replication in trans-
formed T-cell lines. Although these experimental systems allowed rapid
progress in the understanding of the replication of these newly identified len-
tiviruses, some of the information obtained from these studies in fact was
misleading, because these strains have characteristics and replication prop-
erties that are different from those found in most primary HIV strains. In
This work is supported by the James McLaughlin Fellowship Fund and by Public Health
Service grants R24 59656, R01 38414, and R21 46250.
* Corresponding author.
E-mail address: mrfergus@utmb.edu (M.R. Ferguson).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 1 5 - X
612 M.R. Ferguson et al / Clin Lab Med 22 (2002) 611–635
fact, some of these early clones were deficient in some of the accessory genes
or had gene products that exhibited different properties from those in pri-
mary strains.
An important aspect of HIV replication is the requirement for cellular
activation for efficient replication. This activation results in increases in effi-
ciency of reverse transcription, integration, and virus gene expression, in
part caused by induction of cellular proteins that are involved in HIV rep-
lication. The persistence of HIV replication in individuals in whom chronic
infection has established, and the limited effect of currently available antire-
troviral therapies, make the continued investigation of HIV replication crit-
ical for determining potential new targets for novel antiretroviral therapies.
Molecular biology of HIV-1

Virion and genome structure of HIV-1
The HIV-1 virion is spherically shaped and approximately 100 nm in
diameter. The core of the virion is composed of nucleoproteins complexed
with two copies of 9-kilobase single-stranded genomic RNA molecules, and
RNA-dependent DNA polymerase (reverse transcriptase [RT]) [7]. A lipid
bilayer envelope traversed by glycoprotein spikes surrounds this core
(Fig. 1) [8]. Protein products have been identified for at least 10 open read-
ing frames within the genome of HIV-1; however, only three genes are com-
mon to all retroviruses. Two of these genes, gag and env, encode structural
Fig. 1. Structure of the HIV-1 virion. The virion proteins indicated are labeled by size.
NC ¼ nucleocapsid; MA ¼ matrix; PR ¼ protease; RT ¼ reverse transcriptase; IN ¼ integrase;
CA ¼ capsid; TM ¼ transmembrane. The virion also contains Vpr and Nef (not shown).
M.R. Ferguson et al / Clin Lab Med 22 (2002) 611–635 613
proteins, whereas the pol gene encodes enzymatic proteins necessary for re-
verse transcription, integration, and proteolytic processing of viral proteins.
The gag gene product is a 55-kd polyprotein (p55Gag), which is posttrans-
lationally cleaved and modified to form the mature capsid protein matrix
(MA, p17), nucleocapsid (NC, p7), and capsid (CA, p24), as well as the
smaller core proteins p1, p2, and p6. Together p24 and p7 form the major
components of the virus core in which p24 outlines the core and p7 is
directly associated with the RNA genome [9]. The protein domains of Gag
each play different roles in the life cycle of the virus. During assembly of the
virus in host cells, the N-terminal MA domain of Gag targets the protein to
the plasma membrane and serves to incorporate the membrane-spanning
envelope proteins into the newly forming virions [10,11]. After entry of the
virus into cells, MA dissociates from the membrane to expose a nuclear
localization sequence, which allows the transport of viral proteins into the
nucleus and enables HIV-1 to replicate in cells [12]. MA is a myristoylated
protein that facilitates targeting of Gag-Pol polyproteins to the host cell
membrane, and allows association of Gag and Pol with budding progeny
virions [13,14]. The C-terminal NC domain of Gag contains two copies of
the highly conserved cysteine-histidine motifs, which are influential in recog-
nition and incorporation of viral RNA into the particle [15–17]. Removal of
this region during splicing of most messenger RNAs results in omission of
subgenomic viral transcripts from virus particles. The CA domain is located
in the central region of the gag gene (Fig. 2); CA may play a role in particle
assembly [18,19] and packaging of the cellular host protein cyclophilin A
[20,21]. Morphologically, mature CA (p24) forms the distinctive conical core
of the virus that encapsulates the viral RNA-protein complex, and may
solely provide structural stability of the virion. Finally, a small proline-rich
protein, p6, is a C-terminal cleavage product of Gag, which may be impor-
tant for detachment of maturing virus particles and for packaging of other
viral proteins within the virion.
The pol gene encodes highly conserved proteins that perform enzymatic
functions required for HIV-1 DNA synthesis (RT, p66–p51); integration
(IN, p32); and processing of polyprotein precursors (PR, p10) [22]. The pol
Fig. 2. Proviral genomic organization of HIV-1. Location of regions encoding precursors for
known virion proteins is shown.
mRNA is an unspliced genomic mRNA, which is first translated into a Gag-

Pol fusion protein (p160Gag-Pol) [9]. Both Pol and Gag are encoded by the
same mRNA but translated into different proteins through initiation in
alternative reading frames [9]. Viral PR is responsible for processing the Gag
and Gag-Pol precursors. RT is responsible for catalyzing conversion of viral
RNA into DNA (reverse transcription) [23], whereas IN plays a key role in
inserting viral DNA into the host cell chromosome [24,25]. All antiretroviral
drugs approved for use in the United States have targeted products of the
pol gene. The first five drugs approved were directed toward RT, and cur-
rently 10 of 16 approved drugs target RT, whereas the remaining 6 interfere
with the function of PR.
The env gene encodes a glycosylated precursor protein (gp160), which is
cleaved to an extracellular glycoprotein (gp120) involved in recognition and
binding to target cell receptors, and a smaller hydrophobic transmembrane
protein (gp41) involved in membrane fusion. gp120 is noncovalently linked
to gp41, and can be shed from the surface of the virion [26]. These proteins
are crucial for recognition, binding, and entry into target cells.
Accessory genes
In HIV-1 and in the primate lentivirus simian immunodeficiency virus
(SIV), there are complex regulatory systems not found in other retroviruses,
directed by nonstructural proteins encoded in six overlapping open reading
frames, termed accessory genes (see Fig. 2, Table 1). These proteins seem to
play an important role in the viral life cycle. Some of these gene products
also confer adaptive properties that can promote viral persistence. The pre-
cise roles of these novel genes in the virus life cycle, however, are not entirely
understood. Two of these accessory HIV-1 genes (tat and rev) are required
for replication in all host cells [27,28]. Mutations in the other four genes
have variable effects, depending on the experimental system, and their roles
are less defined.
HIV-1 life cycle

Replication of HIV-1, as seen with other retroviruses, involves reverse
transcription of the RNA viral genome to form a double-stranded DNA
intermediate, which can be integrated stably into chromosomal DNA,
resulting in the provirus (Fig. 3). HIV-1 genetic sequences are present for the
lifetime of the cell. The early steps of replication lead to establishment of
infection in target cells, but the virus may be dormant in some cell types, and
require activation for viral gene expression. The late phase of replication
includes transcription and processing of viral RNA, translation and modifi-
cation of viral proteins, and release of progeny virions by budding through
the cell plasma membrane.
Table 1
Accessory proteins of HIV-1
Gene Product Function
tat Transcriptional transactivator Transcriptional activator, which up-regulates
all viral proteins
rev Regulator of virus protein Nuclear RNA export factor, which facilitates
expression the export of unspliced and singly spliced
viral mRNAs to the cytoplasm
nef Negative factor? Numerous effector functions: down-
regulation of cell surface CD4 and MHC-1;
enhance virion infectivity; effects on cellular
signal transduction and activation
vpr Viral protein R Weak transactivator; nuclear import; G2
arrest in cell cycle
vif Virion infectivity factor Enhances infectivity of viral particles.
vpu Viral protein U Enhances virion release from cell; selective
degradation of CD4 in the ER
From Wang WK, Chen MY, Chuang CY, Jeang KT, Huang LM. Molecular biology of
human immunodeficiency virus type 1. J Microbiol Immunol Infect 2000;33:131–40; with
permission.
Binding and entry

The entry of HIV-1 into target cells is through fusion of the virus enve-
lope with the target cell membrane. The initial step of entry involves a
high-affinity binding of the surface envelope glycoprotein gp120 with the cel-
lular receptor, CD4, which is present on the surface of a subset of helper T
lymphocytes and on monocyte-macrophages [9]. This results in conforma-
tional changes in gp120 and exposure of new epitopes that allow interaction
with a coreceptor. In the mid-1990s, the a and b chemokine receptors were
demonstrated to be HIV-1 coreceptors. A series of incompletely character-
ized processes ensue, including insertion of the N-terminal fusion peptide of
gp41 into the target cell membrane and interaction of adjacent gp41 heptad
repeats, which bring viral and cellular membranes into proximity so that
fusion can occur. A more detailed description of HIV-1 entry is found else-
where in this issue.
Reverse transcription
Following receptor-coreceptor interactions and subsequent fusion
between viral and cellular membranes, the viral core enters the cytoplasm
of the cell. Once inside the cell, virion-associated RT directs synthesis of a
linear double-stranded DNA copy of the single-stranded viral genome in
a complex process known as reverse transcription (Fig. 4). The process of
reverse transcription begins with annealing of transfer RNA (tRNAlys) to
a specific primer binding site at the 5¢ end of the viral genome. Replication
proceeds through the 5¢ R region and then stops (see Fig. 4a). RNA
Fig. 3. Retrovirus life cycle. Following binding to the surface of host cell, the viral core enters
by virus-cell membrane fusion. A double-stranded DNA copy is synthesized from genomic
RNA by virion-associated reverse transcriptase. The viral core is transported to the nucleus,
and the provirus is formed by integration into the host genome. Viral RNA can be expressed
from the provirus, typically following cell activation, and viral proteins are synthesized on host
ribosomes. Progeny virions are formed by budding through the membrane of infected cells.
(From Turner BG, Summers MF. Structural biology of HIV. J Mol Biol 1999;285:1–32; with
permission.)
upstream of the primer binding site is then degraded by the RNAseH activ-
ity of RT, and the nascent viral DNA ‘‘jumps’’ and reanneals at the 3¢ R
region (see Fig. 4b). DNA synthesis then proceeds until a complete minus
strand is synthesized (see Fig. 4). During minus strand synthesis, RNAseH
activity of RT degrades the RNA component of the RNA-DNA hybrid
leaving the portion that is complimentary to the 3¢ terminus to serve
as a primer for positive-strand DNA synthesis (see Fig. 4c), which proceeds
after a second template jump (see Fig. 4e). The complete double-stranded
viral DNA copy contains duplicated terminal U3RU5 regions (termed long
Fig. 4. Proposed mechanism for reverse transcription and strategy for polymerase chain
reaction analysis. RNA (thick lines) is converted to double-stranded full-length DNA. The
tRNA primer binds to the primer binding site (solid circle) and primes minus strand
polymerization to the end of the R (repeat) region to from RU5 DNA. RNA upstream of the
primer binding site is degraded by RNAseH activity found in reverse transcriptase, and the
RU5 DNA undergoes the first template switching event by hybridizing with R sequences at
the 3¢ end of the RNA. There is a second priming event and a second template switch with
degradation of the RNA component of RNA-DNA hybrids during the course of viral DNA
synthesis. U5 and U3 sequences are duplicated during reverse transcription to form the long
terminal repeats found in each end of the full-length viral DNA. The right side of the figure
shows the predicted reactivity of various polymerase chain reaction primer pairs for analysis of
DNA intermediates of reverse transcription.
terminal repeats [LTRs]), which are necessary for subsequent integration

[29]. Reverse transcription occurs within a viral preintegration complex
comprised of the Gag matrix protein (p17), Vpr [12,30], and integrase [31]
that is actively transported to the cell nucleus.
Genetic variation
A hallmark of HIV-1 replication is the emergence of dramatic genetic
variability, which results in rapid viral genetic evolution. This can give rise
to immune escape variants, drug-resistant variants, and variants with the
ability to replicate more efficiently [32].
There are two mechanisms that contribute to viral genetic variation. One
is the introduction of mutations as a result of nucleotide misincorporation
by the error-prone RT, and the other is by recombination resulting from
template strand transfers during reverse transcription [33,34]. Because the
RT enzyme lacks proofreading ability, nucleotide incorporation errors are
manifested in the progeny virions (see Fig. 4). The mutation rate of 10)5 (one
mutation in every virus each replication cycle), combined with a high rep-
lication rate (estimated production of over a billion new virus particles
every day in patients with AIDS), means that every nucleotide position in
the virion can be changed multiple times every day [35]. Because mutations
are random, most are lethal, and others are benign. Occasionally, mutations
that confer a growth advantage occur, and through selection become a pre-
dominant genetic species. The virus population evolving from this selec-
tion is termed the quasispecies, which are genetically distinct but related
virus strains arising from one or a few progenitor strains.
Recombination has also contributed to the heterogeneity of HIV-1. Ret-
roviruses contain two copies of genomic RNA per virion. If a cell is dually
infected with different virus strains, recombination between genomes during
RT could produce virions containing heterodimeric RNAs as a result of
template switches between the two RNA strands during minus strand syn-
thesis [36,37]. Full-length HIV cloning has shown frequent recombination
between subtypes, such as AG or BCD, and others in Africa, where different
subtypes coexist in the same population [38]. Recombination probably also
occurs between subtype B strains, but is difficult to demonstrate.
Genetic variability is driven by factors that select for new mutants, par-
ticularly in vivo, including the ability to replicate rapidly, to escape neutral-
ization or other immune clearance mechanisms, and to resist actions of
antiretroviral drugs. Persistence of HIV-1 is enhanced by viral evolution,
driven by a high replication and mutation rate, which allows Darwinian
selection of genetic variants with growth advantages. Genetic variation is
greatest in the env region [39,40] reflecting the importance of this region for
immune evasion and for cell tropism and fusion [41–43].
Integration
Following transport of the preintegration complex from the cytoplasm to
the nucleus, the viral integrase protein catalyzes integration of the full-
length viral cDNA into the host chromosome. This process is required for
replication, and involves recognition of specific sequences within the LTRs
of viral cDNA by the integrase protein, followed by colinear insertion into
chromosomal DNA, resulting in the formation of the provirus. Viral genes
are replicated along with the host chromosomal DNA, and can persist for
the life of the cell. Integrase mutants of HIV-1 do not form the provirus, and
infectious virus is not produced [44–46]. HIV-1 (and other lentiviruses) dif-
fers from other classes of retroviruses in their ability to replicate in non-
dividing and dividing cells. This requires transfer of the preintegration
complex through the nuclear pore complex, a process itself requiring pack-
aging of the HIV DNA intermediate into a specific conformation containing
a centrally located DNA overlap termed the central DNA flap [47]. Other
functions specific for replication in nondividing cells may be supplied by
some of the accessory gene products, notably Vpr.
Expression of viral proteins

The genes in the integrated HIV provirus are only expressed in cells in
which cellular transcription factors have been induced. Expression of
HIV-1 genes is directed by complex regulatory systems, which involve cis-
acting viral elements, and both cellular and viral proteins. Whereas estab-
lishment of infection is mediated largely by viral factors, the latter steps
of the HIV-1 life cycle require host factors to perform synthesis, processing,
and translation of viral mRNA on host ribosomes. Signals for initiation of
transcription are found in the 5¢ LTR region, which is formed by duplication
of the terminal ends of the viral genome during reverse transcription (see
Fig. 4). All HIV-1 transcripts are under control of a weak promotor located
on the LTR region, and gene expression is largely dependent on binding of
transcription factors to the regulatory regions. The core enhancer domain is
located upstream of the coding region and is different among various HIV-1
subtypes [48]. Interaction sites for several cellular transcription factors that
promote HIV infection have been identified in the LTR region. The newly
synthesized viral mRNA can undergo complex patterns of splicing, with
overlapping reading frames. Gene expression is sequential, and in early
infection most viral mRNAs found in the cytoplasm have undergone multi-
ple splicing events and encode mainly for Tat, Nef, and Rev. Later, the pre-
dominant mRNA species are unspliced and singly spliced RNA. This
sequence of gene expression is controlled by Rev, which regulates the ratio
of spliced and unspliced RNA through interaction with the Rev-response
element (RRE) located in the env region of the full-length transcript. Mature
HIV-1 proteins are generated from precursor peptides following cleavage by
viral and cellular proteases. The Env protein is synthesized at the endoplas-
mic reticulum, glycosylated and cleaved into gp120 and gp41 at the Golgi
complex, and directed to the plasma membrane. p55Gag and p160Gag-Pol
polyproteins are translated from unspliced RNA.
Assembly and egress

The Gag polyprotein precursor p55gag (p17, p24, p2, p7, p1, and p6)
contains the necessary elements for the assembly and release of virus-like
particles from many eukaryotic cell types. Cytoplasmic assembly of the
virions is initiated by interaction of p55gag and p160gag–pol with the pack-
aging sequence (W site) in the genomic (unspliced) RNA through p7gag.
This region is removed from all other forms of RNA by splicing, ensuring
that only complete genomes are packaged into virions. The MA protein
(p17gag) inserts into the cell membrane in trimeric form, a process that is
strongly dependent on the presence of myristic fatty acid residues on its N-
terminal domain. The nucleoprotein complex is transported to the plasma
membrane, where the membrane-bound MA mediates oligomerization
and assembly of Gag units, an intermediate that is dependent on the pre-
sence of p2 region at the C-terminus of CA. MA also associates with the
membrane-attached domain of Env and initiates budding. Budding from

the plasma membrane is promoted by late domains located close to the
N-terminus of p6. Late domains have been found in different retrovirus
Gag proteins and are thought to associate with cellular factors, such as
clathrin-associated adapter complexes, and the cellular ubiquitination
machinery [49]. After the virion is assembled and late during budding, the
final processing of the polyproteins by the viral protease takes place, to
generate the independent HIV enzymes and structural proteins. The virion
matures into an infectious particle with rearrangement of the structural
proteins condensing into a cylinder-shaped organization.
Effects of accessory genes on the life cycle

The complex regulatory system involving HIV-1 accessory gene products
has the potential to enhance and restrain viral replication. These nonstruc-
tural proteins have evolved to interact with cell replication mechanisms, and
in many cases require cellular cofactors for their effects. As the precise func-
tions of these seven accessory genes are better defined, insights in the HIV-1
replication process will be gained, particularly the interaction with cellular
factors.
The trans-activator of transcription (tat) gene encodes a 14-kd protein
using exons in two distinct regions of the HIV-1 genome, and is synthesized
early after infection [28,50]. The Tat protein enhances HIV-1 gene expres-
sion by interacting with cellular factors and binding to trans-activation–
responsive RNA sequences located in the 5¢ end of all HIV-1 transcripts
[51]. Tat transactivation may increase HIV-1 gene expression by stabilizing
mRNA transcripts and facilitating elongation [52,53].
The regulator of expression of virion proteins (rev) gene product encodes
another multiply spliced gene [27,28]. The Rev protein binds to HIV-1
RNAs through the RRE sequences located within the env gene [54–56], and
acts at the posttranscriptional level to facilitate the cytoplasmic accumula-
tion and translation of RRE-containing RNAs [57]. Consequently, Rev is
required for the expression of structural proteins, and indirectly down-
regulates its own expression, and that of Tat and Nef.
The nef gene encodes a 27-kd myristoylated phosphoprotein with no
enzymatic activity. It is present in primate lentiviruses but its functions are
still under active investigation. This protein is expressed early in the viral life
cycle and localizes in the plasma and nuclear membrane of infected cells [58].
Nef has been shown to be associated with virulence in various animal mod-
els, including AIDS-like pathologic effects in transgenic mice expressing this
molecule [59]. Contradictory results have been seen with studies of Nef
expression in various cell culture systems, and with in vivo studies. In early
studies, a functional nef gene was associated with a modest decrease in effi-
ciency of viral spread in cell culture as compared with nef-deficient viruses,
and the gene product was named negative factor (Nef) [60,61]. In other
experiments, culture of HIV-1 and SIV strains in peripheral blood lympho-

cytes (PBLs) show little differences in replication between wild-type and Nef-
deficient strains [62]. In contrast, monkeys infected with SIVmac239 strains
encoding a functional Nef protein had high levels of virus replication and
exhibited dramatic clinical progression, whereas animals infected by SIV
strains having point mutations in nef quickly reverted the mutation, giving
rise to comparable levels of virus replication [63]. Similarly, Nef-deficient
HIV-1 strains exhibited attenuated replication and a diminished T-lympho-
cyte depletion in severe combined immune deficiency (SCID) mice harbor-
ing a reconstituted human hematopoietic system [62]. HIV-1 sequences
containing deletions in nef were found in multiple blood samples over 10
years in several patients with nonprogressive HIV-1 infection, suggesting
that nef-deficient strains are attenuated [64,65], although most of these
patients later underwent clinical progression. Most of Nef effects are medi-
ated by direct interaction of Nef with host cell proteins [66]. Expression of
Nef leads to reduced expression of some cell membrane proteins including
CD4 [67] and major histocompatibility complex–I [68], which may reduce
the possibility of superinfection and protect the infected cell from a cyto-
toxic immune response, respectively. Nef also regulates expression of
CD25 (interleukin [IL]-2 receptor) and CD28, modulating the activation
state of the cells. Finally, Nef triggers apoptosis of neighboring cells by
up-regulation of fas ligand in the cell membrane [69], protecting the infected
cell from the immune system, and possibly affecting total cell counts. On the
other hand, Nef blocks apoptosis mechanisms triggered by external recep-
tors, such as Fas and tumor necrosis factor–a receptors, and also by the
internal bcl-2 pathway [70].
The viral infectivity factor (vif ) gene encodes a highly basic 23-kd protein
found both in the cytoplasm and the plasma membrane, which colocalizes
with HIV-1 Gag protein [9]. This late gene product is essential for the spread
of HIV-1 in PBLs and in primary macrophages [71], and in some but not all
established cell lines [72,73]. Although viral particle formation is not
affected, mutations in vif can decrease infectivity by as much as 1000-fold
[73,74], but this is dependent on cell type. Several possible mechanisms can
be envisioned to explain how virus-associated Vif can regulate viral infectiv-
ity. First, it is conceivable that because of its affinity to viral RNA and Gag,
Vif has a critical role in stabilizing viral nucleoprotein complexes. The func-
tion of Vif would be to facilitate proper assembly or maturation of compo-
nents of viral cores [75]. Such a mechanism would be consistent with the
observation that Vif-defective particles exhibit reduced stability of their
nucleoprotein or reverse transcription complexes [76,77] and are impaired
in the reverse transcription of their genomes [78]. Alternatively, it is possible
that Vif, because of its ability to associate with viral nucleoprotein com-
plexes and the cytoskeleton [79,80], functions as an adapter to link the viral
nucleoprotein or preintegration complex to a cellular transport pathway to
facilitate its transport to the nuclear membrane [75]. Cells undergo a rapid
change in their cytoskeletal organization immediately following infection by

HIV-1, and the effect of Vif on the cytoskeleton is microtubule dependent
[75]. HIV, like other viruses, uses an active transport mechanism for nuclear
targeting of its nucleoprotein complex. The accumulation of gag-pol precur-
sor polyproteins in Vif minus–infected cells suggests a proteolytic activity, or
a role in reverse transcription [81], because Vif-mutant virions are impaired
in viral DNA synthesis during the early stages of reverse transcription [82].
The viral protein R (vpr) gene encodes a 15-kd protein that is associated
with the nucleocapsid protein p6 and is present within HIV-1 virions in
abundant quantities. Seemingly diverse functions have been attributed to
Vpr, and it is likely to play important roles at various stages of the HIV rep-
lication cycle. Although early studies implicated Vpr as dispensable for rep-
lication in dividing cells, such as CD4þ lymphocytes and transformed cell
lines, it seems to be important for HIV infection of nondividing macro-
phages. This function is likely a result of the effects of Vpr on nuclear import
of HIV-1 DNA preintegration complexes. Although complex interactions
between Vpr, the nuclear localization signal–containing MA protein, and
the cellular nuclear localization signal–binding and -docking proteins karyo-
pherin a)b are important for nuclear targeting [83], recent studies have
shown that Vpr may facilitate nuclear entry of the preintegration complexes
by inducing transient physical disruptions in nuclear membrane architec-
ture, allowing mixing of nuclear and cytoplasmic contents [84]. By releasing
important cell cycle regulators into the cytoplasm, these nuclear disruptions
are also likely to facilitate arrest of dividing cells in the G2 phase of the cell
cycle, another well-documented function of Vpr [84], which in itself contrib-
utes to transcriptional transactivation from the HIV-1 LTR and various
other promoters. Several reports have also implicated Vpr in induction of
apoptosis, a function that may contribute to HIV-mediated CD4þ T cell
depletion [83]. Vpr has also been shown to be important in vivo, because
rhesus monkeys infected with SIVmac239 containing a vpr deletion had low
levels of viremia and prolonged survival [85]. Rapid reversion of vpr point
mutations in three of five animals was associated with progressive disease
[86], suggesting an important role for Vpr in HIV pathogenesis.
The virion protein U (vpu, approximately 9 kd) gene is found in HIV-1,
but not in HIV-2 or SIV, and the Vpu protein of HIV-1 functions in virus
particle maturation and release [87–89]. Vpu can down-regulate CD4
expression, and is necessary for intracellular dissociation of gp160 and
CD4 through degradation of CD4 in the endoplasmic reticulum [9,90,91].
Although not essential for replication, Vpu may play a role in proper and
efficient virion assembly and release [88,92,93]. Vpu is able to enhance virion
infectivity by promoting the budding of virions from plasma membrane [9].
Vpu may also facilitate export of virus capsid proteins [94].
The virion protein x (vpx, approximately 12 kd) gene is not present
in HIV-1, but is present and produced in large amounts by HIV-2 and
most SIV strains [95]. The vpx gene is not essential for viral replication, but
mutations can modestly impair virus production [96–98]. Because of the

similarity to vpr sequence, vpx is thought to have arisen as a duplication
of vpr [99]. The Vpx protein is also present in the virion outside the core
structure, suggesting a role in virus penetration or uncoating [100].
Cellular activation
Effects of cellular activation in vitro
The replication of HIV can be markedly induced in activated cells, as
observed in cell lines, PBLs, and macrophages. Cellular activation, through
a variety of means, can overcome restrictions placed on quiescent, unstimu-
lated cells. Productive infection of primary T lymphocytes by HIV-1 is
dependent on proliferation of the infected cell. Nonproliferating, quiescent
T cells can be infected by HIV-1 and harbor the virus as a labile, partial
reverse transcript. Reverse transcription can be completed with subsequent
integration if mitogenic stimulation occurs before the labile structure is
degraded [101,102]. In vitro, HIV-1 requires activated T cells for a produc-
tive infection; however, only a small numbers of circulating T cells are in the
activated state. In normal individuals there are few potential target cells for
HIV-1 infection. Although quiescent T lymphocytes can be infected by HIV-
1, in most cases infection is aborted in these resting cells, limiting spread of
infection during the early years of clinical infection.
In a T-cell line latently infected with HIV-IIIB (ACH-2) [103,104], there
is low-level viral gene expression before activation, predominantly singly
and multiply spliced mRNAs, which is similar to primary infection [104].
On cellular stimulation with phytohemagglutinin or phorbol esters, there
is first an increase in the regulatory mRNAs, followed by an increase in the
unspliced RNAs [104,105]. The ordered appearance of mRNA transcripts
seen during acute in vitro infection [106] may also be invoked in infected
cells on release from the latently infected state after activation.
Cytokine treatment has been shown to induce up-regulation of HIV-1
expression in chronically infected promonocyte clones. A clone of the pro-
monocyte cell line U937 chronically infected with HIV-1, U1, showed minimal
expression of HIV-1. Virus expression was markedly up-regulated following
treatment of PBL with phytohemagglutinin-induced supernatant containing
multiple cytokines or by recombinant macrophage colony–stimulating fac-
tor alone [107]. Primary macrophages infected with HIV-1JR-FL exhibit
dramatic increases in HIV-1 replication in vitro after treatment with the
recombinant growth factors granulocyte-macrophage colony–stimulating
factor, macrophage colony–stimulating factor, or IL-2 [108]. This stimula-
tion seems to be associated with activation of cellular transcription factors
[103,108–110], which have also been shown to have stimulatory effects on
HIV-1 replication in vitro. Many of these soluble factors can be produced
from CD4þ lymphocytes and mononuclear phagocyte in response to both
live and inactivated HIV-1, and have been shown to be increasingly elevated
in asymptomatic HIV-positive patients as they progress to AIDS [111–115].
CD4þ lymphocytes show rapid, high-level expression of HIV-1 [116,117],
whereas mononuclear phagocytes tend to show lower-level, more chronic
production typical of a smoldering infection [41,118].
The transcription of HIV-1 also has been shown to be induced by ultra-
violet irradiation and exposure to natural sunlight [119,120], X-radiation
[121], and other chemical compounds that cause DNA-damage [119]. The
promiscuous nature of HIV-1 promoter-enhancer, similar to that of other
viruses, permits a number of intracellular and extracellular signals to con-
vert HIV-1 from low-level to high-level replication.
In vivo immune activation and HIV-1 replication

Not only is immune activation important for the regulation of HIV-1 rep-
lication, but immune activation over time also promotes more efficient viral
replication ultimately leading to the progression of HIV disease. Soluble
mediators of immune activation, such as b2-microglobulin, neopterin, and
tumor necrosis factor–a [122–124], increase over time in patients with
HIV-1. Cellular markers of activation (CD38, HLA-DR, and CD45RO) are
also observed in higher frequency with disease progression [125–128]. Tran-
sient HIV induction (‡ fourfold) was seen at 1 or 2 weeks postvaccination in
10 of 20 HIV-infected patients who received the influenza vaccine; similar
increases were not seen in 15 nonvaccinated controls [129]. In a similar
study, patients who showed normal in vitro T-cell proliferative response
to stimulation by vaccine antigens, or patients who mounted significant
serologic responses after vaccination, manifested higher viral load increases
in response to vaccination [130]. This phenomenon has been subsequently
shown for other vaccinations, including tetanus [131], pneumococcus
[132], and hepatitis B [133] (Table 2). Influenza vaccinations also have been
shown to increase viral loads in HIV-positive pediatric patients [134,135],
but this increase was significant only in those patients who were not under-
going antiretroviral therapy (see Table 2).
Attempts to stimulate T-cell proliferation with IL-2, a known stimulator
of viral replication in vitro, also resulted in HIV induction [136]. The linkage
of vaccine- and IL-2–induced increases in HIV-1 replication and clinical
Table 2
Potential sources of activation-induced HIV-1 up-regulation in vivo
Vaccines Infection Other
Influenza Herpes simplex virus Transfusion
Tetanus Varicella zoster virus Cytokine treatment (interleukin-2)
Pneumococcus Mycobacterium tuberculosis
Hepatitis B
progression has not been proved, but levels of viral replication (plasma HIV
RNA) are correlated with rates of disease progression and increases in virus
load [137,138].
Potential reservoirs for HIV-1 include various types of long-lived infected
cells in various locations of the body. For example, replication-competent
HIV-1 can be recovered from cells in seminal fluid, central nervous system,
and lymph nodes [139]. During the early stages of HIV infection, although
symptoms are absent and viral replication in peripheral blood mononuclear
cells is low, substantial levels of HIV replication can be documented in lym-
phoid tissue [140]. One potentially stable reservoir is composed of latently
infected memory CD4þ T cells carrying integrated provirus. Postintegration
latency seems to result from the reversion of productively infected CD4þ T
lymphocytes to a resting memory state in which there is minimal transcrip-
tion of viral genes [141,142].
Models for studying HIV-1 replication

The goal of model systems is to mimic the in vivo environment to inves-
tigate HIV-1 replication. A variety of model systems have been developed,
including both in vitro and animal models.
Cell culture systems

Cell model systems have been useful for investigating factors important
for control and regulation of HIV replication, but they have limitations.
Although cell lines yield reproducible effects owing to their clonal origin,
important differences between these transformed, continuously proliferat-
ing cells and primary cells may provide misleading results. Among primary
cells, there are important differences in their ability to support replication
(Table 3). Primary cell systems may yield more relevant data, but they are
more variable, and are subject to factors related to in vitro culture systems,
such as fetal calf serum, adherence and activation of macrophages, and
Table 3
Restricted HIV-1 replication in primary cells
Entry Reverse transcription Integration Expression
Resting CD4þ Unrestricted Labile, partially formed, — —
lymphocyte extrachromosomal
DNA
Activated CD4þ Unrestricted Rapid and complete Cell division- High
lymphocyte dependent
Vpr-
independent
Circulating Restricted — — —
monocytes
Differentiated Phenotypically Slow but complete Vpr-dependent Variable
macrophages restricted
donor cell differences [41]. Given these limitations, however, both of these sys-
tems have been useful to elucidate features of stimulation from postintegra-
tion latency and HIV pathogenesis.
HIV was first recovered through coculture of peripheral blood mono-
nuclear cells and T-cell lines, but primary cell systems were not initially used
as an in vitro model system for studying HIV replication. Most of the studies
reported in the mid-1980s examined replication of the prototypic HIV strain
IIIB in T-cell lines, and although useful information was obtained, there are
fundamental differences between replication of T-cell line adapted and pri-
mary HIV strains. T-cell line–adapted strains are restricted from replication
in mononuclear phagocytes, but almost all primary strains are able to repli-
cate to some extent in these cells. Adaptation of HIV to grow in T-cell lines
results in genotypic changes that alter biochemical properties of the HIV
envelope. This can lead to greater CD4-induced shedding of noncovalently
linked gp120 much more readily than primary strains [143–146], and gp120
density seems to be much higher for primary strains. To model HIV-1 repli-
cation in vivo better, culture systems need to study infection of primary
human cells with primary virus strains. The disadvantage to this model is
variability in donor target cells, however, which makes results less consistent
than with cell lines. In particular, there can be marked differences in suscept-
ibility to infection between cells from different donors, and the limited num-
ber of cells available from one donor restricts the complexity of experiments.
Primate models
To understand fully the pathogenesis of AIDS, in vivo model systems
that involve interaction of HIV replication and the immune response are
essential. Although a variety of animal systems have been used to study ret-
rovirus pathogenesis, most standard animal model systems cannot be
infected by HIV-1, and many of those that can be infected fail to display
pathology after infection. Some investigators have relied on animal viruses
related but genetically distinct from HIV-1 that can cause AIDS-like disease
in their hosts. Study of the primate lentivirus SIV in a variety of monkeys
has proved to be an excellent model to investigate the contribution of viral
and host factors to disease progression [147]. The best primate models
involve SIV infection of rhesus macaques or HIV-1 infection of chimpan-
zees. Attenuated SIV has been shown to produce low levels of plasma RNA
and exhibit and cause limited depletion of CD4þ lymphocytes, which then
results in a reduced capacity to cause disease [147]. Some pathogenic forms
can cause disease, however, mostly SIVcpz. In addition, viruses isolated from
macaques that have progressed to simian AIDS are similar in phenotype to
HIV strains frequently detected late in HIV infection in humans and like-
wise exhibit more rapid replication and greater cytopathogenicity. Maca-
ques also develop central nervous system disease, and this may be a useful
model for the study of neuropathogenesis of HIV [148].
Like HIV, SIV strains exhibit tremendous genetic diversity, particularly

in the hypervariable env regions, but there are important differences. For
example, the V3 loop is different in SIV and does not seem to be important
in determining viral phenotype as other variable regions [149]. It is not as
clearly a determinant of tropism or chemokine coreceptor usage, and it is
rare to see SIV X4 isolates. Other differences between HIV and SIV in other
genes seem to affect replication and disease progression in monkeys, which
limits the usefulness of the SIV-macaque model to study HIV replication
and pathogenesis. One approach to address this limitation has been to use
chimeric viruses comprising both SIV and HIV regions, which can mimic
certain properties of HIV yet be suitable for in vivo testing in simian systems
[150]. Chimeric viruses may also be useful for vaccine assessment, if it can be
established that immune responses to these viruses resemble those found in
natural HIV-1 infection in humans.
SCID mouse models

The lack of suitable animal models for the in vivo study of HIV-1 repli-
cation, particularly the expense of simian models, has prompted investiga-
tors to take advantage of mice with SCID. These animals are unable to
undergo immunoglobulin or T-cell receptor rearrangement and are unable
to elicit an effective immune response [151]. Intraperitoneal injection of
human peripheral-blood leukocytes into SCID mice results in varying
degrees of long-term engraftment of these specialized human tissues
[152,153]. These animals can be infected by HIV-1 after intraperitoneal
injection, and T lymphocytes are depleted by HIV infection [154]. Important
limitations of this model relate primarily to the limited number of human
cell types that are inoculated and the marked phenotypic differences in
human cells in this model compared with those circulating in the blood
[155]. Human adult lymphocytes in these reconstituted animals are generally
activated and anergic to further stimulation, in contrast to human periph-
eral blood, in which more than 99% of cells are quiescent.
Another model using SCID mice involves implantation of human fetal
thymus and liver into the renal capsule [152]. A better representation of the
human hematopoietic system ensues after this implantation, and these ani-
mals also can be infected by HIV [152]. HIV-1 infection results in depletion
of T lymphocytes and human thymus tissue in these animals, which allows
assessment of mechanisms of immune destruction caused by HIV-1 infection
[156–158].
Summary
The HIV-1 is a formidable pathogen with establishment of a persistent
infection based on the ability to integrate the proviral genome into chroni-
cally infected cells, and by the rapid evolution made possible by a high
mutation rate and frequent recombination during the viral replication.

