BASIC OF BIOPHARMACEUTICS

“HPV VACCINE REPORT”

By:

1. BREVMANA (114117507)
2. FARID AZWAR ANAS (114116508)
3. NI PUTU AYU RISKADEWI (114117516)
4. WAHYUNI (114117506)

Master Of Clinical Pharmacy

Faculty Of Pharmacy University Of Surabaya

2018
 HUMAN PAPILLOMA VIRUS VACCINE

1. INTRODUCTION

To complete assignment of Novel Drug Delivery Technology, we choose one of topic

entitled: “Human Papilloma Virus Vaccine”. In this report, we explain several aspects
of Human Papilloma Virus Vaccine start from formulation, history, production,
pharmacology, adverse effect, biosimilars, cost, Human Papilloma Virus Vaccine until
usage of Human Papilloma Virus Vaccine in Indonesia. We hope that the report would
be useful for the readers.

2. FORMULATION

The most promising approach for the prophylactic HPV vaccine has been proven to use
of virus-like particle (VLP) technology.VLPs made from the main L1 capsid protein
alone has been demonstrated to be both morphologically and antigenically similar to
authentic papillomavirions (15–17). Genetic engineering techniques can be used to
express these proteins, which undergo self-assembly to form a highly immunogenic
empty viral capsids (18). Current clinical data show that HPV vaccines based on
HPV16, HPV16/18 or HPV6/11/16/18 L1 VLPs and formulated either with aluminium
salts alone or AS04 are highly immunogenic and show up to 100% effectiveness
against persistent HPV infection and development of lesion in the cervix, (19,20).
Although L1 VLP vaccines are highly immunogenic and induce antibody responses in
amost all vaccinated subjects, protection of formal immune substitutes has not been
established in humans yet. Otherwise, in some animal models humoral immunity post-
vaccination has been demonstrated to be very important for protection against
papilloma virus infection.

(21) In the past 10 years, a new adjuvant systems has been designed to trigger a higher
immune response and a longer duration of protection against infection (22,23). Several
immunogenicity studies carried out in rats, guinea pigs, monkeys, and humans have
demonstated the effectiveness of immunological adjuvants such as MPL (3-O-desacyl-
4 - monophosphoryl lipid A), a non-toxic derivative of LPS (lipopolysaccharide), to
potentiate both specific antibodies and cellular immune responses to vaccination. MPL
is able to directly activate the main innate immune mechanisms, including the
activation of antigen presenting cells and induce the pro-inflammatory cytokines, such
as TNF and IL-12, which will increase the adaptive immune response, which induces
T helper cells and B cell responses (24–26). Based on the MPL’s adjuvant potential,
Glaxo Smith Kline (GSK) Biologicals developed an ASO4-appointed adjuvant system
consisting of aluminium salts and MPL. The adjuvant capacity of AS04 has been
evaluated during the development of several candidate vaccines, including herpes
simplex, hepatitis B, and HPV16/18 L1 VLPs. Both immunogenicity data and efficacy
data have shown benefit of AS04 vaccine formulations (20,27,28)

3. HISTORY

Cervical cancer is the second most common cancer among women worldwide, with an
estimated 493,000 new cases, and 274,000 deaths in 2002. About 83% of cases occur
in developing countries, where cervical cancer accounts for 15% of cancers in women,
with risk before age 65 of 1.5%, while in developed countries it only accounted for
3.6% of new cancers, with cumulative risk (ages 0–64) of 0.8% (1). Tracking of
papilloma virus studies resulted in identification of specific HPV types as causative
agents for cervical cancer, other anogenital cancers, and a subset of oropharyngeal
carcinoma. This investigations began with a search for the viral etiology of cervical
cancer, but by the late 1960s and 1970s the results of serological studies showned a
role for type 2 human herpes simplex virus (HSV 2) in this cancer (2,3). The current
group of author can not confirm these findings, and look for alternative virus
candidates. A number of anecdotal reports on the malignant conversion of genital warts
(condylomataacuminata), spread among medical literature from the previous 100 years,
attract attention. Furthermore, the possible causal role of papillomavirus infections for
cervical cancer was postulated, and initial efforts are initiated to characterize viral DNA
in genital warts (4,5). These and other studies have initial consequences of finding the
heterogeneity of the papillomavirus family (6)(7), currently calculating nearly 106
genotypes completely sorted. However, the eventual isolation of HPV DNA from
genital warts, labeled II PV 6(8), and from laryngeal papillomas (HPV 11) two years
later(9), does not produce positive data for these viruses in cervical cancer. However,
their use of DNA in hybridization experiments, carried out in less stringent condition,
allowed the subsequent cloning of HPV 16 (10) and HPV 18 DNA (11), the two types
of papillomavirus most commonly found in cervical cancer. This allows further
experiments to be carried out that would prove the role of these papillomaviruses in
causing this malignancy.

