The Journal of Infectious Diseases

MAJOR ARTICLE

Condylomata Acuminata (Anogenital Warts) Contain

Accumulations of HIV-1 Target Cells That May Provide
Portals for HIV Transmission
Jeffrey Pudney,1,a Zoon Wangu,5,a Lori Panther,3 Dana Fugelso,4 Jai G. Marathe,2 Manish Sagar,2 Joseph A. Politch,1 and Deborah J. Anderson1,2
1Department of Obstetrics and Gynecology and 2Department of Medicine, Boston University School of Medicine, and 3Department of Medicine and 4Department of Surgery,

Beth Israel Deaconess Medical Center, Boston, and 5Division of Pediatric Infectious Diseases and Immunology, UMass Memorial Children’s Medical Center, Worcester,
Massachusetts

Background.  Condylomata acuminata (anogenital warts [AGWs]) are prevalent in human immunodeficiency virus (HIV)–

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infected individuals and sexually active populations at risk for HIV acquisition and have been associated with HIV transmission. We
compared AGW specimens to control tissue specimens for abundance, types, and location of HIV target cells and for susceptibility
to HIV infection in vitro, to provide biologic evidence that AGWs facilitate HIV transmission.
Methods.  We used immunohistologic staining to identify HIV target cells in AGW and control specimens. We also inoculated
HIV in vitro into AGW and control specimens from HIV-negative men and assessed infection by means of TZM-bl and p24 assays.
Results. CD1a+ dendritic cells, CD4+ T cells, and macrophages were significantly more abundant in the epidermis of AGW
specimens than control specimens. These HIV target cells also often appeared in large focal accumulations in the dermis of AGW
specimens. Two of 8 AGW specimens versus 0 of 8 control specimens showed robust infection with HIV in vitro.
Conclusions.  Compared with normal skin, AGWs contain significantly higher concentrations of HIV target cells that may be
susceptible to HIV infection. Condylomata may thus promote HIV transmission, especially in the setting of typical lesion vascularity
and friability. Prevention or treatment of AGWs may decrease the sexual transmission of HIV.
Keywords.  Condylomata acuminata; anogenital warts; HPV; HIV; lymphocytes; dendritic cells.

Human papillomavirus (HPV) is one of the most prevalent sex-
ually transmitted pathogens in humans; most sexually active
adults are infected with at least 1 strain of HPV during their
lifetime [1–3]. Of >100 known types of HPV, 13 are classified
as high risk (ie, cancer causing). Low-risk HPV strains 6 and 11
are associated with the occurrence of condylomata acuminata
(anogenital warts [AGWs]), benign epithelial lesions that often
develop at sites that are vulnerable to abrasion or injury during
sexual intercourse [4, 5]. Anal warts are observed primarily in
individuals who engage in receptive anal intercourse, but they
may also occur in men and women with no such history [6].
Genital warts are usually found under the foreskin or on the
shaft of the penis in men and on the external genitalia/introitus
in women [7]. AGWs are often asymptomatic and may not be
Received 7 May 2018; editorial decision 27 July 2018; accepted 16 August 2018; published
online August 20, 2018.
aJ. P. and Z. W. are co–first authors.
Correspondence: D. J. Anderson, PhD, Department of Obstetrics and Gynecology, 670 Albany
St, Ste 516, Boston University School of Medicine, Boston, MA 02118 (Deborah.Anderson@
BMC.org).
The Journal of Infectious Diseases®  2019;219:275–83
© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
DOI: 10.1093/infdis/jiy505
brought to medical attention until they cause discomfort, pru-
ritus, or bleeding.
AGWs are prevalent in groups at high risk for HIV acqui-
sition and in HIV-infected individuals. In seronegative men
who have sex with men, the prevalence of low-risk HPV
infection associated with AGWs is about 20% [8, 9]. A recent
meta-analysis showed that the prevalence of AGWs in HIV-
uninfected populations at high risk for HIV acquisition in
sub-Saharan Africa (ie, female sex workers and men and
women attending sexually transmitted disease [STD] clinics)
ranges from 2.4% to 14% [10]. HIV-infected men and women
have a higher prevalence of HPV infection, AGWs, and pre-
malignant and malignant lesions, compared with age-matched
uninfected controls [11].
A number of recent studies have shown an increased risk of
HIV acquisition in individuals with HPV infection [12–17] and
those with AGWs [18, 19], and it has been speculated that HPV
may enhance HIV acquisition because of inflammation and
an increased numbers of HIV target cells at the infection site
[20]. HPV clearance, rather than HPV infection, has also been
implicated as a factor in HIV acquisition [17, 21, 22]. AGWs
could also be a site of enhanced HIV shedding and transmission
from HIV-infected individuals to uninfected partners; however,
studies to date have not associated HPV infection or AGWs
with HIV transmission.