HIV-1 has a variety of novel genes that facilitate viral persistence and regula-
tion of HIV replication, but this virus also usurps cellular machinery for
HIV replication, particularly during gene expression and virion assembly
and budding. Recent success with antiretroviral therapy may be limited by
the emergence HIV drug resistance and by toxicities and other requirements
for successful long-term therapy. Further investigation of HIV-1 replica-
tion may allow identification of novel targets of antiretroviral therapy that
may allow continued virus suppression in patients of failing current regi-
ments, particularly drugs that target HIV-1 entry and HIV-1 integration.
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Clin Lab Med 22 (2002) 637–649
HIV-1 genotypic and phenotypic resistance

George J. Hanna, MD
Department of Medicine, Infectious Diseases Division, University of Pittsburgh, Scaife Hall,
Suite 818C, 3550 Terrace Street, Pittsburgh, PA 15261, USA
Human immunodeficiency virus type 1 infection is associated with high

rates of virus replication in an untreated individual. Because the viral reverse
transcriptase (RT) lacks proof-reading capability, the high replication rate
allows the evolution of considerable genetic diversity even within the popu-
lation of HIV-1 in a single infected individual [1]. If a limited drug-selective
pressure is imposed on this heterogeneous population, pre-existing strains of
HIV-1 with decreased susceptibility to the drug are favored and predomi-
nate unless replication of virus is curtailed by potent antiretroviral activity
[2]. Every approved antiretroviral agent used as monotherapy will eventu-
ally select for resistant virus. In contrast, the use of multiple agents in com-
bination brings about potent suppression of virus replication, thereby
severely diminishing the opportunity for resistance to evolve, and allowing
long-term control of viremia. Despite highly active antiretroviral therapy,
however, breakthrough viremia and drug resistance can still occur. This
article focuses on the use of antiretroviral drug resistance assays in the clin-
ical management of HIV-1 infection.
Antiretroviral resistance assays

Two types of antiretroviral resistance assays are currently available to
clinicians. Genotypic assays determine the presence of specific mutations
that confer decreased susceptibility to antiretroviral agents. Phenotypic
assays determine directly the drug inhibitory concentration of the virus
when it is cultured in the presence of the drug.
Genotypic assays
Genotypic assays are the more commonly used assays. Typically, a
patient’s plasma is used to isolate viral RNA. This step is followed by
This work is supported by Grant No. AI-01696 from the National Institutes of Health.
E-mail address: georgehanna@post.harvard.edu (G.J. Hanna).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 0 7 - 0
638 G.J. Hanna / Clin Lab Med 22 (2002) 637–649
reverse transcription and polymerase chain reaction to amplify the genes of

interest (the protease for the protease inhibitors; the amino-terminal part of
the RT for the RT inhibitors). Nucleotide sequencing of this product is usu-
ally performed by dideoxynucleotide cycle sequencing [3]. Alternatively,
methods based on oligonucleotide hybridization, such as with high-density
oligonucleotide arrays (GeneChip, Affymetrix, Santa Clara, CA) [4] and the
more limited line probe assay (LiPa, Innogenetics, Ghent, Belgium) [5] may
be used, but currently remain investigational. This step is followed by trans-
lation into the amino acid sequence and assessment of whether specific
resistance mutations are present. Assay kits that integrate sequencing with
sequence alignment, editing, and interpretation have recently become avail-
able (TrueGene HIV-1 Genotyping Kit and OpenGene DNA Sequencing
System, Visible Genetics, Ontario, Canada; and ViroSeq HIV-1 Genotyping
System, PE Biosystems, Foster City, CA) [3]. Genotypic assays can be per-
formed within 2 days of obtaining a specimen, but practical turnaround time
for clinicians can be up to 3 weeks.
One limitation of genotypic assays is their difficulty in detecting minority
populations of resistant viruses. If drug pressure is withdrawn after resist-
ance mutations have appeared and dominated the virus population, these
mutations often fade when assessed by population sequencing [6]. Currently
available sequencing methods detect minorities reliably only down to 15% to
25% of the population. The presence of resistant populations at lower levels
is not detected reliably by genotypic assays, even though these minority pop-
ulations may be clinically relevant. For example, the reintroduction of a
drug to which resistance is found in a significant minority can result in rapid
failure of that drug to suppress viremia. Another limitation of genotypic
assays is the complexity of interpreting the resistance mutations, particularly
when multiple mutations are present, and if one mutation affects the pheno-
typic expression of another mutation. Examples of these situations are pre-
sented later.
Phenotypic assays
Currently used phenotypic resistance assays use recombinant vectors in
which the amplified genes of interest (derived from a patient specimen, as
is done for genotypic assays) are inserted. These reconstructed vectors are
then used to infect cell lines that are grown under varying concentrations
of drugs, and the antiviral potency of these drugs on the virus is then deter-
mined [7,8]. Antiviral potency is typically expressed as the drug concentra-
tion required for inhibition of virus in vitro by 50% (50% inhibitory
concentration [IC50]). A resistant virus has an increase in the IC50, and the
level of resistance can be expressed as the fold increase in IC50. Because of
the complexity of these assays, they are only performed in highly specialized
laboratories. Currently in the United States, the most common phenotypic
assays used are the Phenosense assay (ViroLogic, South San Francisco, CA)
G.J. Hanna / Clin Lab Med 22 (2002) 637–649 639
and the Antivirogram assay (Virco, Baltimore, MD). Phenotypic assays

consume more time and are more expensive compared with genotypic
assays. Turnaround time to clinicians may be up to 4 weeks.
Phenotypic resistance assays have similar limitations to genotypic assays
in the detection of minority species. An added limitation is the complexity of
interpreting the fold increase in IC50. What fold increase in IC50 is necessary
to call a virus resistant to a drug? Initially, technical cutoffs were used to
determine a resistance phenotype. These cutoffs were based on the variabil-
ity of repeated measurements of the same specimen by a particular assay (ie,
the precision of the assay). This was up to 2.5-fold for the PhenoSense assay
[8] and up to fourfold for the Antivirogram assay [7]. These cutoffs, how-
ever, do not necessarily predict clinical response to antiretroviral therapy.
More recently, biologically based cutoff values have been proposed [10].
These are based on the range of drug IC50 of wild-type isolates never
exposed to the drug. Values outside this normal range (the mean plus two
standard deviations) are then considered to be resistant to the drug. Using
this concept, the cutoff values for resistance are very different for different
drugs. For the protease inhibitor amprenavir, this is 2.5-fold, whereas for
the nonnucleoside RT inhibitor (NNRTI) delavirdine, this is 10-fold [9,10].
These cutoffs, however, may still not be sufficiently predictive of clinical
response. There is a need for clinical cutoffs that determine the fold increase
in IC50 that predicts escape from inhibition by levels of drug present in vivo.
There are accumulating data on this topic. For example, an analysis of data
from several studies in which the nucleoside RT inhibitor abacavir was
added to stable antiretroviral therapy demonstrated that if baseline virus
had less than 4.5-fold increase in abacavir IC50 compared with wild-type
virus, more than 70% of subjects had a clinical virologic response, whereas
if baseline virus had greater than or equal to 6.5-fold increase in IC50, less
than 20% had a virologic response [11]. Based on this study, the clinically
validated cutoff for abacavir resistance was set at 4.5-fold increase in IC50.
Similar studies are required to determine clinically relevant cutoffs for other
antiretroviral agents.
Recent developments in resistance assays

A recently introduced resistance assay is the VirtualPhenotype (Virco,
Baltimore, MD). Here, a patient’s plasma specimen is actually used to per-
form genotyping (not phenotyping) through dideoxynucleotide cycle
sequencing. Once the amino acid sequence is determined, however, the
patient specimen is compared with a database of several thousand previ-
ously genotyped and phenotyped HIV-1 specimens [12]. For each drug of
interest, matches in this database are sought at specific codons that are
informative for resistance to that drug. From all the matches of genotypes
within the database, the geometric mean IC50 is determined and reported
as the virtual phenotype. The VirtualPhenotype provides a probabilistic
estimate of the true phenotype. The VirtualPhenotype is more expensive

than a regular genotype, but less costly and time-consuming than a pheno-
type. Although it promises to provide an enhanced ability to predict resist-
ance, especially in settings of complex mutational patterns, clinical trials are
necessary to demonstrate whether it provides more useful information than
other genotypic assays, and whether it performs as well as actual phenotypic
assays.
Another recently introduced concept in the field of antiretroviral re-
sistance testing is the inhibitory quotient. Blood levels of drugs vary consi-
derably among different subjects taking the same drug. Furthermore,
pharmacologically enhanced therapy (ie, the use of one drug to inhibit the
metabolism of another drug and thereby increase its blood levels) has
become commonplace, especially with protease inhibitors. Using this strat-
egy, even viruses with low-level resistance to a protease inhibitor may be
clinically suppressed with higher levels of the same drug in a pharmacolog-
ically enhanced setting, and the usual cutoff values for phenotypic resistance
may not be relevant [13]. The inhibitory quotient seeks to characterize the
relationship between drug exposure in the individual and drug susceptibility
of the virus. One method of calculating the inhibitory quotient is the ratio of
measured trough drug concentration in blood (Ctrough) to drug susceptibility
(such as IC50) of a virus [14]. Several factors can affect the measurement of
the inhibitory quotient, however, and may limit the ability to compare
inhibitory quotients of different drugs in the same patient. Factors that alter
the inhibitory quotient by affecting Ctrough include the extent of protein
binding of the drug (because only the unbound drug is biologically active)
and the units chosen (eg, microgram per milliliter versus nanometer). Mea-
surement of susceptibility can also vary depending on such factors as the cell
line used for susceptibility assays, and whether susceptibility is expressed as
IC50, IC90, or IC95. Nevertheless, use of an inhibitory quotient is a promis-
ing means of relating in vitro phenotypic assay results to pharmacologically
achievable levels of drug in a patient.
Cross-resistance within antiretroviral drug classes

A detailed review of resistance patterns and cross-resistance profiles for
each drug is beyond the scope of this article. Other reviews are available
on this rapidly evolving topic [15–17], and several internet websites provide
updated information on resistance mutations. These include the Stanford
HIV RT and Protease Database (http://hivdb.stanford.edu/), the Inter-
national AIDS Society-USA website (http://www.iasusa.org/resistance_
mutations/index.html), and a resistance mutation database supported by
Mediscover (http://www.mediscover.net/mutationintro.cfm). In this section,
important principles and recent developments in drug resistance and cross-
resistance are presented.
Nucleoside RT inhibitor resistance

Nucleoside RT inhibitors vary markedly in the time course in which they
induce resistance and in the complexity of the resistance patterns that they
elicit. During suboptimal therapy with zidovudine, there is an ordered
appearance of mutations over several months [18–20]. A change in RT
codon 70 from Lys to Arg (K70R) is typically the first mutation seen. Later,
T215Y appears, often with disappearance of K70R. During continuing sub-
optimal therapy, other RT mutations, such as M41L, D67N, reappearance
of K70R, L210W, T215F, and K219Q or E accumulate, conferring progres-
sively increasing zidovudine IC50. In contrast, suboptimal therapy with lam-
ivudine results in high-level resistance much more rapidly, often within 2
weeks of failed therapy, conferred by the single mutation M184V [21].
Viruses with zidovudine resistance mutations were initially thought to be
susceptible to other nucleoside RT inhibitors based on older phenotypic
assays with relatively poor precision. The introduction, however, of another
thymidine analogue, stavudine, has demonstrated that the same resistance
mutations can be selected during virologic failure of stavudine [22,23]. Fur-
thermore, in zidovudine-experienced but stavudine-naive patients, these
same mutations predicted poor virologic response to stavudine therapy
[24]. These mutations are sometimes referred to as thymidine analogue muta-
tions. Very early studies of virus isolates from patients experienced with
zidovudine but naive to the nucleoside RT inhibitors didanosine and zalci-
tabine, however, suggested that for every 10-fold decrease in zidovudine sus-
ceptibility there was approximately a twofold decrease in susceptibility to
didanosine and zalcitabine [25]. Furthermore, zidovudine-experienced
patients (often with demonstrated zidovudine resistance) have worse viro-
logic responses to therapy with other nucleosides compared with drug-naive
patients [26]. These observations suggest that these mutations should be
thought of more broadly as nucleoside analogue mutations (NAMs).
Until recently, the mechanism of resistance conferred by NAMs has
remained obscure. RT with the lamivudine-resistance mutation M184V has
decreased binding to lamivudine-triphosphate (the biologically active form
of the nucleoside analogue). RT with NAMs, however, has similar binding
to zidovudine-triphosphate as wild-type RT. Recent work has suggested
novel mechanisms of resistance associated with NAMs [27]. Certain NAMs,
such as the combination D67N/K70R, confer on the RT better proofreading
excision of chain terminators, such as zidovudine. Although zidovudine is
incorporated into the growing DNA strand, the RT is more likely to excise
it and proceed with another attempt at incorporating the correct natural
nucleotide. Other NAMs, such as T215Y or the combination T215F/
K219Q, confer greater processivity (ie, better efficiency of DNA polymeriza-
tion) onto the RT. These observations may help elucidate why zidovudine-
experienced individuals with NAMs-containing viruses also are more likely
to fail other nucleosides.
NNRTIs
Certain RT resistance mutations can confer high-level resistance against
all currently approved NNRTIs. The most serious of these is the K103N
mutation, caused by a single nucleotide change, and seen as the most com-
mon mutation during failure with delavirdine or efavirenz [28]. Other
NNRTI resistance mutations, however, do not confer class-wide cross-
resistance. For example, the Y181C mutation, commonly selected by nevir-
apine, does not confer (by itself) high-level resistance to efavirenz [29]. Also,
some mutations at RT codon 190, such as G190A selected by nevirapine and
G190S selected by efavirenz, may actually confer greater susceptibility than
seen in wild-type virus (hypersusceptibility) to delavirdine [30]. Whether the
detection of these mutations after early failure with a NNRTI (when a lim-
ited number of resistance mutations are expected) will allow the successful
use of another NNRTI during salvage with two other active agents remains
to be determined.
Protease inhibitors
Resistance mutations in protease are frequently classified as primary or
secondary. Primary resistance mutations confer significant in vitro resis-
tance to protease inhibitors by themselves. They are not found as naturally
occurring polymorphisms. Different protease inhibitors typically select dif-
ferent primary mutations. For example, saquinavir typically selects for pro-
tease L90M and less often for G48V [31], indinavir selects for one of several
possible mutations at codon 82 and less frequently at codons 46 or 84 [32],
and nelfinavir usually selects for D30N and rarely for L90M [33]. Viruses
with only a primary resistance mutation may remain susceptible to some
protease inhibitors. For example, the nelfinavir-associated D30N does not
confer cross-resistance to the other approved protease inhibitors, and suc-
cessful salvage with other protease inhibitors after virologic failure to nelfi-
navir is likely [34].
Secondary resistance mutations by themselves confer either very little or
no resistance to protease inhibitors. When seen on a background of primary
resistance mutations, however, they confer higher levels of resistance than
seen with the primary mutation [32]. Different protease inhibitors have a
largely overlapping spectrum of secondary mutations. In fact, the accumu-
lation of several secondary mutations on a background of a primary muta-
tion often leads to cross-resistance to several other protease inhibitors.
Unlike primary protease resistance mutations, secondary mutations may
be observed as naturally occurring polymorphisms, and their detection
(without a primary mutation) does not necessarily imply drug selection
[4]. In fact, most antiretroviral-naive patients harbor virus with at least one
secondary mutation. Several recent retrospective studies have examined
whether the presence of secondary protease resistance mutations at baseline
is associated with a poorer response to protease inhibitors [35–37]. For most
secondary mutations, no adverse associations were noted. Two studies sug-

gested that some secondary mutations may be associated with poorer viro-
logic response to protease inhibitor therapy, but the specific codons
implicated were different: codons 71 and 93 in one study [37] and codons
10 and 36 in another [36]. At this time, the presence solely of secondary
resistance mutations does not consistently alter the likelihood of virologic
response. Prospective studies should help clarify this issue.
Hypersusceptibility
In contrast to resistance, hypersusceptibility is the presence of signifi-
cantly greater susceptibility to a drug than expected. Hypersusceptibility
can exist within a single drug class. For example, among the NNRTIs, as
mentioned previously, the nevirapine-resistance mutation G190A confers
hypersusceptibility to delavirdine. Among the protease inhibitors, a nelfinavir-
(and more rarely indinavir-) selected mutation N88S confers hypersuscept-
ibility to amprenavir [38]. Hypersusceptibility can also occur between
classes. Viruses with several NAMs are often hypersusceptible to NNRTIs
[39]. The clinical relevance of hypersusceptibility is currently an area of
active study. Some retrospective studies have shown than the use of efavir-
enz in subjects with NNRTI-hypersusceptible virus results in better than
expected virologic responses compared with efavirenz use in subjects with
virus of normal NNRTI susceptibility [40,41].
Interactions between resistance mutations

The effect of one resistance mutation on the phenotypic expression of
another resistance mutation is often unpredictable. For example, several
mutations in RT have been shown to reverse the zidovudine resistance con-
ferred by some NAMs. These include the lamivudine resistance mutation
M184V [42], the didanosine resistance mutation L74V [43], and the nevira-
pine resistance mutation Y181C [44]. These effects can make the interpreta-
tion of genotypic test results challenging. Although these observations
initially fueled hope that dual resistance to zidovudine and any of these
drugs would be rare, it is now clear that HIV-1 can use different pathways
to become resistant to these combinations. For example, during suboptimal
treatment with combination zidovudine and lamivudine, M184V emerges
rapidly, whereas NAMs emerge slowly (more slowly than seen with failing
zidovudine monotherapy [45]). Initially, virus is highly resistant to lamivu-
dine and remains sensitive to zidovudine. Dually resistant virus, however,
does eventually emerge through one of several pathways. The virus may
accumulate additional NAMs thereby abrogating the reversal of zidovudine
resistance conferred by M184V [46]. The virus may gain a mutation in far-
ther regions of RT (such as codon 333), which on a background of NAMs
also lessens the effects of M184V on zidovudine-resistance reversal [47].
Finally, newly recognized mutations, such as RT E44D and V118I, can
confer lower-level resistance to lamivudine (approximately 10-fold, com-

pared with greater than 100-fold with M184V) [9]. Unlike M184V, however,
these mutations do not reverse zidovudine resistance.
Clinical testing for antiretroviral drug resistance

There are several clinical situations in which resistance testing may be
useful. It is clear that drug regimens may fail because of reasons unrelated
to the development of drug-resistant virus. These may include poor adher-
ence to the treatment regimen, poor absorption of the drugs, or high metab-
olism of the drug. In these situations, there is negligible drug exposure in the
blood, and consequently little antiviral activity. Resistance testing here dis-
closes wild-type virus. Even when failure is caused in part by the evolution
of resistance, the virus seen during virologic failure may not be resistant to
all drugs in the regimen. For example, several studies have now demon-
strated that early failure of a three-drug regimen that contains lamivudine
is usually associated with resistance to only the lamivudine component (with
M184V) and not the other components [48,49]. Resistance testing in these
situations suggests that the other components may retain activity if they are
included in a salvage regimen. Support for this strategy from clinical trials,
however, is currently lacking. Finally, when there is resistance to a drug, the
specific resistance pattern cannot be predicted with certainty for all agents
[50]. In these situations, resistance testing is necessary to predict the specific
mutation pattern and the associated level of cross-resistance to other drugs
in that class.
Several prospective controlled randomized trials of resistance testing
have been completed or are underway. Two early trials of genotypic resis-
tance testing, accompanied by expert interpretation of the test results, dem-
onstrated relatively small (approximately 0.5 log10) but statistically
significant decreases in plasma viral load in subjects receiving resistance
testing compared with those receiving standard care in up to 6 months of
follow-up [51,52]. A more recent trial attempted to separate the effects of
genotypic testing from the provision of expert advice [53]. Subjects were
randomized to get genotyping with expert advise, genotyping alone, expert
advise alone, or neither after failure of an antiretroviral regimen. Those who
received genotyping with expert advise fared best in terms of plasma viral
load declines at week 24, followed by those who received either genotyping
alone or expert advise alone. Those who received neither fared the worst.
This trial demonstrates the added value of expert advice in conjunction with
genotypic resistance testing in constructing successful salvage regimens.
Prospective controlled trials examining phenotypic resistance testing have
not demonstrated consistent improvement in plasma viral loads after the use
of phenotypic assays thus far [54,55]. It is important to note that these trials
used technical IC50-fold increase cutoffs, based on the precision of the
assays, and not drug-specific clinically validated cutoffs. Whether the use of
recently proposed drug-specific clinical cutoffs for IC50-fold increases dem-
onstrates improved virologic outcomes after use of phenotypic assays re-
mains to be determined.
Currently, HIV-1 drug-resistance testing is recommended after treatment
failure of antiretroviral regimens [16,56]. These situations include a minimal
plasma viral load decline in the first few weeks of therapy, failure to achieve
long-term plasma viral load suppression, or breakthrough viremia after suc-
cessful suppression of plasma viral load. Drug-resistance testing should also
be recommended for pregnant women both to control viremia in the woman
and to decrease the possibility of perinatal transmission. Based on the
results of the genotypic testing trials mentioned previously, genotypic resis-
tance testing was found to be cost-effective when performed after treatment
failure [57].
Should treatment-naive patients have resistance testing before starting
their first antiretroviral regimen? Although there is a lack of consensus on
recommending testing in all drug-naive patients, resistance testing is
strongly suggested in patients presenting with acute HIV-1 infection [16].
In these patients, the prevalence of resistance has been in the range of 5%
to 26% in recent years in the United States [58]. Furthermore, successful
treatment at this point in the disease course may preserve antiviral CD4þ
cells crucial for the long-term immunologic control of viremia. Among
patients with longer length of HIV-1 infection (chronically infected pa-
tients), there have been little data on the prevalence of resistance mutations
in the United States. A study in Boston of antiretroviral-naive, chronically
infected patients presenting for care in 1999, however, showed that 18%
harbored viruses with drug-selected resistance mutations [59]. A larger study
in 10 other cities in the United States also demonstrated that the prevalence
of genotypic resistance was 10% [60]. Because genotypic resistance testing
is likely to be cost effective in drug-naive patients if the prevalence of resis-
tance mutations at baseline is greater than 4% [57], screening drug-naive
patients for antiretroviral drug resistance before deciding on initial therapy
may be clinically advantageous in specific settings.
Summary
Antiretroviral failure caused by the development of drug resistance in
HIV-1 is an increasingly common clinical problem. Two types of resistance
assays are available to clinicians. Genotypic assays determine the presence
of mutations associated with drug resistance. The interpretation of muta-
tions is often complicated, however, and may require expert opinion. Pheno-
typic assays provide a direct measure of the drug susceptibility of the virus.
The magnitude of increase, however, in viral drug inhibitory concentration
that is predictive of clinical drug failure remains unknown for several
antiretroviral drugs. The mutational patterns underlying resistance to each
antiretroviral drug are often diverse, and cross-resistance patterns with-

in each of the currently available classes are complex. Currently, resis-
tance testing is recommended for patients who have virologic failure on an
antiretroviral regimen. Furthermore, testing should also be considered in
treatment-naive patients when the prevalence of transmitted drug-
resistant virus is expected to be high.
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Clin Lab Med 22 (2002) 651–680
HIV-1 reservoirs
Roger J. Pomerantz, MD, FACP
The Dorrance H. Hamilton Laboratory, Center for Human Virology,
Division of Infectious Diseases, Department of Medicine, Thomas Jefferson University,
Philadelphia, PA 19107, USA
In most infected individuals, HIV-1 replicates high levels throughout the

duration of infection, including the clinically quiescent phase of disease. The
level of this active viral replication correlates directly with disease progres-
sion and survival [1–3]. The advent of combination therapeutics for HIV-1
(ie, highly active antiretroviral therapy [HAART]) has led to dramatic
reductions in viral replication in vivo and morbidity and mortality, at least
in the developed world [4–6].
A significant portion of therapeutically naive HIV-1–infected individuals
treated with HAART develop undetectable plasma HIV-1–specific virion
RNA levels, as defined by a detection limit of 400 to 500 copies per milliliter,
with somewhat fewer patients obtaining less than 50 copies per milliliter
[4,6]. This now allows both clearer investigations of classic questions in
human retrovirology, and generates new clinical problems to analyze. For
example, with the ability of HAART to inhibit HIV-1 viral load (virion-
associated RNA) to undetectable levels in the blood plasma and genital
fluids of many infected individuals, one can now formally study whether
proviral-harboring genital tract cells can transmit HIV-1 sexually, without
cell-free virions. Mechanisms of proviral persistence and cryptic viral replica-
tion can now be addressed without the noise of active virally producing cells
and high levels of cell-free virions [7].
HIV-1 latency
Interest in retroviral latency or, more precisely, persistence preceded the
AIDS epidemic. Nevertheless, the understanding of restricted retroviral
This work is supported in part by USPHS grants AI46289 and AI38666 to Roger J.
Pomerantz.
E-mail address: roger.j.pomerantz@mail.tju.edu (R.J. Pomerantz).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 0 5 - 7
652 R.J. Pomerantz / Clin Lab Med 22 (2002) 651–680
replication in vivo has significantly increased in recent years, using HIV-1 as

a model [8]. It is critical, at the outset, to define the concepts of cellular per-
sistence or latency in vivo for retroviruses. Many studies have demonstrated
that latency for HIV-1, as defined as no viral expression in an entire
untreated infected individual, does not exist at the organism level in any
stage of disease [3,9,10]. In most HIV-1–infected individuals, some cultivable
virus may be recovered at all stages of disease [3,9,10]. Nevertheless, data
have been reported, using a wide variety of techniques, demonstrating that
cells exist in the infected individual that harbor the HIV-1 provirus but
express little or no viral RNA and produce few or no virions [9,11,12]. As
such, latency or persistence at a cellular level exists in vivo, and the numbers
of latently infected cells may vary based on the stage of disease.
HIV-1 replication
The retroviral life cycle consists of binding and internalization of virion
particles to target cells, reverse transcription of viral RNA to DNA, and
then production of the provirus on integration of viral DNA into the host
cell’s genome. The provirus may be transcriptionally stimulated to produce
viral mRNA and subsequent viral proteins leading to morphogenesis of new
virions. The HIV-1 life cycle contains many possible sites for restricted rep-
lication, both before and after proviral integration (Fig. 1) [8]. Because HIV-1
infects in vivo both CD4þ T lymphocytes and monocyte-macrophages
[13,14], the virus may maintain cellular latency by different mechanisms in
differing cell types. The major, but not sole, cellular reservoir for HIV-1
in the peripheral bloodstream is the CD4þ T lymphocyte. Monocytic cells,
which are the main viral reservoir in most solid tissues, may be fundamen-
tally different in their replication of HIV-1 [13,14].
Various states of HIV-1 cellular latency before integration exist in cell
cultures and in vivo. Studies have demonstrated that CD4þ T lymphocytes,
not activated by mitogens, do not allow productive replication of HIV-1 in
cell culture. In one report, incomplete HIV-1 reverse transcription occurred,
as measured by the polymerase chain reaction (PCR), leading to unstable
partially reverse-transcribed HIV-1 DNA intermediates [15,16]. This may
be caused by low levels of deoxyribonucleoside triphosphates in resting
CD4þ T lymphocytes [17]. The completion of reverse transcription and inte-
gration of these intermediates could occur after infected cells are stimulated
with mitogen. It was suggested that this viral DNA intermediate may sur-
vive in resting T lymphocytes, in vivo, and provide a form of preintegration
latent infection. Further data suggest that the efficiency of the completion of
viral DNA production, from partial reverse transcripts, is rather poor in
some cell types [17,18]. In other studies, evidence was provided that unsti-
mulated CD4þ T lymphocytes in cell culture can fully reverse transcribe
HIV-1–specific RNA but the HIV-1 DNA produced in this process did not
R.J. Pomerantz / Clin Lab Med 22 (2002) 651–680 653
Fig. 1. HIV-1 reservoirs in vivo. This schematic illustrates known and potential sites for HIV-1
residual disease during virally suppressive highly active antiretroviral therapy (HAART). These
include low-level productively infected CD4 T cells, transcriptionally active but nonproductive
CD4 T cells expressing mainly multiply spliced viral RNA, and latently infected resting CD4 T
cells. Other potential sites include follicular dendritic cells to which virions are found and may
exist in this form for significant time periods, in vivo. Potential sanctuaries for both cryptically
replicating and latently infected cells are demonstrated behind various blood-tissue barriers.
Macrophages that may be infected latently or cryptically replicating are also illustrated and
other potential diverse cell types for HIV-1 residual disease are listed on the left of the figure.
(From Pomerantz RJ. Residual HIV-1 infections. AIDS 2002;15:1201–11; with permission.)
integrate into the host genome, because the preintegration complex does not
undergo cytoplasmic to nuclear transport. Only after cellular stimulation by
phytohemagglutinin was the HIV-1 double-stranded DNA able to integrate
and productively express progeny virions [19,20]. Unintegrated HIV-1 DNA
species were demonstrated in peripheral blood lymphocytes of certain
HIV-1–infected individuals [19]. Stimulation of these cells with mitogens in
cell culture led to integration of the viral DNA. It has been proposed that unin-
tegrated, linear HIV-1 DNA structures may function as a reservoir of latent
HIV-1 infection in resting CD4þ T lymphocytes in vivo [19]. In vitro studies
of an inducible form of HIV-1 DNA in quiescent CD4þ T lymphocytes,
which has also been shown to be stable, may have potentially significant
in vivo consequences in understanding lentiviral pathogenesis [21]. None-
theless, several studies have suggested somewhat divergent findings regard-
ing HIV-1 infection of naive and resting T cells in vitro or in vivo [22–27].
Resting CD4þ T lymphocytes may also be relatively poor sites for HIV-1
replication because of cell cycle-specific inhibition of reverse transcription
[16], and the lack of a potentially critical cellular factor (NFAT) required for
completion of reverse transcription during productive HIV-1 replication [28].
Cell lines have been selected from the survivors of lytic HIV-1 infections
that maintain HIV-1 in the restricted but integrated state and constitutively
produce very low levels of the virus [29–31]. These cell lines can be stimu-
lated to increase HIV-1 expression with a variety of exogenous compounds.
Most of these compounds seem to act by activation of the transcription
factor, nuclear factor-jB. Two HIV-1 latently infected cell lines have been
extensively characterized, the U1 monocytic and the ACH-2 T-lymphocytic
lines. These lines have been used as model systems to explore certain aspects
HIV-1 postintegration latency in cell culture. In the baseline unstimulated
state, these cells express multiply spliced HIV-1–specific RNA, as compared
with productively infected cells in which all three HIV-1 RNA species are
expressed in nearly equivalent amounts. This RNA expression pattern
undergoes a switch to mainly synthesis of unspliced transcripts on stimula-
tion of these cells. Cells expressing mainly or solely multiply spliced viral
RNA have also been demonstrated by the author’s group and others in ini-
tial studies of viral persistence [32–34].
As noted previously, restricted replication of HIV-1 in vivo can be pro-
duced through a wide variety of molecular mechanisms. For instance, cells
might contain proviral DNA but lack any viral RNA expression. Partially
defective viral genomes, demonstrated in certain cells in vivo, might also
lead to latent cellular infections [3,35,36]. Importantly, studies have demon-
strated that the number of cells in the peripheral blood of HIV-1–infected
persons who harbor the proviral genome is much greater than the numbers
of cells that express high levels of HIV-1–specific RNA [9].
Cellular HIV-1 persistence, defined as proviral latency or residual low-
level viral replication, may have a further level of control, in which the
quantity of HIV-1 production within a cell may be based not only on the
cell type, but also affected by the location within the body of a particular cell.
The control of HIV-1 proviral latency in monocyte-macrophages may sig-
nificantly differ, based on whether the monocytic cell is in the central ner-
vous system (CNS), the bone marrow, or the liver. The CD4þ T lymphocyte
infected with HIV-1 may differ in its level of viral expression depending on
its location in a lymph node or whether it is found in the peripheral blood,
with differences in cellular activation parameters. Cellular persistence of
HIV-1 may be based on multiple levels of complexity, tied to the molecular
form of latent infection, the cell type, and the location of the infected cell
within an HIV-1–infected individual [8].
It has been demonstrated that HIV-1 replicates at a rapid rate in infected
individuals, with a virion T1/2 in plasma of less than a few hours [35]. Most
of this viral replication (up to 99%) occurs in activated and productively
infected CD4þ T lymphocytes in the peripheral blood and lymphoid tissue.
Nevertheless, using viral decay characteristics in patients initially treated
with HAART, second- and third-phase decay of plasma viremia is shown
to occur, secondary to long-lived infected cells (eg, tissue-bound macro-

phages) and latently infected CD4þ T lymphocytes, respectively [35].
HIV-1–infected individuals will probably require suppressive HAART for
prolonged periods of time (significantly greater than several years) if one is
going to consider viral eradication, at least in most patients. This may also
be greatly hindered by infection in other in vivo tissue compartments and
sanctuary sites. Tissues that maintain blood:tissue barriers, secondary to
microvascular endothelial cell tight junctions, may also hinder penetration
of certain antiretroviral agents. These compartments potentially include the
testes, the CNS, and the retina.
Residual HIV-1 disease during HAART

Persistently infected, nonactivated CD4þ T lymphocytes have been dem-
onstrated in the peripheral blood of HIV-1–infected individuals [36]. Of
importance, these proviral-harboring resting CD4þ T lymphocytes have
now been demonstrated in infected individuals treated with HAART,
with undetectable viral RNA in blood plasma. It has also been shown that
replication-competent viruses can be recovered from these proviral-positive
cells, after CD8þ T lymphocyte depletion in vitro [7,37–39]. In addition,
this latent reservoir is established soon after primary HIV-1 seroconversion
[40], and can be activated by proinflammatory cytokines and bacterial prod-
ucts in vitro and potentially in vivo [41,42]. A latent reservoir of low-level
or nonreplicating HIV-1 has also been described in HIV-1–infected children
treated with suppressive HAART [43]. In a short study by Poggi et al
[44], suppressive HAART initiated before primary HIV-1 seroconversion
was shown to be unable to halt the development of a replication-competent
virus reservoir in CD4þ T lymphocytes. One initial study suggested, indi-
rectly by quantitating total HIV-1 DNA and subtracting integrated HIV-1
DNA, that some low-level viral replication may still take place in certain
of these persistently infected cells, because unintegrated viral DNA may
be present [39]. It was also clear that further studies were necessary to anal-
yze the potential for cryptic viral replication in these cell populations. It had
been shown previously that very low-level viral transcription may take place
in lymph nodes of selected patients on effective HAART [45].
Preliminary studies suggest that HIV-1 may be present in circulating
monocytes, in addition to CD4þ T lymphocytes, of infected individuals but
that this is ablated by suppressive HAART [46,47]. Clearly, further studies
on monocyte-macrophage populations during suppressive HAART are crit-
ical in understanding their potential impact in HIV-1 latency and replication
during suppressive HAART.
Recent data suggest that the virus species in the latent reservoir of CD4þ
T lymphocytes in HIV-1–infected individuals on suppressive HAART are
CCR5-tropic strains. This further suggests that these are archival viruses
that were present from soon after primary infection, because CCR5-tropic
viruses are the strains transmitted both sexually and parenterally [48]. In this
study, it was also demonstrated that the latent virus is present predomi-
nantly in resting memory (CD45ROþ) cells and at significantly lower levels
in resting naive (CD45RAþ) cells. These findings seem based on the low but
detectable levels of CCR5 found on memory but not naive CD4þ T lym-
phocytes. The low, but present, infected resting naive cells are likely caused
by resting memory cells, which contain integrated HIV-1 provirus, reverting
to a naive phenotype [49]. It may be less likely that direct infection of resting
naive cells occurs in vivo.
Of note, activation of CD8þ T lymphocytes seems to normalize in
patients who are HIV-1 infected on suppressive HAART. This was shown
to correlate with the level of infectious provirus in tonsillar biopsies of
patients treated with HAART. As such, activation parameters in T lympho-
cytes may be based on low-level viral replication in lymphoid tissue in
patients on suppressive HAART [50]. In a study of patients on suppres-
sive HAART, immunohistochemical staining revealed some productively
infected CD4þ T lymphocytes and macrophages in lymphoid tissue. As
such, although hyperplastic changes in the lymph nodes and activation of
T lymphocytes decrease with ongoing suppressive HAART, this study sug-
gested that there may be a pool of viral replication in lymphoid tissue [51].
In addition, the gut-associated lymphoid tissue seems to be an important site
for early HIV-1 replication in mucosal lymphoid tissues [52–54]. Mucosal
lymphoid tissue, with high baseline levels of T-lymphocyte activation,
may be another critical reservoir outside of the peripheral blood for HIV-1
residual disease in patients on suppressive HAART. Of note, suppressive
HAART leads to rare viral RNA-positive cells in the anal mucosa of infected
men but has little effect on proviral DNA in this region [50].
Recent data suggest that monocytes may also harbor latent provirus in
patients on virally suppressive HAART [55]. Thymic cells, especially in
young HIV-1–infected individuals, may also be a critical site for HIV-1
latency [56]. Nonimmune cells, such as renal cells, may be a reservoir for low
levels of HIV-1 in vivo [57].
The replication-competent viruses, isolated from proviral-harboring
CD4þ T lymphocytes in patients on HAART, with undetectable viral RNA
in blood plasma, were demonstrated to have little or no antiretroviral resis-
tance mutations [37]. Because resistance mutations in the reverse transcrip-
tase (RT) and protease genes of HIV-1 are correlated with ongoing viral
replication [58], this suggests that these viral strains may represent archival
species from soon after primary seroconversion. Data from these studies
also suggest that although defective proviruses accumulate in CD4þ T
lymphocytes in vivo (ie, viral graveyards) [59], there still exit replication-
competent proviruses in persistently infected cells, which may hinder at-
tempts at eradication and reseed the body if HAART is discontinued in yet
undetermined time periods after primary infection. Continued extensive
analysis of in vivo decay times of these persistently infected cells is now

of critical importance (see later).
HIV-1 reservoirs outside the peripheral immune system

To investigate initially the impact of HAART on HIV-1 replication in the
male genital tract, the author’s laboratories collected peripheral blood and
semen fluids, which were simultaneously collected from seven HIV-1–
infected men receiving triple antiretroviral therapy for significant time
periods, with repeatedly undetectable viral RNA levels in the peripheral
blood plasma [7]. The viral RNA in the blood and seminal plasma on the
days of sample collection were analyzed by quantitative RT-PCR, whereas
the cell-associated HIV-1 proviral DNA from the peripheral blood mono-
nuclear cells (PBMCs) and seminal cells were evaluated by quantitative
DNA-PCR. Both the viral RNA levels in the blood plasma and seminal
fluid of these patients were demonstrated to be below 50 copies per milli-
liter. This suggests that HAART used in these individuals could not only
potently inhibit viral replication in the bloodstream, but also in the seminal
fluids. Importantly, cell-associated viral DNA was detected in all patients’
PBMC samples. Of note, cell-associated proviral DNA was also detected
in the seminal cells from four patients.
Because cell-free HIV-1 in the seminal plasma was very low or absent in
these patients, researchers then examined whether HIV-1 DNA-positive
seminal cells could harbor replication-competent virus. Using a coculture
system, HIV-1 replication was found in seminal cell samples from two indi-
viduals. Since this report, further cases have demonstrated this seminal
reservoir (unpublished results).
Replication-competent viruses were also isolated from three phyto-
hemagglutinin- and interleukin-2–stimulated, CD8þ T-lymphocyte–depleted
peripheral blood lymphocyte samples. To investigate whether the replication-
competent viruses recovered from seminal cells were further capable of
sexual transmission, the sequences in the V3 loop of the gp120 envelope (env)
region and the replication phenotype of the recovered viruses were exam-
ined. According to the net amino acid charges in the V3 loop (ie, macro-
phage-tropic: charge þ4 or less; and T-cell line tropic: charge greater than
þ4) and the growth pattern in cell culture [60], seminal cell-derived virus
isolated from these patients was macrophage- or dual-tropic. Because most
HIV-1 strains detected during sexual transmission are macrophage- or
dual-tropic (using chemokine HIV-1 coreceptors CCR5 or both CCR5 and
CXCR4, respectively) [61–66], these results indicate that the replication-
competent viruses isolated from the seminal cells are potentially capable
of initiating primary infection and transmission to a sexual partner,
although these patients were on HAART and viral RNA in the blood
plasma was at clinically undetectable levels.
It has been reported that RT inhibitor-resistant and protease inhibitor-

resistant HIV-1 can be transmitted between sexual partners [67–73]. To
determine the drug sensitivity of the replication-competent HIV-1 viruses
and proviral DNA from the seminal cells and PBMCs, RT and protease
regions were sequenced. No drug resistance mutations were detected in
either the recovered replication-competent virus from the seminal cells or
seminal cell-associated proviral DNA in these patient’s seminal or PBMC-
derived latent viruses, as had been reported previously for the latent reser-
voir in PBMCs of patients on suppressive HAART [37].
In this report [7], replication-competent HIV-1 was isolated from the
seminal cells of HIV-1–infected men receiving HAART and demonstrated
that, in addition to the resting CD4þ T lymphocytes in peripheral blood and
lymphoid tissues, there is a reservoir in the male genital tract for HIV-1 rep-
lication in vivo. This phenomenon could be caused by the decay at a very
slow rate of certain cells harboring HIV-1 proviral DNA in the male genital
tract. Theoretically, if no antiretroviral-resistant viral mutants develop dur-
ing HAART, reinfection of cells by HIV-1 would not continue in the micro-
environments, in vivo, where the antiretroviral agents can obtain inhibitory
levels. Because reinfection is consistently inhibited, the decay of HIV-1–
infected cells would continue. If the life span of certain cells harboring intact
HIV-1 proviral DNA is long, however, they may shed viruses in select cir-
cumstances. Low-level replication of HIV-1 could still occur. Because the
blood:testes barrier may prevent certain antiretroviral agents from effec-
tively entering testicular tissue, a partial drug sanctuary could also exist
[74]. Other sites of tissue-blood endothelial barriers, including the brain and
the retina, have also been demonstrated to harbor HIV-1–infected cells
[75,76]. Also, a potential reservoir for HIV-1 in certain patients on suppres-
sive HAART may be the renal tubular epithelial cells of the kidney [77].
A plasma membrane-localized drug transporter, entitled P-glycoprotein,
has been shown to decrease the penetration of protease inhibitors across cer-
tain blood:tissue barriers. As such, substances that block this drug trans-
porter increase protease inhibitor concentrations across the blood:brain
and blood:testes barriers [78,79]. This may be an important pharmacologic
approach to target relative viral sanctuary sites with higher levels of anti-
retroviral agents. Although certain antiretroviral agents do not penetrate
the blood:brain barrier efficiently [80], other agents do enter the CNS at rela-
tively high levels [81,82], and most patients on virally suppressive HAART
have concomitant decreases in cerebrospinal fluid levels of HIV-1 RNA to
undetectable [81–84].
Although some studies show relatively poor penetration of certain
antiretroviral agents into the male genital tract [85,86], most (but interest-
ingly not all [87]) patients on virally suppressive HAART also have corre-
sponding seminal fluid HIV-1 RNA at clinically undetectable levels [88–94].
This pattern was also demonstrated in the cervicovaginal fluids of HIV-1–
infected women on suppressive HAART [95]. As described previously,
virally suppressive HAART does not clear the genital secretions of cells har-
boring proviral species [7,96] or replication-competent virus [7]. The clinical
importance of residual HIV-1 in genital secretions requires further analy-
sis, because a recent study has demonstrated that, in at least one cohort,
heterosexual transmission was rare in untreated HIV-1–infected individuals
with plasma viral loads below 1500 copies per milliliter [97]. Nevertheless,
this may differ in patients requiring antiretroviral therapy to decrease viral
loads to this threshold level.
Although HIV-1 RNA was below detectable levels in the seminal plasma
in the HIV-1–infected men in the author’s study, reinfection of cells in the
male genital tract tissues and fluids could also still occur at very low levels
(ie, covert or cryptic replication), as may take place in lymphoid tissue [45].
In this scenario, the life span of the cells harboring HIV-1 proviral DNA
could still be relatively short. It remains to be clarified which specific cells
in the genital tract of HIV-1–infected men receiving HAART harbor repli-
cation-competent HIV-1. Of note, seeding of the male genitourinary tract
may occur not only from the peripheral blood, but also through viral reser-
voir sites in the genitourinary tract itself, including the prostate [98,99].
HIV-1 replication during virally suppressive HAART