4. PRODUCTION

World prediction about the need for HPV prophylactic vaccines for women aged 10-
25 years in developing countries reach 15 trillion doses.(12) Two virus-like protein
(VLP) HPV vaccines such as VLP 16/18 and VLP 6/11/16/18 that have been marketed
in the world today are produced using yeast and baculovirus expression systems. Both
HPV prophylactic vaccines are difficult to distribute in poor and developing countries
because of the high price.(13)Thus the production of large-scale HPV vaccines is
rquired to meet the needs of the world in the future.(12)

The HPV vaccines is currently made by pharmaceutical companies in industrialized

countries which will first introduce the vaccine at relatively high prices in the industrial
market and private-sector markets in developing countries. There is discussion about
the introduction of the HPV vaccines to public sector markets in developing countries
at lower prices, but this will depend on financial demand and commitment from the
public sector. These companies also discuss local production options with developing
country governments and private producers, but whether it will result in low-cost local
production is unknown. The intellectual property (IP) situation related to independent
production in developing countries are not clear (important Ips are held by public sector
entities), and it would be useful to prepare IP “maps” to assist local producers decide
whether local production development of HPV vaccine is an attractive option for them.
Alternative ways to make HPV vaccines that do not use virus-like particle (VLP)
technologies are currently being explored, but they represent technologies at very
different stages of development and therefore have years of research and development
in front of them(14).

5. PHARMACOLOGY (PHARMACOKYNETICS, PHARMACODYNAMICS)

Pharmacology

First cancer vaccine of a type cancer (2006). Contains antibodies to the human
Papillomavirus type 6,11,16 and 18, the cause of cervical cancer and is especially
recommended for women from 16-26 years. The dose: cur from 3 injections of 0.5mL
according to schedule 0-2-6 months (59).

Pharmacokinetics

Pharmacokinetics is a common term for the distribution, absorption, metabolism, and

excretion of a drug in the body. While all of these processes contribute to a drugs eff
ectienes(29).

Test pharmacological medicine and biodistribution of somebody's papillomavirus

(HPV) 16L1 polymer vaccinum delivered in human endogenous animal
virus envelope macromolecule (HERV)-expressing baculovirus (AcHERV) and
people of naked cellular inclusion vaccinum.
HPV16L1 gene was administrated as a naked plasmid or in AcHERV to mice via
intravenous and intramuscular routes.
HPV16L1 cistron was extracted and assayed by quantitative period of
time enzyme chain reaction, that determined to possess a detection limit of
fifty copies/µg genomic polymer. Mean residence times of
HPV16L1 in AcHERV were 4.8- and 272.2-fold higher than naked HPV16L1 DNA
vaccines after intramuscular and intravenous administration, respectively. Naked
HPV16L1 DNA levels 1 month after injection were >3 orders of magnitude lower in
each tissue tested than AcHERV-delivered HPV16L1, which was retained in most
tissues without specific tissue tropism. AcHERV-delivered HPV16L1 administered
intramuscularly persisted at the injection sites.
However, the amount of copy numbers in muscle were low (1,800/μg genomic
DNA) once one month, and undetectable once six months.
HPV16L1 delivered via AcHERV resides longer in the body than HPV16L1 in naked
form. The lack of tissue tropism ensures the safety of AcHERV vectors for further
development (57)

Pharmacodynamics

Pharmacodynamics of HPV Vaccine, based on an analysis of several studies, the HPV

vaccine should ideally be given to girls and boys at average age of 9-12 years. The aim
is to provide immunity to HPV infection before the vaccine recipient is actively
engaged in sexual intercourse. The HPV vaccine will work better if given as a teenager,
than when given after adulthood.