HIV Target Cells in Anogenital Warts  •  JID 2019:219 (15 January) • 275

 HIV primarily infects cells that express CD4, the high-affin- AGW lesions (n = 40) and archived normal genital skin spec-
ity HIV receptor, and either CCR5 or CXCR4, which serve as imens from men and women with no signs of AGWs (n = 26)
coreceptors that participate in viral fusion events [23]. Cells that were included as controls.
express these receptors in anogenital skin, CD4+ T cells, mac-
Clinical Information
rophages, and CD1a+high epidermal dendritic cells (DCs; also
Patient age, sex, race/ethnicity, and lesion type/location were
called Langerhans cells) are primary target cells in HIV sex-
obtained from coded pathology reports. Subjects ranged in age
ual transmission [24, 25]. CD1a+ DCs are especially important
from 19 to 70  years (median, 38  years). All AGW specimens
because they reside in the superficial epidermis and are usually
used for the study were classified as low-grade dysplasia. Thirty-
the first HIV target cell to encounter HIV [26]. Few studies have
six percent of subjects were HIV infected (23 of 59 with anal
described HIV target cell populations in AGWs, and the results
warts and 10 of 32 with genital warts). All HIV-infected subjects
are varied. Mild inflammatory infiltrates have been described
had been prescribed antiretroviral drugs, although one third
in AGWs, along with CD4+ T cells in the stroma [20]. CD1a+
had detectable HIV in blood at their last clinical visit.
DCs have been described to be either unchanged in number
Detailed clinical information was available for the subjects
or depleted in AGWs [20, 27, 28]. Regressing AGWs have

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been described to contain infiltrates of CD4+ and CD8+ T cells
[29, 30] and increased numbers of CD1+ DCs [22].
The purpose of this study was to systematically compare the
abundance, types, and location of HIV  target cells in AGW
specimens to those in anatomically matched control skin speci-
mens from a large collection of samples from HIV-infected and
HIV-negative individuals. HIV target cells (CD1a+ DCs, CD4+
lymphocytes, and macrophages) and cells expressing HIV core-
ceptors (CCR5 and CXCR4) were detected by immunohisto-
chemical staining. Presence and numbers of HIV target cells in
AGW specimens were correlated with tissue site, HIV status,
and other clinical information, as well as with markers of acute
inflammation (CD15+ granulocytes) and wart regression (CD8+
T cells and macrophages). We also used an anti-HIV p24 anti-
body to detect HIV-infected cells in AGW and matched con-
trol tissue specimens from HIV-infected individuals, and we
conducted a pilot study of freshly collected biopsy tissue sam-
ples from HIV-seronegative men to determine whether AGWs
are more susceptible to HIV infection in vitro as compared to
matched normal control tissue specimens.
MATERIALS AND METHODS
Immunohistochemical Study
Tissue Samples
This study was approved by the Institutional Review Board
(IRB) of Boston University Medical Center (BUMC; Boston,
MA). Archived samples of biopsied warts from 91 subjects
with AGW (59 with anal warts and 32 with genital warts) were
available for analysis from the BUMC Pathology Department.
After medical records were reviewed for relevant information,
samples were coded and patient identifiers were eliminated to
preserve confidentiality.
Low-grade anal wart specimens from 47 men and 12 women
and genital wart specimens from 5 men and 27 women were
analyzed. Most specimens from women were from vulvar con-
dylomata; all specimens from men were from penile warts.
Normal skin specimens located outside the margins of the
with anal warts. Many had received topical therapy for their
lesions, including imiquimod (19%), trichloroacetic acid (8%),
or podophyllum (13%); 19% had completed treatment with ≥2
agents, while 4% had completed treatment with ≥3. Few patients
(8%) had a history of systemic steroid or immunomodulatory
therapy in the 6  months prior to anal wart resection. Only 2
subjects had received an HPV vaccine. Ten subjects had a his-
tory of an STD in the past 6 months, with chlamydia reported
in 6%, gonorrhea in 4%, syphilis in 2%, herpes simplex virus
infection in 6%, and trichomoniasis in 2%.
Immunohistochemical Analysis
All tissue specimens were fixed in formalin and embedded in
paraffin. Sections were cut at 5 µ, and mounted on glass slides.
The immunocytochemistry protocol has been described in
detail elsewhere [31]. Primary antibodies were obtained from
Dako, (Carpintaria, CA; CD1a, CD68, and p24), Biocare
Medical (Concord, CA; CD4, CD8, and CD15), and Novus
Biologicals (Littleton, CO; CXCR4 and CCR5). Antibodies
were detected with a biotinylated secondary anti-mouse/rabbit
antibody and alkaline phosphatase–labeled streptavidin and
were visualized with fast red substrate that stains positive cells
red (Dako). Since cells were often unevenly distributed and/or
located in clusters, the following semiquantitative scoring sys-
tem was used to assess cell density: +, 1–10 positive cells per
40× high-power field (HPF); ++, 11–50 cells/HPF; +++, 51–100
cells/HPF; and ++++, >100 cells/HPF [32].
HIV Infection of AGWs In Vitro
Nine HIV-uninfected men undergoing surgery for removal of
anal warts at Beth Israel Deaconess Medical Center (BIDMC;
Boston) were recruited for a prospective HIV infection study.
The protocol was approved by the BIDMC IRB, and informed
consent was obtained from all subjects.
HIV infection of explant tissue specimens was conducted fol-
lowing an established protocol [33]. The Q23.ENV.17 envelope
expression vector (National Institutes of Health AIDS Reagent
Program) was transfected into 293T cells, and the supernatant