The author hypothesized that both latently infected cells containing non-
defective but quiescent provirus and low levels of viral replication could
account for the phenomenon of replication-competent virus within the
PBMCs and seminal cells of these infected individuals [7]. Viral replication
could occur, at least in some cell types within these individuals, at such low
levels that the virus would not be detectable in peripheral bodily fluids but
would take place at cryptic levels, which might infect small numbers of cells
in situ surrounding the microenvironment of an infected cell covertly repli-
cating the virus. Two important articles clearly demonstrated this finding in
at least some patients on suppressive HAART. Using complementary tech-
niques, a study by Zhang et al [100] demonstrated that in two out of eight
patients on suppressive HAART, modest but reproducible evolution of
sequences in the viral envelope region occurred over time. Further stud-
ies have clearly demonstrated that, during what seems to be full suppres-
sion of HIV-1 in the plasma by clinical assays, there is in most patients
ongoing viral replication as shown by evolution of envelope sequences in cel-
lular reservoirs [101,102]. As in other previous studies [7,39], and an article
by Furtado et al [103], these archival viral strains did not demon-
strate antiretroviral resistance mutations, even in those strains cryptically
replicating. Unfortunately, the study by Zhang et al [100] showed that the
in vivo half-life of infectious residual HIV-1 led to an estimate of at least
7 to 10 years of standard HAART viral suppression necessary for possible
viral eradication in even the best case scenarios. Interestingly, the data of
Furtado et al [103] demonstrated a quasi-steady state of viral mRNA and inte-

grated proviral DNA in their patients analyzed after 300 days on HAART.
Studies by Furtado et al [103] used a sensitive measure of the footprints
of cryptic replication, evaluating HIV-1 long terminal repeat (LTR) DNA
circles, which are formed by self-ligation of proviral DNA, after transport
to the nucleus, by cellular nuclear ligases. Because two LTR circles were
demonstrated in most patients on suppressive HAART and based on these
moieties’ potentially short in vivo half-lives, these viral DNA species reveal
the existence of at least low-level viral replication at some time in the recent
past. As such, this cryptic replication might ‘‘reset the virologic clock’’ by
infecting previously uninfected cells in localized microenvironments. Of
note, not all patients in either of these studies were shown to have changes
in proviral envelope sequences or continuous presence of LTR circles sug-
gestive of ongoing viral replication. As such, latently infected cells seem to
exist in some patients, potentially intermixed with cells that are cryptically
replicating virus. Outgrowth of HIV-1 from CD8þ T-lymphocyte–depleted
PBMCs in patients on HAART may actually represent outgrowth of a sub-
set of transcriptionally active viral strains [104].
The article by Furtado et al [103] also illustrated a further complexity
toward understanding the residual HIV-1 disease in patients treated with
seemingly effective HAART. In these studies, multiply spliced viral RNA
was shown to be present in certain cells, out of proportion to the unspliced
RNA used by the virus in generating structural viral proteins and as
genomic RNA for new virions. As such, this represents transcriptionally
active but nonproductive infections of these cells. This extends previous data
from both in vitro cell line model systems [29] and in analyses of certain
asymptomatic individuals before HAART [32–34]. Another study also has
suggested that in patients on suppressive HAART, there exist in the peri-
pheral blood CD4þ T lymphocytes with low levels of viral transcriptional
activation, but not cell-surface adherence of HIV-1 virions [105]. A recent
preliminary study has also shown that cell-associated HIV-1 RNA in
PBMCs was sustained for several years after treatment with suppressive
HAART, when begun during primary HIV-1 infection. In this study unde-
tectable levels of plasma viremia were demonstrated using an assay that is
stated to quantitate down to three copies of viral RNA per milliliter. As
such, viral replication seems to continue in even the best-case scenarios, in
which primary HIV-1 infection is treated aggressively with HAART and
with little to no detectable plasma viremia [106]. Another recent study has
demonstrated that influenza vaccination in HIV-1–infected individuals on
suppressive HAART can, in some subjects, cause a transient increase in
plasma HIV-1 RNA, as it had been shown previously in patients not on
HAART [107]. This was significantly more common in patients who had less
than 400 but greater than 50 copies per milliliter of plasma viral RNA, as
compared with those who had levels of plasma HIV-1 RNA below 50 copies
per milliliter. In some of the patients, especially those with greater than 50
but less than 400 copies per milliliter of plasma viral RNA, these studies sug-
gested that in vivo mobilization of latently infected cell reservoirs may still
occur with cell stimulation during relatively potent HAART. Nonetheless,
stimulation of ongoing, low-level viral replication may also have contributed
to these findings.
Recent data from Haase’s laboratory [108] have shown that certain
CD4þ T lymphocytes found to be HLA-DR negative, and not activated
or stimulated, were positive for low levels of viral RNA in the lymphoid tis-
sue of HIV-1–infected patients on suppressive combination antiretroviral
chemotherapy. As such, this suggests that there may be a spectrum of cell
types in a relatively inactive state that can still express low levels of viral
RNA. A spectrum of viral infection in CD4þ T lymphocytes, ranging from
completely latent provirus through low levels of viral RNA to the final state
of productive active viral expression, probably occurs in different cells
throughout the body of infected individuals, both with and without
HAART [108]. A recent study has demonstrated that circulating lympho-
cytes have both patterns of ongoing HIV-1 RNA expression and latent
infection during suppressive HAART. These two cell populations seem to
correlate with plasma viral load and are stable at a steady state during
HAART [109]. A recent preliminary study has demonstrated that HIV-1
in patients on suppressive HAART may infect resting CD4þ T lymphocytes
after production from small numbers of activated T cells and monocytes in
the peripheral blood, reseeding HIV-1 latent reservoirs in peripheral blood
and potentially lymphoid tissue [110].
In addition, recent studies have suggested that HIV-1 infection of resting
human CD4þ T lymphocytes may be stimulated by specific cytokines and
may not always require antigen-directed T-cell activation [111]. These data
suggest that the high levels of proinflammatory cytokines in the lymph
nodes of HIV-1–infected individuals may be sufficient to allow at least
low-levels of HIV-1 infection of previously resting CD4þ T lymphocytes.
Based on these articles and other recent studies in patients analyzed dur-
ing the era of HAART, one can now rationally approach the residual HIV-1
disease after seemingly effective HAART. To further extend the understand-
ing of residual cryptic HIV-1 replication during suppressive HAART, the
author studied all patients initially referred to his clinics on HAART with
less than 50 copies per milliliter of viral RNA in plasma (ie, undetectable
by ultrasensitive clinical assays), using a supersensitive laboratory-based
RT-PCR assay, which can quantitate to five copies per milliliter and de-
tect but not quantitate viral RNA even below this level [17,112,113]. The
22 patients represented an immunologically diverse group, with CD4þ T-
lymphocyte counts between 100 and 1270 cells/mm3, and 21 of 22 were treated
with at least three antiretroviral agents. The stability of the patients’
HAART regimens was an important criterion of this cohort [88].
In addition, virion RNA levels were evaluated in the genital secretions
from each of these patients. As has been demonstrated previously, the levels
were lower than those found in peripheral blood plasma. Twelve patients
had negative results for virion RNA levels in genital secretions, including
both women analyzed in this study. Nonetheless, eight patients demon-
strated detectable viral RNA in seminal fluid.
This study demonstrated that, in a cohort of patients with undetectable
viral RNA (less than 400 copies per milliliter) for between 5 months and
several years on HAART and with less than 50 copies per milliliter of viral
RNA in peripheral blood plasma at the time of these analyses, all subjects
had low but detectable levels of HIV-1 RNA in blood plasma [88]. This was
surprising in that these data revealed that viral expression could not only be
shown by viral replication in selected cell types within patients on suppres-
sive HAART but by actual virion production within blood plasma. This
correlated with data presented by several groups, which showed low levels
of ongoing viral replication in selected patients by analysis of PBMCs and
lymphoid tissue [45,87,100,103,114]. The author’s studies expanded and
extended on these data. Using specific techniques, the cryptic replication
shown in certain cell types was demonstrated also to be quantifiable using
peripheral blood plasma analysis.
Of importance, this viral replication may not only infect uninfected cells
in the local microenvironment of viral-producing cells, but may infect cells
at a distance within the body. As noted previously, the author’s group and
others have demonstrated HIV-1 replication by episomal HIV-1 infection
intermediates, two LTR circles, in patients who were suppressed for many
months to years on HAART as assessed by less than 500 and in many
patients less than 50 copies per milliliter of plasma viral RNA (110a). This
further extends, on an intracellular level, the ongoing cryptic or covert rep-
lication, which occurs in most patients on suppressive HAART [115].
Only low levels of virion RNA were found in the genital secretions of the
HIV-1–infected individuals in the author’s study. Fifty-five percent of these
patients with less than 50 copies per milliliter of viral RNA in peripheral
blood plasma demonstrated no detectable viral RNA in genital secretions,
as shown by the author’s assay system. Nonetheless, the viral levels in
peripheral blood plasma do not take into account potential viral replication
in other body fluids, including cerebrospinal fluid and the interstitial fluid
between cells in certain solid tissue. In addition, the pathogenetic impor-
tance and potential for sexual transmission from highly viral-suppressed
individuals of very low level cell-free virion RNA, found in genital secretions
of 10 patients in these studies, is unknown and requires further study [88].
Several studies have shown that suppression of viral replication below cer-
tain limits of detection with clinical assays is associated with longer virologic
response, as compared with those not fully suppressed. This has been demon-
strated when comparing continuously decreasing limits of plasma HIV-1
RNA quantitation from 400 to 50, and now to less than 20 copies per milli-
liter [116–118]. Plasma viral load suppression below 20 copies per milliliter
has been demonstrated to yield a more long-term antiretroviral response in
patients, as opposed to those individuals who only obtain viral suppression

below 500 copies per milliliter in plasma [116]. Whether a clear clinical differ-
ence exists between viral suppression below 50 but above 20 copies per milli-
liter, as compared with below 20 copies per milliliter, requires further study.
A recent case report has suggested that a decreasing CD4þ T-lymphocyte
count in an HIV-1–infected patient on HAART might have been caused by
extremely low levels of viral replication (ie, less than 20 copies per milliliter of
plasma viral RNA) [119]. In this case, altering of the antiretroviral regimen
led to an increase in CD4þ T lymphocytes and a potential decrease in even
this very low level of viral RNA, to below 5 copies per milliliter in the plasma,
with a concomitant decrease of low-level, cell-associated viral RNA deter-
mined in a tonsillar biopsy [119]. A patient similar to this case has also been
observed at the author’s center (unpublished data). Further studies are nec-
essary to determine clearly whether CD4þ T-lymphocyte depletion can occur
in some patients at extremely low plasma viral RNA levels while on HAART.
A very interesting study has shown that when early combination therapy
is started after vertical transmission of HIV-1 from mother to infant, no
detectable plasma virus (below 50 copies per milliliter) could be found in the
plasma of certain of these children and, most importantly, labile episomal
two-LTR intermediates of HIV-1 could not be detected in their PBMCs.
It is instructive to suggest that there may be a significant difference in treat-
ment of early vertical infection versus early horizontal infection, relative to
residual HIV-1 disease [120].
A recent study in six patients has demonstrated the rate of HIV-1 re-
bound in peripheral blood plasma after discontinuation of HAART [121].
The median time to obtain 500 copies per milliliter of plasma viral RNA,
from a level of below 50 copies per milliliter, was approximately 10 days
after stopping therapy. Using regression analysis, it was suggested that ap-
proximately 10 copies per milliliter of viral RNA in peripheral blood plasma
could have been the baseline level in these patients. Of importance, this is
remarkably close to the author’s study’s mean viral load in peripheral blood
plasma in the 22 patients studied in the present analyses (ie, 17 copies per
milliliter). Because most [121,122], but possibly not all [123], patients
rebound with high levels of plasma viral RNA when standard suppressive
HAART is discontinued, even when less than 50 copies per milliliter have
been demonstrated for significant time periods in their peripheral blood
plasma, this suggests ongoing viral replication and potentially infectious
virions still present in peripheral blood plasma and possibly other body fluid
compartments. If 3000 mL of blood plasma is considered an approximation
of that found in most humans, this suggests that in the patients in the
author’s recent study there was a mean level of 51,000 virion RNA copies
in blood plasma during suppressive HAART [88]. Other new approaches,
including intracellular HIV-1 mRNA quantitation, may be helpful in eval-
uating the ongoing persistent viral replication in patients on suppressive
HAART [124].
The source of rebound virus after discontinuation of HAART remains

controversial. This is a key issue for understanding residual HIV-1 disease
pathogenesis [125,126]. Of note, an acute seroconversion syndrome may
occur during viral rebound in patients interrupting HAART [127,128].
In a small recent study, viral rebound occurred in eight patients on viral
suppressive HAART after discontinuation of antiretrovirals [129]. In this
study by Garcia et al [129], it was shown that viral load rebounds in patients
on HAART after discontinuation of the antiretroviral therapy, but in these
patients the plasma viral RNA returned to undetectable levels following
reintroduction of the same treatment regimen. Preliminary data in this study
also suggested that in certain patients in whom HAART was discontinued
and then reinitiated, there were specific new impairments of several immu-
nologic parameters associated with the control of HIV-1 infection. An
article by Neumann et al [130] has demonstrated that there is no deleterious
effect when HAART is reinitiated in patients in which there was viral
rebound after an interruption of therapy. This suggested that targeted inter-
ruption studies may be performed without significant risk to the study indi-
viduals, although these findings are somewhat at variance with the data
presented by Garcia et al [129].
The low-level viral replication in patients on suppressive HAART may
take place constantly or in bursts. Viral replication in patients on suppressive
HAART may have some constant parameters, in comparison with untreated
patients or those with resistant virus and high plasma HIV-1 RNA. One
study has shown a constant mean viral copy number per infected cell in lym-
phoid tissue, regardless of the plasma HIV-1 RNA level [131]. Of importance,
viral reservoir decay characteristics in one recent analysis have suggested that
60 years are necessary for patients treated with suppressive HAART to con-
tinue therapy for potential viral eradication [132], although this is controver-
sial (see later). Unfortunately, even this may represent best-case scenarios for
some patients, because this analysis did not fully take into account low-level
viral replication, which occurs in most patients on HAART, as demonstrated
in the author’s study [88] and by others [45,87,100,103,114].
It is still unclear which cell types produce the residual virions in patients
on suppressive HAART. Sequencing of virion RNA at these low levels is
extraordinarily difficult but this may be approachable with new technologies
being developed. Of note, it is not clear whether this very low level viral rep-
lication leading to virion particles in the peripheral blood plasma in patients
on suppressive HAART comes from peripheral blood lymphocytes, tissue-
bound macrophages, lymph nodes, or other compartments that may be
relative drug sanctuary regions (eg, CNS, retina, and testes in men) [133].
Further studies are necessary to understand the in vivo molecular mecha-
nisms yielding residual HIV-1 disease in patients on what is now clinically
considered to be fully suppressive HAART.
Importantly, in a preliminary study [134] it was demonstrated that the
pool of latently infected CD4þ T lymphocytes did not completely account
for rebounding HIV-1 plasma virions in patients in whom HAART was dis-
continued. The envelope region of the rebounding virus in the bloodstream
was shown to be genetically different from the replication-competent virus,
which could be obtained from the reservoirs of latently infected resting CD4þ
T lymphocytes. These data also strongly point to the possibility that other
HIV-1 reservoirs may be involved with viral persistence during HAART.
A study by Hlavacek et al [135] has attempted mathematic modeling to
evaluate dissociation of HIV-1 virions from follicular dendritic cells (FDC)
in lymph nodes during suppressive HAART. It had previously been shown
by Cavert et al [45] that HIV-1 virions, which are bound to FDCs, decrease
after HAART but this may take up to 30 months in some cases to lead to
complete eradication on the FDC network. The Hlavacek et al [135] study
extends these data and suggests that, using a mathematic model, a biphasic
decrease takes place with the second phase being quite long, which may take
up to several years for complete virion ablation on suppressive HAART. Of
note, this study mainly evaluated complement proteins on HIV-1 virions
leading to trapping of virions on FDCs by binding to complement receptors.
The authors note that there are a relatively large number of potential con-
founding variables that may complicate this model, including structural de-
gradation of virions and uptake of virions by antigen-presenting cells. This
binding of virions to FDCs may also occur by the newly described dendritic
cell-specific HIV-1 binding protein, DC-SIGN [136]. Virions bound to
FDCs may be important in negatively affecting attempts at HIV-1 eradica-
tion. Approaches have begun to be developed to increase detachment of
HIV-1 from the follicular dendritic cells in lymphoid tissue [137].
Recent mathematic modeling of ongoing HIV dissemination during
HAART is supported by the virologic data in studies demonstrating resid-
ual HIV-1 replication [138]. In another interesting mathematic model by
Ferguson et al [139], the authors suggest that antigen-induced stimulation
of CD4þ T lymphocytes can generate differences in the decay of HIV-1
plasma viral load in patients on suppressive HAART. This model also sug-
gests that although HAART can lead to dramatic suppression of HIV-1 in
many patients, there is nevertheless ongoing residual HIV-1 replication. This
approach has significant similarities to the modeling by Grossman et al
[138]. These studies may have impact on clinical research and care, because
they suggest that most patients on HAART, at least within the first few
years of therapy, have prompt viral rebound if HAART is discontinued,
based on ongoing viral replication at low levels beyond the detection limit
of most clinical assays of plasma viral load. For the design of viral eradica-
tion studies of HIV-1, these data also suggest that there are two important
and possibly unrelated mechanisms of viral persistence during HAART.
One mechanism may be true proviral latency in quiescent CD4þ T lympho-
cytes and other cell types [140]. In addition, other data have demonstrated
that ongoing low-level viral replication must also be approached [88].
Although stimulatory therapy of latently infected cells may decrease the
pool of persistent virally infected cells in patients on suppressive HAART

[10], intensification therapy to truly ablate viral replication may be an
important initial approach in any attempts at viral eradication.
Persistence of HIV-1 in reservoir sites during effective HAART is of crit-
ical importance with regard to developing approaches to eradicate HIV-1
infection in the human host. New techniques, including real-time PCR and
molecular beacons, have become useful in evaluating residual disease during
HAART of HIV-1–infected individuals [141]. It is important to recognize
that the definition of clinically undetectable plasma HIV-1 RNA has
changed with the increased sensitivity of the technologies (ie, 1000 to 400,
and then to 20 to 50 copies per milliliter). There is still the critical need for
the use of new research techniques that can be used in clinical microbiologic
laboratories with which to determine the effects of novel therapies in addi-
tion to standard HAART in the treatment of residual HIV-1 disease.
Clearly, HIV-1 latency or persistence must be analyzed on a variety of
cellular and molecular levels. The cell types that harbor latent proviral
species, including resting CD4þ T lymphocytes, certain monocyte-
macrophage populations, and possibly other cell types, require further
investigation. In addition to CD4þ T lymphocytes, there may also be
a viral reservoir in patients on suppressive HAART in double-negative
T lymphocytes (ie, lacking CD4 and CD8 receptors) [142]. As described
previously, possible sanctuary or reservoir sites behind blood:tissue barriers,
such as the CNS, testes, and retina, are also critical areas for further study.
A short recent study has demonstrated that HIV-1 proviral DNA decays
during effective and suppressive HAART [143]. Only those patients who
were treated during the early asymptomatic phase, however, had the most
dramatic decrease in HIV-1 DNA copies in peripheral blood CD4þ T lym-
phocytes. This study also demonstrated that replication-competent virus
could be detected in all phases of disease during suppressive HAART. This
article may suggest, indirectly, that ongoing replication may occur more
commonly in late-stage patients versus early asymptomatic patients treated
with suppressive HAART [143].
Ongoing viral replication has also been shown to complicate analysis of
the latent HIV-1 reservoir. Recent studies have suggested that some patients
with less than 50 copies per milliliter of blood plasma RNA on HAART still
have low-level bursts of viral replication leading to more than 50 copies per
milliliter in transient spikes [144]. It was shown that in this cohort of patients,
the mean decay half-life of latent replication-competent proviral reservoirs is
higher in patients with transient RNA spikes compared with those patients
who consistently maintain plasma HIV-1 RNA levels at below 50 copies per
milliliter as followed longitudinally. As such, there may be a viral reservoir,
which in a study described previously was shown to have a very long half-life
and would require greater than 60 years of suppressive therapy before latent
reservoir eradication [132], which may actually be caused more by ongoing
subclinical viral replication rather than slow decay of a truly latent proviral
state. The more recent study [140] suggests at least some further optimism,
because the mean half-life of approximately 6 months of the latent viral res-
ervoir in patients who have no spikes above 50 copies per milliliter of plasma
viral RNA may allow eradication in this very relatively select group of pa-
tients if fully suppressive HAART is able to be maintained for a time period
significantly less than 60 years. This assumes that even in these patients
there exist no other viral reservoirs either in peripheral blood, lymphoid tis-
sue, or other solid tissues. Transient spikes or blips of plasma viral RNA in
patients on virally suppressive HAART were shown in one trial not to be
associated with lower CD4þ T-lymphocyte counts [145,146]. A preliminary
study has added still further complexity by showing that in acute HIV-1 sero-
conversion there is a biphasic decay of latent HIV-1 in resting CD4þ T lym-
phocytes. This may be caused by differing decay rates of preintegration
versus postintegration latent viral DNA forms [147].
A preliminary but interesting and important study has evaluated the
selection of antiretroviral resistance mutations in the latent reservoir of
HIV-1 during suppressive HAART. In this study, there were some drug
resistance mutations that developed in vivo in patients with frequent meas-
urements of HIV-1 plasma RNA levels below 50 copies per milliliter. This
is a very important study because it suggests that the low-level, ongoing viral
replication during suppressive HAART may in some cases lead to the
progression of resistance to antiretrovirals in the parental viral strain that
initially infects an individual [148]. Nevertheless, only those patients with
transient blips or spikes of detectable plasma viremia developed resistance
mutations during suppressive HAART [148].
Residual HIV-1 disease must be understood at the most detailed molec-
ular and cellular levels. Only in this way can potential HIV-1 eradication
therapies be rationally designed [149].
Acknowledgments
The author thanks Drs. Ashley Haase, Ian Frank, and Robert Silicano
for critical discussions. The author also thanks Ms. Rita M. Victor and
Ms. Brenda O. Gordon for excellent secretarial assistance.
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HIV-1 entry and entry inhibitors

as therapeutic agents
Linda D. Starr-Spires, PhD,
Ronald G. Collman, MD*
Pulmonary, Allergy and Critical Care Division,
University of Pennsylvania School of Medicine, 522 Johnson Pavilion,
36th and Hamilton Walk, Philadelphia, PA 19104–6060, USA
Combination antiretroviral therapy for HIV-1 infection introduced in the

last half-decade has resulted in dramatic control of viremia in most patients
and remarkable improvement in morbidity and mortality. A sizable propor-
tion of infected individuals, however, fail to benefit from these agents or
experience only temporary benefit because of the emergence of resistant
strains, unacceptable drug toxicity, or difficulties with compliance. Although
any step in the viral replication cycle is a potential target for therapeutic
intervention, currently available drugs target only two of these steps: reverse
transcriptase inhibitors block the stage early after viral entry when the viral
RNA genome is copied into DNA, and protease inhibitors block the protein
processing step carried out by the viral protease enzyme late in the replica-
tion cycle as virus leave the host cell. New strategies that target other steps
of infection are critically needed. The last several years have yielded exciting
advances in the understanding of how HIV-1 enters its target cell, which
have led to the development of promising agents that block viral entry and
are likely to be the next group of antiretroviral drugs to become clinically
available. This article addresses new insights into the mechanism of
HIV-1 entry, and focuses on prototype agents under study as representative
approaches that target specific steps of the entry process. More detailed
information of the structural biology and mechanisms of entry may be
found in several excellent recent reviews [1–4].
This work was supported by grants AI 07632, AI 35502, and MH 61139 from the National
Institutes of Health.
E-mail address: collmanr@mail.med.upenn.edu (R.G. Collman).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 1 1 - 2
682 L.D. Starr-Spires, R.G. Collman / Clin Lab Med 22 (2002) 681–701
HIV-1 entry
The entry of HIV-1 is mediated by the viral envelope glycoprotein (Env),
which interacts sequentially with two cell surface receptors, binding first to
CD4 and then to one of two alternative chemokine receptors, CCR5 or
CXCR4. These interactions are associated with complex structural changes
in Env that lead to fusion between the viral and cell membranes and enable
entry. The HIV-1 entry process may be viewed as a series of discrete albeit
overlapping steps involving intermolecular interactions and structural rear-
rangement initiated by receptor binding and culminating in merging of the
viral and cellular membranes and fusion pore formation (Fig. 1). Remark-
ably, similar mechanisms seem to be shared by highly divergent viruses,
Fig. 1. The HIV-1 fusion process according to current models. (A) The Env glycoprotein is
comprised of surface (gp120) and transmembrane (gp41) subunits that assemble as a trimer,
with the gp120 variable regions screening important conserved structures and the gp41
hydrophobic fusion peptide protected. Env attaches to the principal receptor CD4, which
induces conformational changes in gp120 that unmask the coreceptor binding site (B), enabling
binding to the chemokine receptor (C). The sequential binding of gp120 to CD4 and a
chemokine receptor induces structural changes in gp41 that extend the coiled-coil helical heptad
repeat domains to form a prehairpin intermediate (D). The N-terminal hydrophobic fusion
peptide inserts into the target cell membrane, allowing gp41 to span between the virus and cell
membranes. The gp41 heptad repeats then folds into a six-helix bundle (trimer of hairpins),
bringing the N- and C-terminal domains together and membranes in apposition (E). Contact
between the membranes allows mixing of the outer leaflets, resulting in hemifusion (F), which
then proceeds to development of a fusion pore (G). For clarity sake, gp120 is omitted from
panels F and G. (Reproduced from Doms RW, Moore JP. HIV-1 membrane fusion: targets of
opportunity. J Cell Biol 2000;151:F9–F13 by copyright permission of The Rockefeller
University Press; and Zimmerberg J, Chernomordik LV. Membrane fusion. Adv Drug Deliv
Rev 1999;38:197–205; with permission.)
L.D. Starr-Spires, R.G. Collman / Clin Lab Med 22 (2002) 681–701 683
including HIV-1, influenza, and Ebola (reviewed in [1,3,4]). Although differ-

ences exist between their envelope glycoproteins and the triggers that lead
to structural changes needed for fusion, much of the understanding of
HIV-1 entry has been developed based on insights gleaned from other models.
Env and receptor structure

Env is a type I integral membrane protein encoded by the virus and syn-
thesized by the host cell of origin. Env is formed as a polyprotein precursor
(gp160) that is heavily glycosylated and cleaved by cellular proteases into
two subunits that remain noncovalently associated. The N-terminal domain
forms the gp120 surface subunit that mediates attachment to the receptor
complex on target cells, whereas the C-terminal region forms the gp41 trans-
membrane subunit that is principally responsible for fusion. Env oligomer-
izes on the virion surface into trimeric spikes, with the gp41 extracellular
region containing the oligomerization domain.
Although gp120 is the most exposed viral protein, it offers a rather poor
target for antibodies because it is heavily glycosylated and because it is highly
mutable, varying greatly between different individuals and within an infected
person both over time and in different tissue compartments. Interspersed
among five relatively conserved constant domains (C1 to C5) of gp120 are five
domains that differ so greatly that they are termed hypervariable regions (V1 to
V5) and comprise a large proportion of the exposed surface area. The variable
regions form loop-like structures by intramolecular disulfide bonds and retain
a large degree of flexibility, whereas the conserved regions comprise the gp120
core. This core contains an inner domain that interacts principally with gp41,
and an outer domain that interacts mainly with CD4. Between the inner and
outer domains is a highly conserved bridging sheet, which comprises the
principal chemokine receptor binding site but is either not formed or not
exposed until gp120 binds to CD4 [5–7]. The gp41 is composed of an ecto-
domain, a membrane-spanning region, and a long cytoplasmic tail. The non-
covalent association between surface subunit and transmembrane subunit
involves interactions between discontinuous sequences of the aminotermini
and carboxytermini of gp120 that make up the inner domain and the gp41 ecto-
domain [8]. Within the gp41 ectodomain are the critical structures involved in
fusion, including a pair of heptad repeats that undergo structural reorganiza-
tion during the fusion process, and an N-terminal hydrophobic region known
as the fusion peptide that is normally buried within the glycoprotein and is
exposed only after initial triggering events during the fusion cascade [9].
CD4 is the principal receptor used by all known natural isolates of
HIV-1. CD4 is expressed on cells of myeloid lineage including T lympho-
cytes, monocytes and macrophages, thymocytes and dendritic cells (DC),
and this is the principal factor that determines HIV-1 tropism for these cells.
Normally serving as the T-cell receptor that interacts with major histocom-
patibility complex class II on antigen-presenting cells, CD4 is a member of
the immunoglobulin gene superfamily and contains four extracellular disul-

fide-bonded globin-like domains. The gp120 interaction site is located on
the N-terminal domain (D1), in a region different from the major histocom-
patibility complex class II binding site [10,11]. CCR5 and CXCR4 can be
engaged only after gp120 first binds to CD4, hence the term coreceptors. The
coreceptors belong to the seven transmembrane (7TM) G protein coupled
receptor superfamily, and have common structural characteristics of an
N-terminal extracellular domain, three extracellular loops, three intracellu-
lar loops, an intracellular cytoplasmic tail, and seven membrane-spanning
regions. These molecules normally serve as receptors for chemokines, which
are small (8 to 15 kd) structurally related cytokines involved in chemotaxis,
cellular activation, and, more recently recognized, embryologic development
(reviewed in [12]).
Although all HIV-1 isolates analyzed use CCR5, CXCR4, or both, entry
in vitro can also be facilitated by many related chemokine or orphan recep-
tors (molecules that resemble other members of this family but for which no
ligand has yet been identified). The alternative coreceptors include CCR2b,
CCR3, CCR8, CX3CR1, GPR1, GR15, STRL33-CXCR6, and others.
These molecules, however, are used by limited numbers of strains, generally
support entry less efficiently than the principal coreceptors, and it remains
uncertain whether they have any relevance to entry and infection in vivo
[13,14]. In contrast to HIV-1, some isolates of simian immunodeficiency
virus can engage CCR5 without first binding CD4 and enter cells in a
CD4-independent manner [15]. This finding, combined with the observation
that some strains of feline immunodeficiency virus use CXCR4 for entry
without using CD4 [16], have led to speculation that the chemokine receptor
is actually the original primordial immunodeficiency virus receptor, with
CD4 use emerging as a more recent evolutionary acquisition.
Mechanism of fusion
The initial contact between HIV-1 and the target cell involves binding by
Env on the virion to cell surface CD4 (see Fig. 1). Attachment to the CD4
induces conformational changes in the gp120 that exposes a previously cryp-
tic highly conserved site within gp120 (bridging sheet) that serves as the
coreceptor binding site. This process involves loops of the V1, V2, and V3
gp120 domains, which undergo structural rearrangements that move them
away from the buried coreceptor binding site [7,17]. Not only do the hyper-
variable loops limit immunologic recognition of the virus, but they also
serve to obscure the critical chemokine receptor binding site. The gp120
coreceptor binding region is now available to interact with the secondary
receptor, either CCR5 for macrophage-tropic variants or CXCR4 for T-cell
line–tropic variants (discussed later). These conformational changes take
place rapidly and are estimated to take less than 1 minute for CXCR4-using
strains and approximately 4 minutes for those that use CCR5 [18]. This
CD4 triggering, which enables gp120 to engage the chemokine receptor, is

required by all naturally occurring HIV-1 strains.
Interaction of gp120 with its cognate chemokine receptor then triggers a
series of structural changes in gp41 that ultimately lead to fusion [9,18–21].
Just how gp120 communicates these structural changes in gp41 is not clear.
The Env glycoprotein (gp120-gp41 complex) forms a trimer on the surface
of virions, which is held together by an oligomerization motif located in the
extracellular domain of gp41 immediately adjacent to the membrane. In its
native or nonfusogenic conformation, each gp41 ectodomain monomer con-
tains (from N- to C-terminus) a hydrophobic fusion peptide; a leucine zipper-
like 4-3 repeat that is termed the heptad repeat 1 (HR1) or N-peptide; a
hinge region; and a second leucine zipper-like 4-3 repeat termed heptad
repeat 2 (HR2) or C peptide. Individually, HR1 and HR2 adopt helical
conformations. Together, they spontaneously fold on themselves in an anti-
parallel manner, resulting in a hairpin configuration. When three monomers
associate to make up a mature gp41 trimer, these three sets of antiparallel
HR1 and HR2 domains are bundled together into what is termed a six-helix
bundle, such that the three N-peptides form an inner coiled-coil with a fusion
peptide at each amino terminus and the three C peptides nestle into hydro-
phobic grooves formed by the inner coiled-coil [9]. The C terminal region of
each gp41 serves to anchor the mature protein into the viral membrane by
the transmembrane subunit regions and intracellular tail. This configuration
also serves to sequester the N-terminal hydrophobic fusion peptide in the
center of the gp41 trimer.
Sequential binding of gp120 to CD4 and a coreceptor induces conforma-
tional changes in gp41 that enable the hydrophobic fusion peptide to
become inserted into the target cell membrane, bridging the virion and tar-
get cell membranes, in a structure known as the prehairpin intermediate
[9,22]. In this structure the gp41 C-terminal transmembrane domain is
anchored in the viral membrane, the N-terminal fusion peptide is anchored
in the target cell membrane, and the intervening HR1 and HR2 peptides
span the distance between. Experimental observations indicate that the pre-
hairpin intermediate is generated rapidly after chemokine receptor binding,
probably in less than 1 minute, but may exist for as long as 15 minutes [18].
Once the fusion peptide engages the target cell membrane, the HR1 and
HR2 peptides refold, acquiring their hairpin conformation (forming a
trimer-of-hairpins), and resulting in juxtaposition of the two membranes
[22]. Once the membranes are contiguous, mixing between the outer leaflets
occurs (hemifusion), followed by the development of a fusion pore that en-
ables entry of the core into the target cell (reviewed in [4]) [23].
Chemokine receptors, HIV-1 tropism, and pathogenesis

All HIV-1 isolates use CD4, but they differ according to the coreceptor
used for entry [24–27]. Coreceptor choice is a principal determinant of target
Fig. 2. Entry coreceptor use in HIV-1 pathogenesis. A schematized model of disease

progression is shown on top, with coreceptor phenotypes of blood virus isolates shown below.
New infections are nearly always associated with CCR5-dependent (R5) macrophage (M)-
tropic variants. In 40% to 50% of infected individuals, CXCR4 using X4 T-cell line (T)-tropic
(or R5X4 dual-tropic) variants emerge, and their appearance is associated with rapid disease
progression. Entry coreceptor usage plays an important role in person-to-person transmission
(CCR5) and accelerated pathogenesis (CXCR4).
cell tropism, which is regulated largely at the level of entry, and viral tropism
plays an important role in pathogenesis (Fig. 2). CCR5 and CXCR4 are the
only coreceptors clearly identified as relevant to HIV-1 infection in vivo.
Macrophage-tropic HIV-1 strains infect primary human macrophages and
CD4 lymphocytes in vitro but not CD4þ transformed cell lines. Macro-
phage-tropic isolates are relatively noncytopathic (non–syncytia-inducing
[NSI]) and use CCR5 as a coreceptor for entry (R5 strains). In contrast, T-cell
line–tropic isolates infect CD4þ lymphocytes and cell lines but not macro-
phages in vitro; are cytopathic (syncytia-inducing [SI]); and use CXCR4 for
entry (X4 strains). Dual-tropic HIV-1 isolates can infect all three target cells
and have the capacity to use both CCR5 and CXCR4 for entry (R5X4
strains). The region of Env that determines tropism and coreceptor selectivity
lies mainly within variable domains of gp120, particularly the V3 region
[25,28]. Interestingly, the region of Env that determines which coreceptor is
engaged is not the principal region that actually engages the coreceptor.
New HIV-1 infections are characterized almost exclusively by the pres-

ence of non–syncytia-inducing macrophage-tropic R5 strains, even if the
person from whom infection was acquired harbored SI variants that use
CXCR4 [29,30]. CCR5 use is necessary for person-to-person viral trans-
mission or, possibly, for infection to become established in a new host. The
reason for this stringent CCR5 requirement in person-to-person transmis-
sion remains unknown, but is true for both sexual and blood transmission
[31–33]. NSI R5 isolates are also the variants found throughout the course
of infection [34]. In 40% to 50% of patients, SI viral variants emerge that
have the capacity also to use CXCR4 [35–38]. These X4 or R5X4 HIV-1
strains evolve through mutations in the envelope gene and coexist along
with the R5 variants [34,39]. Although CXCR4-using variants do not arise
in all infected individuals, their appearance is closely associated with very
rapid immune destruction and accelerated progression to AIDS. The reason
that the acquisition of CXCR4 usage (termed the phenotypic switch or core-
ceptor switch) facilitates disease progression is not entirely clear. Potential
explanations include a higher proportion of CD4 cells that are CXCR4-pos-
itive and available for infection, or a mechanism to escape from the suppres-
sive effects of naturally produced CCR5-binding chemokines that might
otherwise interfere with the viral life cycle.
Additional surface molecules involved in HIV-1 entry