However, if you have not received or have not yet received the HPV vaccine at the age
of 9-12 years, the HPV vaccine can be given to women aged 13-26 years. HPV vaccines
can also be given to women who have been sexually active. However, keep in mind
that this vaccine cannot treat HPV infections that are happening.

Clinical studies

Immune Responses to Gardasil employing a 2-dose schedule in people 9-13

years older
A clinical test showed that among women United Nations agency received a pair
of doses of HPV vaccinum half dozen months apart, protein responses to
the four HPV varieties, one month once the last dose were non-inferior to those
among young ladies United Nations agency received three doses of
the vaccinum at intervals half dozen months.
At Month 7, within the Per Protocol population, the reaction in women aged 9-13
years (n=241) United Nations agency received a pair of doses of Gardasil
(at zero, half dozen months) was non-inferior and numerically higher to
the reaction in ladies aged 16-26 years (n=246) United Nations
agency received three doses of Gardasil (at zero, 2, 6 months).
At thirty six month follow-up, the Greenwich Time in women (2 doses, n=86)
remained non-inferior to the Greenwich Time in ladies (3 doses, n=86) for
all four HPV varieties.
In the same study, in women aged 9-13 years, the reaction once a 2-dose schedule
was numerically below once a 3-dose schedule (n=248 at Month 7; n=82 at Month
36). The clinical connexion of those findings is unknown.
Duration of protection of a 2-dose schedule of Gardasil has not been established
(58).
6. ADVERSE EFFECT

Although the benefits of the vaccine are proven, risks and adverse effects associated
with these vaccines include erythema (4–25%); pain (36–71%); swollen (0–21%); and
pruritis (0–4%) (30). Besides these physical effects, there are potential social barriers
that need to be addressed if the general population accepts the HPV vaccine. One such
barrier is that the vaccines must be given to individuals before they became sexually
active. Another obstacle is an indication for vaccination is preventing the possibility of
sexually transmitted diseases. Approval and consent to such a procedure could face
resistance for a variety of cultural, personal, and religious reasons. Finally, even though
only a half of the population (i.e., women) is at risk for cervical cancer, most children
(boys and girls) must be vaccinated to reduce the incidence of disease worldwide (31)

Like all other vaccines, vaccination against HPV could have side effects, but most of
which are not giving serious effect. Non-serious side-effects include swelling, pain,
erythema, fever, headache, and nausea. Serious effects include recovery with sequelae,
life threatening events, admission to hospital, and death. In Australia, a country where
HPV vaccination has been included in the national immunization program since 2007,
9% of reported adverse effects after HPV vaccination are classified as serious. This is
similar to the percentage of all serious adverse events reported after immunization,
which is 8% (32)

7. BIOSIMILARS

Cervarix (Glaxo Smith Kline; London) is a two-valence human papilloma virus (HPV)
vaccine, consist of virus-like particles from the major capsid L1 proteins truncated from
HPV types 16 and 18, produced in the recombinant baculovirus expression system and
insect cell line Hi-5 Rix4446 comes from Trichoplusiani.(33)

Gardasil® and CervarixTM are both composed of HPV L1 proteins which

spontaneously assemble themselves into VLPs. CervarixTM is designed to prevent
infection by HPV-16 and 18, the two types that cause 70% of cervical cancer.
Gardasil® targets the same two of cancer causing types and, besides, is intended to
prevent infection by HPV-6 and 11, which causes 75–90% of external genital warts.
Both vaccines must be refrigerator and given by intramuscular injection in the deltoid
area, but slightly different at the time of the second dose. Every VLP type is generated
and purified separately and during final formulation the different types are mixed.
Besides valence, another difference between the two vaccines is adjuvants choice.
Different aluminum salts are used in two vaccines. The Gardasil® vaccine only uses
an aluminum adjuvant (aluminum hydroxyphosphate sulfate), while the CervarixTM
adjuvant system, called AS04, consist of a detoxified form of lipopolysachharide
(LPS), monophosphosphoryl lipid A (MPL), and aluminum hydroxide. Aluminum salt-
based adjuvants typically induce a type of Th2 response and it was observed when
Merck’s aluminum adjuvant were combined with HPV VLPs. However, MPL activates
innate immune responses via toll-like receptor molecules and can induce a mixed
Th1/Th2 differentiation patterns in human T cells (25,34). Th1 responses are tipically
sought in therapeutic vaccines designed to produce cell-mediated immune responses,
in contrast to prophylactic vaccines designed to produce antibody responses. Th1 and
Th2 responses induce antibody responses which usually have different ratios of specific
immunoglobulin types and IgG subtypes, but there is currently no evidence that various
antibody species differ in HPV neutralizing potential. Glaxo Smith Kline (GSK) has
published that VLP antibody titers in women are about 2-fold higher when their VLP
is formulated in AS04 than simple aluminum hydroxide. A more complete review of
adjuvants and immune responses is discussed completely in this monograph by Stanley
M et al. (35) . There has been no direct comparison between VLPs adjuvanted with
AS04 and aluminum salts of Gardasil® to date.
8. USAGE IN INDONESIA