276 • JID 2019:219 (15 January) • Pudney et al

was used to infect PHA-activated peripheral blood mono- 100
Cell density
nuclear cells for production of HIVQ17/23 [34] viral stock. The
80 1+
tissue culture infectious dose (TCID50) was determined by the

% Tissues w/Cells
2+
TZM-bl assay [35]. Biopsy specimens from AGWs and normal 60
3+
perianal tissue (control) were transported on ice to BUMC.
40 4+
Tissue specimens were cut into sections (2  mm × 2  mm ×
1 mm), and replicate pieces were transferred to 24-well tissue 20

culture plates. A  total of 103 TCID50 of HIV or tissue culture 0

medium was added, and samples were incubated overnight. On G A G A G A G A G A G A
Skin Wart Skin Wart Skin Wart
day 1, tissue explants were washed and resuspended in fresh
CD1a+ DC CD4+ T CD68+ MØ
tissue culture medium. On days 3, 7, 11, and 14 of culture,
HIV-target cells in Epidermis
supernatants were harvested and stored at −80°C. Supernatants
were tested for the presence and amount of infectious HIV,
Figure  1.  Summary of the concentration of human immunodeficiency virus
using TZM-bl cells [36]; HIV infection was assessed by the

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Galacto-Light Plus kit (Applied Biosystems). In addition, HIV
p24 concentrations were measured in supernatants by using the
Alliance HIV-1 p24 Antigen enzyme-linked immunosorbent
assay kit (PerkinElmer).
Statistical Analysis
For initial analysis, differences in the abundance of HIV tar-
get cell populations in AGW versus control specimens were
analyzed by 1-way analysis of variance (ANOVA) with post
hoc testing by the Fisher protected least significant difference.
This was followed by analysis of HIV  target cell populations
in AGW specimens versus tissue-matched control samples by
2-way ANOVA, with HIV-1 serostatus (a between-subjects
factor) and tissue site (a within-subjects factor) as indepen-
dent variables. When >1 sample was available from an indi-
vidual participant, the mean of sample values was used. Data
were log transformed before ANOVA, to control for nonnor-
mal distribution and heterogeneity of variance. Correlations
between numbers of different cell types in AGW specimens
were performed using Spearman rank correlation coefficients.
Discrete, categorical data were analyzed by the Fisher exact
test (or the Freeman-Halton extension) or the McNemar test.
Differences or associations were considered to be statistically
significant at a P  value of <.05. StatView (version 5.0.1; SAS
Institute, Cary, NC) statistical software was used for data
analysis.
RESULTS
Characterization and Enumeration of Cell Populations by
Immunohistochemical Analysis
The focus of this study was the identification and enumera-
tion of principal HIV  target cells (CD1a+ DCs, CD4+ T cells,
and macrophages) in the epidermis (ie, the top layer) of AGW
specimens and site-matched normal skin specimens. An initial
analysis indicated that the number of these cells was similar
between anal and genital control specimens and between anal
and genital wart specimens and that AGW specimens contained
(HIV)  target cells in the epidermis of anogenital warts and normal control skin.
Types and numbers of cases: anal (A) warts, n = 51; genital (G) warts, n = 30; A skin,
n = 26; G skin, n = 40. Differences in cell distribution between A skin and G skin,
and between A warts and G warts were not significant (Fisher Exact test, Freeman-
Halton extension). A and G warts had significantly higher concentrations of all three
HIV target cell types (CD1a+ DCs, CD4+ lymphocytes and CD68+ macrophages) than
did control A and G skin (all P  ’s < 0.01).
significantly more HIV target cells than normal anogenital skin
specimens (P < .01; Figure 1).
We also described HIV  target cells in the dermis and used
other phenotypic markers (CCR5 and CXCR4 [HIV corecep-
tors], p24 [HIV-infected cells], CD15 [granulocytes], and CD8
[CD8+ T cells]) to further characterize the HIV target cells and
identify possible cofactors related to their abundance (described
below).
Normal Anogenital Skin Specimens
Because the distribution of HIV  target cells was similar in
normal genital and anal skin specimens, descriptions of these
2 sites have been combined. Histologic findings in normal
anogenital skin specimens are shown in Figure  2A. CD1a+
DCs were present in low-to-moderate numbers (score, ++ or
less) in the epidermis and were rarely observed in the dermis
(Figures 1 and 3A). CD4+ T cells and CD68+ macrophages were
rarely observed in the epidermis (Figures 1 and 4A) but were
frequently observed in low-to-moderate numbers in the dermis
(Figures  4A and 5A). No CD15+ granulocytes and few to no
CXCR4+ or CCR5+ cells were observed in normal skin spec-
imens. Low numbers of CD8+ lymphocytes were observed in
the stratum basalis of the epidermis and were scattered in the
dermis.
AGW Specimens
Because the distribution of HIV target cells was similar for gen-
ital and anal wart specimens (Figure 1), specimens from these
sites have been combined in the descriptions specified below.
Characteristic histologic features of AGW specimens are shown
in Figure 2B–D.