Although CD4 and the chemokine receptors comprise the essential entry
pathway, additional molecules can facilitate the process. Most prominent
among these are DC-SIGN (DC-specific intercellular adhesion molecule
[ICAM]–3 grabbing nonintegrin) and DC-SIGN-R [40–44]. DC-SIGN is a
type II membrane protein with a C-type lectin domain that is expressed
on the surface of myeloid dendritic cells (DC), present in mucosal epithelia.
DC are responsible for the surveillance, capture, processing, and presenta-
tion of antigen to T cells, and are believed to have an important role in
transport of HIV-1 from the mucosa to secondary lymphoid organs [45].
DC-SIGN binds ICAM-3, which is hypothesized to stabilize the interaction
between DC and T cells during the process of antigen presentation [41]. DC-
SIGN also binds gp120 with high affinity, and HIV-1 adheres to mucosal
DC by DC-SIGN. Furthermore, HIV-1 binding to DC-SIGN seems to sta-
bilize the virus and enable it to remain infectious longer [42]. DC may pick
up virus at mucosal surfaces through DC-SIGN–gp120 interactions, and
stabilize and transport it to the lymphoid tissues, where it encounters suit-
able target cells for infection. In addition to DC, DC-SIGN is expressed
by placental cells, peripheral blood mononuclear cells (PBMC), endothelia,
and certain cell lines [46,47]. DC-SIGN-R is a related molecule that seems to
have similar features although somewhat different distribution [40,43]. Of
note, HIV-1 binds to the oligosaccharide-recognition lectin domain of
DC-SIGN and can be blocked by carbohydrates, such as mannan [48].
Given its potential role in mucosal [49] and transplacental transmission [50],
and sensitivity to carbohydrate inhibition, DC-SIGN represents a potential
target for inhibitory therapy, although no such agents have yet been
identified [44].
Leukocyte function-associated antigen–1 (LFA-1) is a cellular integrin
molecule that enhances HIV-1 syncytia formation, virion infectivity, and
viral resistance to neutralization [51]. This effect is related to the presence
on virions of ICAM-1, a natural ligand for LFA-1, which is of cellular
origin and selectively incorporated as virions exit the host cell. Interactions
between virion ICAM-1 and target cell LFA-1 seem to stabilize the virus–
target cell interaction and enhance Env–receptor-mediated fusion [52,53].
Although the relevance of this mechanism to pathogenesis is uncertain, it
is probably a factor (in addition to gp120 structure and variability) contrib-
uting to the resistance of HIV-1 to antibody neutralization and limited
effectiveness of humoral immune responses in vivo. Another cellular protein
incorporated into virions is cyclophilin A. HIV-1 (but not simian immuno-
deficiency virus) specifically incorporates cyclophilin A during virion matu-
ration, and cyclophilin A is required for efficient progress through early
steps of infection [54,55]. Although the exact mechanism involved remains
uncertain, recent studies suggest that cyclophilin A becomes associated with
the virion outer membrane, where it facilitates attachment to target cells
[56]. It has been proposed that this attachment is mediated by cyclophilin
A binding to cell surface heparans or through interaction of cyclophilin A
with the cell surface receptor CD147 [56,57].
The entry of HIV-1 can also be enhanced by specific antibody or anti-
body and complement. In certain viral infections, notably dengue virus,
antibody or complement-mediated enhancement is well recognized and can
significantly worsen illness [58]. Virions bound by antibody or antibody and
complement attach to target cells that express Fc or complement receptors,
facilitating entry into target cells or even enabling entry into otherwise non-
permissive cells. Numerous studies have demonstrated antibody or comple-
ment-mediated enhancement of HIV-1 infection in vitro [59,60]. Although
some groups reported that this could serve as an alternative pathway of
CD4-independent entry [61], in most cases it seems to stabilize virus–target
cell interactions and facilitate entry through traditional pathways [62].
Although entry enhancement through this pathway was described over a
decade ago, it remains uncertain whether it contributes to pathogenesis [63].
In addition to the proteins involved in viral entry, the organization of
these molecules in the cell membrane and constituents of the membrane also
plays a role. Cholesterol and sphingolipids localize to microdomains on the
outer leaflet of the cell membrane known as lipid rafts, which are highly
organized domains resistant to nonionic detergents at low temperatures
[64]. In addition to lipids, certain surface proteins specifically localize to
rafts, including CD4 and the chemokine receptors, although they seem
to localize to distinct raft domains [65,66]. The interaction of HIV-1 Env
with the cell surface triggers lateral membrane diffusion, redistribution, and
clustering of CD4 and chemokine receptors, enabling the subsequent inter-
action with coreceptors [66]. Treatment of cells with b-cyclodextrin depletes
membranes of cholesterol (without affecting cell viability) and blocks
infection by HIV-1 in a manner that can be reversed by replenishment of the
cholesterol [66,67], probably by preventing the structural reorganization of
the receptor complex. Of note, this seems to be a bidirectional process
because budding of immature virions from the surface of infected cells
occurs specifically at the site of lipid rafts [68].
Entry inhibitors
Spurred on by both the successes and limitations of current highly active
antiretroviral therapy, new insights into viral entry have led to considerable
effort to target this step of the viral life cycle. A number of entry antagonists
are under development, and this field is rapidly evolving. Several promising
agents have entered clinical trials at the time of this article, but the history of
HIV drug discovery teaches that many strategies that initially seem promis-
ing ultimately do not succeed. Because the specific agents that will reach the
clinic may differ from those most promising at this point, this article focuses
on several prototypes that highlight specific approaches by which therapeu-
tic agents might target the individual steps in the entry process including
(1) CD4-mediated attachment, (2) CCR5 engagement, (3) CXCR4 engage-
ment, and (4) gp41-mediated membrane fusion.
CD4-based inhibitors
Attempting to block viral entry is not a new approach. Following the iden-
tification of CD4 as the principal viral receptor, considerable effort was
directed over a decade ago at developing agents that disrupt this interaction.
Recombinant soluble CD4 protein was shown to bind Env, prevent attach-
ment to the cellular receptor, inhibit entry, and effectively neutralize HIV-1
in vitro [69–71]. As a result, various CD4-based proteins entered clinical stud-
ies. Disappointingly, all were shown to be without significant clinical benefit in
vivo. Further investigation demonstrated that, although laboratory-adapted
strains of HIV-1 were indeed highly sensitive to soluble CD4 neutralization,
primary isolates (those obtained from infected people) were relatively resistant
[72]. The structural basis for this relative resistance is complex, but at least in
part involves a lower affinity between CD4 and oligomeric Env from primary
isolates as compared with laboratory-adapted isolates (which is not reflected
in affinity measurements between CD4 and monomeric gp120) [73]. Recently,
however, the use of CD4 to neutralize HIV has received renewed attention.
PRO 542 is a recombinant protein in which four copies of the CD4 outermost
two domains (D1D2) are fused to the Fc portion of human IgG2 [74,75].
Although the reason that this tetravalent molecule is more potent than
monomeric CD4 is not known, it has broad, potent neutralizing capabilities,

with antiviral activity against HIV strains from both clade B (the major North
American and European subtype of HIV-1) and outside clade B (responsible
for most infections elsewhere in the world) [75,76]. Phase I studies in HIV-
infected adults and children indicate that it is well tolerated, without toxicity,
and reduced both plasma RNA and viremia levels in patients [77,78]. This
agent is currently in phase II clinical trials.
CCR5 inhibitors
CCR5 is the coreceptor used by HIV-1 variants responsible for nearly all
cases of person-to-person transmission and the maintenance phase of infec-
tion, and so is an attractive candidate for blocking. Enthusiasm for CCR5
antagonists is heightened by an experiment of nature showing that, theoret-
ically, it is likely to be well tolerated and effective. Approximately 10% of
CCR5 alleles among whites carry a mutation encoding a 32-base pair dele-
tion (CCR5D32) that leads to a frameshift and lack of CCR5 expression
[31,32]. As a result, about 1% of these individuals are CCR5D32 homozy-
gous and completely lack CCR5 expression, whereas 18% are heterozygous
for the mutant allele and express lower levels of CCR5. In vitro, cells from
CCR5D32 homozygotes are resistant to R5 variants of HIV-1, although
they are permissive for X4 and R5X4 strains [79]. These individuals are very
highly (albeit not completely) resistant to becoming infected with HIV-1
even if repeatedly exposed [32], which proves both that R5 variants are
responsible for transmission or establishment of infection, and that blocking
CCR5 can block infection in vivo. Furthermore, infected heterozygotes
show markedly slower progression of disease [80], suggesting that even par-
tial blocking of CCR5 may impact disease progression in vivo. Finally, indi-
viduals who lack CCR5 expression seem completely normal, indicating that
interfering with this molecule is likely to be well tolerated. Of note, CCR5
is probably dispensable in part because it serves as a receptor for three
members of the highly redundant CC chemokine family (macrophage
inflammatory protein [MIP]-1a, MIP-1b, and regulated upon activation,
normal T cell expressed and secreted [RANTES]) and both MIP-1a and
RANTES can signal through other receptors.
Because b-chemokines block R5 HIV-1 entry (and provide a mechanism
by which CD8 cells normally suppress R5 HIV-1 strains [81]), several modi-
fied chemokines were developed with antagonist activity [82]. Small mole-
cule antagonists are far more attractive than proteins, however, and small
molecule agents that block 7TM receptors comprise the largest group of
pharmaceuticals in clinical use. TAK-779 was the first nonpeptide small
molecule CCR5 antagonist [83]. TAK-779 inhibits infection by many
(although not all) R5 primary and laboratory-adapted strains (IC50 ¼ 1.6 to
3.7 nm) and is completely inactive against X4 strains [84]. A more broadly
active small molecule nonpeptide CCR5 antagonist, SCH-C (SCH351125),
is active against R5 primary isolates from multiple clades and is effective

both in vitro and in vivo in severe combined immune deficiency mice
reconstituted with human hematopoietic tissue (SCID-hu Thy-Liv mice)
[85]. SCH-C is a true antagonist because it blocks RANTES and MIP-1a–
induced CCR5 activation and chemotaxis. SCH-C exhibits low inhibitory
concentrations (IC50 ¼ 0.4 to 9 nm) and has 50% to 60% oral bioavail-
ability and a serum half-life of 5 to 6 hours. This molecule is specific for
human CCR5 and has no cross-reactivity with macaque CCR5, which
prevents efficacy evaluation using simian immunodeficiency virus model
systems [85]. SCH-C is currently being evaluated in phase I to II human
clinical trials.
Although appealing for the reasons discussed previously, blocking CCR5
in HIV-1 infection raises an important concern unique to this target. Expe-
rience teaches that HIV-1 exposed to antiviral therapy inevitably develops
drug resistance. For reverse transcriptase and protease inhibitors, resistance
is associated with mutations that enable normal protein function while pre-
venting interaction with the inhibitor. For entry inhibitors, resistance muta-
tions in gp120 may do the same, allowing gp120 to interact with the same
cellular receptor but in a manner that is insensitive to the drug. Because
HIV-1 often evolves from CCR5 to CXCR4 use in vivo, however, it is con-
ceivable that this might occur under pressure of CCR5 blockade. Because
this coreceptor switch is associated with (and probably causes) rapid disease
progression in individuals with SI strains, it is possible that blocking CCR5
could encourage the emergence of X4 variants and actually accelerate dis-
ease progression. In one study that selected viruses resistant to CCR5 block-
ade using SCH-C in primary PBMC in vitro, resistant viruses did not use
CXCR4 and continued to rely on CCR5 in an SCH-C–resistant manner [86].
Whether coreceptor evolution might result in vivo from CCR5 blocking
remains unknown, but this concern highlights the importance of using any
new agent in combination with other drugs that inhibit the replication cycle,
because only by limiting the rounds of viral replication can the likelihood
of mutation and resistance be minimized.
CXCR4 antagonists
Both peptide and nonpeptide antagonists have been developed to target
CXCR4-mediated viral entry. ALX40-4C is an oligocationic peptide origi-
nally designed to mimic the basic domain of HIV-1 Tat and competitively
inhibit Tat-TAR interactions [87]. Unexpectedly, its inhibitory effect was
limited to SI variants and the V3 region of gp120 was found to be the prin-
cipal determinant of ALX40-C sensitivity. Subsequent studies showed that
in fact it blocked infection by inhibiting gp120 interaction with CXCR4,
possibly through electrostatic interactions [88]. Clinical trials indicated the
compound was well tolerated but further development has not been pursued
[89,90]. Synthetic polymeric constructs are another peptide construct based
on the consensus sequence of the apex of the gp120 V3 loop (GPGRAF) [91].
Synthetic polymeric constructs have X4-specific anti–HIV-1 activity when
assembled in multibranched constructs, and seem to block by interacting
with CXCR4 [92]. Synthetic polymeric constructs were tested in early
clinical trials but have not been pursued recently.
Bicylcams are the first nonpeptide small molecules CXCR4 antagonists
[93] and one of these agents, AMD3100, is being evaluated in clinical trials
[94,95]. AMD3100 is highly specific for CXCR4 and inhibits X4 HIV-1
infection and binding and signaling by the normal ligand, stromal-derived
factor (SDF-1a). AMD3100 is active at low concentrations (IC50 ¼ 1 to
10 ng/mL) and blocks HIV-1 both in vitro and in vivo in a SCID-hu
Thy-Liv mouse model [96]. AMD3100 has low oral bioavailability and
requires parenteral administration. In phase I studies, AMD3100 by intra-
venous infusion was well-tolerated and had a half-life of 3.6 hours [97].
Given that its activity is restricted to X4 strains, AMD3100 is likely to be
useful only for the fraction of patients in late-stage disease with predomi-
nantly X4 or R5X4 variants. Unlike CCR5, which is part of a redundant
b-chemokine network, CXCR4 has only a single known ligand, SDF-1a,
which itself has no other receptor. Although congenital absence of CCR5
is well tolerated, the lack of either CXCR4 or SDF-1a during embryogenesis
in knockout mice results in lethal cardiac, neurologic, vascular, and hema-
tologic abnormalities [98–100]. Although the consequences of pharmaco-
logic inhibition differ from the developmental absence of a molecule, it
remains to be determined whether blocking CXCR4 is as well tolerated as
the evidently dispensable CCR5 receptor.
Fusion inhibitors
Clarifying the specific steps involved in fusion has led to the development
of agents that interfere with the conformational changes required. T20
(known also as DP178) is a synthetic peptide corresponding to the C peptide
(HR2) region of the gp41 ectodomain (residues 643-678) [101]. As described
previously (Fig. 1), gp120 binding to CD4 and the coreceptor causes gp41 to
assume a transient prehairpin intermediate conformation, which inserts the
fusion peptide into the target cell membrane. The gp41 N-terminal (HR1)
and C-terminal (HR2) peptides then associate in an antiparallel manner
to form a hairpin structure and pack together within the trimer as a six-helix
bundle. The C peptide mimetic T20 is believed to associate with the N-ter-
minal peptide in this unfolded prehairpin intermediate, and disrupt the sub-
sequent interaction between HR1 and HR2 required for fusion (Fig. 3). In
vitro, T20 exhibits strong fusion inhibition (EC50 ¼ 1 ng/mL). Even though
T20 targets gp41 (rather than gp120), it is about 10-fold more active for X4
than R5 strains and determinants within gp120 regulate T20 sensitivity
[102], underscoring the complex interactions between surface and transmem-
brane subunits of Env. In a small phase I and II study, T20 administered
Fig. 3. Structure-based mechanism of a prototype fusion inhibitor, T20. The prehairpin

configuration assumed by gp41 following Env engagement of CD4 and coreceptor bridges
between the two membranes. Fusion results from the N- and C-terminal heptad repeats (HR1
and HR2) assuming a hairpin configuration and assembling into a trimer of hairpins. The C
peptide HR2 mimetic T20 associates with gp41 N-peptide HR1, preventing hairpin formation
and blocking fusion. (Modified from Eckert DM, Kim PS. Mechanisms of viral membrane fusion
and its inhibition. Annu Rev Biochem 2001;70:777–810 Ó 2001 by Annual Reviews, www.
Annual Reviews.org; and Doms RW, Moore JP. HIV-1 membrane fusion: targets of opportunity.
J Cell Biol 2000;151:F9–F13; with copyright permission of The Rockefeller University Press.)
intravenously at a dose of 100 mg twice daily showed clinical efficacy, with

approximately 2-log reduction in plasma viremia [103]. Phase III clinical tri-
als are under way. Barriers to clinical use of T20 include the need for paren-
teral administration, and the difficulty and cost of manufacturing large
quantities of this peptide agent. Of note, in addition to fusion inhibition,
T20 has a number of other biologically active properties including chemo-
taxis and immune activation [104].
A different approach has been described to lock gp41 into the prehairpin
intermediate configuration and block entry. Termed 5-Helix, this construct
is designed to mimic the structure of the gp41 trimer-of-hairpins and con-
tains three N-peptide coiled-coil regions and two C peptide regions attached
by peptide linkers [105]. Elimination of the third C peptide exposes a hydro-
phobic groove generated by two of the N-peptide regions, making it available
for binding to an exposed C peptide region of a prehairpin fusion interme-
diate. 5-Helix exhibits broad-spectrum activity in vitro against R5 and X4
strains from diverse clades at nanomolar concentrations, although like T20
the activity was greater against X4 strains. As a complex protein, 5-Helix has
limited potential as a therapeutic agent itself but offers a useful model for the
development of other fusion intermediate-targeted therapeutics.
Gene therapy and other approaches

In addition to pharmacotherapy, HIV-1 entry has the potential to be tar-
geted by such methods as gene therapy and immunomodulation. Genetic
approaches developed to down-regulate coreceptor expression in vitro and
make cells resistant to entry have included intracellular immunization (the
intracellular expression of single-chain antibodies directed at the chemokine
receptor) [106,107]; antichemokine receptor ribozymes (which interfere with
message expression) [108,109]; and intrakines (chemokines modified to be
retained in the endoplasmic reticulum that bind and trap their cognate
receptors) [110,111]. Similarly, a retrovirally expressed membrane-bound
form of T20 has been shown also to block infection in vitro [112]. Obvi-
ously, gene therapy approaches to block HIV-1 entry are subject to the same
general limitations as all gene therapies, such as efficient delivery and persis-
tence in vivo, and these approaches are much less developed and further
from clinical application than is entry-targeted pharmacotherapy. In vitro,
T-cell stimulation with immobilized anti-CD3 and anti-CD28 antibodies
leads to CCR5 down-regulation and resistance to infection [113]. Studies
of ex vivo T-cell costimulation followed by reinfusion in HIV-infected sub-
jects have been initiated, but clinical benefit remains to be determined [114].
Summary
Defining the mechanisms of HIV-1 entry has enabled the rational design
of strategies aimed at interfering with the process. This article delineates
what is currently understood about HIV-1 entry, as a window through
which to understand what will likely be the next major group of antir-
etroviral therapeutics. These exciting new approaches offer the promise of
adding viral entry to reverse transcription and protein processing as steps
to block in the viral life cycle.
Several principles learned with other antiretroviral drugs are sure to be
valid for entry antagonists, whereas other considerations may be unique
to this group of agents. There is no agent to which HIV-1 has not been able
to acquire resistance and this is likely to remain the case. Multiple rounds of
viral replication are required to generate the genetic diversity that forms the
basis of resistance. Combination therapy in which replication is maximally
suppressed will remain a cornerstone of treatment with entry inhibitors, as
with other agents. Furthermore, the coreceptor specificity of some entry and
fusion inhibitors argues that combinations will likely be needed to broaden
the effective range of susceptible viral variants. Finally, the targeting of mul-
tiple steps within the entry process has the potential for synergy. The fusion
inhibitor T20 and CXCR4 antagonist AMD3100 are synergistic in vitro at
blocking infection of PBMC with clinical isolates [115] and T20 combined
with the CD4 inhibitor PRO 542 have synergistic in vitro effects, with more
than 10-fold greater inhibition of R5, X4, and R5X4 strains than either
agent alone [116].
Entry antagonists raise other, unique issues. As discussed previously, the
theoretic concern exists that blocking CCR5 could enhance the emergence
of CXCR4-using variants and possibly accelerate disease. So far, in vitro
selection for variants resistant to the CCR5 antagonist SCH-C in PBMC
(which express both CCR5 and CXCR4) has resulted in mutants that were
resistant to the blocker but still used CCR5. Alternatively, because many
HIV-1 strains have the capacity to use several other chemokine or orphan
receptors for entry, blocking both CCR5 and CXCR could lead to a variant
that uses one of these other molecules in place of the principal coreceptors,
although data in vitro so far suggest that this is unlikely [13,14]. This new
class of antiviral drugs offers great promise but also novel concerns, and
careful analysis of viruses that arise with their use in vivo is essential.
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Clin Lab Med 22 (2002) 703–717
Neuropathogenesis of central nervous

system HIV-1 infection
Dennis L. Kolson, MD, PhD
Department of Neurology, University of Pennsylvania Medical Center, Room 280C
Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104–6140, USA
The neuropathogenesis of HIV-1 infection of the central nervous system

(CNS) has been intensively studied, yet it remains incompletely understood.
The virus enters the CNS early after systemic infection, establishes a viral
reservoir, and remains largely inaccessible to antiretroviral therapies. In cer-
tain individuals, slowly evolving cognitive dysfunction becomes manifested
years after infection, resulting in frank dementia (AIDS dementia complex
[ADC]) in approximately 10% of individuals, with less severe cognitive dys-
function in up to 50% of all infected individuals. Neuropathologic and neu-
roimaging studies demonstrate that reversible metabolic disturbances occur
in neurons and glia early after CNS invasion and that progressive, irre-
versible neurodegeneration may precede overt clinical deterioration by
years. In vitro studies demonstrate that HIV-1 infection of macrophages and
microglia, the endogenous brain macrophages, releases soluble factors that
induce neuronal cell death. The viral and host factors that modulate neuro-
nal cell death in vivo are only partly understood. Evolving therapies directed
toward preventing neurodegeneration in HIV infection include CNS-pene-
trating antiretroviral drugs and pharmacologic agents that protect neurons
from effects of HIV-1–infected macrophage neurotoxins. Major unanswered
questions concerning the pathogenesis and prevention of HIV-1–induced
CNS degeneration include (1) which cellular and viral factors are primarily
responsible for HIV-1–induced neurodegeneration in vivo; (2) which surro-
gate markers of HIV-1 infection in the CNS (eg, viral load or cytokines) can
predict risk for neurodegeneration; and (3) can antiretroviral drugs or
specific CNS-targeted agents protect against HIV-1?
This work is supported by grants NS37651 and NS27405 from the National Institutes of
Health.
E-mail address: kolsond@mail.med.upenn.edu (D.L. Kolson).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 0 9 - 4
704 D.L. Kolson / Clin Lab Med 22 (2002) 703–717
The CNS as a viral reservoir for HIV-1

The invasion of the CNS by HIV-1 occurs early after infection and virus
replication within the CNS is essential for the development of ADC
(reviewed in [1–3]). Productive viral replication within the brain occurs only
in perivascular monocyte-derived macrophages and endogenous microglia
[4–6]. Although the number of infected macrophages does not correlate
closely with the degree of dementia [7], the state of activation of macro-
phages and microglia may indeed correlate with dementia severity [8].
HIV-1 can be demonstrated consistently within the brain of patients with
ADC and, less consistently, within the cerebrospinal fluid (CSF) [9]. Because
the percentage of productively infected macrophages and microglia is low
(5% to 20%; [5]) and because they are variably distributed throughout the
brain, neuronal cell death and dysfunction likely result from release of solu-
ble factors from infected macrophages and other cells (see later; reviewed in
[1,3]). It is likely that proinflammatory cytokines and neurotoxic metabolites
and viral gene products released by infected macrophages are expressed in
brain parenchyma shortly after infection and throughout the course of infec-
tion. This suggests that neuronal cell damage may begin long before neuro-
logic symptoms appear. It is unclear, however, whether a threshold of viral
replication (viral load) or accumulation of proinflammatory cytokines and
neurotoxins must be reached before cells are lost.
Viral load, CD4 count, and neurologic dysfunction

The relationship between viral load in the systemic circulation, CSF, and
brain parenchyma and the development of neurologic symptoms in HIV-1
infection remains controversial. In a large cohort comprised of patients from
the Multicenter AIDS Cohort Study and Johns Hopkins HIV Neurology
Clinic, McArthur et al [10] demonstrated that brain parenchymal RNA lev-
els and CSF RNA levels correlated in demented subjects and that CSF and
plasma RNA levels were correlated in patients with CD4 counts less than 200
copies/mm3. In a later study with the Multicenter AIDS Cohort Study
patient cohort, Childs et al [11] found that plasma RNA levels were predic-
tive of ADC and sensory neuropathy. Similarly, Hengge et al [12] showed
that both plasma and CSF viral load positively correlated with severity of
ADC in a controlled cohort of 100 patients (73 with ADC, stages 1 to 3).
Importantly, however, in 25 control patients followed longitudinally for 13
months in the Hengge et al [12] study, CSF HIV-1 RNA levels failed to pre-
dict the development of ADC. Di Stefano et al [13] also found a correlation
between CSF viral load and neurologic impairment in a study of HIV-1 iso-
lates from paired blood and CSF specimens from a cohort of 33 patients.
Some patients also suffered from cytomegalovirus encephalitis or progressive
multifocal leukoencephalopathy (PML), however, and neurologic symptoms
were classified only as motor, behavioral, or cognitive impairment [13].
D.L. Kolson / Clin Lab Med 22 (2002) 703–717 705
Several other studies have failed to confirm a correlation between CSF

viral load and severity of neurologic impairment. Brew et al [14] found no
correlation between ADC severity and plasma viral load in 19 patients with
ADC, although a significant correlation between ADC severity and CSF
viral load was seen. Importantly, there was no correlation between CSF and
plasma viral load, suggesting significant compartmentalization of virus rep-
lication in the CNS and periphery. In a series of 41 HIV-1–infected patients
(11 with HIV encephalitis [HIVE], 12 with other CNS diseases, and 19 neu-
rologically normal), Bossi et al [15] found no correlation between CSF viral
load and presence of HIVE. The different conclusions reached in these stud-
ies may reflect different antiretroviral regimens, sensitivities of the RNA
assays, sample size, and demographics of the control groups. Considering
these differences, the data provided by well-controlled studies provide ample
evidence that high CSF viral load is a significant risk factor for the develop-
ment of ADC, and that inhibition of HIV replication within the CNS might
be expected to lessen the severity of progression of ADC.
Neuropathology of ADC
The major pathologic features of ADC are brain atrophy with varying
degrees of neuronal loss (up to 50% of cortical pyramidal neurons) and neuro-
nal apoptosis [16–22]. In addition, altered cortical neuronal dendritic spine
density and morphology, microglial nodules and multinucleated giant cells,
myelin pallor, astrocytic proliferation, and (in pediatric cases) basal ganglia
calcifications are common (reviewed in [23]). Another prominent finding is the
perivascular accumulation of macrophages, believed to be recruited from cir-
culating monocytes [24,25]. Release of toxic factors (see later) from infected
and noninfected macrophages is thought to be the primary inducer of neuro-
degeneration that is commonly associated with neuronal dropout and
apoptosis in the brain in both pediatric and adult patients (reviewed in [26]).
Evidence for neuronal apoptosis in HIV-1 infection

Neuronal apoptosis in HIV-1–infected brain has been demonstrated by a
number of investigators using brain tissue from both AIDS and pre-AIDS
patients [16,17,19–22]. Gelbard et al [22] demonstrated apoptotic neurons
colocalizing with HIV-infected perivascular macrophages in the basal gan-
glia and cerebral cortex in seven of seven autopsied pediatric brains and
zero of nine seronegative controls. Although quantitative correlation with
neurologic functioning was not attempted, the investigators concluded that
neuronal apoptosis was greater in patients with HIVE or progressive
encephalopathy in comparison with patients without similar pathologic or
clinical findings. In a concurrent publication, Adle-Biassette et al [19]
detected apoptotic neurons in the temporal and frontal cortices of 12 of 12
patients with AIDS, 1 of 4 asymptomatic HIV-seropositive controls, and
0 of 5 HIV-seronegative controls; the relative abundance of apoptotic cortical

neurons was believed to be higher in patients with cortical atrophy. Petito
and Roberts [16] also detected neuronal (and astrocytic) apoptosis in five
of eight brains with HIVE and one of seven brains from patients with AIDS
but without HIVE, and suggested that the frequency of neuronal apoptosis
is directly correlated with the severity of HIVE. In a later study, An et al
[21] found apoptotic cells with neuronal morphology in 6 of 10 cases of
HIVE, 1 of 8 cases of AIDS without CNS disease, and in 4 of 36 asymp-
tomatic HIV-seropositive cases. This study was again consistent with the
hypothesis that neuronal apoptosis may occur in pre-AIDS patients, but
that increasing severity of pathologic abnormalities in the brain is associated
with increasing prevalence of apoptotic neurons and perhaps glia. Finally, a
more recent study of 40 brains by Adle-Biassette et al [20] showed neuronal
apoptosis in 18 of 20 patients with AIDS and 2 of 10 HIV-seropositive
asymptomatic patients. This study extended earlier reports to conclude that
the severity of neuronal apoptosis in HIV-infected brain correlates with the
presence of cerebral atrophy and local microglial activation, but not with
clinical dementia. One very recent study also reported a significant level of
astrocytic apoptosis in the basal ganglia of patients with ADC and dem-
onstrated a significant correlation between rapid neurologic progression and
level of basal ganglia astrocytic apoptosis [27].
Several conclusions may be derived from examination of published neu-
ropathologic studies: (1) neuronal brain damage, including apoptosis and
morphologic alterations, is common in pediatric and adult patients with
AIDS, but less common in asymptomatic HIV-seropositive individuals;
(2) the severity of microscopic neuronal damage generally correlates with
the severity of brain atrophy; and (3) regional differences in the degree of
neuronal damage reflect the relative degree of brain macrophage infection
or activation. Prevention of neuronal damage through inhibition of HIV-1
replication in brain macrophages, prevention of immune activation of brain
macrophages, or prevention of neuronal apoptosis through neuronally tar-
geted agents may protect against development of ADC.
Mechanisms of HIV-1–induced neurodegeneration

HIV infection of macrophages within the CNS
With occasional exceptions, viruses isolated from the CNS of patients
with ADC are macrophage-tropic (M-tropic), consistent with pathologic
evidence supporting productive infection in cells of the macrophage lineage
in patients with ADC [28,29]. M-tropic strains generally use the CC chemo-
kine receptor, CCR5 (R5 isolates), whereas T-tropic strains use CXCR4 (X4
isolates) as their primary entry cofactor [30]. Not all R5 isolates produc-
tively infect macrophages [29]. Dual-tropic strains can infect either cell type
and are generally R5/X4 in phenotype. Recent data have shown that some
X4 isolates may also infect macrophages by CXCR4 [31]. Throughout the

course of infection, R5 isolates are predominate in plasma, CSF, and brain
tissue of most patients, although emergence of X4 isolates occurs in some
[29,32,33]. Studies comparing viral sequences from the CNS with those from
blood have, in general, shown distinct species in the two compartments [34–
36]. Recent evidence suggests HIV-1 envelope (env) sequence-specificity in
brain isolates as a determinant of neurovirulence [37–39]. Several other
studies, however, failed to find brain-specific HIV-1 env sequences [39–41].
Although it is clear that HIV infection of macrophages in vivo is associated
with neuronal apoptosis, it remains unclear whether HIV-1 strains with selec-
tively enhanced ability to induce neuronal apoptosis evolve within the CNS.
HIV envelope as a determinant of neurotoxicity

Several studies have focused on the role of env in inducing neuronal cell
death, including apoptosis [37,42–44]. In an early study, Power et al [37]
examined env sequences from postmortem brains of ADC and non-ADC
AIDS and demonstrated an M-tropic consensus sequence within the V3
loop of env. In addition, there were multiple amino acid differences in enve-
lopes from brain and spleen isolates from the same individual. This sug-
gested that there is little env sequence divergence within the CNS and that
viral determinants for neuroinvasiveness or neurovirulence are located with-
in env. These env sequences were cloned into an infectious and otherwise
isogenic molecular clone, and such clones containing env sequences from
patients with ADC induced greater neurotoxicity in vitro [44], again sug-
gesting that CNS-derived M-tropic strains induce neuronal death through
infection of brain macrophages.
In related studies, Ohagen et al [42] examined neuronal and astrocytic
apoptosis in human fetal brain cultures infected with HIV isolates from
peripheral blood (89.6, SG3, and ADA) and brain (YU2, JRFL, DS-br,
RC-br, and KJ-br). Low, but significant, levels of apoptosis were induced
by the blood isolates but not the brain isolates. Furthermore, apoptosis
determinants mapped primarily to the V3 region of env, and apoptosis was
independent of the level of virus replication. This study demonstrated that
R5/X4, X4, and R5 HIV isolates could induce neuronal apoptosis in vitro,
albeit to varying levels. The chemokine receptor use pattern of these blood
isolates (89.6: R5/X4; SG3: X4; and ADA: R5) suggested that increased
ability to induce apoptosis was associated with the strain’s ability to use
CXCR4, suggesting that evolution of expanded chemokine receptor use
(eg, acquisition of the ability to use CXCR4) may be associated with
increased neurovirulence and neurotoxicity. A more recent study extended
these findings to suggest that increased fusogenicity of the viral envelope
may account for increased neurovirulence [43]. In addition, non–macro-
phage-tropic X4-restricted T-cell–tropic virions (MN, IIIB, and Lai) may
induce neuronal apoptosis through interaction with CXCR4 [45–47]
Fig. 1. Potential HIV-associated neuronal apoptosis pathways. In vitro studies suggest

activation of the intrinsic (mitochondrial) apoptosis pathway in neurons by activation of the
N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Activation of the extrinsic
(death receptor family) pathway may occur by actions of tumor necrosis factor (TNF)–a and
Fas ligand (Fas-L) although experimental evidence for Fas-mediated neuronal apoptosis in
HIV-1 infection is lacking. Apoptosis induced by gp120 or stromal cell-derived factor 1–a
(SDF-1) by neuronal CXCR4 activation may result in activation of p38 mitogen-activated
kinase (MAPK), although the specific cascade of apoptosis effectors involved in such apoptosis
remains to be elucidated Akt-P ¼ phosphorylated Akt/Protein kinase B; NO ¼ nitric oxide;
Apaf-1 ¼ apoptotic protease activating factor 1.
(Fig. 1). Furthermore, envelope toxicity may be seen in the context of whole
virions or as free gp120 [47]. These observations suggest that both M-tropic
and T-tropic strains may ultimately induce neuronal apoptosis through dis-
tinct mechanisms throughout the course of infection, although the ability of
strains to do so varies considerably. This also suggests that the CNS is likely
vulnerable to HIV-induced neuronal injury even early after infection.
Other HIV-associated factors that induce neuronal apoptosis

Essentially all studies that have addressed mechanisms of HIV-1–induced
neuronal apoptosis are based on in vitro model systems; studies of neuronal
gene expression in vivo related to apoptosis pathways are very limited
[17,48,49]. A number of studies have focused on the ability of recombinant
HIV-1 proteins or replicating virus to induce neuronal damage in mixed
neuronal-glial cell cultures (reviewed in [3]). Recent studies have focused
on the effects of replicating virus strains isolated directly from patients or

strains derived by recombinant cloning techniques [42,44,50]. In vitro
evidence supports a role for the HIV-1 envelope (gp120); other viral pro-
teins (Tat, Vpr, and Nef); proinflammatory cytokines; nitric oxide; platelet-
activating factor; arachidonic acid; metabolic amines; and other cellular
factors (chemokines, such as stromal cell-derived factor–1a) released by
HIV-1–infected or –activated macrophages, as mediators of neuronal death
rather than direct infection of neurons (reviewed in [2,26,51]). Some of these
proteins (eg, Tat and gp120) may show synergistic toxicity [52]. Although
the mechanism of HIV-1–induced neuronal apoptosis remains controversial,
stimulation of N-methyl-D-aspartate (NMDA)–type glutamate receptors may
be a final common pathway of injury to neurons (see Fig. 1). NMDA receptor
activation may be induced by glutamate and other released excitotoxins from
infected macrophages and macrophages stimulated with gp120, proinflam-
matory cytokines, chemokines, Tat, Vpr, and other soluble factors [50–55].
In vitro studies suggest that the intrinsic (mitochondrial) apoptosis path-
way is activated in neurons by such soluble macrophage neurotoxins, result-
ing in cytochrome c release and subsequent caspase (9, 3) activation and
apoptosis (see Fig. 1) [56]. In addition, a role for nitric oxide–induced neuro-
nal cell damage under such exposure is widely supported, although such a
mechanism may involve both receptor-mediated effects and direct membrane
damage with mitochondrial injury by the intrinsic apoptosis pathway [57–59].
The gp120 may induce neuronal apoptosis, either directly by interaction with
neuronal chemokine receptors (eg, CXCR4 [45,46]) or indirectly by stimulat-
ing release of NMDA receptor-stimulating neurotoxins from glial cells
[58,60–62], perhaps by interaction with either CXCR4 or CCR5 [46,47,63].
Stromal cell-derived factor–1a may also induce neuronal apoptosis by direct
interaction with CXCR4 [45,47]. Whether chemokine receptor activation and
subsequent apoptosis are mediated through specific downstream effectors of
the intrinsic or extrinsic apoptosis pathways is currently unknown. Evidence
for activation of the extrinsic apoptosis pathway in neurons in HIV-1–asso-
ciated neuronal death is limited. Although tumor necrosis factor–a has been
implicated in HIV-1–associated neuronal death and ADC, it remains unclear
whether this occurs by direct activation of the extrinsic apoptosis pathway
[64–66]. In contrast, there is little evidence for HIV-1–associated Fas-medi-
ated neuronal apoptosis in vitro or in vivo, although one study has identified
expression of Fas ligand on reactive astrocytes, lymphocytes, and occasional
macrophages in HIV-infected brain tissue [67].
In summary, in vitro studies of HIV-1–induced neuronal death and apop-
tosis have shown that (1) HIV-1 isolates of X4, R5, and R5/X4 phenotypes
can induce neuronal death through infection of macrophages with concom-
itant immune activation of noninfected macrophages [42,44,50]; (2) the
neurotoxic effects of macrophage activation and HIV-1 infection in combi-
nation are greater than the effects of infection alone; (3) chemokine recep-
tors on glial cells (astrocytes and macrophages) and on neurons may
mediate apoptosis induction in the presence of HIV envelope (recombinant

or virion-associated); and (4) neurotoxicity resulting from macrophage
infection or exposure to viral proteins is largely abrogated by NMDA recep-
tor antagonists. Effective neuroprotection against HIV-1 in the CNS likely
depends on suppression of virus replication in macrophages and agents that
have direct protective actions on neurons (eg, NMDA receptor blockers).
Neuroprotection against HIV-1

Impact of highly active antiretroviral therapy on neurologic dysfunction
Evidence suggests that resting CD4þ T cells are a latent HIV-1 reservoir in
patients on highly active antiretroviral therapy (HAART) despite plasma
viremia below detectable limits (\50 to 500 copies/mL) [68–70]. A pool of
latently infected, resting CD4þ T cells is established early (10 days) after
symptoms of primary infection, despite early initiation of HAART [71]. These
observations suggest that even early initiation of HAART is unlikely to elim-
inate reservoirs of HIV-1 in all cellular compartments, including the brain
[72]. Despite the failure of HAART to eliminate latent HIV-1 reservoirs,
patients may experience significant reconstitution of immune function [73].
The impact of HAART on the incidence and clinical course of neuro-
cognitive dysfunction in AIDS remains unclear, although accumulating
evidence suggests significant benefit. Several early studies suggested that
cognitive deficits may be partially and temporarily reversed by zidovudine
monotherapy in children and adults [74–76]. Although CSF penetration
of most antiretrovirals is poor [77], early institution of HAART might
reverse or prevent clinical manifestations of ADC and certain signature
metabolic abnormalities indicative of neuronal and glial cell dysfunction
detected by proton MR spectroscopy (1H-MRS) [78]. A case study by
Gendelman et al [79] demonstrated rapid decline in CSF and plasma viral
load and CSF neurotoxins (tumor necrosis factor–a, quinolinic acid, and nitric
oxide) with a concomitant improvement in cognitive function in an anti-
retroviral therapy–naive patient presenting with cognitive impairment. In an
1H-MRS study by Chang et al [78], 16 patients with mild-to-moderate ADC
(stages 1 to 2) were studied before and after 3 months of HAART, which
resulted in reversal of abnormalities of the choline-creatine and myoinositol
ratio in frontal cortex and basal ganglia. Importantly, these changes corre-
lated with improvement in clinical staging according to the HIV dementia
scale [80]. Combination antiretroviral therapy with or without protease
inhibitors has also been shown to improve tests of psychomotor speed per-
formance in HIV-infected individuals [81]. A recent epidemiologic study
suggested, however, that although the overall incidence of ADC may be
decreasing because of institution of HAART, there is a relative increase in
ADC with respect to other AIDS-defining illnesses, despite consistent
increases in CD4 cell counts [82]. This suggests that HAART may provide
less protection against ADC than other AIDS-defining illnesses, possibly
because of consistently poor penetration of antiretrovirals into the CNS or a

delayed effect on viral replication in the CNS. Along these lines, Ellis et al [83]
have shown that the kinetics of the decline in CSF HIV RNA levels in patients
starting antiretroviral therapy is increasingly independent of the plasma
RNA response with advanced disease, especially in patients with ADC. This
suggests that specific targeting of the CNS with antiretrovirals may be neces-
sary to suppress effectively HIV replication in the CNS compartment.
Other neuroprotective strategies against HIV-1