According to the World Health Statistics Report 2010 of the World Health
Organization (WHO), Indonesia has a population of 227,345,000. Part of the
population at risk for cervical cancer, women aged 15 years and over, is 79.14 million.
Present estimates show that every year 13,762 women are diagnosed with cervical
cancer and 7493 die from the disease. Cervical cancer ranks second most frequently
cancer in women in Indonesia, after breast cancer.

In cases of cervical cancer in Indonesia, the highest prevalence type of HPV are HPV
16, 18, and 52. The most prominent two HPV types in this study are HPV-16 (35–
41.9%) and HPV-18 (28–43%) (13,36). HPV-18 has a relatively higher prevalence in
the general population and in cases of cervical cancer, compared to other parts of the
world (37,38).

9. COST

Vaccine costs in Indonesia are still relatively expensive. The high costs of the vaccine
is named as the reason against HPV vaccination by 53.3% of the participants. With the
required amount of 3 vaccines costing between 2.250.000 and 3.000.000 Indonesian
Rupiah (USD 167.24–222.99), and the median monthly family income is USD 89.20,
HPV vaccination can not be reached by most people in Indonesia. That could be a
reason why people indicated that they believed the government should be responsible
to pay the vaccines (66.9%), or that the government and the parents had to share the
costs (22.4%). Nearly two-thirds (63.3%) of the participants would be willing to
contribute around 15% of their monthly family income to vaccinate their daughters
against HPV (32).

10. HUMAN PAPILLOMAVIRUS VACCINE DEVELOPMENTS

a. Peptide-based vaccines
Vaccination with tumor-specific peptides generates neutralizing antibodies and can
activate antigen specific CTLs. Since HPV 16 E6 and E7 peptides are retained in
cervical cells and are regardered important for malignant transformation, vaccination
with HPV 16 E6- or E7-derived peptidescan increase tumor-specific CTL production.
Peptides encoded by HPV 16 E6 and E7 that are known by human CTLs in a HLA-
A2restricted fashion have been recognized for use in vaccinating patients with HPV
16–positive cervical carcinoma. In practical applications, peptides and most cancer
antigens are often less immunogenic (39). To control this limitation, they can be altered
to increase their immunogenicity by modifying of the peptide amino acid sequence,
their relationship with immunostimulatory molecules, or coadministration of
adjuvants.

Van Driel et al. (40) conducted a phase I-II vaccine trial with HPV 16 E7 participant
of HLA-A*201–positive patients suffering from HPV 16–positive cervical carcinoma
who were refractory to conventional treatment. This vaccine consist of two HPV 16 E7
peers (aa E711-20, sequence YMLDLQPETT and aa E786-93, sequence TLGIVCPI)
representing two CTL epitopes with a high binding affinity for HLA-A*201 molecules,
emulsion before being used in Montanide ISA 51, a mineral oil–based adjuvant similar
to incomplete Freund’s adjuvant. Four vaccinations were given subcutaneously every
three weeks with an escalation doses from 100 µg to 1000 µg peptide per vaccination.
Of the 19 patients who underwent the study, 2 had stable disease for 1 year, 15
progressed, and 2 experienced tumor-regression after subsequent chemotherapy. The
good news is there are no adverse side effects. This study shows that HPV 16 E7
peptide vaccination is feasible even in a group of patients with terminal disease.
However, the application of peptide based vaccines is limited by MHC restriction and
the need to define the CTL epitopes.