HIV Target Cells in Anogenital Warts  •  JID 2019:219 (15 January) • 277

 A A
B C D
Figure 2.  Morphology of normal anogenital skin specimens (A) and anogenital
wart (AGW) specimens (B–D) stained by hematoxylin eosin (40× original magni-
fication). The epidermal layers (stratum corneum [SC], stratum granulosum [SG],
stratum spinosum [SS], stratum basalis [SB]), dermis, and dermal papillae [DP]) are
labeled. AGW specimens can demonstrate abnormal thickening of the SS (acantho-
sis; B), thickening of the SC (hyperkeratosis; C), and/or thickening of the SG (hyper-
granulosis; C). AGW specimens also characteristically show parakeratosis (nuclei
in the SC) and perinuclear cytoplasmic necrosis (koilocytes; D).
A B
C D
Figure  3.  CD1a+ dendritic cells detected by immunohistologic staining (red) in
normal anogenital skin specimens (A) and anogenital wart specimens (B and C). A,
Normal vulvar skin specimen demonstrating a few CD1a+ cells in the epidermis and
fewer in the dermis (20× original magnification). B, Vulvar wart specimen displaying
hyperkeratosis and acanthosis with numerous CD1a+ cells in the epidermis (40×
original magnification). C, Clitoral wart specimen with pronounced hyperkeratosis
and numerous CD1a+ cells in the stratum corneum (SC) and stratum spinosum (40×
original magnification). D, Penile wart specimen showing focal accumulations of
CD1a+ cells in the dermis (40× original magnification).
CD1a+ DCs
Approximately half of the AGW cases had abundant (score,
+++ or greater) CD1a+ DCs in the epidermis (Figure  1). In
many samples, these cells were dispersed throughout the width
of the stratum spinosum (Figure  3B), whereas in others they
278 • JID 2019:219 (15 January) • Pudney et al
B
C D
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Figure 4.  CD4+ lymphocytes detected by immunohistologic staining (red) in nor-
mal anogenital skin specimens (A) and anogenital wart specimens (B–D). A, Normal
anal skin specimens demonstrating few CD4+ cells, with most located in the der-
mis (20× original magnification). B, Vulvar wart specimen with CD4+ T cells present
in a localized area of the epidermis containing abundant koilocytes (40× original
magnification). C, Anal wart specimen with large focal accumulations of CD4+ T
cells in the dermis (20× original magnification). D, Penile wart specimen with focal
accumulations of CD4+ T cells in the dermis (20× original magnification). SC, stratum
corneum.
A B
C D
Figure  5.  CD68+ macrophages detected by immunohistologic staining (red) in
normal anogenital skin specimens (A) and anogenital wart specimens (B–D). A,
Normal vulvar skin specimen with CD68+ cells located primarily in the dermis (20×
original magnification). B, Vulvar wart specimen showing CD68+ cells infiltrating
koilocytes in epidermis (40× original magnification). C, Penile wart specimen with
numerous CD68+ cells associated with accumulations of lymphocytes in the dermis
(20× original magnification). D, Vulvar wart specimen showing an area of localized
CD68+ macrophage infiltration into the epidermis (20× original magnification). SC,
stratum corneum.
were concentrated in the upper epithelial layers (ie, the stratum
granulosum and stratum corneum; Figure 3C) or the stratum
basalis. CD1a+ DCs were also consistently detected in the der-
mis and/or dermal papillae in AGW samples. In a substantial
number of specimens (20% of anal wart specimens and 28% of
genital wart specimens), large numbers were observed in the
dermis in clusters associated with focal accumulations of lym-
phocytes (Figure 3D).
CD4+ T Lymphocytes
Twenty percent of AGW specimens had moderately high num-
bers (score, ++ or greater) of CD4+ lymphocytes in the epi-
dermis (Figure  1). They were often detected in regions with
abundant koilocytes (Figure 4B). CD4+ T cells were consistently
detected in the dermis of AGW specimens and were often abun-
dant. In some samples, CD4+ T cells were organized in a layer
just beneath the base of the epidermis; in others, they occurred
as distinct focal accumulations in the dermis. These accumu-
lations varied in number from few to numerous and varied in
size from small to very large (Figures 4C and 4D). Focal accu-
mulations of CD4+ T cells in the dermis usually also contained
numerous CD8+ T cells, macrophages, and CD1a+ DCs.
CD68+ Macrophages
Twenty percent of AGW specimens contained moderately high
numbers (score, ++ or greater) of CD68+ macrophages in the
epidermis (Figure 1). These cells were found either throughout
the epidermal layer or in localized areas often associated with