Current clinical trials: NMDA receptor and free radical antagonists
Randomized, blinded, placebo-controlled therapeutic efficacy studies in
ADC are few in number, although two such studies are currently underway
(AIDS Clinical Trials Group study [ACTG] 301 and ACTG 5090). Previous
studies of abacavir (reverse transcriptase inhibitor; 99 patients followed for
4 months) and nimodipine (calcium channel antagonist; 30 patients followed
for 2.5 months) showed no clear benefit ([84] reviewed in [85]). As for these
earlier studies, additional strategies for protecting the CNS against effects of
HIV-1 are evolving from in vitro studies of mechanisms of HIV-induced
neuronal cell death. The most widely investigated and verified pathway of
HIV-induced neuronal death occurs by NMDA receptor overstimulation
(reviewed in [86]). Early observations indicated that HIV-1 replication in
cells of the monocyte-macrophage lineage release factors that ultimately
result in neuronal NMDA receptor overstimulation, followed by neuronal
cell death [87]. This ‘‘final common pathway’’ of cell death may be triggered
by glutamate and other related NMDA receptor agonists released by macro-
phages that are either infected by HIV-1 or stimulated by many other solu-
ble factors released by neighboring infected macrophages [53]. NMDA
receptor antagonists, such as memantine, have been shown to protect neu-
rons in vitro against such damage [52,54], and memantine is currently being
tested for efficacy in ADC in a large clinical trial (ACTG 301).
Another promising target for neuroprotection against HIV-1 is the cellular
pathway leading to production of nitric oxide and related free radicals. Neuro-
nal toxicity of nitric oxide released from either infected or immune-activated
macrophages may be prevented by free radical scavengers, antioxidants, or
agents that block free radical production ([58,88,89], reviewed in [26]). One
such drug, selegiline (L-deprenyl), an inhibitor of monoamine oxidase B and
an effective antiparkinsonian agent, has shown the potential for therapeutic
benefit in HIV-associated cognitive impairment [90]. It is also currently under-
going large-scale clinical testing in patients with ADC (ACTG 5090).
Future targets
Additional cellular targets for neuroprotection against HIV-1 are rapidly
being identified. Among the most intensively investigated are neuronal and
glial pathways involving chemokine receptors and other seven transmem-

brane G protein–coupled receptors, which are linked to p38 mitogen-
activated protein kinase (p38 MAPK). In vitro studies indicate that certain
chemokines (RANTES, fractalkine, and macrophage inflamatory protein-
1b [MIP-1b]) can protect cultured cortical neurons against gp120–induced
apoptosis by interaction with specific neuronal chemokine receptors (CCR5
and CX3CR1-fractalkine receptor) [63,91]. These protective actions seem to
be mediated by chemokine receptor-linked activation of the cellular serine-
threonine protein kinase, Akt–protein kinase B (Akt/PKB), in neurons,
which promotes neuronal survival similar to that of other known acti-
vators of (Akt/PKB) [91,92]. This class of neuroprotective agents may offer
additional means of neuroprotection against HIV and other brain insults.
On the other hand, under some circumstances, activation of the CXCR4
chemokine receptor on neurons may have proapototic effects [45]. Recent
studies suggest that such effects represent a CXCR4 death-signaling path-
way mediated by neuronal p38 MAPK and further suggest that targeted
inhibition of p38 MAPK may offer neuroprotection against HIV [26,63].
These and other neuronal signaling pathways involved in apoptosis are
likely to be major therapeutic targets for neuroprotective drug design
strategies against ADC.
Summary
Neuronal damage and death are consistent pathologic findings in the
brains of patients with ADC, and multiple cell model systems have demon-
strated neurotoxicity through the effects of HIV-1 infection in macrophages
and microglia. Brain MRI studies (1H-MRS) indicate that reversible neuro-
nal cell dysfunction occurs early during the course of HIV-1 infection, long
before overt symptoms of ADC appear. Epidemiologic studies suggest that
a high viral load in the CNS is a major risk factor for ADC and that
HAART may significantly reduce, but not eliminate, the risk of developing
ADC. Targeted adjunctive therapies administered early are likely necessary
to maximize CNS protection against HIV, and rational approaches to such
therapy are rapidly evolving through in vitro analysis of the mechanisms of
HIV-associated neurotoxicity. Soluble factors released by infected cells may
directly or indirectly damage neurons and induce apoptosis at the level of
NMDA subtype of glutamate receptors, and NMDA receptor antagonists
represent a major therapeutic option currently under intense clinical inves-
tigation. Likewise, drugs with antioxidant or free radical scavenging effects
offer another rational approach to adjunctive therapy and are also under
intense clinical scrutiny. Finally, agents that inhibit neuronal death-signaling
pathways (eg, p38 MAPK inhibitors) and that stimulate cell survival path-
ways (eg, Akt/PKB) may represent the next investigational step in designing
anti-ADC therapies.
Acknowledgments
The author thanks members of his laboratory and the AIDS Clinical
Trials Unit of the University of Pennsylvania for their support. In addition,
many investigators from the NeuroAIDS Research Consortium have shared
information that has contributed to this article.
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Clin Lab Med 22 (2002) 719–740
Immune reconstitution
Drew Weissman, MD, PhDa,
Luis J. Montaner, DVM, MSc, DPhilb,*
a
Division of Infectious Diseases, University of Pennsylvania, 522B Johnson Pavilion,
Philadelphia, PA 19104, USA
b
HIV-1 Immunopathogenesis Laboratory, Immunology Program, The Wistar Institute,
3601 Spruce Street, Philadelphia, PA 19104, USA
The term immune reconstitution has been used to describe the recovery of
immune function following effective HIV therapy. Early reports on the out-
comes of potent antiviral therapy document a rise in CD4 T-cell count, which
initially was assumed to indicate an increase in overall immune responsive-
ness. Subsequently, it was documented that increases in CD4 count also
included an increase in naive T-cell subsets and a renewed responsiveness
to recall antigens. As explored in this article, the clinical and basic immu-
nologic changes that represent immune reconstitution span a variety of func-
tions and outcomes. This article reviews the outcomes of effective treatment
on immune function and the repair of immune organs and addresses new
therapies that aim to enhance HIV-specific immunity. Overall, the onset of
long-term suppression of viral replication with antiviral therapy has opened
a new field of interventions that clearly rests on the ability of the immune sys-
tem to recover from damage caused by viral replication.
Immune reconstitution: clinical outcomes

Increase in CD4 count
With the advent of effective control of viral replication through the use of
multiple retroviral drugs (highly active antiretroviral therapy [HAART]) the
This work was supported by National Institutes of Health grants HL62060 and AI45318 to
DW; and Philadelphia Foundation (Robert I. Jacobs Fund), M. Stengel-Miller, H.S. Miller Jr.,
AIDS funds from the Commonwealth of Pennsylvania, and National Institutes of Health grants
AI47760, AI44304, and AI34412 to LJM.
E-mail address: montaner@mail.wistar.upenn.edu (L.J. Montaner).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 1 2 - 4
720 D. Weissman, L.J. Montaner / Clin Lab Med 22 (2002) 719–740
age of repairing immune function in HIV disease became a possibility. The

main goal of HAART is to achieve long-lasting suppression of viral replica-
tion. A secondary goal in the setting of little, or no, viral replication is repair
of the immune system including regaining lost functions and antigenic spe-
cificities. The treatment of people during acute HIV infection differs from
chronically infected subjects and is dealt with in a separate section. The
extent of immune function repair in chronically HIV-infected individuals
treated with HAART has been evaluated in multiple studies (reviewed in
[1]). These data demonstrate that partial, but not complete, recovery of host
immunity does occur. The most noticeable alteration in immune function
observed was an increase in CD4 counts, which was found in almost all indi-
viduals with complete suppression of viral replication and also in people
who achieved partial control of viral replication. This increase in CD4 count
correlated with a reduced incidence of opportunistic infections [2].
Following the initiation of HAART, a biphasic increase in CD4 counts is
observed. An initial rapid increase over the first 4 to 12 weeks is observed
and likely is caused by a redistribution of existing T cells, both CD4þ and
CD8þ, from lymphoid tissue [3]. A component of this initial increase may
also be from a reduction in activation-induced apoptosis [4]. This increase
in CD4þ T cells is mainly characterized by CD45ROþ memory cells. A
second, slower rise in CD4 counts, caused by de novo proliferation, is also
observed. These cells are also comprised of CD45RAþ, CD62Lþ naive cells
[5,6]. It is of interest to note that HAART has been observed to render ben-
efits even without a complete suppression of viral replication, suggesting
that reconstitution of immune function or CD4 count may not be dependent
on complete viral suppression [7–9]. Conversely, a lack of increase of CD4
despite viral suppression may be associated with higher baseline CD4 levels
or a larger first-phase decay of viral replication [10,11].
Repair of immune organs: lymphoid tissue and thymus

HIV is a disease of lymphoid tissue. T cells present in blood represent
only 1% to 2% of the total population, with most T cells localized in tissues.
Dysfunction and destruction of immune organs begins early in HIV disease.
Initially, lymphoid hyperplasia is observed that leads to lymphoid involu-
tion and finally fibrosis with disease progression [12]. Viral clearance after
HAART is similar in blood and lymphoid tissue and an improvement in
lymphoid architecture is found with effective HAART [13,14]. Increased
organization of germinal centers and follicular dendritic cell (DC) networks
and a decrease in activation markers of lymphoid cells is observed. This
improvement in lymphoid architecture is also found in late-stage individuals
who demonstrated fibrotic lymph nodes before starting HAART.
T-cell populations are believed to be restored through a combination of
thymus-dependent regeneration and peripheral expansion of mature T cells.
In two studies of immune reconstitution following HAART using multiple
D. Weissman, L.J. Montaner / Clin Lab Med 22 (2002) 719–740 721
methods to measure thymic regenerative pathways (thymic CT scans; T-cell

receptor excision circle, a marker of T-cell thymic education that is diluted
with T-cell division levels; and naive T-cell analysis) it was determined that
adequate HAART led to increased thymic production [15,16]. Younger sub-
jects with more thymic reserve demonstrated an increase in cells likely derived
from the thymus. Older subjects, with lower thymic reserve by CT scan, dem-
onstrated diminished telomere length and increased peripheral T-cell activa-
tion, markers of peripheral expansion. These data supply a marker and
mechanism for the variable immune reconstitution observed with HAART.
Other studies have found improvement in thymic histology and surrogate
markers of thymic function with effective HAART, further supporting
immune reconstitution through improved or increased thymic function.
Timing of therapy: acute versus chronic infection

The institution of HAART therapy remains a controversial topic, but a
major difference in treatment outcome can be identified when treatment is
begun at the time of seroconversion. The difficulty of identifying recently
HIV-infected subjects, however, remains a concern. Two questions can be
raised concerning the immune reconstitution goals of treatment of HIV-
infected subjects: (1) Can HIV-specific T-cell responses be restored; and (2)
Will improved HIV-specific T responses result in better control of HIV? The
objectives of therapy can be arranged by the desired outcome. The first objec-
tive is to reduce HIV replication and allow the immune system to repair itself.
This does not seem to be a viable approach if treatment is delayed because the
damaged caused by HIV-infection does not seem completely to be reversible
[1]. The next objective is, can one combine viral replication inhibition with
tools that repopulate, restore, or repair the damaged immune system? This
approach is at the center of major research efforts using immune adjuvants,
cytokines, and vaccines in combination with HAART.
The institution of effective HAART during the early stages of HIV infec-
tion results in a higher level of immune reconstitution compared with treat-
ment during chronic infection, including stronger anti–HIV antigen CD4þ
T-cell responses [17,18]. The institution of HAART before seroconversion
results in potent CD4 helper responses but a more limited (fewer epitopes
recognized) CD8þ T-cell response compared with treatment after serocon-
version. Following the preseroconversion treatment subjects for one year
revealed continued control of viral replication and a continued restriction
in epitopes recognized by CD8þ T cells, but interestingly their viral genetic
diversity was more limited. CD4þ T-cell HIV-specific responses were also
increased after 1 year in subjects treated before seroconversion [19,20]. The
treatment of preseroconversion subjects with other therapies including stra-
tegic treatment interruption (STI) also seems to have better results. A study
of eight subjects treated with STI during acute infection demonstrated that
all achieved long-term control of viral replication in the absence of HAART
[20]. It is generally accepted that treatment of acute HIV infection before

seroconversion and the beginning of the destruction of the immune system
has superior results. Maintaining suppressive treatments at any stage of the
disease results in the restoration of some level of CD4þ T-cell proliferative
responses to HIV and recall antigens in specific patient subsets [5,18,21–23].
Clinical immune reconstitution and disease management in chronic infection

The institution of antibiotic prophylaxis after the development of AIDS
is an accepted clinical practice. Although the measurement of T-cell–specific
immune responses in vitro is a difficult task, the results of which are often
fraught with misinterpretation and are subject to the sensitivity and specif-
icity of the assay, the clinical observation that an individual who requires
antimicrobial suppression of diseases, such as Pneumocystis carinii pneumo-
nia, Mycobacterium avium-intracellulare, and cytomegalovirus (CMV), no
longer requires such treatment after effective HAART suggests that immune
reconstitution has occurred (reviewed in [17]). In addition, the observation
that after starting HAART a number of individuals can develop a syndrome
attributed to immune responses to an ongoing infection, as described later,
supports the observation that immune reconstitution has taken place.
Indeed, a corollary of the loss for the requirement for prophylaxis against
opportunistic pathogens with effective HAART in chronic infection is the
appearance of heightened clinical manifestations of infection observed soon
after the institution of HAART, known as immune reconstitution syndromes.
This outcome is directly associated with immune responses to an ongoing
infection, which supports the clinical assessment that immune reconstitution
has occurred. Immune-induced reactivation of pathogen-induced pathology
(ie, inflammation driven) has been observed with multiple pathogens in
many different organ systems [24]. For example, certain subjects with
advanced immunosuppression and ongoing infection with M avium complex
develop local adenitis following HAART. Biopsy specimens from these sub-
jects (Fig. 1) show a reorganization of immune responsiveness and appear-
ance of granuloma, which are not observed in the absence of HAART in
HIV disease stage-matched subjects [25]. The appearance of multinucleated
giant cells, which require antigen-specific CD4þ T cells for formation, sug-
gests that the institution of HAART led to the regaining of previously lost
or suppressed immune responses.
Pediatric HIV infection

Pediatric infection and treatment are associated with similar clinical
changes to those observed in HIV-infected adults, with certain differences.
Specifically, pediatric subjects sustain higher levels of naive T-cell subsets
than adults before and after therapy in association with an active thy-
mus. HIV-infected children treated with HAART have a more rapid and
pronounced increase in naive T cells that can be seen as early as 4 weeks
Fig. 1. An AIDS patient with clinically silent Mycobacterium avian infection developed focal
adenitis in a cervical lymph node (A) associated with granuloma formation (B) after the
introduction of highly active antiretroviral therapy. (From O’Mahony C. Focal adenitis
developing after immune reconstitution with HAART. Int J STD AIDS 2000;11:685–6; with
permission.)
after the start of treatment. Unlike adults, significant increases in naive

CD4þ T cells can be observed in children with no naive cells before treat-
ment [26]. Studies suggest that children, even at the most advanced stages
of HIV disease, can restore their CD4 counts better and more rapidly than
adults [27]. More specific markers of immune reconstitution are also ob-
served in children after effective HAART. Untreated HIV-infected children
often are unable to respond adequately to vaccines. The institution of
HAART followed by immunization with measles- or tetanus-containing
vaccines demonstrated improved T- and B-cell responsiveness [28,29],
demonstrating memory cell immune reconstitution. In addition, in a
very small trial, immune responsiveness to a neoantigen after HAART was
observed demonstrating naive cell function [30]. HAART has also been
associated with increases in T-cell receptor excision circles [31] and thymus
volume [32], suggesting improved thymus function. In regards to HIV-
specific responses, studies have analyzed outcomes of stable suppression but
have described limited recovery of recall responses [33].
Immune reconstitution: improvements of immune cell function

and HIV immunity
The main immune defect in HIV disease is a quantitative and qualitative
loss of CD4þ T cells. This is measured by decreased delayed-type hyper-
sensitivity, decreased ability of T cells to proliferate in response to specific
antigens (lymphoproliferative response [LPR]), and decreased production
of cytokines that support cell-mediated immunity (interleukin [IL]-2, IL-12,

and interferon [IFN]-c). Because CD4þ T cells are intricately involved in
most adaptive and certain innate immune responses, this defect is associated
with broad immune dysfunction (Fig. 2). The reversal of defects in immune
cell function, with control of viral replication, is not globally observed. Cer-
tain studies have demonstrated an improvement in delayed-type hypersensi-
tivity responses to some recall antigens after 12 weeks of HAART. Increases
in LPR to CMV, Candida, and Mycobacterium tuberculosis, but not to all
recall antigens, was observed within the first 24 to 48 weeks of effective
HAART. A major and consistent observation is that little or no increase
in LPR to HIV antigens has been observed even after 2 years of effective ther-
apy of advanced chronically infected subjects [5,21,34]. This inability to
develop responses to HIV is a major concern because studies of recently
infected subjects demonstrated that HIV-specific CD4þ T-cell responses
were lost very quickly after acute infection, except in long-term nonprogres-
sors (LTNP) [35].
Immune reconstitution and innate immunity

Studies on HAART-mediated immune reconstitution have focused on
the analysis of T-cell functions, with limited attention to the direct effects
Fig. 2. Immune cell impairments associated with HIV-1 infection and pathogenesis.
IL ¼ interleukin; LPS ¼ lypopolysaccharide; IFN ¼ interferon.
on antigen-presenting cells (APCs) and natural killer (NK) cells. Studies on

APC, such as macrophages, also remain limited but include studies showing
therapy can lead to an increase in IL-12 and decrease in IL-10 secretion fol-
lowing lypopolysaccharide (LPS) stimulation [36].
DC subsets
The DC are members of a distinct family of bone marrow–derived cells
with a characteristic morphology identified in lymphoid and nonlymphoid
tissues of many species. They represent a minor population, generally com-
prising less than 1% of peripheral blood cells or of total cells in lymphoid
and most nonlymphoid tissues. Because they are powerful stimulators of
primary T-cell responses and the most effective APC, DCs are critical in ini-
tiating T- and B-cell responses [37]. Stimuli, such as viruses, LPS, double-
stranded RNA, bacterial DNA, CD40 ligand, and prostaglandin E2 in
combination with tumor necrosis factor–a and IL-1b are associated with
changes in DC phenotype and function, such as up-regulation of HLA-DR
(necessary for CD4þ T-cell activation) and B7 (T-cell costimulatory mole-
cules necessary for optimal T-cell activation) expression and decreased
endocytic activity [38]. DCs are also critical cytokine producers (eg, IL-12
and IFN-a) participating in the orchestration of the innate immune response
against pathogens [39,40].
Functional impairment of DC during HIV infection has largely been
focused on their decreased ability to stimulate antigen-specific or allogeneic
T-cell responses [41] and to be activated to secrete immune modulatory cyto-
kines (IL-12 and IFN-a). A decrease in IFN-a production, associated with
the onset of opportunistic infections, was an early observation in HIV-
infected individuals [42]. Using cell sorting of bulk DC (as defined by the
lack of expression of markers for T, B, NK, and monocytic cells and expres-
sion of CD4þ and HLA-DRþ), it was later demonstrated that a selective
loss of IFN-a production was associated with decreased accessory cell func-
tion of DC in HIV infection [43]. In addition, recent reports have shown a
decrease of one subset of DC that produces most IFN-a in late-stage
patients [44], in primary HIV-1 infection [45], in patients with high viral load
[46], and those with AIDS who develop opportunistic infections or cancer
[47]. It still remains to be determined how and when DC functions are recov-
ered following suppressive HAART on longitudinal analysis and whether
changes in subsets of DC differ between early and advanced disease. Among
the limited studies that have measured the effects of antiviral therapy on DC
function, viral suppression following monotherapy with zydovudine has
been associated with an increase in number of myeloid dendritic cells
(MDC) and mixed lymphocytic reaction (MLR) activity [48]. The authors’
observations have expanded this area by showing differential deficiency and
recovery of the CD11cþ, CD123þ, and BDCA-2þ DC subsets and of IFN-a
secretion in HIV-1 chronic infection during antiviral therapy. Specifically,
the authors have observed a loss of CD123þ or BDCA2þ DC subsets and
IFN-a secretion that is not recovered under therapy, whereas levels of

CD11cþ DC are increased under suppressive HAART (J. Chehimi, PhD,
Luis J. Montaner, personal communication, 2002). Taken together, DC sub-
sets are differentially reconstituted following HAART.
NK cell subsets
A dysfunction of NK cells is present in the course of HIV-infection, as
indicated by decreases in NK cell numbers [49–53], major histocompati-
bility complex nonrestricted cytotoxicity [54], and antibody-dependent cell-
mediated cytotoxicity [49,55–57]. In addition to NK cells’ role in antiviral
immunity, NK cells produce cytokines and chemokines with antiviral activ-
ity [58,59] and IFN-c, which may contribute directly to HIV control and dis-
ease progression. IFN-c secretion by NK cells also contributes to adaptive
type-1 responses, by IFN-c priming of IL-12 production by APC associated
with Th1-type T-cell differentiation [60–63]. Studies that have addressed
recovery of NK subsets following antiviral therapy have largely focused
on changes in the frequency of mature NK phenotypes and cytotoxicity
showing that both functions can be increased [64–70]. It remains undeter-
mined, however, to what extent IFN-c–secreting NK cell subsets decrease
in frequency, and whether or not they increase following suppressive ther-
apy as shown for T-cell IFN-c responses [71]. The authors’ observations
in this area confirm increases in mature NK cells and cytotoxicity within
NK subsets following therapy, yet still show a sustained impairment in the
ability of NK subsets to secrete IFN-c (L. Azzoni, PhD, Luis J. Montaner,
personal communication, 2002). Although NK cells seem to recover, they
still harbor some functional impairments following HAART.
Effect of immune reconstitution on T-cell repertoire and activation

Reversal in expression of activation antigens
HIV is a disease of immune activation. This is characterized by increased
expression of proinflammatory cytokines and markers of activation on T cells
(eg, CD38, TNFRII, and HLA-DR) [72]. Furthermore, HIV infection leads
to functional and apoptosis-related changes in both CD4þ and CD8þ T cells
[73]. This includes a decrease in the T-cell molecule CD28 (more pronounced
in the CD8 T-cell subset) that binds B7 on APC and delivers an important
activation signal to T cells, and an increase in CD95 expression associated
with induction of fas ligand (CD95L)–mediated apoptosis. The institution
of effective HAART leads to a decrease in activation markers, a decrease
in CD38 expression, and an increase in CD28 expression [69,74,75].
Reversal of perturbations in Vb repertoire

The T-cell receptor repertoire in HIV infection shows increased skewing
with disease progression in both CD4þ and CD8þ T cells. These types of
analyses measure the usage of each variable beta chain of the T-cell receptor,
alone or in combination with complement determining region–3 length het-

erogeneity, which is formed during recombination of the individual Vb gene
families. In progressive HIV infection, alterations in the repertoire are
observed ranging from the absence of usage of certain Vb chains to the pre-
dominance of certain lengths of complement determining region–3 at the
expense of others. These changes are postulated to correspond to a loss of
recognition of antigenic specificities. The CD8þ T-cell repertoire expands
rapidly after HIV infection, which is an indication of the formation of oli-
goclonal cell populations that recognize HIV antigens. A drastic reduction
and skewing in CD8þ T-cell Vb repertoire is observed with disease progres-
sion. After the institution of HAART, CD8þ T-cell repertoires can remain
skewed even after 6 months, but in individuals with higher CD4 counts at
the start of therapy and continued viral suppression, a slow restoration of
CD8þ TCR Vb repertoire is observed. The skewing in the CD4þ T-cell
Vb repertoire improves slowly after starting HAART, although not all stud-
ies agree on the degree of improvement [76–78]. The T-cell repertoire seems
to respond slowly to viral suppression suggesting that lost immunologic spe-
cificities may be regained.
Immune reconstitution and de novo humoral immune responses

Although HIV-infected persons require special consideration because of
concern that their response to vaccine may be abnormal, the vaccination
and antibody response of HIV-infected persons against tetanus, diphtheria,
pertussis, poliomyelitis, hepatitis B, influenza, and pneumococcal disease
has been recommended [79]. The ability to respond to immunization is a
reflection of host immune competence in HIV infection. Response to immu-
nization with antigens, such as tetanus, hepatitis A, and keyhole limpet
hemocyanin (KLH) is improved with suppression of HIV replication and
diminished immune activation [80]. Indeed, it has recently been proposed
to the Food and Drug Administration that in vivo responses to immuniza-
tion reflect a better assessment of the host’s ability to respond to and control
a microbial challenge and a direct quantitative assessment of a therapy-
induced immune reconstitution or modulation [81].
Immune reconstitution and therapies directed at enhancing

HIV-specific immunity
One area that is increasingly targeted for clinical development is the
potential of immune-based therapies to complement existing antiviral drugs
[82–85]. Although clinical studies have yet to show longitudinal data in
chronically infected persons to support a direct role of immune function
in the control of HIV-1 in vivo, much speculation has focused on the poten-
tial for increasing immune function and HIV-specific responses to acquire
better viral control and delay disease progression.
Immune reconstitution and adaptive cell-mediated responses

HIV control and cell-mediated responses
Infection with HIV almost invariably leads to an impairment of the
CD4þ T-cell response and this impairment is theorized to limit the efficacy
of the antiviral CD8þ T-cell response (reviewed in [86,87]). The loss of
immune fitness (eg, impairment of APC and T-cell function) and suppres-
sion of an HIV-1 cell-mediated response in progressive disease differ from
a small subgroup of HIV-infected individuals, the LTNPs in whom immune
fitness and high CD4þ and CD8þ T-cell antiviral responses are correlated
with viral suppression [88]. The confounding effects of viral and genetic fac-
tors that result in LTNP status make it difficult to conclude whether the lev-
els of antiviral cellular immunity present in these individuals are the reason
for, or the consequence of, their viral control. Direct evidence for the role of
cell-mediated immunity in the control of viral replication is limited to data
in the simian immunodeficiency virus (SIV) animal model where depletion
of CD8þ T cells resulted in a measurable increase of viral replication and
rapid progression of disease [89]. Nevertheless, high-level anti–HIV-specific
immune responses are correlated with both a delay in the progression to
AIDS in chronically infected individuals [90] and with protection from infec-
tion in high-risk exposed individuals [91]. Studies of acute infection and
early treatment further support a correlation between the presence of HIV-
specific responses (helper and cytotoxic) and a decrease in plasma viremia
[20,23,92,93]. These observations support the potential for HIV-specific
CD4þ and CD8þ T-cell–mediated responses to suppress HIV-1 in vivo and
support therapeutic approaches to increase these responses.
HAART and reversal of defects in T-cell HIV-specific

and recall antigen–specific immunity
Although current studies indicate that recovery of T-cell memory
responses in chronic infection may require as long as 6 months following
antiviral therapy [34,94], recent data from acute seroconverters indicates
an immediate recovery of T-helper responses following HAART, suggesting
differences in immune reconstitution between early and late stages of disease
following therapy [35]. In addition, data from the authors’ laboratory dem-
onstrated the potential to recover T-cell cytokine secretion within 8 weeks of
suppressive treatment, suggesting differential recovery kinetics for antigen-
specific responses [95] versus global impairments on immune function (ie,
polyclonal T-cell cytokine secretion).
Early treatment outcomes in acute infection have led to the recovery of
HIV-specific CD4þ T-cell proliferative responses and higher levels of
HIV-specific CD8 responses [23,93]. In chronically infected persons, the pro-
longed suppression of HIV results in recovery of immune function and
responses against some recall antigens, although in contrast to acute in-
fection it does not consistently result in the recovery of an HIV-specific
proliferative response [5,96]. The authors’ observations and those of others,

however, support the finding that in cases of virologically suppressed
patients with high CD4 counts a higher frequency of CD4 HIV-specific re-
sponders is observed, suggesting that HIV-specific responses can be reconsti-
tuted following treatment during the early stages of infection [17,18,97]. On
the other hand, increases in LPR to CMV, Candida, and M tuberculosis but
not to all recall antigens were observed within the first 24 to 48 weeks of
effective HAART in patients who started therapy with low CD4 counts.
Unfortunately, no increase in LPR to HIV antigens has been observed
even after 2 years of effective therapy in these subjects (Fig. 3). It remains
unknown what is the irreversible change that occurs at low CD4 counts that
determines the differential recovery of HIV-specific LPR between subjects
with high and low CD4 counts, despite recovery of responses to other recall
antigens. As expected, this impairment in recovery of HIV-specific responses
has become a target of therapeutic vaccines and other approaches, such as
treatment interruption, as discussed later.
Immune reconstitution and HIV-specific neutralizing antibodies

HIV control and HIV neutralizing antibodies
The role of neutralizing antibodies (Nab) in controlling viral replication
in vivo has been the subject of considerable scrutiny. Although Nab are
potent inhibitors of HIV replication in vitro, their absence during the acute
phase of infection has led many to believe that they play a minor role in
immune control. Recent studies in experimental animal models have shown,
however, that virus-specific antibodies can clear circulating virus particles
rapidly [98] and protect macaques from infection [99,100]. These protective
or disease-ameliorating effects of passively administered antibody were even
more pronounced when animals were challenged through vaginal [101] or
oral [102] routes. These data suggest that anti-HIV antibodies may have
an effect on viral set-points. Additional evidence that Nab are involved in
controlling viral replication in vivo comes from studies of LTNP who have
broadly cross-reactive Nab that clearly distinguish them from individuals
who have progressive disease [103–105].
HAART and reversal of B-cell activation

With regard to immune reconstitution and HIV-1 Nab, previous studies
have indicated that both nonspecific and antigen-specific antibody levels are
sensitive to HIV-1 viral load. Patients receiving potent antiviral therapy
show a decline in the number of antibody-secreting cells and the titers of
binding antibodies [106]. This indicates that ongoing antibody production
is dependent on continual viral replication (analogous to the effect on cell-
mediated responses). More recently, it has been shown that HAART ther-
apy does not affect Nab titers and that intermittent therapy with HAART
can enhance autologous Nab levels [107,108].
Fig. 3. Model representation of changes in recall responses and HIV-specific responses between
different stages of HIV-1 infection. Three panels showing longitudinal changes following highly
active antiretroviral therapy treatment for viral load (dark dotted line), recall responses (Candida
albicans, tetanus; dashed line), CD4 HIV-specific T cells (solid line), CD8 HIV-specific T cells
(light dotted line) are presented. The top panel represents changes in acutely infected treated
subjects (\4 months from infection); the middle panel shows treatment during mid chronic
infection; and the bottom panel shows treatment during late-stage disease.
Immune reconstitution and specific adjunct therapies

Cytokines
The primary immune defects induced by HIV, in addition to its infection
and destruction of CD4þ cells, include defects in production of immune
cells, other viral protein effects on uninfected immune cells, and infection
and impairment of thymic and bone marrow cells. To this end, cytokines
have been used clinically to enhance existing immune function, boost out-
put and production of immune cells, as a direct antiviral agent, and to
regulate the expression of unwanted cytokines and immune effectors. Multi-

ple cytokines have also been used as adjuvants with vaccines to promote
immunity.
At the time of writing of this article, IL-2 is in phase III trials to deter-
mine its value in the treatment of HIV infection. Many earlier trials demon-
strated that IL-2 substantially increased CD4 counts (reviewed in [109]).
IL-2, has also been demonstrated to improve specific immune functions.
These include the modulation of activation markers [110] and the restora-
tion of CD8þ T-cell perforin expression, which is deficient in HIV-infected
individuals. Perforin is an important effector molecule in CD8þ T-cell medi-
ated lysis of target cells and its absence also signals a defect in cytotoxic lym-
phocyte maturation [111]. The increase in CD4þ T cells by intermittent IL-2
therapy seems to be composed of peripheral expansion instead of increased
thymic output [112]. This peripheral expansion of CD4þ T cells also does
not seem to restore or increase anti-HIV responses in IL-2 and HAART-
treated chronically or acutely infected subjects [113].
Various other cytokines have undergone or are being investigated for use
in the treatment of HIV infection. IFN-a inhibits multiple steps of the HIV
life cycle and a randomized trial demonstrated that modest antiviral activ-
ities occurred with daily administration [114]. IL-12 is a critical cytokine
in the priming of Th1-type immune responses and its production is deficient
in peripheral blood mononuclear cells (PBMC) from HIV-infected individ-
uals [115,116]. Phase I trials with IL-12 demonstrated increases in NK and
CD8þ T cells but no effect on viral load or CD4 counts [117].
Vaccines
Therapeutic vaccination holds promise in its ability to induce responses
to HIV that are reduced or not found in progressors. The current ap-
proaches, difficulties, and controversies in HIV vaccine development are
described elsewhere in this issue.
The SIV macaque model system has been used to model the effect of vac-
cines on immune reconstitution. Many studies have demonstrated that the
presence of pre-existing immunity induced by vaccines ameliorates disease
progression (reviewed in [118,119]). The addition of cytokine adjuvants
enhances this effect as demonstrated by immunization of macaques with a
DNA vaccine encoding Gag, Env, and an IL-2-Fc fusion protein before
SHIV 89.6P challenge. Immunized monkeys demonstrated potent cytotoxic
lymphocyte responses, stable CD4þ T-cell counts, a low to undetectable
viral set-point, and no disease progression [120]. These studies demonstrate
that although vaccination does not prevent infection, it establishes an
immune response that alters disease course. The results of prophylactic and
therapeutic vaccination against SIV in the macaque model system are quite
impressive and the translation of these approaches to human trials may
open up new avenues of treatment and research.
Immune reconstitution and HIV-1 therapy interruption

The STI represents the stopping and starting of antiviral therapy and the-
oretically can serve two purposes. The first is to limit toxic drug exposure
and the second is to act as an autovaccination. By stopping HAART and
allowing viral replication to proceed for a short period of time, followed
by the reintroduction of HAART, it is hoped that HIV-specific immune
responses can be induced or enhanced. As proposed, an STI strategy could
be compared with an ‘‘attenuated-live vaccine’’ approach, where viral anti-
gens are autologous and the ‘‘attenuation’’ is provided by reinitiation of a
suppressive treatment following the period of viral replication.
In both HIV and SIV infection, treatment early after infection followed
by an interruption in treatment has been associated with increased antiviral
responses (both cellular and humoral); control of viral replication in the
absence of reinitiated treatment; and in the SIV model, protection from
homologous challenge [20,108,121,122]. To increase antiviral immune
responses, the reinitiation of therapy may be critical because these responses
may eventually be lost (HIV-specific CD4 T-cell response) if therapy is not
started because of high levels of viral replication. Indeed, case reports,
patient histories, and longitudinal studies in acute and chronic infected
patients by the authors’ group and others support an association with
increased cell-mediated antiviral responses and treatment interruption epi-
sodes ([20,122–128], SSIT study, B. Hirshel, www.shcs.ch). Furthermore, a
recent report of two sequential 4-week STIs followed by an open-ended STI
in 10 early chronic infection subjects resulted in a delay of viral doubling
times and a lower viral set-point at 12 months off therapy than the viral load
before starting HAART [127]. The approach is complicated by multiple
unknown variables, however, such as how long to stop treatment, how high
to allow viral replication to proceed, and what benefits or risks are inherent
to this strategy when compared with a therapeutic HIV vaccine approach.
Safety and HIV therapy interruption

The main risks associated with STI are the potential for irreversible viral-
mediated CD4þ T-cell loss and the onset of viral mutations that make it
resistant to the reinitiated treatment. To date, reports have emphasized the
favorable outcomes of treatment interruption [20,108,122,123,125–131].
Only sporadic reports of development of resistance have emerged, such as
1 of 128 patients on the SSIT study inclusive of over 500 monitored STIs
(patient on 3TC, ZDV, NFV developed multiple resistance mutations by
23 weeks on a nonsuppressive regimen, B. Hirshel, www.shcs.ch). With
regard to CD4 count, a drop in CD4 count from pre-STI levels but above
pre–antiretroviral therapy levels has also been reported at 12 months off
therapy from a Spanish cohort following two 4-week STIs with 24 weeks
of treatment in between. In relation to serious adverse events, a recur-
rence of the acute retroviral syndrome has been recorded following an
unmonitored treatment interruption concurrently with the administration

of an influenza vaccine [132].
Summary
Immune reconstitution in HIV-infected patients remains a potential
mechanism to explain delayed disease progression and increased survival
following suppressive therapy. Many discrepancies remain to be studied.
Is an immune response to HIV protective? Why are anti-HIV CD4
responses lost so quickly in progressors and how can they be restored? What
is the damage to the immune system that occurs early in disease and why can
it not be overcome by simply controlling viral replication? Will management
of immune reconstitution be used in future adjunct treatment strategies
(vaccine or STI)? Because HAART is not the answer to long-term manage-
ment of HIV throughout the world, the recovery of immune function and
it’s potential to control viral replication remains a key goal in the long-term
management of HIV-infected persons.
Acknowledgements
The authors acknowledge the graphics assistance of Christine DeLauren-
tis, input by Lynn Morris, and useful discussions by Drs J. Chehimi, and
L. Azzoni.
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Clin Lab Med 22 (2002) 741–757
Antivirals against HIV-1

Ian Frank, MD
Infectious Diseases Division, 502 Johnson Pavilion, University of Pennsylvania,
Philadelphia, PA 19104-6073, USA
Clinical illness caused by HIV infection results from the progressive

decline of CD4þ T lymphocytes and immune dysregulation associated with
viral replication. In untreated individuals, CD4þ T lymphocytes decline
by approximately 50 to 100 cells/mm3 annually. As counts decline, typically
asymptomatic patients are at increased risk for a variety of opportunistic
infections or malignancies associated with impaired cellular immune func-
tion or the inability to make appropriate immune responses following expo-
sure to neoantigens. When an individual’s CD4þ T lymphocyte count falls
below 200 cells/mm3, or s/he develops one of a specified number of oppor-
tunistic infections or malignancies, that individual is considered to have
AIDS. Morbidity and mortality associated with HIV infection increased
rapidly following the recognition of this syndrome in 1981, and by 1994,
AIDS was the leading cause of death in the United States among men and
women ages 15–45. The antiretroviral therapy era began in 1987 following
the Food and Drug Administration (FDA)-approval of zidovudine, a
nucleoside analog reverse transcriptase inhibitor; however, it was not until
1996, when HIV viral load assays were developed that could quantify the
copy number of HIV RNA present in plasma, that investigators and clini-
cians could appropriately evaluate the antiviral efficacy of therapy. This
important technical breakthrough and the availability of HIV protease
inhibitors led to a dramatic decline in the morbidity and mortality associ-
ated with the disease [1], as demonstrated in Fig. 1.
Pathogenesis of HIV infection and the rationale for treatment

Following acute HIV infection, high levels of virus in plasma decline to a
stable level of approximately 10,000 to 1,000,000 copies HIV-1 RNA/mL,
This work was supported by grant P30 AI45008 from the National Institutes of Health.
E-mail address: franki@mail.med.upenn.edu (I. Frank).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 1 6 - 1
742 I. Frank / Clin Lab Med 22 (2002) 741–757
Fig. 1. Decline in deaths attributable to HIV infection. (From Palella FJ Jr, Delaney KM,
Moorman AC, et al. Declining morbidity and mortality among patients with advanced human
immunodeficiency virus infection. HIV outpatient study investigators. New Eng J Med 1998;
338:853–60; with permission.)
following the development of an HIV-specific immune response. The HIV-

specific immune response controls HIV replication incompletely, and this
stable concentration of virus in plasma, termed the viral load set point,
represents an equilibrium between virus production and clearance. Approxi-
mately 107 to 108 CD4þ T lymphocytes are productively infected with
HIV at any point in time, and 100 times that number contain HIV DNA
[2,3]. The majority of virus in blood is produced from activated CD4þ
T lymphocytes. Evidence suggests that the viral load set point is determined
by the number of productively infected cells, rather than the efficiency of
virus clearance. The half-life of virus clearance is approximately 6 hours,
irrespective of the viral load, and approximately 1010 virions are produced
daily [3]. Therefore, HIV is a dynamic infection, even during periods when
the patient is asymptomatic. The half-life of infected CD4þ T lymphocytes
is approximately 1.6 days [3–5], and epidemiologic evidence demonstrates
that the rapidity of the decline in CD4þ T lymphocyte counts is directly
related to the plasma viral load [6]. HIV disseminates widely throughout the
body following acute infection, and can be isolated from brain and spinal
fluid, nodal and gut-associated lymph tissue, and semen and cervicovaginal
fluid. HIV sequence evolution appears to occur in an independent fashion in
the central nervous system and semen compared with evolution in blood [7–9],
suggesting that either these tissue compartments restrict virus circulation or
that the virus has adapted to preferentially replicate in these sites.
Compared to other polymerases, HIV reverse transcriptase has relatively
poor fidelity. As reverse transcription progresses, mutations are introduced
into the proviral DNA sequence, occurring at a rate of approximately one
mutation per full length reverse transcript [10]. Progeny virions released
from cells infected with a mutant virus contain that mutation. Given the
amount of virus replication occurring on a daily basis, viruses with single
I. Frank / Clin Lab Med 22 (2002) 741–757 743
mutations at each nucleotide in the HIV genome and many double mutants
are being generated. Specific mutations within the segments of the genome
that encode for the enzymes that are the targets of the antiretroviral drugs
can impart relative resistance to the effects of the medication. Therefore,
infected individuals are believed to harbor viruses that are resistant to med-
ication, even before they are started on therapy. A virus with these muta-
tions is often less fit than the wild type, sensitive virus, which means its
replication rate is slower. Thus, this mutant virus is present in a relatively
low copy number compared with the wild type virus, and usually cannot
be detected by the clinical assays used to test for resistant viruses without
the selective enhancement of this population of viruses that occurs after
therapy is instituted.
A further challenge to successful therapy is the ability of HIV to establish
latent infection. Replication-competent, integrated HIV can be cultured
from resting, memory CD4þ lymphocytes taken from patients with viral
loads below quantifiable levels for several years [11–13]. This cellular reser-
voir of integrated, latent HIV has a long half-life, suggesting that antiretro-
viral therapy would need to be continued for decades to clear this population
of infected cells. Therefore, HIV infection is not thought to be a curable
condition given the current therapeutic approaches.
Mechanisms of action of antiretroviral agents

There are currently 16 FDA-approved drugs to treat HIV infection, in
addition to two fixed-drug combinations of individual agents (see box
below). These drugs work by inhibiting one of two viral enzymes, reverse
transcriptase or protease, and can be divided into three separate classes.
Nucleoside and nucleotide analog reverse transcriptase inhibitors act as
chain terminators when incorporated into proviral DNA. Each of the agents
in this class contains a modification of the 3¢-OH group of the ribose com-
ponent, thus preventing the formation of the 3¢-5¢ phosphodiester bond
required for the addition of another nucleotide onto the elongating DNA
molecule. These agents are anabolized within cells after their absorption,
and compete with native nucleosides for the enzymes responsible for their
phosphorylation into their triphosphate, active form. Tenofovir, the sole
nucleotide reverse transcriptase inhibitor in this group, already contains a
single phosphate group. The intracellular pharmacokinetics of these drugs
are not fully understood and may differ substantially from their plasma
pharmacokinetics. For example, the half-life of lamivudine is approximately
5 hours in plasma, while the half-life of lamivudine-triphosphate, the active
form of the drug, is approximately 12 hours.
The nonnucleoside reverse transcriptase inhibitors are polycyclic
compounds that bind to reverse transcriptase in proximity to the enzyme’s
catalytic domain, thereby stoichiometrically interfering with the enzyme’s
function. Although active against HIV-1, these drugs lack activity against
FDA-approved antiretroviral agents