Human papillomavirus 16 E6 protein generated by cervical cancer tumor cells are

immunogenic which is poor in patients, but the protein is a tumor-specific antigen in
which therapeutic vaccine strategies can be directed (41). To explore E6 helper T-
epitope immunogenicity (MHC class II processing), Azoury-Ziadeh et al. (41)
immunizing C57BL/6 (H-2b) mice with overlapping peptides that stretch all 158 amino
acid sequence. Two peptides, 60-VYRDGNPYA-68 and 98GYNKPLCDLL-107,
given a proliferative response in lymph node cells. One of these peptides stimulated T-
cells from a number of strains from different mice MHC class II haplotypes which
indicate MHC restriction promiscuity. The inventions have implications for
incorporation of E6 into a therapeutic vaccine.

Viral-like particles vaccines because E6 and E7 are selectively maintained and

expressed during the development of malignancy, most vaccine strategies focus on
these two viral targets (42). Otherwise, the virion capsid proteins L1 and L2 are
expressed in differentiated squamous epithelial cells but are not considered to be
expressed in proliferating cells (42). Yet papillomaviral virions is very immunogenic
when systemically inoculated. High levels of L1 expression produces efficient L1
selfassembly into viral-like particles (VLPs) that resemble genuine viral capsids, both
antigenically and structurally. Moreover, coexpression of L1 and L2 leads to VLPs
containing both proteins, with the ratio of 30:1 L1:L2 which is similar to synthetic
virions. VLPs contsist of L1 are only able to induce neutralizing antibody levels that
are comparable to those caused by authentic virions. Coexpression of L2 increases VLP
production four- to 100-times in vitro but does not improve neutralizing antibody titers
after VLP vaccination (43). The antigenic characteristics of VLPs and the ability to
generate preparative amounts in non-mammalian cells has led to several vaccine trials.

An approach to raise the efficacy of VLPs may be include antigens from nonstructural
viral proteins. Experiments on animal papillomaviruses show that immunity to E1, E2,
E6, or E7 produces fewer papillomas and can induce regression of papilloma.
Immunization with chimers of L1/L2 VLPs with full-length HPV 16 E7 protein
combined to the L1 carboxyl terminus not only obtained production of high-titer
neutralizing antibodies not seen with the parental L1/L2 VLPs, but also protected mice
from tumor challenge with the TC-1 cell line (42). Parents of L1/L2 VLPs do not
protect the mice from tumor formation. No protection was monitored when the CD8+-
depleted mice were vaccinated with HPV 16 L1/L2 VLPs and challenged with tumor
cells (42). Conversely, VLP vaccination actually protected the CD4+ cutted mice from
tumor challenge. Therefore, the authors conclude that the anti-tumor response induced
by the VLPs is mediated by CD8+ CTLs [37]. But, neutralizing antibodies are still
considered important to prevent HPV infection in human body.

Marais et al. (44) discussed the need for an animal model to show protection against
HPV challenge. The papillomaviral species prohibits the ability to easily infect
laboratory animals with HPV. Their study explained the development of a recombinant
HPV-16 L1—vaccinia virus challenge model in mice to show protection in HPV 16
VLP-vaccinated mice. Inoculation of BALB/c and C57BL/6 mice with HPV-16 L1
VLP produced in HPV VLP-specific T cell proliferative responses characterized by the
production of both Th1 and Th2 CD4+ cytokines (IFNγ and IL-4, respectively), and
provided protection against challenge virus from recombinant vaccinia virus that
expresses HPV-16 L1.

When VLPs enters the cytosol of infected cells, the capsid proteins L1 and L2 are
processed in class I MHC path. Rudolf et al. (45) showed that HPV 16 capsid proteins
stimulated a MHC class I–limited CTL response with human peripheral blood
lymphocytes in vitro. In addition, they demonstrated the presence of at least one HLA-
A*201 limited CTL epitope within the HPV-16 capsid proteins. These results indicated
that VLPs can induce a HPV-16 capsid protein-specific immune response in humans.
The results provided here clearly indicate in the presence of CTL epitopes in the
papilloma virus capsid proteins L1 and L2. Generally, the L1 protein is expressed only
in differentiated epithelial cells, which represent only a small proportion of the infected
cells in a lesion. However, these cells produce new virus particles, and eradication of
these cells would prevent further spread of viral infection. This shows a possibility use
for VLPs in treatment vaccines.