koilocytes (Figure 5B). CD68+ macrophages were also consist-
ently present in the dermis and dermal papillae of AGW speci-
mens, ranging in concentration from few to very abundant. For
some samples, CD68+ macrophages were associated with focal
accumulations of lymphocytes in the dermis (Figure  5C) and
were sometimes observed infiltrating from dermal aggregates
into the epidermis (Figure 5D).
CXCR4+ Cells
This HIV coreceptor can be expressed by a variety of cell types found
in AGWs, including CD4+ T cells, granulocytes, and keratinocytes
[37]. In 11 specimens, CXCR4 was expressed on granulocytes or
CD8+ lymphocytes in the epidermis. Varying concentrations of
CXCR4+ cells resembling lymphocytes and macrophages were also
detected in the dermis. CXCR4+ keratinocytes were occasionally
detected in both genital and anal wart specimens in basal layers of
the epithelium surrounding dermal papillae.
CCR5+ Cells
The CCR5 HIV coreceptor can be expressed on a number of
cell types found in AGWs, including memory T cells, macro-
phages, granulocytes, Langerhans cells, and keratinocytes [38].
Only 12% of AGW specimens had large numbers of CCR5+
cells in the epidermis; most of these cells were granulocytes and
were observed in the stratum spinosum. Many anal wart spec-
imens but few genital wart specimens had a large number of
CCR5+ lymphocytes and macrophages in the dermis; however,
few CCR5+ cells were detected in the focal accumulations of
HIV target cells present in the dermis.
HIV Target Cells in Anogenital Warts  •  JID 2019:219 (15 January) • 279
HIV p24+ Cells
HIV-infected Jurkat cells were used as a positive control, and
all stained positive with the p24 antibody (data not shown).
No cells definitively positive for p24 were observed in 52 AGW
samples from 31 HIV-infected patients. Most HIV-infected
subjects were receiving antiretroviral drugs, which may have
suppressed p24 expression by HIV-infected cells.
CD15+ Granulocytes
CD15+ granulocytes were assessed as a marker of acute inflam-
mation [39]. Notable concentrations were observed in 17% of
AGW specimens (10 anal specimens and 6 genital specimens).
They often appeared in large focal accumulations in the dermis
and/or in the stratum corneum and stratum spinosum; they
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were often observed infiltrating koilocytes.
CD8+ Lymphocytes
CD8+ lymphocytes were assessed as a potential marker of
cell-mediated immunity associated with wart regression [29].
Approximately one third of AGW specimens had high con-
centrations of CD8+ lymphocytes in the epidermis, localized
throughout the layer or concentrated in the stratum basalis.
Many AGW specimens (54% of genital specimens and 33% of
anal specimens) also had high concentrations of CD8+ lym-
phocytes in the dermis, mostly in focal aggregates. There was
concurrence between CD8+ lymphocyte concentrations in the
dermis and epidermis.
Comparison of HIV Target Cell Numbers in AGW Versus Donor-
Matched Control Specimens
An in-depth statistical analysis was performed for cell counts
in the epidermis of paired wart and control skin samples. Anal
wart specimens (from 39 patients) had significantly higher
concentrations of all 3 HIV  target cell types than did donor-
matched control anal tissue specimens (Table 1). Anal wart spec-
imens had elevated concentrations of CD1a+ cells (P = .0001),
with HIV-infected subjects having the highest concentrations
(P = .03 for the interaction between tissue site and HIV infec-
tion status). Anal wart specimens also had significantly higher
concentrations of CD68+ macrophages (P < .0001), CD4+ T cells
(P = .0006), and CD15+ granulocytes (P = .0002) than control
anal skin specimens. Anal wart specimens from HIV-uninfected
subjects had higher concentrations of CD8+ cells than did those
from HIV-infected subjects (P = .02 for the interaction between
tissue site and HIV infection status). Elevated concentrations
and focal accumulations of HIV target cell populations in anal
wart specimens were not associated with use of topical therapy
or recent history of an STD.
Paired genital wart and control skin samples were available
from 13 women. Genital wart specimens contained significantly
higher concentrations of CD1a+ (P = .0006), CD68+ (P = .015),
CD4+ (P  =  .026), and CD8+ (P  =  .002) T cells in the epider-
mis than did donor-matched control tissue specimens; HIV-1
Table 1.  Human Immunodeficiency Virus (HIV) Target Cells, Granulocytes, and CD8+ T Cells in the Epidermis of Anogenital Wart Specimens Versus Donor/
Site-Matched Control Tissue Specimens (CS) From HIV-Infected and Uninfected Subjects