Reverse transcriptase inhibitors
Nucleoside/nucleotide analogs
Zidovudine (Retrovir)
Didanosine (Videx)
Zalcitabine (Hivid)
Lamivudine (Epivir)
Stavudine (Zerit)
Abacavir (Ziagen)
Tenofovir (Viread)
Fixed dose zidovudine þ lamivudine (Combivir)
Fixed dose zidovudine þ lamivudine þ abacavir (Trizivir)
Nonnucleosides
Nevirapine (Viramune)
Efavirenz (Sustiva)
Delavirdine (Rescriptor)
Protease inhibitors
Saquinavir (Invirase, Fortovase)
Ritonavir (Norvir)
Indinavir (Crixivan)
Nelfinavir (Viracept)
Amprenavir (Agenerase)
Lopinavir/ritonavir fixed dose (Kaletra)
The generic name of the drug is followed by the trade name.
Drugs are listed in order of FDA approval, except for the fixed
dose formulations of the nucleoside reverse transcriptase inhibi-
tors, which are listed at the end of that category.
HIV-2. These enzymes are substrates of isoenzymes of cytochrome P450

(CYP450), and in addition, are potential enzyme inducers and inhibitors.
Therefore, there is the potential for drug–drug interactions when these
agents are combined with many other classes of drugs used to treat multiple
medical conditions.
The third class of agents is the protease inhibitors. These agents were
originally designed as peptidomimetics of the enzyme’s substrate, though
nonpeptidomimetic agents have also been developed. These agents bind
within the cleavage domain of the protease, thereby inhibiting the enzyme’s
cleavage of fusion precursor polypeptides that are the translation products
of the viral gag and pol genes. Poor solubility and bioavailability have
hampered the development of these drugs. Similar to the nonnucleoside
reverse transcriptase inhibitors, these agents are substrates, inducers, and
inhibitors of CYP450 isoenzymes. This property is occasionally taken
advantage of therapeutically. Ritonavir, the most potent inhibitor of
CYP450 in the class, is often combined with other protease inhibitors to
improve the pharmacokinetic profile of the companion agent, reduce the pill
burden or the number of daily doses of drug, or eliminate the food restric-
tion required of indinavir. Unfortunately, this pharmacokinetic boosting
may increase the incidence of adverse events.
Constructing combinations of antiretroviral agents

Several factors are considered when selecting an antiretroviral combina-
tion for a patient, including the patient’s medical history and lifestyle,
HIV treatment history, viral load, CD4þ T lymphocyte count, and clinical
laboratory parameters. These factors influence practitioners to recommend
different combinations based on potency, convenience of the pill-taking
regimen, possible adverse events, and the ability to maintain therapeutic
options if resistance to one or more of the components of the combination
develops.
Antiretroviral therapy treatment guidelines have been developed to estab-
lish standards for patient care [14]. The consensus of the experts that drafted
these documents suggests that antiretroviral naı̈ve patients be started on a
combination of two nucleoside reverse transcriptase inhibitors plus a third
agent, consisting of an additional nucleoside reverse transcriptase inhibitor,
a nonnucleoside reverse transcriptase inhibitor, or a protease inhibitor. A
combination of three drugs is typically required to construct a combination
sufficiently potent to achieve the therapeutic goal of reducing the viral load
to below quantifiable levels [6], which is necessary to prevent the selection of
resistant virus. The recommendations published in the guidelines are based
upon the historical conventions of antiretroviral drug development. No data
exists that suggests that the use of three-drug combinations is optimal for
each patient. Studies including combinations of a single nonnucleoside
reverse transcriptase inhibitor and one protease inhibitor or a combination
of two protease inhibitors demonstrate that alternative approaches to ther-
apy may be as effective [15,16]. Some clinicians prefer to use four-drug com-
binations in patients with high viral loads (arbitrarily defined as >100,000
copies HIV-1 RNA/mL) because data suggest that three-drug combinations
may not be sufficiently potent in these patients [16]. In addition, there is no
data that suggest an advantage of a combination of agents that targets two
viral enzymes or uses two classes of agents that target a single enzyme over
a combination that uses multiple agents from one class to target a single
enzyme.
Antiviral and immunologic effects of therapy

There is a rapid, first-phase clearance of viruses from the circulation fol-
lowing the administration of an antiretroviral combination, which is attrib-
utable to the clearance of both cell-free viruses and productively infected
CD4þ T lymphocytes [4]. During the initial 2 to 4 weeks of therapy, a
patient’s viral load may decline by two log10 copies of HIV-1 RNA/mL or
more. A rapid rate of virus clearance and productively infected cells from
lymph tissue has also been demonstrated [2]. Following this rapid, initial
clearance of virus, the viral load continues to decline at a slower rate. This
is thought to be due to the slower clearance of virus from infected macro-
phages or longer-lived CD4þ T lymphocyte populations, as well as clear-
ance of virions bound to dendritic cells. Assuming the combination is
adequately potent and the patient has adhered to the regimen to experi-
ence its full antiviral effects, the viral load will decline to below quantifiable
levels, currently accepted as 50 copies HIV-1 RNA/mL, the threshold of sen-
sitivity of the most recently developed and available commercial assays. The
time it takes for a patient’s viral load to decline to less than 50 copies HIV-1
RNA/mL depends on that patient’s baseline viral load. It may take up to 6
months for the viral loads of a patient with high baseline viral load levels to
decline to undetectable levels. Patients who achieve a viral load of less than
50 copies HIV-1 RNA/mL have maintained their viral load below this level
for 3 years or longer (Fig. 2) [17]. The majority of patients with viral loads
less than 50 copies/mL have quantifiable viral loads using more sensitive
research assays [18]. In addition, the evolution of proviral sequences in
peripheral blood mononuclear cells from individuals on therapy with plas-
ma viral loads below quantifiable levels is further evidence of ongoing viral
replication [19]. Therefore, achieving a viral load below quantifiable levels
does not mean virus replication has been completely inhibited. In addition,
patients may have an occasional viral load ‘‘blip,’’ which is an increase in
viral load to a low, quantifiable level, followed by a return of the viral load
to less than quantifiable levels. Single, isolated blips are not a harbinger of
the imminent emergence of resistant virus; however, multiple isolated blips
or sequential blips suggest that virus replication will not remain adequately
controlled.
Following the initiation of antiretroviral therapy, immune function
improves with respect to cell number and function. CD4þ T lymphocyte
counts increase rapidly during the first month of therapy (many patients
experience an increase of 100 cells/mm3), followed by a slower increase
in cell counts over time [20]. There is restoration of naı̈ve as well as memory
CD4þ T lymphocyte populations [21]. Repopulation of CD4þ T lym-
phocytes results from proliferation of cells in the circulation following a
decrease in virus replication, as well as an increase in thymus mass and
function [22,23]. CD4þ and CD8þ lymhoproliferative responsives to recall
antigens also improve, such that prophylactic therapy for the primary
Fig. 2. Stability in proportion of subjects with viral loads less than 500 and less than 50
copies/mL after 3 years of treatment. (From Gulick RM, Mellors JW, Havlir D, et al. 3-year
suppression of HIV viremia with indinavir, zidovudine, and lamivudine. Ann Int Med 2000;
131:35–9; with permission.)
prevention of an opportunistic infection or the secondary prevention of a

relapse of a previous infection may be discontinued once the CD4þ T lym-
phocyte count exceeds certain critical thresholds that differ for individual
opportunistic infections; however, some patients who begin on therapy with
low CD4þ T lymphocyte counts (<100 cells/mm3) may fail to experience
an increase in cell number to normal or near normal levels. In addition,
immune responses to neoantigens are not restored in many patients, regard-
less of their cell counts, and HIV-specific immunity generally does not
improve [24,25]. Whether or not immune function can be restored to a level
that is comparable to that of an HIV-uninfected individual (assuming inhib-
ition of virus replication is maintained) will require additional observation.
Challenges to successful therapy

Adherence
Suboptimal adherence to pill-taking is the most common reason for the
failure of an antiretroviral combination to maintain suppression of virus
replication and for the selection of virus resistance [26,27]. Pill-taking is an
enormous challenge for HIV patients. Historically, three-drug antiretroviral
combinations frequently required patients to take medication three times a
day, though most patients now receive combinations that are taken twice a
day. Some regimens are more complicated because certain drugs require pill-
taking either with food or on an empty stomach. Studies suggest that the
risk of virologic failure increases substantially for patients who fail to take
95% or more of their doses of medication [28]. Because HIV patients are
typically young when started on therapy, and because treatment for this
uncurable disease will be required for decades, patients must adhere to their
regimen diligently.
Complications of antiretroviral therapy

An additional challenge to adherence is the potential for drug-related
adverse events. Among the nucleoside reverse transcriptase inhibitors, zido-
vudine frequently causes headaches and nausea when initially administered;
long-term side effects include anemia and myositis. Didanosine, zalcitabine,
and stavudine are associated with peripheral neuropathy after long-term
use. All of the agents in this class can cause pancreatitis, alone or together
with hyperlactatemia or lactic acidosis, though evidence suggests that stavu-
dine most likely causes these adverse effects. A systemic hypersensitivity
reaction occurs in 3% to 5% of patients started on abacavir; this reaction can
lead to fatal anaphylaxis if a patient has this reaction and is readministered
abacavir after its discontinuation. Each of the nonnucleoside reverse tran-
scriptase inhibitors can cause a rash shortly after their initiation; in the case
of nevirapine, this may lead to Steven-Johnson syndrome. Nevirapine also
may cause an elevation in hepatic transaminases. Patients on efavirenz com-
monly experience dizziness, lightheadedness, or sleep disturbances, which
may or may not be transient. As a class, the protease inhibitors frequently
cause gastrointestinal disturbances, including nausea, vomiting, dyspepsia,
and diarrhea.
Of greater concern than the adverse events described above is the fact
that a significant percentage of patients on antiretroviral combinations
experience a constellation of metabolic abnormalities, including insulin
resistance, dyslipidemia, and body fat redistribution [29]. Body fat redistri-
bution manifests itself as (1) fat wasting of the extremities, buttocks, and face,
or (2) central fat accumulation, including visceral adiposity, enlargement of
breasts or gynecomastia, increased neck circumference, dorsicocervical fat
pads (buffalo humps), and lipomas. These various manifestations of fat
redistribution are often referred to as lipodystrophy. The exact prevalence
of this syndrome is not known, though several studies suggest that it occurs
in half or more of protease inhibitor recipients [30,31]. Most studies describe
an association between fat redistribution and hyperlipidemia and abnormal-
ities of glucose metabolism, including elevated serum and plasma glucose,
fasting insulin, and C-peptide levels [29]. In addition, insulin resistance has
been correlated with the extent of visceral adiposity, as well as the magni-
tude of fat wasting. There is no epidemiologic evidence suggesting that HIV
patients with this syndrome are at increased risk of cardiovascular or cere-

brovascular disease [32]; however, the pattern of dyslipidemia observed,
including elevated triglycerides, total cholesterol, VLDL cholesterol, and
low levels of HDL cholesterol, has led to the concern that these patients will
be at increased risk for heart disease and stroke following longer periods of
therapy. Many HIV patients now also take a lipid-lowering medication to
treat these effects.
HIV resistance
As stated above, resistance to antiretroviral therapy develops as a conse-
quence of the virus’s rapid replication rate and its ability to mutate. Virus
can be tested for resistance by both genotypic and phenotypic assays. Geno-
typic assays report mutations in the portion of the HIV genome encoding
reverse transcriptase and protease that deviate from the consensus wild type
sequence and that are associated with a change in resistance phenotype.
Phenotypic assays compare the amount of drug required to inhibit a given
amount of virus replication relative to wild type virus and are typically
expressed as a fold-change in the IC50 value (the concentration of drug
required to inhibit 50% of virus replication.
The rapidity with which a resistant virus will emerge in a patient on ther-
apy depends on several factors, including the extent of virus suppression, the
number of mutations required for the resistant virus to develop, and the
impact of the mutations on the virus’ replicative fitness (ie, efficiency of rep-
lication). A resistant virus is inevitably selected if a patient continues on an
antiretroviral combination that fails to reduce and maintain the viral load
below quantifiable levels. An increase in the viral load from nadir levels
occurs concurrently with the selection of resistant virus. A single mutation
confers 50-fold to 100-fold resistance to lamivudine or a nonnucleoside
reverse transcriptase inhibitor [33]. Consequently, when these drugs are used
in a failing combination, resistance can emerge rapidly, usually within 1 or
2 weeks. Resistance to other nucleoside reverse transcriptase inhibitors and to
the protease inhibitors tends to develop more gradually, although only a sin-
gle mutation is required for phenotypic resistance to didanosine, nelfinavir,
and amprenavir [34,35]. Continuing a failing combination when the virus is
resistant to a single drug will result in sequence evolution and the further
selection of resistant viruses, even in patients with viral loads of less than
1,000 copies HIV-1 RNA/mL [36].
A virus that is resistant to an agent in a particular class of inhibitors may
be cross-resistant to other agents within the same class. As a virus becomes
resistant to more drugs within a class, or as additional mutations accumu-
late that confer increasing resistance to a single agent within a class, the
virus becomes more broadly cross-resistant to other drugs within the class.
Some mutations induce broad cross-resistance against an entire class of
agents. For example, a K103N mutation in the reverse transcriptase genome
confers resistance to all of the nonnucleoside reverse transcriptase inhibi-

tors, and the accumulation of three or more or a set of six mutations asso-
ciated with resistance to zidovudine generally confers resistance to the entire
group of nucleoside reverse transcriptase inhibitors [37].
If an antiretroviral combination is changed or treatment is discontinued
following the emergence of a resistant virus, the mutant virus may decrease
in frequency, the wild type virus may emerge, and the virus can appear to
regain its sensitivity to a particular agent; however, in this case, some of the
virus will become archived in latently infected cells [38], and if the agent that
initially selected for the resistant virus is reintroduced, the mutant virus rel-
ative to that agent will rapidly re-emerge. If a virus with a particular muta-
tion is relatively fit compared with a wild type virus, the resistant virus may
persist despite the discontinuation of the drug that selected it. For these rea-
sons, once resistance is selected, the virus has traveled down an evolutionary
path from which there is no return.
Resistance is heterotypic; multiple viral clones from a single patient have
different genotypes and will therefore have different sensitivity profiles [39].
In addition, certain combinations of mutations may resensitize resistant
virus to a particular drug [37]; and other mutations may confer hypersus-
ceptibility to an agent (a reduction in IC50 levels compared with wild type)
[40]. For these reasons, genotypic resistance testing does not always predict
phenotypic resistance.
Resistant viruses can be transmitted; genotypic or phenotypic resistant
viruses have been detected in 10% or more of patients with acute HIV infec-
tion [41]. These patients either respond more slowly or fail with their initial
regimen. In addition, that resistant virus can persist even when the patient
remains untreated. For example, a recent study examined six patients with
acute HIV infection who had phenotypic resistance to all of the nonnucleo-
side reverse transcriptase inhibitors [42]. Although these patients were not
treated, viruses in five of the six patients remained resistant to nonnucleoside
reverse transcriptase inhibitors for more than 1 year. This was due to the
fact that the nonnucleoside reverse transcriptase inhibitor resistant virus
had similar replicative fitness compared with the wild type virus, or these
patients were infected with a pure nonnucleoside-resistant virus such that
conversion to a sensitive virus required a back mutation to the wild type
genotype.
Deciding when to initiate therapy

Early clinical trials demonstrated that zidovudine could prolong survival
in patients with CD4þ T lymphocyte counts less than 200 cells/mm3 with
and without previous AIDS-defining clinical conditions [43] and could pre-
vent disease progression to AIDS in asymptomatic individuals with CD4þ T
lymphocyte counts less than 500 cells/mm3 [44]. Following the development
and commercial availability of the viral load assays, these tests were used
together with CD4þ T lymphocyte count determinations to predict the

risk of clinical disease progression. Under the therapeutic paradigm that
emerged, asymptomatic patients with viral load and CD4þ T lymphocyte
counts that placed them at significant risk of AIDS-defining conditions
within the next 3 years, if left untreated, were recommended therapy [6];
however, before the discovery of HIV latency, some HIV investigators pro-
moted a ‘‘hit hard, hit early’’ approach, suggesting that potent antiretroviral
combinations be started when a patient’s CD4þ T lymphocyte counts are
high in the hopes that infection could be eradicated. These more aggressive
treatment philosophies have recently given way to a more cautious treat-
ment paradigm outlined in Table 1 [14]. This shift followed the recognition
and increasing concerns that surround the metabolic complications of ther-
apy, the high rate of treatment failure in patients started on initial therapy in
clinical practice [45], and the increasing prevalence of resistant viruses in
newly infected individuals. In addition, recent epidemiologic studies of
patients who receive potent combinations of agents have demonstrated that
treatment can be delayed without clinical consequences until CD4þ T
lymphocyte counts reach 200 to 350 cells/mm3 [46]; however, the lower a
patient’s CD4þ T lymphocyte count is below 200 cells/mm3, the less likely
that individual will achieve and maintain a viral load that is below the
threshold of detection on therapy.
A special circumstance exists for patients with acute HIV infection.
Early evidence suggests that HIV-specific helper cell lymphoproliferative
responses can be maintained if a patient is diagnosed with acute HIV infec-
tion and started on therapy within 2 or 3 months of their initial symptoms
[25]. HIV-specific helper responses are lost if therapy is delayed beyond that
point. Although the reasons for this are not clearly established, HIV prefer-
entially infects the HIV-specific, activated CD4þ T lymphocyte populations;
therefore, early therapy may help to preserve these cells.
Monitoring patients
Patients who are not started on antiretroviral therapy typically have their
viral loads and CD4þ T lymphocyte counts monitored at 3- or 4-month
intervals, and the need for therapy is reassessed accordingly.
For those started on therapy, many practitioners obtain a viral load 2 to
4 weeks after treatment is initiated. This is done to confirm that the patient
is receiving some antiviral effect, but also to reinforce the importance of
adherence, and to demonstrate to the patient the benefit that he or she is
receiving. For an asymptomatic patient, a reduction in viral load or increase
in CD4þ T lymphocyte count will be the only indication that the medica-
tions are having a beneficial effect. A viral load that is not 0.3 log10
copies/mL lower than the baseline value indicates no antiretroviral re-
sponse; in practice, however, a fall in viral load of at least 1 log10 copies/mL
Table 1
Indications for the initiation of antiretroviral therapy in the chronically HIV-1 infected patient
Clinical CD4þ T Plasma
category cell count HIV-1 RNA Recommendation
Symptomatic Any value Any value Treat
or AIDS
diagnosis
Asymptomatic <200 cells/mm3 Any value Treat
Asymptomatic 200 to 350 Any value Treatment is generally offered,
cells/mm3 though controversy exists about
treating patients with viral
loads <10,000 copies/mL
Asymptomatic >350 cells/mm3 >55,000 Some experts would recommend
copies/mL therapy, given that the 3-year risk
of developing AIDS in untreated
patients is >30%, though some
would defer treatment and
monitor closely
Asymptomatic >350 cells/mm3 <55,000 Most experts would defer therapy
copies/mL and monitor, given that the
3-year risk of developing AIDS
in untreated patients is <15%
Adapted from Guidelines for the use of antiretroviral agents in HIV-infected adults and
adolescents. Available at: http://www.hivatis.org. Accessed 2002.
would be expected if a patient were taking medication reliably and the virus
was sensitive to the combination prescribed. Some practitioners monitor the
viral load at monthly intervals until the viral load has declined to a less than
quantifiable level, while others will monitor the viral load at 2- or 3-month
intervals once evidence for an adequate response to therapy has been gath-
ered. Once a patient’s viral load has declined below quantifiable levels, the
viral load is typically monitored at 3- or 4-month intervals.
CD4þ T lymphocyte monitoring is often done in conjunction with viral
load testing, though it may not be obtained as frequently during the few
months on therapy. Laboratory tests that are monitored at regular intervals
for safety evaluations include complete blood counts, liver and kidney func-
tion tests, and triglyceride and cholesterol levels. Other tests are obtained
according to the safety profiles of the drugs used in the combination.
Resistance testing is recommended for patients experiencing virologic
failure. For patients failing therapy, the resistance test should be obtained
while patients continue on their current combination, because the selective
pressure of therapy is lost once therapy is discontinued or modified, and a
wild type virus may outcompete a resistant virus that is not yet detected. Both
genotypic and phenotypic resistance testing have been shown to improve
therapeutic outcomes when used to enhance antiretroviral decision-making
[47,48]. There is no data supporting the use of one type of resistance test
over the other. Discordant reports of virus susceptibility when genotypic
and phenotypic tests have been performed on a single specimen have been
noted [49], and the two types of assays differ with respect to strengths and
weaknesses. Both tests may fail to detect minority subpopulations of resistant
viruses; a resistant virus must comprise 10% to 20% of the viral population
to be detected, and neither test may be able to detect a resistant virus if the
viral load is less than 5,000 copies/mL. Algorithms that link mutational pat-
terns with resistance to specific drugs may not predict virus phenotypes
accurately because of the presence of new mutations not previously identi-
fied as causing resistance or the effects of combinations of mutations that
may resensitize a resistant virus. Phenotypic assays are limited because they
may not be sensitive enough to detect phenotypic decreases in susceptibili-
ties that are less than 2 to 4 times greater than a wild type virus. In addition,
mutations associated with antiviral failure may occur before a phenotypic
change in virus susceptibility, which also suggests that these assays may not
be sensitive enough to detect small changes in a virus phenotype that are
clinically significant. One of the greatest obstacles to resistance testing is the
paucity of data that can accurately predict whether or not a drug remains
clinically active when a certain level of phenotypic change in virus suscep-
tibility has occurred, or a certain set of mutations has developed. For these
reasons, resistance tests are better predictors of which drugs will not work
rather than which drugs will work.
Resistance testing is also suggested for patients diagnosed with acute HIV
infection. As stated above, resistant virus can be transmitted, and patients
may not have an optimal response to therapy when placed on a combination
to which the virus is resistant. There is ongoing debate concerning the value
of obtaining a resistant test on patients starting on therapy with long estab-
lished infection. Some reports suggest that resistant virus can be detected in
a large enough minority of these patients to prompt some clinicians to
obtain these tests in this setting as well.
Modifying antiretroviral therapy

Antiretroviral therapy is modified because of toxicities or failure of the
combination to maintain suppression of HIV replication. When an agent
produces a toxicity that requires treatment modification, in general, the drug
causing the toxicity is replaced with another agent within the same class of
inhibitors.
Antiretroviral failure is defined as a failure to suppress plasma HIV RNA
to undetectable levels after 4 to 6 months of treatment, or a rebound of plas-
ma HIV RNA from undetectable levels to repeatedly detectable levels. Viral
loads may be elevated in the setting of a clinical illness or a recent vaccina-
tion, so a viral load test obtained under these circumstances should not be
used to define treatment failure. As stated above, resistance testing should
be obtained in the setting of virologic failure to assess therapeutic options.
Continuing a combination that has failed will result in the accumulation of

additional mutations and a more broadly cross-resistant virus. The selection
of a new combination is driven by the patient’s treatment history and ther-
apeutic options. Broad cross-resistance exists among the nonnucleoside
reverse transcriptase inhibitors; therefore, patients with a nonnucleoside
resistant virus will not benefit from other drugs within the class. Some
degree of cross-resistance between agents among the other two classes of
inhibitors also exists; for that reason, resistance testing is an important
adjunct to help select different drugs with a class where some resistance has
emerged. Patients initiated on a combination that did not include a protease
inhibitor are often prescribed a second combination containing a single pro-
tease inhibitor or a protease inhibitor boosted with ritonavir plus two differ-
ent nucleoside reverse transcriptase inhibitors. Patients who have failed a
second combination are likely to have virus that is cross-resistant to a num-
ber of drugs, and these patients should ideally receive a four-drug combina-
tion that contains a ritonavir-boosted protease inhibitor or two protease
inhibitors. The third antiretroviral combination represents the last oppor-
tunity to achieve an undetectable viral load given the agents that are avail-
able today.
The future of antiretroviral therapy

There are a many new antiretroviral agents in development. New gener-
ations of nucleoside and nonnucleoside reverse transcriptase inhibitors and
protease inhibitors are being developed that are easier for patients to take,
have more favorable toxicity profiles, and are active against a virus that is
resistant to other agents with these classes. Drugs that target novel targets
are also being studied. Several candidate inhibitors of HIV integrase have
progressed through animal toxicology studies and will soon enter phase I tri-
als in humans. In addition, drugs that inhibit the fusion of HIV to lympho-
cytes and monocytes are being studied. One such agent, T-20, binds to the
HR-1 domain of the viral envelope glycoprotein gp-4 thereby preventing the
formation of a hairpin structure needed for viral and cellular envelope
fusion, is currently in phase III clinical trials, and may be clinically available
by the end of 2002. Other fusion inhibitors inhibit virus entry by binding to
CCR5 or CXCR4, cellular chemokine receptors required for HIV fusion
that define virus as either macrophage- or lymphocyte-tropic. Agents that
inhibit HIV replication by interfering with viral regulatory proteins, inhibit
virus uncoating, RNAase H activity, or capsid protein polymerization, or
bind to zinc fingers are at earlier stages of development. In addition, HIV
vaccine candidates are being tested as potentially therapeutic agents.
HIV therapeutics is one of the most rapidly evolving fields in medicine. It
is the hope that continued progress will further erode the numbers of people
dying from this disease and create the hope of a normal life expectancy for
infected individuals. While this is a realistic goal for residents of the devel-
oped world, approximately 95% of individuals infected with HIV live in
underdeveloped countries where these drugs are unavailable, save for a
small portion of wealthy individuals who pay for medication out of pocket.
The greatest challenge facing the HIV epidemic today remains the delivery
of HIV treatment to the entire world.
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Clin Lab Med 22 (2002) 759–772
Pediatric HIV infection and treatment

Paul Palumbo, MD
Department of Pediatrics, UMDNJ-New Jersey Medical School,
185 South Orange Avenue, Newark, NJ 07103, USA
Human immunodeficiency virus has been recognized as a significant

human pathogen for at least two decades and continues to pose persistent
challenges to prevention and treatment efforts. Press releases and epidemio-
logic studies have documented its penetration into every corner of the globe
with by far the highest infection burden in sub-Saharan Africa. Worrisome
data from the densely populated regions of the Indian subcontinent, South-
east Asia, and China suggest relatively unchecked expansion. This pandemic
has impacted all levels of society, but possibly none as seriously as our
future hope: infants, children, and young adults. Some communities in
sub-Saharan Africa have been documented with rates of infection among
women of child-bearing age as high as 25% to 35%, which also happens
to be the approximate risk for HIV transmission from mother to newborn.
In the developed world with drug access and health care infrastructure, it
has been difficult to develop and maintain prevention strategies for the long
term, evidenced by recent surges in new infection among youth and gay
communities [1,2]. Although substantial strides have been made in the ther-
apeutic arena through access to 15 Food and Drug Administration (FDA)–
approved antiretroviral (ARV) agents, there is some concern that only a
temporary holding pattern has been established (at least 50% of treated indi-
viduals currently have evidence of substantial drug resistance with no effec-
tive cure in sight). A common thread underlying the characteristics of the
HIV epidemic is the highly mutable nature and adaptability of the virus,
which poses a clear challenge to development of effective therapy and
vaccines.
This article focuses on issues and challenges of particular relevance to
HIV-infected infants and children. Detailed information regarding individ-
ual ARV agents or recommended multidrug therapeutic regimens is not
provided but several excellent web sites are available and listed below, which
E-mail address: palumbo@umdnj.edu (P. Palumbo).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 1 0 - 0
760 P. Palumbo / Clin Lab Med 22 (2002) 759–772
provide public health service and expert panel recommendations updated on

a regular basis:
HIV/AIDS Treatment Information Service: www.hivatis.org
The HIV/AIDS Treatment Information Service provides information
about federally approved treatment guidelines for HIV and AIDS.
National Pediatric & Family HIV Resource Center: www.pedHIV-
AIDS.org
A nonprofit organization that serves professionals who care for chil-
dren, adolescents, and families with HIV infection and AIDS.
Elizabeth Glaser Pediatric AIDS Foundation: www.pedaids.org
Identifies funds and conducts critical pediatric AIDS research that will
lead to the prevention and treatment of HIV infection in infants and
children.
Revised Guidelines for the Use of Antiretroviral Agents in Pediatric HIV
Infection
Living document (HTML version): http://www.hivatis.org/guidelines/
Pediatric/Text/summary.html
Living document (PDF version): http://www.hivatis.org/guidelines/
Pediatric/Aug08_01/pedaug08_01.pdf
The Pediatric AIDS Clinical Trials Group: http://www.pedhivaids.org/
links/
The preeminent organization in the world for evaluating treatments
for HIV-infected children and adolescents, and for developing new
approaches for the interruption of mother-to-infant transmission.
It has set the standards of care for children infected with HIV and
for the interruption of vertical transmission.
Epidemiology and pathogenetic features of perinatal HIV transmission

Perinatal transmission
Natural history studies of HIV transmission from an infected, pregnant
woman to her fetus, newborn, or young infant suggest a rate of transmission
in non–breast-feeding populations of approximately 25% [3]. Breast-feeding
through the first 6 months of infancy adds an additional 10% to 15% to the
transmission rate. In developed countries where breast-feeding is uncom-
mon among infected women, it is recognized that about one third of trans-
mission occurs during fetal development in utero, whereas most occurs
during the time of delivery. The timing of detection of virus in the newborn
has been hypothesized to differentiate in utero from peripartum transmis-
sion; a positive culture or polymerase chain reaction (PCR) for HIV in the
first 2 days of newborn life suggests in utero acquisition, whereas a delay in
viral detection beyond 1 week of life suggests peripartum transmission [4].
Approximately 30% of perinatal HIV transmission is estimated to occur
P. Palumbo / Clin Lab Med 22 (2002) 759–772 761
in utero, with the remaining 70% at the time of delivery in the natural setting
with no intervention measures [5–7]. Although this concept is generally
accepted by the research and practicing medical community, it has been dif-
ficult to prove rigorously. It has importance for the design of transmission
prevention strategies and because in utero transmission has been associated
with increased risk for rapid disease progression.
One of the most important and broadly impacting studies ever conducted
was the Pediatric AIDS Clinical Trials Group 076 Study, which demon-
strated that perinatal transmission could be reduced from 25% to 8% by the
administration of an oral ARV agent (azidothymidine [AZT]) to a woman
during pregnancy and delivery and to her baby for the first 6 weeks of life
[8]. This intervention approach was quite controversial during study design
in the late 1980s and was successful beyond the hopes of even the more
optimistic. Short-term safety concerns were allayed [9,10] (long-term obser-
vation is ongoing) and widespread implementation subsequent to public
health recommendations in 1994 [11] have dramatically deceased the inci-
dence of perinatal transmission in developed countries [12,13]. The advent
of more potent ARV regimens has been instrumental in reducing transmis-
sion risk to about 1% [14]. Variations on the Pediatric AIDS Clinical Trials
Group 076 regimen have also been evaluated demonstrating success of
shorter-course AZT regimens and single-dose peripartum nevirapine, which
make implementation measures feasible worldwide [15,16].
Transmission risk factors have been identified, with high maternal plasma
virus levels, prolonged rupture of amniotic membranes, and advanced
maternal disease being among the more dominant promoting factors. Cae-
sarian section has been demonstrated to reduce risk significantly and is used
in high-risk obstetric practices with a frequency of 30% to 50% or more [17].
Weighing the risks and benefits of surgical delivery has been somewhat con-
troversial, in particular developing a comfort threshold for vaginal delivery
for HIV-infected mothers.
As the understanding of the biology of HIV replication and the details of
viral interaction with host cells improves, human genetic risk factors for both
transmission and disease progression are being discovered. An example is the
identification of a second target cell receptor (chemokine receptor: CCR5 and
CXCR4) necessary for viral binding and uptake in addition to the primary
receptor: CD4 [18–24]. A 32-base pair deletion in the CCR5 gene exists [25],
primarily in the white population (approximately 5% prevalence), and prob-
ably originating thousands of years ago in North-Central Europe. It has been
associated with HIV nontransmission in homozygous exposed individuals
and with decreased rates of disease progression in heterozygous infected indi-
viduals [26–28]. More importantly, multiple polymorphisms in the promoter
regions of CCR5 have been identified, which have varied and increased prev-
alence in multiple races and which seem to impact risk for HIV transmission
[29–31]. This is a rapidly expanding and complex field in that multiple genetic
polymorphisms almost certainly interact. Given the recent accomplishments
and direction of human genome research intersecting with advances in basic

understanding of HIV, further dramatic developments are expected.
Viral resistance and its impact become a concern as multidrug treatment
regimens become more prevalent. Nevirapine, a nonnucleoside reverse tran-
scriptase inhibitor (NNRTI), has been documented to select for resistance
even after the single-dose regimen used for prevention of perinatal transmis-
sion [32]. Studies have not documented an increased risk for transmission of
ARV-resistant maternal viruses, but the situation is rapidly evolving and
requires ongoing surveillance [33,34]. In addition, when transmission of
resistant virus does occur, it may doom customary initial treatment regi-
mens unless specifically known and addressed.
Despite the availability of transmission prevention measures, implemen-
tation in large populations, especially those with limited health care infra-
structure and resources, remains problematic. In developed countries,
access to health care is also a problem with many metropolitan areas report-
ing 10% to 20% of pregnant women with no or limited prenatal care, pre-
cluding HIV screening and prevention measures.
Newborn diagnosis
Following the identification of HIV as the causative agent of AIDS [35,36]
and with recognition of vertical HIV transmission in the early 1980s [37,38],
diagnostic methods for direct viral detection were developed, serologic
methods being uninformative for infants less than 2 years of age because of
the presence of passively transferred maternal antibodies in all at-risk
infants. Culture-based assays were quickly supplanted by molecular detection
assays predominantly featuring PCR [39]. Currently, DNA PCR, targeting a
segment of the viral gag gene in samples of blood mononuclear cells, and
plasma viral RNA quantitative assays (reverse transcriptase PCR [Roche,
Branchburg, NJ] [40,41]; nucleic acid sequence based amplification [Organon-
Teknika, Boxtel, NL] [42]; and branched DNA [Bayer, Leuerkusen,
Germany] [43]) are the techniques of choice and available commercially.
Serial testing is performed with infection status definable by 2 to 4 months
of age [5,44]. Not all molecular assays are equally sensitive regarding
detection of all the geographic HIV subspecies or clades. Although clade B
is the predominant subtype in the United States, non-clade B subtypes or
circulating recombinant forms are found in 1% to 3% of cohorts [33,45]. DNA
PCR and first-generation reverse transcriptase PCR assays created for
detection of clade B in developed countries may generate false-negative results
for individuals with some non-clade B infections. Assays that target gene
sequences common to all clades either are available or will be released soon.
Viral pathogenesis during infancy

Application of newly developed molecular viral quantitative assays to
specimens from pediatric cohorts revealed the persistence of unusually high
plasma virus levels during the first few years of life when compared with
average levels established for acute and chronic infection in adults [46–49].
This has been associated with a higher proportion of rapid disease pro-
gression in infant cohorts; as many as 30% to 40% develop AIDS or death in
the first 1 to 2 years of life. Multiple hypotheses have emerged to explain this
phenomenon including the presence of higher levels of CD4 lymphocytes
(both naive and activated) in infancy and the absence of a fully mature,
counterbalancing immune system [50]. Recent studies have also pointed out
the unique, potentially high-risk genetic-immunologic background setting of
maternal-infant HIV transmission: approximately 50% of all maternal genes
are shared with the infant, in particular HLA genes, which drive CD4 and
CD8 memory immune response [51]. If virus has evolved to evade maternal
HLA-driven dominant immune responses and is subsequently transmitted,
the infant has a 50% chance of being preprogrammed for nonresponsive-
ness. This is in contrast to adult horizontal transmission wherein hetero-
geneity of genetic alleles ensures mismatches between donor and recipient.
Preliminary studies in small numbers of mother-infant pairs has demon-
strated this phenomenon and linked it with rapid disease progression in the
infant [51]. Further studies are eagerly anticipated to confirm and extend
these findings.
The mid-1990s witnessed breakthrough analyses, which painted an amaz-
ing and informative picture: HIV replicates and turns over at an extraordi-
narily high rate, consistent with a virion half-life of 6 hours and total daily
production of 10 billion particles per infected individual [52,53]. This pro-
duction rate is paralleled by similar output and turnover rates for produc-
tively infected lymphocytes in the steady state. This dynamic balance,
coupled with the relatively high error rate for the genetic copying mecha-
nism of viral reverse transcriptase, resulted in a model incorporating a
diverse genetic pool of virions within any infected individual (quasispecies)
capable of rapid mutation and selection in response to environmental pres-
sures. The latter include host immune response, exhaustion of preferred
target cells, and ARV agents. Recognizing unique infant infection features
of high plasma virus levels, rapid disease progression, and a developing
immune system, it is important yet extremely difficult to quantify viral and
target cell kinetics accurately in this setting. Such studies are ongoing and
currently suggest equivalent dynamic parameters in infancy [54,55].
Another feature of the unique setting of infant HIV infection (ie, acute
infection during maturation of the immune response system) is observed
when infants are treated aggressively in the first few months of life. Rapid
and persistent control of viral replication with highly active ARV regimens
results in both seroreversion (ie, the infant loses maternal HIV-specific
antibody over time) and failure to generate either HIV-specific humoral or
cell-mediated immunity [56]. These infants continue to be HIV-infected, as
evidenced by persistence of proviral DNA within circulating mononuclear
cells and by bursts of viral replication in the event of ARV holidays, but
remain immunologically naive in the context of current testing methodol-

ogy. Children in this setting have been followed for more than 6 years and
are prime candidates for HIV immunization.
A hopeful area for children and adolescent youth, and one of intense
study, is that capacity for thymic immune cell output may be more vigorous
early in life compared with later periods, after which the thymus has under-
gone significant involution [57–60]. This may not be without its downside, in
that increased output may only provide more targets for HIV replication
and ultimate destruction. Destruction of both CD4 and CD8 lymphocytes
in a subset of rapidly progressing infants has been documented and labeled
as a ‘‘thymic deficiency’’ profile [61,62]. Despite this, T-cell receptor excision
circles (TREC) assays, which target quantification of lymphocytes containing
extrachromosomal DNA that are the products of T-cell receptor rearrange-
ment, detect lymphocytes recently released from the thymus and, together
with radiographic assessment of thymic size, support the increased capacity
of thymic youth and the substantial potential for immune reconstitution.
Treatment issues: HIV-infected infant and child

When to initiate therapy
The availability of ARVs, initially the nucleoside reverse transcriptase
inhibitor (NRTI) AZT followed by additional NRTIs and NNRTIs and
protease inhibitors (PIs), created a legitimate demand for treatment of all
HIV-infected children. Although significant benefit was demonstrated for
AZT monotherapy and for combination NRTIs (initially among children
with more advanced disease) [63,64], dramatic improvements in outcome
were observed after the introduction of the NNRTIs nevirapine [65–67] and
efavirenz [68], and the PIs nelfinavir [67,69] and ritonavir [70]. Highly active
ARV therapy was the term coined for combination therapy with agents from
multiple ARV classes.
Complications of pediatric HIV infection being, on average, more fre-
quent than in adults and unique features of pediatric HIV disease manifesta-
tions, such as growth and developmental delay, allowed clinical end point
trials to be conducted with significantly fewer enrollees than usually required
for adult trials. Within the context of these trials, plasma virus levels and
CD4 lymphocyte counts were demonstrated to be predictive of disease pro-
gression or death independently, with improved accuracy when the variables
were used together [46,48]. Plasma RNA levels have been demonstrated to
possess a linear correlation with risk throughout all age groups, whereas
CD4 lymphocyte counts demonstrated age-specific thresholds for disease
progression. It should be emphasized that these were cohort risk analyses and
that there was significant heterogeneity for individuals within the group.
When presented with a newly diagnosed infant or child or one who is
ARV-naive, many factors must be considered in deciding whether (and with
what) to treat. These include the readiness and willingness of the family unit
and the age and health of the child. The ability to differentiate children who
experience rapid versus slow disease progression by clinical and laboratory
parameters is most limited in the first year of life. Because this is also the
period of highest risk, the consensus among experts caring for such children
is to recommend aggressive therapy (two NRTIs combined with either an
NNRTI or a PI at a minimum with some recommending four or five drug
combination regimens).
Once children have reached 1 year of age or older, the option of deferring
treatment can be considered with increasing comfort level as age increases.
The difficulty encountered with this approach is that there are no well-estab-
lished thresholds for clinical and laboratory parameters to assist with this
decision. The difficulties with drug administration (quality of life and multi-
drug regimens in large quantities multiple times per day) and their com-
plications (metabolic, hematologic, and neurologic toxicity; drug–drug
interactions; and ARV resistance) support serious consideration of deferral.
Another feature to be considered is that, at most, two effective multidrug
regimens are available during an infected individual’s lifetime given the cur-
rently available agents. This must be balanced against both the known and
hoped for benefits, in particular the preservation of the immune system
through suppression of viral replication. The public health service supports
the option of therapy deferral for children over 1 year of age who are in
good health and whose CD4 lymphocyte count is in the normal range for
age. Despite these reservations, the availability of highly active ARV regi-
mens and their widespread general use in pediatric populations within devel-
oped countries has resulted in gratifying decreases in morbidity and
mortality over the last 5 years [71,72].
Factors affecting therapy outcome