Jochmus et al. (46), in an effort to develop a vaccine that combines prophylactic and
therapeutic properties, produced chimeric VLPs consist of a C-terminally truncated L1
protein combined to amino acids 1 to 60 of the HPV 16 E7 protein. C57BL/6 mice
were immunized by a single injection of L1∆CE71-60 chimeric VLPs (1–20 µg)
without adjuvant. Immunization of VLP chimeric mice induce neutralizing antibodies
direct against L1 virus-like particles (devoid of the E7 portion) and E7-specific T cells
as measured in vitro. Vaccinated animals are protected against tumor growth after
inoculation of syngeneic HPV 16-transformed tumor (TC-1) cells, and experienced a
therapeutic effect on pre-existing tumors. These data allows the conclusion that
chimeric VLPs are suitable for prevention and treatment of HPV infection.

Nardelli-Haefliger et al. (47) recently showed that nose immunization of anesthetized

mice with HPV 16 VLPs was very effective at inducing both neutralizing
immunoglobulin A (IgA) and IgG in genital secretions, whereas parenteral
immunization induced only neutralizing IgG (48). However, the production of IgA and
IgG in genital secretions fluctuates during the estrous cycle. The authors therefore
investigated how the estrous cycle affected the number of HPV 16 immunoglobulins
induced by VLP immunization. Parenteral vaccination caused levels of IgA and IgG to
fluctuate during the estrous cycle. Conversely, nasal vaccination caused neutralizing
titers of IgA plus IgG throughout the estrous cycle. Their data suggested that mucosal
immunization might be more efficient than parenteral immunization at encouraging
continuous protection of the female genital tract.

Dupuy et al. (49) investigated if the intranasal administration of either HPV 16 L1

protein VLPs or the HPV 16 L1 DNA in combination with cholera toxin gave rise to
systemic and mucosal humoral also cellular immune responses. Intranasally
administered VLPs (with cholera toxin) in vaginal IgA secretory antibodies and
BALB/c mice induced serum IgG, and CD4+ and CD8+ cells in the vagina and spleen.
Activated CD4+ TH1-like T cells are shown to synthesize IFNγ, and activated CD8+
T cells have been shown to be cytotoxic. Only very weak serum IgG and vaginal IgA
responses are found after DNA immunization.

b. DNA Vaccine

Nude DNA vaccines use plasmid DNA to induce humoral and cellular immunity and
represent an attractive approach to produce antigen-specific tumor immunotherapy
(50). DNA vaccines have the following benefits over other vaccines(51). (1) The
vaccines can be easily set on a large scale with high purity (51,52). Genes inserted into
the vector (plasmid) can be easily modified, allowing convenient insertion and removal
of a particular sequences (51). (2) Stable temperature DNA vaccines which allows
economical transportation, especially in underdeveloped countries. They are very
stable relative to other proteins and biologic polymers (52). (3) The immune responses
induced by long-lasting DNA vaccines(51). (4) DNA immunization will not induce an
immune response against the DNA vector itself, so the DNA vaccine repeatedly can be
used. Although efficient viral vectors in delivering genes of interest to targeted cells,
immune recognition after adenoviral vector or vaccinia delivery inhibits re-vaccination
with the same delivery system (50). DNA vaccines are relatively safe and can be
repeatedly given (51). Retroviral vectors have potential virus-associated complications,
including virus replication and insertive mutagenesis (50). But the mode and sequence
of DNA delivery vaccine can affect the effectiveness of immune responses.