Control Specimens Wart Specimens Pa

HIV Negative vs HIV

Variable HIV Negative HIV Positive HIV Negative HIV Positive Positive Wart vs Control

Anal wart vs control specimens

 CD1a+ cells
  Mean ± SEb 1.2 ± 0.2 1.1 ± 0.1 1.9 ± 0.3 2.6 ± 0.2 NS <.0001
  Matched pairs (n) 10 10 10 10
 CD4+ cells
  Mean ± SEb 0.2 ± 0.1 0.3 ± 0.1 0.8 ± 0.1 0.6 ± 0.2 NS .0006
  Matched pairs (n) 14 9 14 9
 CD68+ cells
  Mean ± SEb 0.04 ± 0.04 0.2 ± 0.1 0.7 ± 0.1 0.6 ± 0.1 NS <.0001

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  Matched pairs (n) 12 12 12 12
 CD8+ cells
  Mean ± SEb 0.9 ± 0.1 0.7 ± 0.1 1.6 ± 0.2 0.6 ± 0.1 .002 NS
  Matched pairs (n) 17 12 17 12
 CD15+ cells
  Mean ± SEb 0.03 ± 0.03 0.0 ± 0.0 0.6 ± 0.2 0.4 ± 0.1 NS .0002
  Matched pairs (n) 16 16 16 16
Genital wart vs control specimens
 CD1a+ cells
  Mean ± SEb 1.1 ± 0.1 0.9 ± 0.2 2.9 ± 0.4 2.7 ± 0.5 NS .0006
  Matched pairs (n) 7 5 7 5
 CD4+ cells
  Mean ± SEb 0.1 ± 0.1 0.3 ± 0.1 0.8 ± 0.3 0.8 ± 0.3 NS .026
  Matched pairs (n) 7 6 7 6
 CD68+ cells
  Mean ± SEb 0.0 ± 0.0 0.0 ± 0.0 1.2 ± 0.4 0.8 ± 0.3 NS .015
  Matched pairs (n) 3 4 3 4
 CD8+ cells
  Mean ± SEb 0.9 ± 0.2 0.5 ± 0.2 1.7 ± 0.2 1.4 ± 0.2 NS .002
  Matched pairs (n) 9 4 9 4
 CD15+ cells
  Mean ± SEb 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 NS NS
  Matched pairs (n) 4 4 4 4