Three factors have major impact on the effectiveness and success of a pre-
scribed ARV regimen: (1) adherence to therapy, (2) pharmacokinetic and
dynamic parameters, and (3) ARV resistance.
Adherence to therapy
Most consider it no overstatement to declare that the single most impor-
tant factor in therapeutic outcome is the patient’s ability to adhere to pre-
scribed therapy [73]. HIV-infected children often come from fragile and
overburdened family units whose biologic parents may be ill, deceased, or
otherwise unable to provide care and support. The development of drug for-
mulations appropriate for children has been difficult from both a commer-
cial and technical standpoint. The acidic, relatively insoluble PIs have been
particularly problematic regarding the development of liquid formulations
with the result being either no formulation or those that have been charac-
terized as bitter, gritty, or unpalatable. Most drugs have administration
schedules of two to four times per day with very few possessing pharmaco-
kinetics allowing once-a-day timing. When one considers frequency of
administration with the need for three to five ARVs in combination, not
to mention other pharmaceuticals for infection prevention and support, the
task seems quite daunting. Infants at one end of the age spectrum pose a set
of challenges that take on different characteristics among HIV-infected ado-
lescents. Clinics providing care for HIV-infected children and adolescents
have formed multidisciplinary care teams for medical and behavioral sup-
port and significant research is being conducted into the behavioral manage-
ment of therapeutic adherence issues and into simplification of therapeutic
regimens.
Pediatric ARV pharmacokinetics

Defining an appropriate ARV drug dosage with the aim of toxicity avoid-
ance combined with optimum suppression of viral replication has been par-
ticularly challenging for children of all ages and for pregnant women. The
issue of significant drug-drug interactions adds to the complexity in light
of the need for multidrug ARV regimens. It is clear that industry drug devel-
opment targets adult populations and, although there have been recent
improvements and legislative mandates regarding parallel pediatric devel-
opment, the complexities of drug development for children and pregnant
women are often beyond the scope of industry efforts or FDA requirements.
Pediatricians and obstetricians have been continuously caught in a cycle
of ARV drug FDA approval and availability before comprehensive pharma-
cokinetic data are accessible, frequently resulting in underdosing with its
adverse consequences.
The dosing of drugs for infants and children is clearly not straightforward.
Pharmacologic theory predicts that dosing based on milligram of drug per
kilogram of body weight estimates results in drug exposures lower than those
seen in adults, and the underdosing increases as the size of the child
decreases. The use of milligram of drug per square meter of body surface area
provides substantial correction of this problem, but may still be inadequate
for children under 2 years of age. Past experience with the introduction of
new ARV agents is replete with underdosing problems among young chil-
dren, especially with the NNRTIs and PIs. Following intensive study of drug
pharmacokinetics and ARV effect, drug dosing recommendations are often
established that are age-specific and which, in young infants, may be two
to four times the dosage recommendations for older children and adults on
a milligram per kilogram basis. In a similar manner, different pharmacoki-
netic parameters have been demonstrated among pregnant women compared
with nonpregnant adults for all PIs evaluated. To complicate matters further,
significant drug-drug interactions occur between PIs and NNRTIs and PIs.
Because aggressive therapy, especially among ARV-experienced individuals,
requires combinations of this sort, these drug interactions, which may have
unique features in children, present a difficult challenge.
Antiretroviral resistance
The pervasive problem posed by ARV resistance is intimately linked with
both adherence to therapy and the development of optimal pharmacokinetic
parameters. Given the daily productive replication capacity of HIV in the
steady state (approximately 10 billion virions per day) and the inherent
copying error rate of reverse transcriptase (3 to 5 104), it is not surprising
that a complete repertoire of mutant viral variants are developed very early
in infection. Calculations of the potential for daily generation of de novo
resistance mutations suggests capacity for 22 million single mutations, 3 mil-
lion double mutations, 300 thousand triple mutations, and so on. Not only
do the quasispecies have tremendous capacity to generate new mutations in
response to selective pressure, but existing low-frequency mutations are cer-
tainly present, which can assume dominance under appropriate conditions.
It should not be surprising that ARV resistance is a dominant phenom-
enon among treated individuals. This is particularly true in pediatrics con-
sidering the difficulties with drug administration and adherence and the
understandable use of agents before complete pharmacokinetic data are
available for appropriate age groups. Screening for resistance to ARV, by
means of detecting viral genetic mutations associated with resistance (geno-
typing) or by replication characteristics identified in culture-based systems
(phenotyping), is a common clinical practice and recommended in multiple
settings: pregnancy, acute infection, and therapeutic failure. Although the
results of several adult studies support the current recommendations [74–76],
decision-making remains problematic and there have been no systematic
studies in pediatrics.
Summary
Knowledge regarding the basic mechanisms of pediatric HIV infection
and its prevention and treatment has expanded greatly in the last decade.
Significant questions remain and have been largely refocused to the com-
plexities of a chronic disease process. Management invariably requires spe-
cialists who must keep abreast of a rapidly evolving information base.
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Clin Lab Med 22 (2002) 773–797
CD8þ T-cell immunity to HIV infection

Paolo Piazza, PhDa,*, Zheng Fan, MDa,
Charles R. Rinaldo, Jr, PhDa,b
a
Department of Infectious Diseases and Microbiology, University of Pittsburgh
Graduate School of Public Health, 425 Parran Hall, Pittsburgh, PA 15261, USA
b
Department of Pathology, University of Pittsburgh School of Medicine,
Pittsburgh, PA 15261, USA
Since the first cases were reported in the early 1980s, the AIDS epidemic
has grown to alarming proportions both in the United States and the rest of
the world, particularly in sub-Saharan Africa. The total number of people
living with HIV-1 infection has reached 40 million, with 5 million new infec-
tions in year 2000 alone. In the light of numerous efforts to control the pro-
gression of the disease in infected subjects and the spread of the infection in
the general population, remarkable success has been achieved since 1995
with the use of combination antiviral treatments. Safe, prophylactic and
therapeutic vaccines are still under study, however, and considered to be
many years in the future.
The limited success in controlling HIV-1 infection is because of the
unique propensity of HIV-1 to infect the very cells that are responsible for
fighting it, namely CD4þ T-helper lymphocytes and antigen-presenting cells
(APC). This causes profound perturbations of the immune system. More-
over, HIV-1 replicates to very high numbers (billions of copies per day) and
has the capacity to mutate at very high rates because of the lack of fidelity of
the virus polymerase, with tens of millions of new variants produced every
day [1]. Drug-resistant viruses generated in this way are now well controlled
by multidrug regimens that act by simultaneously targeting proteins that are
crucial for the virus cycle, such as the reverse transcriptase (polymerase) and
the protease. Although pharmacologic treatment is very effective in reducing
the amount of virus to extremely low or undetectable levels, it unfortunately
This work is supported by grants R01-AI-41870, U01-AI-37984, and U01-AI-35041 from

the United States Public Health Service and the National Institutes Health.
E-mail address: paolo@pitt.edu (P. Piazza).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 0 6 - 9
774 P. Piazza et al / Clin Lab Med 22 (2002) 773–797
does not eradicate HIV-1 infection and cannot be prolonged indefinitely

because of its toxicity [2].
Modulation and augmentation of immune control of viral replication
represents a major target of intervention. Antibodies do not seem to confer
protection to the advancing of the disease, even when they have neutralizing
activity in vitro. In this context, cell-mediated immunity has been the object
of intensive research, with a large amount of knowledge having accumulated
since a previous review on this subject appeared in this journal in 1994 [3].
This article focuses on one of the major effector cells that form these adap-
tive responses, the cytotoxic T lymphocytes (CTL), because increasing
evidence points to their critical role in the immune response to HIV-1.
CTL in antiviral immunity

The CTL are important in controlling and ultimately eradicating viral
infections [4]. They are primarily of CD8 phenotype and mediate killing
of virus-infected targets when these express viral antigens on their surface
in the form of short, 9–11 amino acid peptides bound to major histocompat-
ibility complex (MHC) class I molecules. Although MHC class II restricted
CTL of the CD4 phenotype have also been described, they do not seem to
play as critical a role in controlling viral infections. At the molecular level,
antigen recognition is a highly restricted event mediated by the T-cell recep-
tor complex (TCR), an ensemble of proteins expressed on the surface of all
T lymphocytes, representing the T-cell analogue of antibody produced by
plasma cells and B lymphocytes. On recognition of its peptide ligand on the
infected cell, the TCR and its coreceptor CD28 and various signaling mol-
ecules form an immunologic synapse with the peptide-MHC complex. This
leads to cytokine release and lytic activity by the CD8þ T cell.
Broadly speaking, the generation of CTL consists of two distinct phases:
induction and effector. During the induction phase, CD8þ lymphocytes
become ‘‘licensed to kill’’ their cognate targets after they come in contact
with APC, typically dendritic cells (DC) [5]. In their immature state, DC are
highly efficient in capturing and processing foreign antigens. In the classic
endogenous pathway of antigen presentation, proteins produced during
viral replication in APC are proteolytically cleaved in the cytosol by highly
complex structures termed proteasomes. The resulting peptides are then
transported to the endoplasmic reticulum where they complex with MHC
class I molecules and then travel through the Golgi apparatus to the cell sur-
face. In the exogenous pathway, viral proteins are ingested from the extracy-
tosolic environment into endosomal vesicles where they are digested by
proteases and the resulting viral peptides are complexed with MHC class
II molecules before transport to the cell membrane. Because some viruses
do not replicate efficiently in DC, alternative mechanisms to the classic
MHC class I endogenous pathway exist that can induce CD8þ T-cell
responses. Such unconventional pathways use uptake of exogenous antigen
P. Piazza et al / Clin Lab Med 22 (2002) 773–797 775
by DC in the form of virus-infected, apoptotic or necrotic cells followed by

cross-presentation of antigen with MHC class I on the surface of CD8þ
lymphocytes. After encountering antigen, immature DC undergo matura-
tion events induced predominantly by ligation of CD40 with CD40 ligand
(CD40L or CD154) expressed on CD4þ T cells. Mature DC then present
the foreign processed antigens on their surface in association with MHC
class I and II molecules, depending on which pathway of antigen degrada-
tion has been used. It has been shown that DC also express a vast array
of stimulatory molecules, both membrane bound, like CD40, CD80, and
CD86, and secreted, such as interleukin (IL)-12, IL-15, and more recently
IL-2, that are crucial in activating T lymphocytes.
The effector function of CTL, namely the killing of virus-expressing tar-
gets, is performed through the release of lytic granules containing pore-
forming proteins named perforins (cytotoxins) and also proteases, such as
granzymes, that activate apoptosis. The action of perforins is very rapid and
it induces formation of large pores through the cell membrane of the targets
and causes their death by disruption of their osmotic integrity. In addition
to the action of granzymes, CTL can induce apoptosis of the target through
interactions between molecules like CD95 ligand on the surface of the CTL
and CD95 on the target cell. Induction of apoptosis is generally a much
slower process than the action of perforins, because it requires active
involvement of protein synthesis by the target cells. These two killing mech-
anisms, however, are not mutually exclusive and can occur simultaneously.
The killing capacity of CTL has been measured in vitro with cytotoxicity
assays where responder cells are incubated with MHC matched cell targets
expressing viral antigens and a radioactive tracer. The amount of killing is in
proportion to the amount of radioactivity released by the targets. The most
quantitative measure of cytotoxicity is determination of CTL precursor fre-
quencies by the limiting dilution assay. Questions concerning the accuracy
of this assay have recently emerged, however, mainly in relation to the need
to culture responding cells in the presence of growth factors, such as IL-2,
for 1 to 2 weeks before testing. Several highly sensitive, new techniques that
do not require long-term in vitro culture have revolutionized the field of
antiviral cellular immunity, allowing for very accurate and precise determi-
nation of CTL frequencies and activity [6]. When compared with limiting
dilution assay these new methods result in 10 to 100 times’ higher numbers
of antigen-specific, CD8þ T cells [7]. This is probably caused by loss of effec-
tor cells in vitro in the CTL assays caused by a phenomenon termed activa-
tion-induced cell death, which is known to occur with prolonged exposure
to growth factors or antigens, although discrepancies in the results seem to
disappear using improved culture techniques [8]. The most powerful, new
method is tetramer staining, which rapidly enumerates the numbers of
CD8þ cells that express TCR specific for a particular, unique combination
of peptide and MHC molecules by flow cytometry [9]. It does not require in
vitro activation of the T cells and represents a snapshot of the actual
immune status of the subject. Each tetramer is unique for a particular set of
TCR, however, not allowing detection of multiple antigen-specific T cells. In
contrast, the new Elispot and intracellular staining techniques measure the
cytokine-producing function of antigen-specific T cells, taking advantage
of the fact that such cytokines as interferon-c (IFN-c) are produced mostly
by antigen-activated CD8þ T cells. Of importance is that the levels of both
tetramer and IFN-c expression correlate very well with cytotoxic activity in
certain viral infections [7].
A recent technical advance in the field is the use of large libraries of
HIV-1 peptides representing either known CTL epitopes for particular HLA
class I haplotypes [10] or 15 amino acid peptides overlapping by 11 amino
acids for different HIV-1 proteins. This allows for simultaneous delineation
of the quantity and breadth of the CD8þ T-cell response to various HIV-1
proteins. These may be used in a matrix format consisting of multiple pools
of up to 15 peptides each for screening purposes; single peptides are chosen
based on the positive peptides in these pools, and used to fine-map the
epitopes [11,12]. It should be noted, however, that these studies simply add
a large concentration of synthetic peptide to peripheral blood mononuclear
cells, culture for several hours to days, and examine these for cytokine
production. They assume homogeneity and consistency in APC function,
even in longitudinal studies of immunosuppressed individuals, and do not
account for the effects of variations in the number, function, and type of
APC on T-cell responses in the cultures. In this regard, the authors and
others have shown that DC loaded with human herpesvirus 8 (Kaposi’s
sarcoma–associated herpesvirus) or Epstein-Barr virus peptides are superior
inducers of antiviral CD8þ T-cell responses as compared with peripheral
blood mononuclear cells stimulated directly with peptides alone [13,14]. Use
of DC loaded with HIV-1 peptides as APC have also revealed new HIV-1
CD8þ T-cell epitopes [15]. This factor should be considered in future studies
of HIV-1 peptide-specific T-cell responses.
Clinically relevant profiles of disease progression and HIV-1–specific CTL

High-risk HIV-1 seronegative persons
There is a fascinating group of persons who have been exposed to HIV-1
to a high degree and yet do not have antibodies to the virus and remain per-
sistently virus negative (highly exposed, persistently seronegative subjects)
[16]. It is presumed that the protective mechanisms in these persons are crit-
ical to the understanding of immune correlates of protection from HIV-1
infection that are needed to assess the efficacy of prophylactic vaccines.
These highly exposed, persistently seronegative subjects include female com-
mercial sex workers in Gambia and Kenya with an estimate of over 60 epi-
sodes of vaginal HIV-1 exposures per year [17–20], and other individuals
who engage in unprotected sexual contact with HIV-1–infected partners
(exposed seronegatives) [21]. These individuals apparently have genetic
resistance to HIV-1 infection that is not the well-described, homozygous

recessive trait for lack of expression of the CCR5 coreceptor that is found
in about 1% of Western European and North American Caucasians [22].
Anti–HIV-1 CTL and CD8þ T cells that produce a soluble, noncytotoxic
cell antiviral factor (CAF) have been detected in the blood of some of these
highly exposed, persistently seronegative subjects [20,23]. Moreover, the
quality, rather than (or better, in addition to) the magnitude, of CTL
immunity is relevant to conferring protection to HIV-1 infection [24,25].
T-cell responses to a broad array of specific regions of the virus may be crit-
ical for protection from HIV-1 infection. Furthermore, the specificity of the
immunodominant responses differs in highly exposed, persistently seronega-
tive subjects when compared with HIV-1–infected subjects [25].
A provocative finding is that the local anatomic site of the cellular
immune response is a highly relevant factor in conferring protection to mul-
tiple exposures in these individuals. T-cell IFN-c responses to Gag, Pol, and
Nef were higher in cervical mucosa and lower in blood in HIV-1–resistant,
as compared with HIV-1–infected, Nairobi sex workers [17]. A possible
explanation is that the infection is actually present but it is somehow
contained by CTL in the local genital tract. The infection does not become
systemic and remains undetected. Support for the importance of local
immunity in the protection to mucosal challenge to HIV-1 comes from stu-
dies in rhesus monkeys [26]. After simian immunodeficiency virus (SIV)
infection of the colonic intestinal tract, a retrovirus highly related to HIV-1,
protection was found only in those animals that displayed virus-specific
CTL in the lamina propria of the jejunum, whereas it was absent in ani-
mals that had poor localized CMI.
Progressive HIV-1 infection

During primary, acute infection with HIV-1, viremia peaks by 3 weeks,
after which virus production declines very rapidly. After about 3 months,
the plasma levels of virus seem to stabilize and represent what is commonly
referred to as the viral set-point. This value has been shown to correlate with
progression of disease and the length of survival postinfection [27]. The
importance of CD8þ CTL in control of HIV-1 infection has been suggested
by the appearance of anti–HIV-1 CTL in the blood during the acute phase
of HIV-1 infection, coincident with the decrease of the initial, large burst
of virus production to a much lower set-point, and before the advent of
HIV-1–specific neutralizing antibodies [28,29]. Perhaps the most convincing
evidence of the fundamental importance of CD8þ cells in determining the
outcome of early HIV-1 infection, however, comes from nonhuman primate
models where depletion of this cell subset in rhesus macaques infected with
SIV results in a remarkably rapid progression to AIDS [30–33].
It is likely that adaptive T-cell immunity, possibly in concert with anti-
body-mediated cell cytotoxicity and several forms of innate immunity
(eg, natural killer [NK] cells, a and b IFN), control early HIV-1 infection in all
but the most rapid progressors. These latter individuals progress relatively
unabated to AIDS within a few years of infection, which has been related
to a lack of development of anti–HIV-1 CTL responses [34,35]. What fol-
lows immediately after acute infection with HIV-1 in over 90% of infected
individuals is a series of assaults on the immune system that systematically
break down essentially all aspects of antiviral immune control. With only a
few relevant exceptions, CTL ultimately fail to keep HIV-1 infection in
check, and unless aggressive antiviral therapy is used, infected adult subjects
develop AIDS within a median of 10 years because of the progressive immu-
nosuppression induced by the virus. It should be noted that the plasticity of
host immunity leads to multiple, alternative antiviral mechanisms that could
replace this lack of adequate HLA class I restricted CD8þ T-cell reactivity,
particularly CAF produced by CD8þ T cells that directly suppresses HIV-1
replication [36].
There are a multitude of posited mechanisms for lack of CD8þ T-cell con-
trol of HIV-1 infection (Table 1). For many years it has been hypothesized
Table 1
CD8þ T-cell dysfunction in HIV-1 infection
Category of dysfunction Mechanism of dysfunction References
Intrinsic CD8þ T-cell and APC defects Lack of CD4þ TH1 cell help
Low cytokine production [37–40]
Low expression of CD40 coreceptor [44]
for APC
Low levels of perforin [45]
Low expression of CD3f [46,47]
Low expression of CD28 [46,47]
Limited TCR repertoire [52,53]
Association of HLA class I haplotypes [48–50]
with rapid progression of infection
Immune evasion engineered by HIV-1 HIV-1 escape variants
Weak binding of peptide to HLA [64]
class I molecule
Antagonism of peptide for the [68,69]
original epitope (APL)
HLA class I molecule down- [70–73]
modulation
Lysis of CD8þ T cells Fas-FasL–induced apoptosis [76,77]
Expression of CD4 on CD8þ T cells [80]
resulting in HIV-1 infection and
lysis
HIV-1 infection and lysis of CD8þ T [81,82]
cells by non-CD4 receptor
mechanisms
Abbreviations: APL, altered peptide ligands; APC, antigen-presenting cells; TCR, T-cell
receptor complex.
that the relentless assault of continuous HIV-1 replication leads to T-cell

anergy in the host. In this scenario, the host may maintain CD8þ T cells with
specificity for HIV-1 epitopes, but these cells lose their ability to kill virus-
infected targets [24]. This has been shown by the presence of lower numbers
of IFN-c–producing, as compared with tetramer-positive, CD8þ T cells in
HIV-1–infected persons [37–39]. A similar dichotomy between tetramer-
positive and IFN-c–producing CD8þ T cells has been noted in SIV-infected
monkeys [40]. The presence of such nonfunctional, HIV-1–specific CD8þ
T cells has been questioned by Goulder et al [8], who used an improved
assay for CTL precursors to show an extraordinarily high correlation among
HIV-1–specific CTL, tetramer, and IFN-c–producing CD8þ T cells.
The mechanistic basis of T-cell anergy in HIV-1 infection is unclear. A sim-
ilar impairment of antiviral CD8þ T-cell responses has been related to poor
CD4þ T-cell activity in the murine lymphocytic choriomeningitis virus
(LCMV) model [41], comparable with loss of anti–HIV-1 CD4þ T-cell activ-
ity in progressive HIV-1 infection [42]. Indeed, maintenance of anti–HIV-1
CD4þ T-cell proliferative responses is paramount for retention of HIV-1–
specific CD8þ T-cell responses, and negatively correlates with HIV-1 load
[43]. This CD4þ T helper cell effect likely includes CD40L costimulation of
APC and production of IL-2 or other mediators by the TH1 helper subset that
are necessary for activation of antiviral CD8þ T cells. In this regard, gp120
binding to CD4 can down-regulate CD40L expression on these cells, poten-
tially leading to improper signaling of APC [44]. It is also well documented
that IL-2 production by TH1 cells is decreased during HIV-1 infection [24].
The HIV-specific CD8þ T cells from chronically infected persons also
have inherent defects, such as significantly lower levels of perforin than
cytomegalovirus-specific CD8þ T cells [45]. The HIV-1–specific cells with
low levels of perforin express CD27, which is part of the tumor necrosis fac-
tor receptor superfamily present on thymic emigrants, suggesting that these
cells have impaired maturation. Finally, T-cell anergy during HIV-1 infec-
tion has been related to defects in CD8þ T-cell signaling that occur by loss
of surface expression of the CD3 f chain and CD28 coreceptor [46]. Expres-
sion of these molecules can be restored by overnight culture with IL-2,
together with recovery of HIV-1 CTL function [47].
Intriguing evidence is mounting that persons with certain HLA class I
haplotypes and transporter for antigen presentation (TAP) alleles have faster
or slower rates of progression to AIDS [48]. The most striking example of
this is the association of HLA class I alleles B*35 and Cw*04 with rapid
development of AIDS [49]. Interestingly, a subpopulation of the B*35
haplotype individuals has a single amino acid change in their HLA B*35
molecule that is related to more rapid progression to AIDS [50]. In contrast,
HLA B*5701 is associated with low viral load and nonprogression [51].
These associations are presumably related to as-yet-to-be-defined HLA class
I restricted APC and CD8þ T-cell activity. Of further importance is that
slower progression of HIV-1 infection is associated with a broad specificity
of the TCR Vb clonal repertoire for HIV-1 in CD8þ T cells after acute
infection, as compared with narrow anti–HIV-1 TCR repertoires in more
rapid progressors [52,53]. Multiple TCR familial lineages with specificity for
many different viral peptides of various HIV-1 structural and nonstructural
proteins lead to more potent and durable control of HIV-1 infection.
Because the T cell–antigen recognition phase is so fundamental to CTL
function, it is not surprising that it has become the target of a number of
strategies deployed by HIV-1 and other viruses to evade immune control
[54]. The most enduring concept is that, as CD8þ T cells exert constant
pressure on the massive pool of replicating virus, eliminating infected cells
at a very high rate, they must continuously alter their TCR specificity to
accommodate the high rate of mutational changes in HIV-1 [55,56]. Because
of this extremely high mutational rate, there is constant escape of a portion
of replication competent HIV-1 that is not susceptible to control by CD8þ
T cells. Several extensive studies now support that selective pressure of anti–
HIV-1 specific CD8þ T cells leads to stable mutants no longer susceptible to
this form of antiviral control. During both HIV-1 and SIV infections, there
is an early focusing of CD8þ T-cell immunity on certain HIV-1 proteins.
For example, during acute HIV-1 infection, there is no evidence of CD8þ
T-cell reactivity to an HLA A*0201 p17 Gag epitope that is immunodomi-
nant in 75% of HLA A*0201 adults chronically infected with HIV-1 [57].
There is a similar dominant pattern of CTL activity to SIV Tat and Gag
peptide during acute infection in the macaque model that changes to only
the Gag peptide in chronic infection [58].
Most important is that the HIV-1 and SIV mutants, which evolve after
acute infection, are increasingly resistant to CTL activity. CTL are ineffec-
tive against certain gp160 Env mutants that evolve during the first year of
HIV-1 infection [59]. Likewise, in macaques infected with cloned SIV, there
is an early dominance of CD8þ T cells specific for Tat epitopes that declines
over time in concert with changes in Tat sequences and establishment of
chronic SIV infection [60]. A similar loss of CTL reactivity has been recog-
nized in relation to progressive mutations in SIV Env and Nef [61]. Interest-
ingly, HIV-1 escape mutants may not dominate until very late in infection,
because those to HLA B27-restricted, HIV-1 p24 Gag epitopes are not evi-
dent until after 9 years of infection [62]. The serious implications of this
HLA B27 Gag escape mutant have been emphasized by evidence showing
its transmission from infected mother to neonate, with development of CTL
against subdominant Gag epitopes that fail to suppress HIV-1 viremia in the
child [63].
Several mechanisms have been proposed as the basis for how these viral
mutants actually escape immune control. The most straightforward mecha-
nism of this immune evasion is loss of binding of mutant HIV-1 peptides to
their HLA class I restriction molecules, rendering the infected cell invisible
to CD8þ T-cell recognition. An example of this is a cluster of mutations in
a p24 Gag epitope that result in poor binding to their MHC restriction
element, HLA B27 [64]. Mutations in regions of HIV-1 proteins that include
normal proteolytic cleavage sites may also lead to alterations in processing
of peptides that are required for their presentation by HLA class I molecules
[65]. Interestingly, recent evidence indicates that the TCR may not be so
highly restricted in its recognition of unique peptide and MHC class I com-
binations, allowing for a certain amount of flexibility in the reaction to even
large sets of naturally occurring HIV-1 epitope variants [66].
Immune evasion could be caused by evolution of altered peptide ligands
that still bind normally to their respective HLA class I molecules and are
recognized by anti–HIV-1 CD8þ T cells, but are antagonistic, rather than
agonistic, for induction of CD8þ T-cell reactivity. Evidence for this concept
comes from the murine LCMV model, where mice primed with one LCMV
strain maintain a dominant CTL response to this initial antigen after infec-
tion with an LCMV-altered peptide ligand variant [67]. The biologic basis
of this ‘‘original antigenic sin’’ is unknown. Another mechanism of T-cell
anergy to HIV-1 has been described by Purbhoo et al, wherein certain
antagonistic HIV-1 Gag peptide variants compete with their agonist pep-
tides by failing to induce protein tyrosine phosphorylation after TCR bind-
ing [68]. A natural altered peptide ligand variant of gp120 Env peptide has
also been shown to induce anergy in a CD4þ CTL clone specific for the ori-
ginal gp120 peptide [69].
A well-documented loss of T-cell recognition of HIV-1–infected cells in
vitro occurs because of down-regulation of HLA-A and HLA-B molecules
on the surface of infected cells caused by HIV-1–encoded Nef, Vpu, and Tat
[70–72]. This leads to a greatly diminished capacity of CTL to recognize and
lyse HIV-1–infected targets [73]. Nef acts by blocking transport of HLA-A
and HLA-B molecules to the cell surface [74]. Moreover, Nef does not mod-
ulate expression of HLA-C and HLA-E molecules, which inhibit NK cell
lysis by ligation with the killer cell immunoglobulin-like receptors and the
lectin-like CD94-NKG2 receptors of NK cells, respectively [75].
Finally, there are several posited mechanisms for how direct lysis of
CD8þ T cells could result in loss of effector CTL function during HIV-1
infection. HIV-1 and SIV-encoded Nef can up-regulate FasL expression
on infected CD4þ cells, thereby arming them to kill Fas-expressing, HIV-
1–specific CD8þ T cells by Fas-FasL–induced apoptosis [76,77]. There is
also evidence that X4 Env interacts with CXCR4 to cause apoptosis of unin-
fected CD8þ T cells through a caspase-independent mechanism [78], con-
cordant with earlier work showing Env-dependent, ‘‘bystander’’ apoptosis
of CD8þ T cells [79]. Activation of CD8þ T cells, as shown in vitro by stim-
ulation with anti-CD3 and anti-CD28 mAb or by allogeneic dendritic cells,
can also induce expression of CD4 and make these cells susceptible to HIV-1
infection [80]. Furthermore, some isolates of HIV-1 can infect CD8þ T cells
by non-CD4 receptor mechanisms [81]. This has been confirmed by detec-
tion of productive HIV-1 infection in mature CD8þ T cells from patients
with AIDS [82].
HIV-1–infected long-term nonprogressors

In less than 5% of HIV-1–infected adults, there is little or no progression
of HIV-1 infection for more than 7 years [83]. Obviously, this natural
immunity to HIV-1 disease progression could reveal important insights for
development of HIV-1 vaccines. Many but not all of these long-term non-
progressors have relatively high, stable levels of anti–HIV-1 CD8þ CTL
[84–86], CD8þ T cells producing CAF [87] and CC-chemokines [88], and
anti–HIV-1 CD4þ T cells [42]. Active CD4þ TH1 cytokine responses that
modulate CD8þ T cells have also been documented [89]. The basic hypothe-
sis supported by these findings is that ‘‘more is better,’’ in that higher levels
of broadly HIV-1–specific, circulating CD8þ T cells are necessary for con-
tinuous control of persistent HIV-1 infection. An enigma, however, is that
very high anti–HIV-1 CD8þ T-cell frequencies have been noted in persons
with progressive HIV-1 infection and high viral loads [37,51,90]. Moreover,
long-term nonprogressors can eventually undergo progressive HIV-1 infec-
tion [91]. Such persons also can be infected with replication-defective
mutants of HIV-1 [92]. It is not clear what immune responses are controlling
HIV-1 replication in long-term nonprogressors.
Anti–HIV-1 CTL during anti–HIV-1 pharmacologic treatment

and the effects of structured treatment interruptions (STI)
Treatment of HIV-1 infection is currently attained with the use of com-
binations of highly effective antiviral drugs, termed combination therapy or
highly active antiretroviral therapy (HAART) [93]. HAART targets proteins
that are absolutely essential for the viral life cycle, such as the protease and
the reverse transcriptase. The success of this therapy in reducing the viral
load to minimal or undetectable levels is profound. Unfortunately, HAART
does not completely eradicate infection because of the integrated, latent state
of HIV-1 and sequestering of virus in sites that are inaccessible to antiviral
drugs. Also, prolonged exposure to these potent treatment combinations
is associated with several adverse side effects, which include lipodystrophy,
thrombocytopenia, and renal dysfunction [2].
A striking finding is that administration of HAART in the first few
months of HIV-1 infection prevents loss of anti–HIV-1 CD4þ T-cell reactiv-
ity [42], which is crucial to maintenance of antiviral CD8þ T-cell responses.
Such early suppression of the HIV-1 load prevents maturation of anti–HIV-
1 CD8þ T-cell responses, however, possibly because of lack of sufficient pro-
duction of HLA class I immunogenic antigens [94,95]. Indeed, there may be
several pathways for recovery of CD8þ T cells after reduction of viral load
during HAART, involving rapid decline of circulating, activated cells; redis-
tribution of memory cells; and thymic regeneration of naive cells [96,97].
Moreover, HAART results in a decrease or only a temporal enhancing effect
on anti–HIV-1 CD8þ T-cell function in adult [98–100] and pediatric [101]
chronic progressors. Similarly, enhancing effects on anti–HIV-1 CD4þ
T-cell reactivity occur in a minority of persons with chronic HIV-1

infection, even though CD4þ T-cell responses are restored to other patho-
gens [96,102–106]. The loss of HIV-1–specific, CD8þ memory T-cell re-
sponses during HAART, however, is not likely to be linked to anti–HIV-1
CD4þ T-cell dysfunction, as in untreated, progressive HIV-1 infection.
Rather, it seems to be a normal, homeostatic immune control mechanism
caused by the reduction in the viral antigenic load. Consequently, there
is resumption in the circulating number of anti–HIV-1 CD8þ T cells in
some patients who have an increase in viral load after discontinuation of
HAART [107].
New strategies are needed to enhance anti–HIV-1 T-cell immunity to con-
trol completely HIV-1 infection in chronic progressors on HAART. One
such approach is to interrupt treatment in a structured fashion, which results
in rapid resurgence of the patient’s virus [96,108]. This acts as an autologous
vaccine, raising the level of virus above an antigenic threshold for a limited
time so as to stimulate immune responses, with the objective of clearing resid-
ual virus. Suspension and resumption of therapy can vary from a few days to
months, depending on the effect of the interruption on reappearance of virus
and the clinical condition of the patient. Such STI administered in patients
with early infection has resulted in dramatic control of virus replication asso-
ciated with stable levels of functional anti–HIV-1 CD4þ and CD8þ T cells
[109]. The emerging picture in chronically infected patients, however, is that
STI only leads to a very moderate and transient increase of HIV-1–specific
CD4þ and CD8þ responses and does not control viral load [107,109–116].
Highly controlled analyses in the SIV-macaque model support the poten-
tial benefit of early treatment with antiviral drugs and of STI in enhancing
CD8þ T-cell control of infection [117,118]. Indeed, Lifson et al [117] have
documented resistance to rechallenge with homologous and heterologous,
highly pathogenic strains of SIV in animals that are able to control viremia
after early and transient antiretroviral drug treatment. The duration of the
protection is extended for over 1 year and the control of the infection is
mediated, at least in part, by the activity of CD8þ T cells, whereas neutral-
izing antibodies were not observed. The key role of CD8þ T cells was ele-
gantly supported by repeated in vivo CD8þ cell depletions, resulting in a
dramatic increase of plasma virus levels, which after the natural reconstitu-
tion of the CD8þ T-cell pool, return to levels observed during treatment.
Noteworthy is the cross-protection from inoculation with heterologous
virus, a feature difficult to achieve even with conventional vaccines including
live-attenuated virus. In a study by Lori et al [118] a fixed schedule STI (four
cycles of 3 weeks on and 3 weeks off treatment) was compared with contin-
uous HAART therapy, with start of treatment at 6 weeks after inoculation
with a pathogenic strain of SIV. Strikingly, after discontinuation of therapy
in both groups following a total of 21 weeks of treatment, only animals
under STI were able to control viremia. This condition lasted without signi-
ficant changes for 6 months, whereas virus production increased in animals
under uninterrupted HAART to return to the same elevated levels measured

at seroconversion. Furthermore, SIV-specific IFN-c production by CD8þ T
cells was significantly higher in the STI group compared with uninterrupted
treatment, an effect lasting 6 months after permanent interruption of ther-
apy. These studies support the importance CD8þ T lymphocytes in the con-
trol of virus replication when viral loads are kept low by use of HAART
early in the infection, with treatment interruptions making virus available
for restimulation of antigen-specific immunity.
Reversing the defects in anti–HIV-1 CD8þ T-cell reactivity

in chronically infected persons on HAART with immunotherapy
After a dismal outcome in early clinical studies, the concept of using
immunotherapy to restore HIV-1–specific T-cell responses has had a keenly
renewed interest with the advent of HAART. The basic goal is to enhance
the quantity and breadth of the anti–HIV-1 CD8þ T-cell responses beyond
that accomplished by HAART alone, with the hope of improving control of
residual virus (Fig. 1). Many cytokines including IL-2, IL-12, and IL-15 are
being considered for use in augmenting CD4þ and CD8þ T-cell numbers
and function in HAART patients [119]. Several clinical trials have shown
that intermittent or continuous IL-2 therapy increases CD4þ T-cell num-
bers for prolonged periods, with only transient increases in viral load.
Although this treatment usually does not increase numbers of CD8þ T cells,
studies in monkeys suggest that it can enhance anti–HIV-1 CTL function
[120,121].
Given the fundamental role of DC as professional APC in initiating
immune responses, modulation of their function may augment antigen-
specific T-cell responses for immunotherapeutic intervention in HIV-1–
infected patients on HAART. Support for this approach is that DC derived
from culture of blood precursors in IL-4 and granulocyte-monocyte
Fig. 1. The typical CD8þ T-cell response to HIV-1 over the course of chronic HIV-1 infection
and highly active antiretroviral therapy (HAART), with hypothetical effects of immunotherapy
during HAART on this immune function (dotted lines).
colony-stimulating factor retain their HIV-1–specific, APC capacity for

CD8þ T cells even late in progressive, untreated HIV-1 infection [122]. Sub-
sequent studies showed that DC loaded with recombinant HIV-1 proteins
complexed with cationic liposome can use alternative, cytosolic HLA class I
processing pathways for activation of HIV-1–specific, memory CD8þ T cells
from untreated subjects [123]. This model has been applied to persons with
advanced immunodeficiency undergoing HAART, where blood-derived DC
loaded with liposome-complexed HIV-1 proteins and treated with IL-12 in
vitro activate anti–HIV-1 CD8þ T-cell responses, even though their CD8þ T
cells no longer respond to stimulation with HIV-1 antigens presented by
other types of APC [124]. Recent studies have extended this model to show
that DC loaded with liposome-complexed HIV-1 proteins and matured
with CD40L can cross-prime naive anti–HIV-1 CD8þ T cells in vitro (Huang
et al, unpublished data). Similarly, primary anti–HIV-1 CD4þ T cells can be
induced in vitro by TNF-a matured DC loaded with HIV-1 proteins with-
out liposome [125]. Recombinant HIV-1 proteins may represent potent im-
munogens for activation of anti–HIV-1 CD8þ T cells if given with liposome
or other vehicles that allow entry into alternative MHC class I pathways
in DC, and for induction of HIV-1–specific, CD4þ T cells by conventional
MHC class II processing pathways.
These highly specialized APC may also be used in therapeutic strategies
where DC from individuals with chronic HIV-1 infection on HAART are
loaded with antigens derived from their own, autologous virus (Fig. 2). This
differs from the STI model in that the DC are engineered to elicit greater and
broader T-cell responses to residual virus than can occur simply by increas-
ing virus levels in the host with removal of therapy. Initial proof of this con-
cept has come from studies showing that DC loaded with apoptotic cells
infected with an X4 strain of HIV-1 (which preferentially uses the CXCR4
coreceptor) induce anti–HIV-1 reactivity in vitro in both CD8þ and CD4þ
T cells from chronically infected patients on long-term HAART [126]. The
processing of this antigen for presentation to CD8þ T cells may be through
alternative HLA class I pathways in that X4 HIV-1 does not replicate effi-
ciently in DC [127,128]. This model could lead to a therapeutic approach for
enhancing T-cell immunity to autologous HIV-1 by DC that have been
loaded with apoptotic cells containing strains of HIV-1 that are unique to
the individual.
Prevention of HIV-1 infection