Nude DNA does not have clear cell type specificity (50). Therefore it is necessary to
find an efficient route for managing DNA vaccines to correspond target cells. This was
achieved by bulding a chimeric gene, Sig/E7/LAMP-1, where E7 was associated to the
endoplasmic reticulum translocation signal peptide (Sig) on its amino terminal and to
the transmembrane and lysosome targeting domains of the lysosome-related membrane
protein 1 (LAMP-1) on its carboxy terminus, and is inserted into the pCMV neo-
expression plasmid vector (53). This allows antigenic peptides of E7 to complex with
MHC class II molecules and enhances MHC class II presentation. Ji, et al. (50), in the
same laboratory, uing Sig/E7/LAMP-1 DNA to prevent tumor and treat tumor in mice.
The DNA vaccine was delivered by gene gun ballistic bombing into the abdominal
region. In the tumor regression experiments, mice were first challenged with murine
tumor cells which expressing HPV-16 E7 (TC-1 cells) injected subcutaneously to the
left side and then vaccinated with E7 or Sig/E7/LAMP-1 DNA. In tumor prevention
and tumor regression testing, Sig/E7/LAMP-1 DNA produced larger numbers of tumor
cell-specific CD4+ helper T cells and CD8+ CTLs, and increased the in vivo antitumor
effect, compared with wild-type E7 DNA. 60 percent of mice vaccinated with
Sig/E7/LAMP-1 DNA stayed tumor free 60 days after tumor injection. In comparison,
none of the mice vaccinated with wild-type E7 DNA remained tumor free. Results
showed that converting a cytosolic tumor antigen into the endosomal/lysosomal
compartment can greatly increase the in vivo therapeutic potency of nucleic acid
vaccines.

The same researchers (52) evaluated DNA vaccines to prevent and treat liver and lung
metastasis in mice. Tumor was delivered intraperitoneally, given intravenously by the
tail vein (lung study), or injected directly into the liver (liver study). Mice vaccinated
with the Sig/E7/LAMP-1 DNA produced the strongest E7-specific CTL activity,
highest number of E7-specific CD8+ cell precursors, and the highest titers of E7-
specific antibodies compared with the nude plasmid or the E7 DNA plasmid.
Eventhough E7 DNA and Sig/E7/LAMP-1 DNA produced strong antitumor immunity
in the liver and lung metastases models, the Sig/E7/LAMP-1 DNA was stronger under
tight conditions. These data indicated that antigen-specific DNA vaccination had the
potential to be applied to control liver and lung metastases of tumors with tumor-
specific antigens.

Although DNA vaccines have been demonstrated to induce CTL responses, not all
immunized individuals develop CTL. Therefore, the potential of DNA vaccines in
inducing CTL must be increased. Shi et al. (51) explored if a DNA vaccine could be
used to induce an E7-specific CTL response and protect against tumor challenge.
However, because HPV 16 E7 has changing potential, the authors used a mutated E7
which greatly reduced the transforming activity. Also, an unstable protein has greater
potential to produce CTL responses than a stable one. To develop a safe DNA vaccine
with low or no transformation activity and to increase the instability of the E7 protein,
they generated a plasmid that contained an HPV 16 E7 double mutation in the two Cys-
X-X-Cys repetitions (58 Cys → Gly, 91 Cys → Gly) under the direct control of the
cytomegalovirus immediate-early promoter and enhancer. This vaccine expressed a
degraded E7 protein rapidly and induced a significantly stronger E7specific CTL
response and better tumor protection in mice compared to a wild-type E7 DNA vaccine
expressing a stable E7 protein. The HPV 16 E7 mutant vaccine proven protected 100%
of mice against tumor challenge. Eventhough the wild-type E7 DNA vaccine protected
mice against challenge with HPV 16 E7-positive tumor cells, the protection was
incomplete. These data indicate that acceleration of protein degradation leads to
increase antigen presentation in the context of MHC class I and subsequently CTL
induction. But the authors warn that because E7 is not secreted and enters the MHC
class I path when not redirected into the MHC class II pathway, unfortunately no E7-
specific T helper cells are expected to be produced in this model. This might limit
prophylactic use of the vaccine and limit the expansion of E7-specific CTL.