Abbreviations: NS, not significant; SE, standard error.

aBy two-way analysis of variance (main effects).
bData are semiquantitative cell density values.

serostatus was not significant for any of the white blood cell
counts (Table  1). There were no donor-matched tissue speci-
men pairs available from men with genital warts, but data were
compared for penile wart specimens from 5 subjects and normal
penile tissue specimens from 5 unrelated subjects. The epider-
of granulocytes or any of the HIV target cells in the epidermis.
However, all 3 HIV target cell types were often found associated
with CD8+ T cells in focal aggregates in the dermis layer.
Infection of AGW and Control Specimens With HIV In Vitro

mal layer of male genital wart specimens had significantly higher
concentrations of CD1a+ (P = .004) and CD68+ (P = .009) cells
(both P values were determined by the Mann-Whitney U test).
Correlations Between HIV Target Cells, CD15+ Granulocytes,
and CD8+ T Cells in AGW Specimens
Granulocyte concentrations were positively correlated
with CD68+ cell numbers in the AGW epidermis (ρ  =  0.54;
P  <  .0001) but not with CD1a+ DCs or CD4+ lymphocytes.
CD8+ T-cell concentrations were not correlated with numbers
Donor-matched samples of anal warts and normal anal epithe-
lium were obtained from 9 HIV-negative men (89% were white;
age range, 20–29  years). All wart samples were anal intraepi-
thelial neoplasia grade 1–2 on pathologic review. One case was
discarded because of contamination. Two of 8 AGW samples
versus 0 normal tissue samples showed definitive signs of HIV
infection, as evidenced by robust TZM-bl values (>1000 relative
light units on >2 days of culture) and p24 assay confirmation
(Figure 6); because of the small sample size, these data were not
significant (P > .10).