Given the high rate of new infections estimated to take place every day
(tens of thousands), vaccines that halt the spreading of the virus have been the
‘‘holy grail’’ of HIV research since early in the epidemic. The daunting diffi-
culties encountered in their actuation have led some to argue that success is
extremely difficult or actually impossible to achieve [129]. Nevertheless,
Fig. 2. Autologous HIV-1 vaccine in highly active antiretroviral therapy patients. Dendritic
cells (DC) are loaded ex vivo with autologous, apoptotic cells expressing the patient’s endo-
genous strains of HIV-1, then are matured with CD40L and treated with interleukin (IL)-12 or
IL-15 recombinant proteins or expression vectors to enhance activation and survival of T cells.
These mDC are reinfused in the patient to stimulate of CD8þ and CD4þ T cells specific for the
patient’s residual virus. This can be done together with STI or in vivo IL-2 treatment to activate
residual virus and expose HIV-1–infected cells to the CD4þ T cells and CD8þ CTL.
advances in the understanding of both the immunology and biology of HIV,

particularly the role of CTLs and neutralizing antibodies, have led to very
promising results (Table 2) [130,131]. Major efforts are currently focused on
DNA and viral vector-based strategies [132]. These have the important
advantage of resulting in active expression of HIV-1 antigens in the target cells
and can induce more potent CD8þ T-cell responses than those obtained with
natural or recombinant proteins or inactivated whole virus, strategies known
to induce predominantly antibody and CD4þ T-cell responses. Another rea-
son for desiring vaccines that induce strong CD8þ T cell-mediated responses
is that the resulting immunity is much more broadly reactive to different
HIV-1 clades than can be obtained with antibody-induced vaccines [133].
Primate models, in particular the rhesus macaque, are invaluable to deter-
mine vaccine efficacy. The use of chimeric SIV and HIV in the same construct,
termed SHIV [134], allows expression of different HIV-1 proteins to study
their effect on pathogenesis in susceptible macaques. Vaccines that induce
strong and broad-based CTL have the capacity to reduce the viral set-point
in these models [135,136], although in one case CTL responses do not seem to
correlate with protection [137]. Use of immunomodulators, such as cytokines
Table 2
Recent vaccine strategies in human and nonhuman primate models
Mode 1 Virus Vaccine Immune markers Protection Reference
Human HIV CPV with rProtein LA, Nab NC [161]
CPV with rProtein LA, Nab NC [153]
CPV ICS NC [154]
DNA with HAART LP, LA, ELS, LDA NC [151]
Simian HIV Fowlpox LP, LA, E NC [139]
SHIV DNA with IL-2/Ig fusion LP, LA, Nab, ICS, TM þþ [121]
protein
DNA with CpG motifs LP, LA, Nab, E þþ [138]
DNA with IL-2/Ig fusion LP, LA, Nab, ICS, TM þþ [120]
protein
DNA/MVA LA, TM NC [135]
DNA/MVA LP, Nab, ICS, TM, ELS þþþ [143]
DNA/SV LA þþ [158]
Live attenuated LA, Nab þþ [156]
VLP, DNA, rProtein, VAC LP, ELS, Nab, E þ/ [137]
Synthetic peptide cocktail LA, Nab, ELS þþ [159]
VLP LA, Nab [162]
MVA LA, Nab, TM þþ [150]
DNA/MVA/AdV TM þþ [144]
SIV DNA LP, LA, Nab, E þ [155]
DNA LA, LDA, TM þ [152]
DNA/MVA LA, ICS, TM, ELS NC [149]
MVA LA, TM NC [160]
MVA LA, TM þ [136]
BCG LA NC [157]
VAC with IL-12 vector LA, Nab, E þ [140]
Abbreviations: AdV, adenovirus vector; BCG, bacille Calmette-Guerin; CPV, canarypox
viral vector; E, cytokine ELISA; ELS, cytokine Elispot; ICS, cytokine intracellular staining;
LA, lytic assay; LDA, limiting dilution assay; LP, lymphocyte proliferation; MVA, modified
vaccinia virus Ankara vector; Nab, neutralizing antibody; NC, not challenged; rProtein,
recombinant protein; SV, Sendai virus vector; TM, tetramer staining; VAC, vaccinia virus
vector; VLP, virus-like particles; () no protection; (þ) partial protection with lowered viremia;
(þþ) protection with contained viremia and no clinical symptoms; (þþþ) sterile protection
with no viremia and no clinical symptoms.
or bacterial DNA analogues (CpG), that are included in the vector [120,121,
138,139] or separately [140] have resulted in durable CD8þ T-cell responses.
Notably, the efficacy of DNA vaccines has been improved by more
potent gene-expression strategies and use of human codons to elicit more
effective immunity to HIV. Recombinant modified vaccinia virus Ankara
(MVA) has been highly effective at boosting DNA-primed CD8þ T cells
and is relatively safe in that it does not replicate in human cells [141]. This
is a major improvement on the vaccinia and fowlpox virus HIV-1 vectors,
which induce strain-specific immunity that only achieves complete protec-
tion to homologous virus challenge [142]. In contrast, DNA priming and
MVA boosting strategy protected macaques against mucosal challenge with
a highly pathogenic SHIV chimeric virus (strain 89.6P) possibly because

both the DNA and MVA constructs express multiple viral proteins [141].
Priming with a single DNA plasmid vaccine by skin or muscle, followed
by boosting at 24 weeks with MVA, resulted in HIV-specific CD8þ T-cell
responses and complete, sterile control of intrarectal SHIV challenge 7
months later in all animals. These data raise hope that a relative simple mul-
tifactor vaccine can control the AIDS epidemic [143].
Another recently described approach that induces antiviral cellular
responses and reduces virus replication relies on the use of replication-defec-
tive recombinant adenoviral vectors, alone or in combination with DNA
vaccines [144]. Barouch et al [145], however, reported that vaccine strategies
that are centered solely on induction of T-cell immunity may ultimately fail
because of the well-described mechanism of viral escape from CTL. That is,
a single point, escape mutant in a Gag CTL epitope in animals that seemed
to control viremia after initially successful DNA and IL-2 immunization
resulted in a burst of viral replication, disease progression, and AIDS-
related death. These results emphasize the challenge of developing a vaccine
that can be used in the general human population [146].
Summary
Fifteen years after the first, definitive reports of HIV-1–specific, CD8þ
T cells [147,148], there is ample evidence for the importance of these cells
in control of HIV-1 infection. As much is known of their role in the
natural history of HIV-1 infection and their cellular and molecular mechan-
isms of reactivity than of T-cell responses to any other human virus. Indeed,
HIV-1–related research has led the scientific field in revealing many new,
fundamental principles of cellular immunity in the last 15 years. From these
data, there are multiple, posited mechanisms for loss of CD8þ T-cell control
of HIV-1 infection. These include both intrinsic defects in T-cell function
and loss of T-cell recognition of HIV-1 because of its extraordinary genetic
diversity and disruption of antigen presentation.
Efforts have begun on devising approaches to reverse these immune
defects in infected individuals and develop vaccines that induce T-cell im-
munity for protection from infection. Combination antiretroviral drug regi-
mens now provide exceptional, long-lasting control of HIV-1 infection, even
though they do not restore anti–HIV-1 T-cell immunity fully in persons with
chronic HIV-1 infection. Very encouraging results show that such treatment
can maintain normal T-cell reactivity specific for this virus in some per-
sons with early HIV-1 infection. Unfortunately, the antiviral treatment does
not cure the host of this persistent, latent virus. This has led to new strat-
egies for immunotherapeutic intervention to enhance the level and breadth
of the T-cell repertoire specific for the host’s residual virus in persons
with chronic HIV-1 infection. Although the principles of immunotherapy
stem from early in the last century, modern era approaches are integrating
highly sophisticated, molecular and cell biology reagents and methods
for control of HIV-1 infection. The most promising immunotherapies are
autologous virus activated in vivo by STI or administered in autologous
DC that have been engineered ex vivo. There are also compelling rationales
supported by animal models and early clinical trials for use of cytokines and
chemokines as recombinant proteins or DNA to augment anti-HIV-1 T-cell
reactivity and trafficking of T cells and APC to tissue sites of infection.
For prevention of HIV-1 infection, the discouragingly poor results of
vaccine development in the late 1980s and early 1990s have led to very
encouraging, recent studies in monkeys that show partially protective and
possibly sterilizing immunity. Finally, clinical trials of new-generation DNA
and live vector vaccines already have indications of improved induction of
HIV-1–specific T-cell responses. Knowledge of HIV-1–specific T-cell
immunity and its role in protection from HIV-1 infection and disease must
continue to expand until the goal of complete control of HIV-1 infection is
accomplished.
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Clin Lab Med 22 (2002) 799–820
HIV-1 vaccines: the search continues

James P. McGettigan, PhDa, Philip M. McKenna, MSb,
Matthias J. Schnell, PhDa,*
a
Department of Biochemistry and Molecular Pharmacology,
The Dorrance H. Hamilton Laboratories, Center for Human Virology,
Philadelphia, PA 19107-6799, USA
b
Department of Microbiology and Immunology, The Dorrance H. Hamilton Laboratories,
Center for Human Virology, Philadelphia, PA 19107-6799, USA
The HIV-1 is the best-studied virus affecting humans. Since the first
description of AIDS 20 years ago, dramatic progress has been made in the
molecular characterization of HIV-1 and in the fields of immunology, virol-
ogy, and pathogeneses of the HIV-1 infection. This knowledge has resulted
in new antiviral strategies against HIV-1, which have resulted in a dramatic
decrease in mortality among infected humans in developed countries. Lim-
ited progress has been made in the development of a successful HIV-1 vac-
cine to prevent infection, however, and this task is still the major goal to halt
the HIV-1 pandemic. The World Health Organization estimates that 36.1
million people worldwide are living with HIV or AIDS (Table 1). In sub-
Saharan Africa alone, 25.3 million people are living with HIV-AIDS.
Although the incidents of new infections has stabilized in this region of the
world (3.8 million in 2000, compared with 4 million in 1999), other regions
of the world are seeing increases. For example, the number of adults and
children living with HIV or AIDS in Eastern Europe and Central Asia was
420,000 in 1999 and has increased to 700,000 by the end of 2000. Overall,
21.8 million deaths have been attributed to HIV-AIDS. An effective and
affordable vaccine is essential and is the only method to control the contin-
ued spread of HIV-1 worldwide. This article gives an overview about the
development of an HIV-1 vaccine. Tremendous numbers of papers have
been published on this topic during the last 10 years, and this article can
This work was supported in part by Grant No. AI44340 from the National Institutes of
Health.
* Corresponding author. 1020 Locust Street, Suite 335, Philadelphia, PA 19107-6799.
E-mail address: matthias.schnell@mail.tju.edu (M.J. Schnell).
PII: S 0 2 7 2 - 2 7 1 2 ( 0 2 ) 0 0 0 0 4 - 5
800 J.P. McGettigan et al / Clin Lab Med 22 (2002) 799–820
Table 1
Global summary of the HIV/AIDS epidemic, December 2000
People newly infected with HIV in 2000 TOTAL 5.3 million

Adults 4.7 million
Women 2.2 million
Children <15 y 600,000
Number of people living with HIV-AIDS TOTAL 36.1 million

Adults 34.7 million
Women 16.4 million
Children <15 y 1.4 million
AIDS deaths in 2000 TOTAL 3 million
Adults 2.5 million
Women 1.3 million
Children <15 y 500,000
Total number of AIDS deaths since the TOTAL 21.8 million
beginning of the epidemic Adults 17.5 million
Women 9 million
Children <15 y 4.3 million
Adapted from the Joint United Nations Programme on HIV/AIDS (UNAIDS) and the
World Health Organization (WHO) AIDS Epidemic Update, December 2000; with permission.
only touch on the different directions taken toward the development of an

HIV-1 vaccine, and not give a complete overview of the entire field.
What are the required immune responses for a successful HIV-1 vaccine?
Evidence for the importance of HIV-1–specific CD8þ cytotoxic
T-lymphocyte–mediated responses
Even though the requirements for protective immune responses against
HIV-1 are not well defined, growing evidence supports that CD8þ cytotoxic
T-lymphocyte (CTL)–mediated immune responses are critical in controlling
HIV-1 infection [1,2]. This finding is based on several studies showing that
exposed but uninfected individuals have HIV-1–specific CTLs without
detectable antibodies [3–5]. For example, HIV-1–specific CTLs were elicited
from three of six Gambian prostitutes who were repeatedly exposed to HIV
but remain HIV-seronegative, with no evidence of HIV infection by poly-
merase chain reaction or viral culture [5].
There also is a strong correlation between a high frequency of HIV-1–
specific CTLs with a low viral titer and a slow disease progression in chroni-
cally HIV-1–infected individuals [6,7]. Asymptomatic individuals who have
been infected for more than 8 years were compared with individuals who
progressed to AIDS within 5 years postseroconversion. The long-term non-
progressors showed persistent HIV-1 Gag-specific CTL responses over time,
whereas there was a loss of Gag-specific CTLs that coincided with the
J.P. McGettigan et al / Clin Lab Med 22 (2002) 799–820 801
progression to AIDS [6]. Env-specific CTLs were also identified to cor-

relate with disease progression [7]. Patients with higher frequencies of Env-
specific memory CTLs had a median level of plasma HIV-1 RNA about one
third that of the patients with lower frequencies. Patients with low frequen-
cies of Env-specific CTLs in early infection had more rapid decline to less
than 300 CD4þ cells/mm3. Taken together, these results indicate that Gag
and Env-specific CTLs are important in the control of viral loads.
Perhaps the most compelling evidence for the requirement for a cellular
immune response comes from the finding that virus replication was not con-
trolled in rhesus monkeys depleted of CD8þ lymphocytes during primary
simian immunodeficiency virus (SIV) infection [8]. In addition, eliminating
CD8þ lymphocytes from monkeys during chronic SIV infection resulted
in a rapid and marked increase in viremia that was again suppressed with
the reappearance of SIV-specific CD8þ T cells. This was measured using the
p11C, C-M tetramer binding assay, which supports the notion that Gag-
specific CTLs may be important in controlling viremia. This particular
study demonstrated that an effective HIV-1 vaccine should elicit a strong cel-
lular immune response that might clear a primary infection or modulate
viral replication in a newly infected individual.
There are many CTL target epitopes that have been identified on the dif-
ferent structural and regulatory proteins of HIV-1 [9]. As noted previously,
evidence suggests that HIV-1 Gag and Env are excellent targets for a cellular
immune response. One of the major obstacles in the development of an effec-
tive HIV-1 vaccine, however, is the genetic variability of the virus itself.
HIV-1 Gag is one of the most conserved proteins of HIV-1 (HIV Molecular
Immunology Database, Theoretical Biology and Biophysics, Los Alamos
National Laboratory, Los Alamos, NM, 1999), and epitopes that are con-
served among different clades have been described for individuals infected
with HIV-1 [9,10]. Specifically, in vitro–stimulated Gag-specific CTLs from
infected individuals were able to recognize and cross-kill target cells infected
with vaccinia virus expressing HIV-1 Gag from different clades [9]. In fact,
Ugandan HIV-1–positive patients had inducible CTL responses recognizing
HIV-1 Gag from clades A, B, C, and D [10]. Because of its sequence conser-
vation and cross-clade studies, Gag p24 is one of the most likely HIV-1 Gag
proteins to stimulate heterologous Gag immune responses [11].
The envelope protein of HIV-1, which is the predominate target for neu-
tralizing antibodies, is especially variable. Studies have also shown that Env-
specific CTLs from infected individuals were able to recognize and cross-kill
target cells expressing Env from different HIV-1 clades [11]. Env-specific
CTL clones isolated from individuals infected in the United States were
able to recognize, and react with, non–clade B sequences. In addition, of 14
African individuals infected with clade A, C, or G viruses, all demonstrated
cross-reactivity with the US clade B viral constructs. It is not yet clear
whether this recognition is caused by epitope conservation or by cross-clade
recognition; however, it is clear that the potential for a broad-based vaccine
based on the envelope protein is possible. These data suggest that CTL
cross-reactivity exists and that a vaccine based on a single clade of Env or
Gag may be broadly applicable.
It has been reported that the regulatory proteins HIV-1 Tat and Rev are
frequent targets for CTLs in natural HIV-1 infection [12]. These proteins are
expressed early in HIV-1 infection, before Nef down-regulates major histo-
compatibility complex class I molecules on the cell surface, and may be
important targets for a HIV-1 vaccine. Studies in macaques using biologically
active Tat protein, Tat toxoid, or recombinant vaccinia virus expressing Tat
and Rev proteins showed some attenuation from disease [12]. In addition,
the presence of anti-Tat antibodies or Tat-specific cytotoxic lymphocyte
responses was correlated with slow progression in HIV-1–infected individuals
[13,14]. Given the fact that native Tat has been demonstrated to be toxic in
murine models, and that Tat toxioids on their own are not sufficient to block
disease progression [15], additional studies need to be completed to examine
the effectiveness of including Tat as a component of a HIV-1 vaccine.
Evidence for the importance of anti–HIV-1 antibody responses

Early studies in chimpanzees demonstrated a possible correlation
between neutralizing antibodies and protection of HIV-1 infection in chim-
panzees [16]. The titer of neutralizing antibody response not only correlated
with protection from infection, but the neutralizing antibody titer also inver-
sely correlated with the postchallenge virus load [17]. More recently, Mascola
et al [18] used passive transfer studies in rhesus macaques to study the role
of antienvelope antibodies in protection. HIV immunoglobulin and two
human monoclonal antibodies, 2F5 and 2G12, were administered to six rhe-
sus macaques 24 hours before an intravenous challenge with SHIV89.6PD.
Three of the six demonstrated complete protection from infection, whereas
the remaining three animals became SHIV-infected but showed reduced
plasma viremia and near normal CD4þ T-cell counts [18]. They extended
their finding by using the same virus and antibodies, but showed greater
protection after vaginal challenge [19]. These two studies demonstrated that
antibodies can affect subsequent disease course after vaginal and intrave-
nous SHIV challenge. Furthermore, Baba et al [20] reported on the treat-
ment of four pregnant macaques with a triple combination of the human
IgG1 monoclonal antibodies F105, 2G12, and 2F5. All four macaques were
protected against intravenous SHIV-vpu challenge after delivery. In addi-
tion, the infants received monoclonal antibodies after birth and were chal-
lenged orally with SHIV-vpu. No evidence of infection was observed in
any infants during a 6-month follow-up period. Although SHIV-vpu is read-
ily neutralized, replicates poorly in macaques, and has elicited disease in
only a single neonate and after prolonged infection [20], this study suggests
that IgG1 monoclonal antibodies may protect against mucosal challenge in
neonates.
Current HIV-1 vaccine strategies

The killed virus vaccine approach
Most successful vaccines against viral diseases have been composed of
killed or attenuated viruses [21]. Based on the finding by Salk that killed
poliovirus protects against polio, one of the first approaches for an HIV-1
vaccine was the use of inactivated whole virus. These vaccine studies initially
showed some protection in the SIV-macaque model system [22–24].
Unfortunately, later results demonstrated that in most cases protection was
caused by an anticellular response against human leukocyte antigen con-
tained in both the killed and challenge virus, and not to an antiviral
response [25–27]. On the other hand, one study with inactivated SIV did
show the induction of antiviral and not anticellular antibodies and protec-
tion against SIV challenge [22], whereas another trial did not show protec-
tion in the SIV-macaque model even with the induction of antiviral
antibodies [28]. Further preclinical evaluation of the efficacy of the killed
virus approach in comparison with other HIV-1 vaccine approaches in a
more standardized system will clarify if inactivated lentiviruses can be used
as an HIV-1 vaccine.
Recombinant HIV-1 proteins

The HIV-1 envelope protein gp160 is the only viral transmembrane pro-
tein and is responsible for both binding to CD4 and an HIV-1 coreceptor,
and fusion to the host cell membrane. Neutralizing antibodies are exclu-
sively directed against HIV-1 Env and recombinant gp160 is an attractive
candidate for an HIV-1 vaccine. In addition, the application of a recombi-
nant protein is considered very safe and does not contain the risk of incom-
plete inactivation, which is one concern with the use of a killed HIV-1
vaccine. Vaccination studies in chimpanzees have shown that multiple inoc-
ulations with recombinant HIV-1 envelope proteins in adjuvant can induce
immunity and the animals resist challenge with homologous or heterologous
HIV-1 [29,30]. Of note, in both cases the HIV-1 strain SF2 used for chal-
lenge was nonpathogenic for chimpanzees and replicates only marginally
in this animal model [31,32]. Recombinant HIV-1 gp120 or gp160 has been
tested in several human trials and it proved to be quite safe [33–35], but
failed to induce a broad range of neutralizing antibodies [36]. Most of the
volunteers developed virus neutralization antibodies against the homolo-
gous HIV-1 strain and sometimes lower titers of neutralizing antibodies
against other laboratory-adapted strains, but failed to neutralize completely
any primary viral isolate [36]. In addition, 50% of the vaccine recipients lost
the humoral response against HIV-1 within 2 years after vaccination and
none of them developed an HIV-1–specific CD8þ CTL response. This may
explain why some volunteers immunized with HIV-1 recombinant gp120 or
gp160 have been subsequently infected with HIV-1 [37–40].
To enhance the protective potential of recombinant HIV-1 envelope pro-

tein several new approaches are currently under investigation. The use of
new, more potent adjuvants to achieve better immune responses has shown
some success. Comparison of alum, SBAS-1, and ABAS-2 indicates that
ABAS-2 is superior for inducing T-helper responses and the antibodies
induced by SBAS-1 SBAS-2 were long-lasting. All approaches failed, how-
ever, to induce antibodies that neutralized homologous or heterologous
primary HIV-1 isolates from peripheral mononuclear cells (PBMCs) and
no HIV-1 specific CD8þ CTLs were detected [41].
Other approaches to enhance the immunogenicity of HIV-1 envelope
protein were to modify the antigen itself. These strategies include changes
in the structure by deletion of the V2 or V3 loops, or removing certain gly-
cosylation sites to expose important neutralization epitopes [42–46]. More
recently, a new method has been developed to synthesize a secreted form
of the HIV-1 envelope protein as it is present on a viral particle. To produce
these native trimers of the gp120–gp41 heterodimers, a disulfide bond
between gp120 and gp41 was introduced to stabilize the weak interaction
between the two envelope subunits [47].
Besides the HIV-1 envelope protein, the regulatory HIV-1 Tat protein
has been used in vaccine studies. HIV-1 Tat protein is a critical component
in the mechanisms of AIDS pathogenesis [48], and preventive immunization
with Tat protein has been addressed in both animal and human studies
[13,49]. In addition, the presence of anti-Tat antibodies was correlated with
slow clinical disease progression in humans [13]. A recent report from
Gallo’s group, however, questions the use of Tat toxoid as a preventive
vaccine because they failed to protect macaques after an effective immuniza-
tion with Tat toxoid against SHIV89.6PD challenge, and they suggest its use
only as a component in an HIV-1 vaccine [15].
DNA-based vaccines
Another method of immunization against HIV-1 is to inoculate naked
HIV-1 DNA constructs into the host [49–53]. This method has been efficient
in eliciting protective immune response against pathogens other than HIV in
animal studies [54–57]. The strong Rev-dependency of the HIV-1 structural
genes, however, results in low expression levels of HIV-1 Gag or Env by
DNA vaccines [58,59]. Nevertheless, this problem has been addressed at
least for HIV-1 Gag by modification of the Gag encoding sequence and sev-
eral reports have shown that DNA vaccines can generate HIV- and SIV-spe-
cific CTLs and antibodies in mice and nonhuman primates [60]. These data
regarding protection of primates by DNA vaccines encoding HIV or SIV
proteins are somewhat conflicting. A recent report showed the protection
of chimpanzees by DNA immunization with HIV-1 gp160, and gag and pol
sequences [61]; however, an experimental DNA vaccine containing a mix-
ture of plasmids encoding SIV proteins failed to protect rhesus macaques
from SIV challenge [62]. Of note, several vaccine strategies have prevented
infection with slowly replicating lentiviruses but failed to protect from infec-
tion with viruses that replicate at normal vigorous levels [63]. In general, it
seems that DNA vaccines work best in the initial mouse model and they
tend to induce rather weak immune responses in monkeys and humans.
Two recent vaccine approaches in the SIV-macaque model system,
however, indicate the usefulness of DNA vaccines in combination with a
cytokine (interleukin-2 [IL-2]) or as DNA as a prime vaccination in combi-
nation with a viral vector boost. Robinson’s group described a new approach
that combines priming with naked DNA followed by a boost vaccination
with a viral vector as outlined later in the viral vector section [135].
In another approach, Barouch et al [64] used two plasmids encoding SIV
Gag or HIV-1 envelope protein (strain 89.6P) in combination with a plasmid
encoding IL-2, recombinant IL-2 protein, or nothing. Unfortunately, all
immunized monkeys got infected after challenge with SHIV89.6P; in contrast
to the DNA-only approach, both vaccination methods using DNA express-
ing SIV-HIV proteins in combination with IL-2 induced strong immune
responses against HIV-SIV as indicated by stable CD4þ T-cell counts, pre-
served virus-specific CD4þ T-cell responses, and low to undetectable set-
points of viral loads.
Attenuated Lentiviruses
One of the most effective methods to protect from SIV infection is the use
of live, attenuated SIV. A naturally attenuated SIV strain approach pre-
vented disease in macaques by a virulent SIV strain, but was not able to
prevent infection [65,66]. More striking was the finding by Desrosiers et al
[67] that a genetically modified, nef-deleted SIV strain that does not cause
disease in rhesus monkeys induced high titers of antibodies and CTL activity.
Subsequent challenge of the immunized animals with infectious doses of a
pathogenic SIV strain gave protection from infection [67,68]. Moreover, ani-
mals immunized with a nef-attenuated SIV strain resisted a challenge with
virus-infected cells [69]. The finding that immunized animals also resisted
infection with a chimeric SIV containing the envelope protein of HIV-1 strain
89.6 (SHIV), indicated that antibodies were not solely responsible for pro-
tection, and implies the importance of the cellular response [69]. A major
drawback for the use of attenuated lentiviral viruses is the finding that even
nef-deleted SIV can give rise to an AIDS-like disease in both neonatal and
adult macaques [70–72]. Additional concerns for the use of attenuated lenti-
viruses arise from the finding that recombination of live, attenuated SIV with
challenge virus in some cases results in an even more virulent strain [73].
Recombinant live vectors as HIV-1 vaccine vehicles

The finding that rhesus macaques vaccinated with live, nef-deleted SIV
were completely protected against challenge with high doses of live,
pathogenic SIV [67] emphasizes that live vectors, such as recombinant bac-
teria and viruses, are excellent candidates for live vaccines against HIV-1.
Recombinant vectors have the potential to elicit immunity against an
expressed foreign protein through an infection, without the pathogenic
consequences.
Bacterial vectors
Different live vectors based on bacteria have been studied as HIV vac-
cines. The results showed that recombinant Mycobacterium [74,75] and Lis-
teria monocytogenes [76] are able to induce a cellular and humoral response
against HIV proteins in mice and they may prove useful in nonhuman or
human-primate studies. Negative results with live bacterial vectors, how-
ever, have also been obtained. Oral immunization with recombinant Salmo-
nella typhimurium expressing the HIV-2 gag and the gp130 portion of the
envelope either alone or in combination with alum-adjuvant and recombi-
nant gp170 failed to confer protection in rhesus macaques [77].
Viral vectors
A variety of recombinant viral vectors are under evaluation as HIV vac-
cines and are reviewed elsewhere in greater detail [78] (Table 2). The most
widely used recombinant viral vectors are based on DNA viruses (eg, pox
viruses), such as vaccinia virus or canarypox virus expressing HIV-1 genes,
mostly the HIV-1 envelope proteins, gp160, or HIV-1 gag [79–82]. In one of
the first studies, chimpanzees were immunized with recombinant vaccinia
virus expressing HIV-1 envelope protein and challenged with high and low
doses of homologous HIV-1. Although all animals developed HIV-1–specific
antibody and T-cell responses, virus was isolated from lymphocytes of all
challenged chimpanzees, indicating that immunization did not prevent infec-
tion [83]. In contrast to this, immunization of chimpanzees with a vaccinia
vector expressing HIV-1 gp120 protected chimpanzees from low-dose HIV-1
challenge, but only after a boost injection with recombinant gp160. Studies
in rhesus monkeys using vaccinia viruses expressing SIV envelope protein
and a subsequent boost with recombinant gp130–gp170 showed some
protection against SIV challenge [84–87]. Similar results were found with
recombinant, replication-deficient canarypox viruses expressing HIV-2 for
priming and recombinant HIV-2 envelope proteins as a boost [77,88–90].
More recently, several investigators have used modified vaccinia virus
Ankara (MVA), which is replication-deficient in primate cells and a safer
vaccinia viral vector. Macaques immunized with MVA expressing SIV gag,
pol, and env proteins became infected postchallenge, but plasma viremia was
reduced, indicating some benefits of the vaccine [91]. Several phase I and
phase II trials with vaccinia or canarypox virus have been conducted. The
results are somewhat disappointing because only moderate levels of neutral-
izing antibodies against laboratory-adapted viral strains and some cellular,
Table 2
Current viral vectors under investigation
Replication competent in humans and primates
DNA viruses
Poxviridae Vaccinia virus [93,99,137–144]
Adenoviridae Adenovirus [100,105,106,145–148]
Herpesviridae HSV [149]
Papovaviridae SV40 [150]
RNA Viruses
Picornaviridae Poliovirus [151,152], Mengo virus [108,109]
Togaviridae HRV [110,153,154], coxsackie virus [155], VEE [156]
Retroviridae SIV (attenuated) [22,72,157]
Rhabdoviridae Rabies virus [121–124], Vesicular stomatitis virus
[127,128]
Orthomyxoviridae Influenza A virus [158–160]
Replication incompetent in humans and primates
DNA viruses
Poxviridae MVA [91,135,161–166], canarypox virus
[79–81,89,90,95,161,167–178], fowlpox virus [175,179]
RNA viruses
Paramyxoviridae Newcastle disease virus
Replicons (nonreplication competent)
DNA viruses
Adenoviridae Adenovirus [105,106]
RNA viruses
Togaviridae SFV [116], VEE [117,180,181]
Picornaviridae Poliovirus [182]
Abbreviations: HRV, human retrovirus; HSV, herpes simplex virus; MVA, modified vaccinia
virus Ankara; SFV, Semliki Forest Virus; SIV, Simian immunodeficiency Virus; VEE,
Venezuelan equine encephalomyelitis.
Adapted from Schnell MJ. Viral vectors as potential HIV-1 vaccines. FEMS Microbiol Lett
2001; 200:123–9; with permission.
mostly transient, responses were induced [92–99]. Of note, weak responses

failed in both rhesus macaques and chimpanzees to protect from infection.
Recombinant adenoviruses expressing HIV-1 gp160 were first studied in a
dog animal model [100]. Prime-boost experiments with three different re-
combinant adenoviruses (serotypes 4, 5, and 7) showed the induction of
neutralizing antibodies against laboratory-adapted HIV-1 strains. A modified
approach, using three sequential injections of adenoviruses expressing
HIV-1 env and gag followed by three boost injections of recombinant
HIV-1 gp160 protected chimpanzees against challenge with a low dose
of HIV-1 strain SF2 [101,102]. Countering these positive results are obser-
vations showing no protection of rhesus monkeys immunized with recom-
binant adenoviruses expressing SIV env and challenged with pathogenic
SIVmac251 [103,104]. A study in macaques by Merck comparing three
different candidate vaccines based on DNA, MVA, or adenovirus, however,

indicated that the adenovirus-based vaccine induced the strongest T-cell
response. Immunization with the adenovirus expressing SIV Gag provided
better protection against challenge with the highly pathogenic SHIV89.6P
as immunization with MVA or DNA expressing the same antigen as indi-
cated by nearly unchanged CD4 cell counts after challenge in adenovirus-
immunized animals [105]. The question still remains if similar responses can
be achieved in humans in the presence of a high pre-existing immunity
against the adenovirus vector, and research results by Merck indicate that
similar responses as seen in naı̈ve macaques can not be detected in macaques
that have been immunized previously with the vector to mimic a pre-existing
immunity [106].
The expression and immunogenicity of a partial region of HIV-1 was
demonstrated by three members of the Picornaviridae family [107]. Intro-
duction of a 147–amino acid sequence from HIV-1 gp120 into the RNA
genome of Mengo virus induced a humoral and cellular response in mice
[108,109]. Similar results were found with chimeric Rhinoviruses in which
the sequence from the V3 loop of the MN strain of HIV-1 was introduced
[110,111]. These systems are limited, because only small portions of the viral
envelope can be inserted into the viral genome. The same problem was
encountered for other viruses, such as hepatitis B [112]. For this reason, two
different approaches were used for a recombinant poliovirus-based vector.
The construction of a dicistronic replication-competent poliovirus vector
expressing a truncated version of HIV-1 gp120 was developed by Lu et al
[113]. Because of the limited cloning capacity of poliovirus, other groups
suggested the use of replicons [114]. Poliovirus-based replicons expressing
HIV-1 env and gag were used [115], but they are encapsidation defective,
and only persist through one infectious cycle. Poliovirus-based viral vectors
are limited, although suitable for expression of HIV polypeptides, but the
determination of their usefulness as an HIV vaccine awaits further studies.
Two alphavirus-based replicons, derived from Venezuelan equine enceph-
alitis virus and Semliki Forest virus, were used as SIV vaccines. The data
indicated the induction of both a humoral and cellular response, resulting
in a lower viral load in vaccinated animals after challenge with SIV, but
none of the vaccine candidates were able to protect against infection [116–
118]. New methods to engineer the genome of negative-stranded RNA
viruses made these viruses available as viral expression vectors and certain
features, such as no DNA phase and no RNA recombination, suggest their
use as vaccine vectors [119]. For influenza A virus, the hemagglutinin pro-
tein was used as a carrier for small antigenic epitopes of the HIV-1 env gly-
coprotein. Guinea pigs and mice immunized with an influenza chimera,
which contained a V3-loop sequence of HIV-1 env, elicited neutralizing anti-
bodies against the homologous strain of HIV-1 [120].
As mentioned previously, the recombinant influenza-based system also is
limited because it is restricted to small portions of the HIV-1 Env or other
HIV-1 proteins. In contrast, nonsegmented negative-stranded RNA viruses,

such as rabies virus (RV) [121–125], vesicular stomatitis virus (VSV) [126–
128], Sendai virus [129], measles virus [130], or Newcastle disease virus
[131], can be modified easily to incorporate large foreign sequences in their
genome. As an example of this group of relatively new, promising vectors as
an HIV-1 vaccine may serve two members of the Rhabdoviridae family, RV
and VSV. Both vaccine vehicles can express full-length HIV env and gag
stably over multiple passages. Whereas highly attenuated RV-based vectors
were studied in detail in a small animal model, the potency of the VSV-based
vectors was recently analyzed in the SIV-macaque model system. In the case
of RV, the preliminary analysis in mice is very promising and indicated that
a single inoculation with RV expressing HIV-1 Env or Gag induces a vigo-
rous CD8þ mediated T-cell response against the expressed antigen.
As seen in many other approaches [105,117,132,133], VSV-based vectors
failed to induce sterile immunity or clear the challenge virus after the initial
infection. Multiple inoculations with replication-competent VSV vectors
expressing SIV Gag or HIV-189.6 envelope protein protected the immunized
monkey from an AIDS-like disease. Of note, the CD4þ T-cell loss of VSV-
vaccinated animals was high compared with other experimental HIV-1 vac-
cine approaches and only direct site-by-site comparison of different HIV-1
vaccine vehicles, such as RV and VSV, in the SIV-macaque model system
will indicate which is the better vaccine approach.
Prime-boost approaches
Multiple immunizations with the same vaccine, such as DNA, recombi-
nant protein, or viral vector, are commonly used for most immunizations and
are discussed previously. In the case of viral or bacterial vectors, repeated
immunizations are self-limiting by an increasing immune response against
the vector itself. Of note, this problem also exists for vaccines that take
advantage of vectors that are used for immunization against important infec-
tious disease, such as polio virus, measles virus, and pox virus, or viruses that
commonly infect the human population, such as adenoviruses. In fact, a pre-
existing immunity may cause an important obstacle for the use of certain vac-
cine vectors in humans even if they previously showed promise in animal
experiments [106]. To circumvent the problem of the pre-existing immunity,
Rose et al [127,134] used VSV vectors with glycoproteins (G) from different
VSV serotypes. Because VSV contains only one glycoprotein in its envelope,
antibodies raised against G protein during the first immunization do not
neutralize the boost-vector containing another G protein. The question still
remains, however, if a cellular immune response directed against the other
VSV proteins may inhibit a productive second vaccination.
Another method than using the same vector with different G proteins is
to use different viral vectors expressing the same HIV-1 antigen. This new
HIV-1 vaccine approach is currently under investigation in a joint effort of
three laboratories at Thomas Jefferson University (Philadelphia) and Mount

Sinai School of Medicine (New York) using rhabdovirus-, paramyxovirus-,
and influenza virus–based vectors.
Recently, Amara et al [135] published another very efficient HIV-1 prime-
boost vaccine strategy. For this approach, priming with naked DNA
expressing HIV-1–SIV antigens is followed by a booster with recombinant
MVA virus expressing the same antigens. Monkeys immunized by this
method controlled efficiently an intrarectal challenge, but vaccination did
not prevent infection.
Outlook
Within the last years, tremendous effort has been made in (1) the under-
standing of the required immune responses against HIV-1, (2) the techno-
logic tools available to study HIV-1, (3) treatment options to fight HIV-1
infection, and (4) the further development of old and new potential HIV-1
vaccines. Some of these new vaccine strategies are able to induce very strong
immune response against HIV-1–SIV and protected monkeys from an
AIDS-like disease. All these efforts did not result in the Holy Grail, how-
ever, namely an HIV-1 vaccine that protects from infection or at least clear
HIV-1 after infection. A protective HIV-1 vaccine seems to be even more
important after a recent publication indicated that a suboptimal working
vaccine may lead to higher levels of intrinsic virulence and hence to more
severe disease in unvaccinated individuals. The authors indicate that the
evolution of more pathogenic strains can erode any population-wide bene-
fits [136]. It seems to be obvious that over the next few years a more stan-
dardized vaccination challenge model system is needed to compare the
different HIV-1 vaccine approaches. This site-by-site comparison should
include several HIV-1 vaccine strategies that show great promise in small
animal models. This preliminary screening would be helpful to move a large
panel of potentially useful HIV-1 vaccine candidates into clinical trails. In
addition, it may be necessary to combine some strategies to reach the goal
of a protective HIV-1 vaccine.
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Clin Lab Med 22 (2002) xi–xiii

Clinical HIV-1 virology

R.J. Pomerantz, MD, FACP

Roger J. Pomerantz, MD, FACP

New methods for the detection of HIV

ELISA and Western blot testing

* Corresponding author. 1020 Locust Street, Suite 329, Philadelphia, PA 19107–6799.

infant born to an HIV-infected mother is often not helpful. Detection of

negative. If a single band is present, or a combination of bands is present that

Rapid HIV-1 antibody tests

Currently, only one FDA-approved rapid antibody test is commercially

Home sample collection kits

Non–serum-based HIV-1 antibody tests

wider HIV-1 testing in developing countries, where laboratory support may

p24 antigen detection

Nucleic acid–based tests

These three assay methods seem to be equivalent in sensitivity and specif-

cytokines [104]. Also, missing even a few doses of antiretroviral therapy

The diagnosis of acute HIV-1 infection

Detection of HIV-1 infection in infants

[94] Lew J, Reichelderfer P, Fowler M, et al. Determinations of levels of human immu-

HIV-1 RNA and viral load

The use of reverse transcription (RT) followed by polymerase chain reac-

Assay performance issues and problems in interpretation

Pathology. Most laboratories participating in these programs are using

First-generation versus later-generation assays

Variability between HIV groups and subtypes

Sample handling and precision issues

Issues in clinical interpretation

Neonatal and pediatric viral load

Viral load and risk of transmission

Viral load in primary HIV-1 infection

compartment viral loads has yet to be elucidated. The lack of detection

inﬂammatory processes or signiﬁcant lymphocyte counts in saliva [57]. The

Viral load in relation to pathogenesis and antiviral eﬃcacy studies

expressed and secreted (RANTES) or macrophage inﬂammatory protein

HIV treatment guidelines and viral load

therapy. If HIV RNA remains detectable in plasma after 16 to 20 weeks of

Use of HIV-1 viral load testing in blood donor screening

HIV-1 replication cycle

Acquired immunodeﬁciency disease is caused by HIV. HIV types 1 and 2

Molecular biology of HIV-1

mRNA is an unspliced genomic mRNA, which is ﬁrst translated into a Gag-

HIV-1 life cycle

Binding and entry

terminal repeats [LTRs]), which are necessary for subsequent integration

Expression of viral proteins

Assembly and egress

membrane-attached domain of Env and initiates budding. Budding from

Eﬀects of accessory genes on the life cycle

experiments, culture of HIV-1 and SIV strains in peripheral blood lympho-

change in their cytoskeletal organization immediately following infection by

mutations can modestly impair virus production [96–98]. Because of the

In vivo immune activation and HIV-1 replication

Models for studying HIV-1 replication

Cell culture systems

Like HIV, SIV strains exhibit tremendous genetic diversity, particularly

SCID mouse models

mutation rate and frequent recombination during the viral replication.

HIV-1 genotypic and phenotypic resistance

Human immunodeﬁciency virus type 1 infection is associated with high

Antiretroviral resistance assays

reverse transcription and polymerase chain reaction to amplify the genes of