Smahel et al. evaluated a prophylactic effect of DNA vaccination. Hamsters were

injected intramuscularly with 100 µg or 10 to 15 µg HPV 16 E6/E7 plasmid DNA
(p16HHMo plasmid consisting E6/E7 genes) 3 times at 3-week intervals. Ten days
after the third immunization, the hamsters were challenged with Syrian hamster cells
modifed with HPV 16 E6 and E7 and H-ras oncogene. A protective effect was being
observed only in animals inoculated with the higher 100 µg dose of p16HHMo/HPV
E6/E7 plasmid DNA. 11 of 12 hamsters remained without tumor for 50 days. Then, E7
and E6 antibodies were detected in 66.7% and 50% of sera, respectively. All positive
sera showed weak reactivity with peptide.

c. Viral vector vaccines

Mostly, immunization with intact soluble antigen does not activate CD8+ CTLs (54).
Therefore, to stimulate CTL responses to specific protein antigens, approaches focus
on intracellular antigen delivery have been tried (54). One approach was a direct viral
vectors. Viral vectors are very efficient in delivering genes to targeted cells (50). Use
of a recombinant viral vector that carries the genes for HPV 16 E6 and E7 proteins
allows the production of the target antigen in host cells and results in antigen processing
and MHC class I-mediated antigen presentation. This approach induces T-cell
proliferative, antibody, and CTL responses.
The main concerns for vaccines based on viral vectors or DNA injections are safety
problems relating to possibility of DNA integration into the host cell genome, which is
very relevant to HPV 16 E6 and E7 oncogenes with changing potentials (54). This is a
safer approach to use purified antigens in combination with an appropriate
noninfectious delivery system with minimal toxicity to stimulate immune responses
(54). Another concern is that immune recognition following adenoviral vector or
vaccinia delivery inhibits revaccination with the same delivery vector, while retroviral
vectors are potentially toxic due to auxiliary virus replication and insertional
mutagenesis (52).

Recombinant vaccinia viral vector showing HPV E6 and E7 (mutated to abrogate

changing capacity while maintainingthe estimated immunogenic regions) has been
given as a single vaccination to eight latestage cervical carcinoma patients in the United
Kingdom. All patients put on antivaccinia IgG antibody responses, and three of the
eight showed HPVspecific antibody. HPV CTLs developed in one of three evaluable
patients, and two patients were remained clinically healthy 15 and 21 months post-
vaccination. This study showed that HPV recombinant vaccinia viral vaccine could be
used in patients with advanced disease, and this vaccine may be benefits as adjuvant
therapy.

d. Immunomodulation

The success of a cancer vaccine depends, in part, on the induction of a tumor-specific

Th1-type immunity. Hallez et al. (55) evaluated the ability of interleukin-12 (IL-12)
express nonimmunologic murine HPV 16 changed cells to induce Th1-type immunity
(IFNγ production). The goal of their study was to develop a genetically modified tumor
cell–based vaccine capable of driving a protective in vivo immune response directed
against HPV-expressing murine cell lines. They showed that IL-12 expression provided
strong immunogenic properties to nonimmunogenic, HPV 16–changed BALB/c
kidney cells. Mice injected with tumor cells engineered to secrete IL-12 were protected
against the parental tumor and developed long-lasting Th1-like immune responses
directed at the viral encoded proteins. These data indicate that cell lines that expressing
HPV-related proteins and secreting IL-12 can provide a therapeutic important approach
for treating HPV-induced lesions.

Conclussions

The connection between cervical cancer and HPV infection requires HPV vaccine
development. The latest rapid development in papillomaviral vaccine research has
expanded the presented number of viral models (56). Emperically, a multivalent
vaccine that target many HPV types or a multi-vaccine program may be needed to
provide protection from infection or regression of viral lesions. It may be time to
compare vaccine models and immunological tests side by side to determine optimal
HPV vaccines and improve vaccine strategies

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58 International K, No P. Assessment report. 2018;44(June).

59 Susanti N. FARMASI BAB 5 : FARMAKOLOGI BERBAGAI KELOMPOK

OBAT. 2016;

SCORE FORM AND FEEDBACK

Item (Weight factor) Comments Score (0-20)

Structure and organisation
(2)
Content (3)
(Functional use of) figures
and tables (1)
(Use of) references (1)
Language and syntax (2)
Conclusion and reflection
(1)
Final score

Score Grade Weight Description

≥8.0 A 4 Outstanding
7.6-7.99 A- 3.7 Highly competent
7.25-7.59 B+ 3.3 Competent
 6.9-7.24 B 3 Partially
Competent
6.5-6.89 B- 2.7 Marginally
Competent
6.0-6.49 C 2 Insufficient
5.0-5.99 D 1 Highly insufficient
<5 E 0 Fail

h.j.woerdenbag@rug.nl