280 • JID 2019:219 (15 January) • Pudney et al

 100 000
10 000
1000
RLU
100
10
0.1
3 7
Days
Figure 6.  Human immunodeficiency virus (HIV) infection of anal wart specimens and control anal skin explant tissue specimens from HIV-uninfected men in vitro. Two of 8
anal wart specimens, compared with 0 of 8 control tissue specimens, showed high levels of HIV infection in the TZM-bl assay.
DISCUSSION
HPV is highly infectious and is transmitted between persons via
microabrasions during sexual skin-to-skin contact [7, 40]. Most
initial HPV infections occur in adolescence, making early pre-
vention critical. After initial HPV infection, patients may have
clearance of subclinical infection, development of lesions with
subsequent regression, persistence of infection with subsequent
regression, or persistence of infection with development of can-
cer. Most HPV infections are subclinical and are cleared by the
immune system via the cell-mediated immune response before
any clinical disease is observed [41]. Clinical AGWs may also
undergo regression as a result of cell-mediated immunity [29].
Our study demonstrates that the density of principal HIV tar-
get cells (CD1a+ DCs, CD4+ T cells, and CD68+ macrophages) is
significantly higher in AGW tissue than donor/tissue-matched
control skin tissue. Approximately half of AGW specimens had
elevated concentrations of DCs in the epidermis. It is generally
accepted that DCs play a pivotal role in the initial events of HIV
transmission by transferring HIV to CD4+ T cells [26]. A subset
of AGW specimens also had elevated numbers of macrophages
and CD4+ T cells in the epidermis. There was a strong asso-
ciation between numbers of macrophages and granulocytes
in the epidermis, suggesting the recruitment of macrophages
to the superficial layer of AGWs during acute inflammation.
Macrophages and CD4+ T cells were often observed near koilo-
cytes, classical HPV-infected cells with enlarged nuclei and
perinuclear halos, indicating that HIV target cells accumulate
in AGWs at sites of active HPV infection.
Previous studies have described immune cells in human
AGWs, with varying results [29, 42–45]. Two studies that ana-
lyzed naturally regressing warts described increased concen-
trations of lymphocytes and macrophages [29] and DCs [22].
Although we did not specifically study wart regression, approx-
imately one third of our patients with AGWs had elevated num-
bers of CD8+ T cells (a marker of cell-mediated immunity) in
BI-6 Wart
BI-6 Control Skin
BI-7 Wart
BI-7 Control Skin
11 14
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the epidermis and/or massive focal infiltrates in the dermis.
Whereas CD8+ T cells in the epidermis did not correlate with
elevated numbers of HIV target cells at that site, CD8+ dermal
infiltrates contained numerous CD4+ T cells, macrophages, and
DCs, providing evidence that AGW regression may be associ-
ated with the recruitment of high densities of HIV target cells
to the dermis layer.
A recent study demonstrated HIV-1 RNA and episomal DNA
in intraanal high-grade HPV lesions in HIV-infected men who
have sex with men and speculated that receptive anal inter-
course with HIV-infected individuals that have HPV-associated
lesions may increase the risk for HIV acquisition [46]. This is
an intriguing hypothesis, although the presence of HIV nucleic
acid does not necessarily indicate presence of infectious virus.
In our study we investigated evidence of assembly of HIV viral
particles (p24 protein) in AGW tissues from HIV-infected indi-
viduals. We not detect p24-positive cells in AGW from HIV-
infected individuals, potentially due to viral suppression by
antiretroviral drugs. This research should be extended to indi-
viduals who are not receiving antiretroviral therapy, to deter-
mine whether AGWs can be foci of HIV infection. In a related
experiment, we challenged AGW and normal tissue explants
from uninfected individuals with HIV in vitro; 2 AGW spec-
imens and no donor-matched control specimens showed evi-
dence of robust HIV infection, suggesting that some AGWs
may be highly susceptible to HIV infection. We propose that
future studies be conducted to correlate HIV  target cell types
and numbers with HIV infectability in vitro.
In summary, our study demonstrates a significant increase in
the number of HIV target cells in AGWs and provides evidence
of enhanced HIV infection of AGW in vitro. These findings sug-
gest that AGWs may provide portals for HIV transmission and
reinforce the importance of prevention and treatment of AGWs
to control the HIV epidemic. Potential susceptibility of AGWs
to HIV infection provides additional impetus for vaccination
HIV Target Cells in Anogenital Warts  •  JID 2019:219 (15 January) • 281
of children and adolescents before the age of sexual debut with
HPV vaccines that induce immunity to strains associated with
AGWs (ie, HPV types 6 and 11) [47]. Prelicensure trial efficacy,
modeling, and postvaccination surveillance studies of quadri-
valent vaccine programs have already demonstrated the signif-
icant short-term impact of this vaccine on the incidence and
prevalence of AGWs in the United States [48, 49].
The new nonavalent HPV vaccine is expected to show simi-
lar results. Large-scale roll out of HPV vaccines in HIV-endemic
areas such as sub-Saharan Africa could significantly impact the
HIV epidemic in these regions.
Notes
Acknowledgments.  We thank Dr Antonio de las Morenas,
Department of Pathology, Boston University School of
Medicine, for providing access to samples; Oscar Gonzalez,
PhD, Sagar Laboratory, Boston University, for performing p24
assays for the prospective study; Lindsay Bohnert, MS, and
Tesfaldet Tecle, PhD, Boston University, for providing assistance
with immunohistochemical and infection assays; and Linda
Rosen, MSEE, Boston University Clinical Data Warehouse, for
her assistance in obtaining clinical information for the immu-
nohistochemical data set.
Financial support.  This work was supported by the
National Institutes of Health (grant U19 AI096398) and Merck
(IISP grant #37966) both to D. Anderson. Supported in part by
a research grant from Investigator-Initiated Studies Program of
Merck Sharp & Dohme Corp. The opinions expressed in this
paper are those of the authors and do not necessarily represent
those of Merck Sharp & Dohme Corp.
Potential conflicts of interest.  All authors: No reported con-
flicts of interest. All authors have submitted the ICMJE Form
for Disclosure of Potential Conflicts of Interest. Conflicts that
the editors consider relevant to the content of the manuscript
have been disclosed.  sition among circumcised and uncircumcised young men
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