Development and Implementation of

Papillomavirus Prophylactic Vaccines

Ian H. Frazer
This information is current as J Immunol 2014; 192:4007-4011; ;
of June 9, 2014.

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doi: 10.4049/jimmunol.1490012
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Translating Immunology
Development and Implementation of Papillomavirus
Prophylactic Vaccines
Ian H. Frazer
Translation of basic scientific findings into practical pa-
tient outcomes is a significant exercise even when the
goal is conceptually straightforward, as in the develop-
ment of a vaccine for an infectious disease. Recognition
of the association of cervical cancer with papillomavirus
infection encouraged development of a vaccine to help
with prevention of this very common cancer, causing
over 250,000 deaths each year worldwide. To introduce
a vaccine program, it was however necessary to develop
a technology for making viral Ag, demonstrate that sys-
temic immunization could provide mucosal surface pro-
tection in the genital tract, develop assays for vaccine
potency, and understand enough about the epidemiology
and natural history of the infection to plan effective in-
tervention strategies. This process took ∼25 years. The
major hurdle, now that effective vaccines are available, is
to ensure their deployment in the countries where they
are most needed. The development and deployment of
human papillomavirus vaccines demonstrate the benefits
of collaborative research activity across the globe, and
between academia and industry, to translate scientific
discoveries into public health benefits. The Journal of
Immunology, 2014, 192: 4007–4011.
A
n association of viral infection with development of
proliferative skin lesions in animals was recognized
almost as soon as viruses were first described as fil-
terable agents causing disease (1). Evidence for association of
viral infection with specific cancers in humans took longer, as
causality could not be tested according to Koch’s postulates. It
is now accepted that nearly 20% of the global burden of cancer
in humans can be attributed to viral infection (2). One family
of viruses, papillomaviruses (PVs), is recognized as respon-
sible for a quarter of virus-associated human cancers and 5%
of the global cancer burden. One major cancer, cervical can-
cer, occurs only as a consequence of human PV (HPV) infec-
tion of the cervix and is responsible for over 250,000 deaths
annually (3).
From the perspective of the viral immunologist, PVs are
difficult to work with, as they are species specific and can only
replicate in differentiating epithelia. Unlike most viruses, PVs
have not been grown in monolayer cell culture, as their life
University of Queensland Diamantina Institute, Translational Research Institute, Uni-
versity of Queensland, Woolloongabba, Queensland 4102, Australia
Address correspondence and reprint requests to Dr. Ian H. Frazer, University of Queens-
land Diamantina Institute, Translational Research Institute, 37 Kent Street, Woolloon-
gabba, QLD 4102, Australia. E-mail address: i.frazer@uq.edu.au
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1490012
The Journal of
Immunology
cycle requires differentiating epithelial cells, and even in
organotypic culture it has proven hard to produce more virus
out than was provided as the infecting inoculum (4). These
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issues delayed research on HPVs, as although it was recog-
nized that the Shope (cottontail rabbit) PV was able to induce
cancers in rabbits (5), it was not possible, until the advent of
the molecular era, to determine that there were more than two
HPVs responsible for skin and genital warts. Because warty
lesions almost never turned malignant, and serology for PV
infection was difficult in the absence of quality Ag material
(6), the association of HPV infection with cancer had to wait
for molecular cloning and hybridization technology. This
allowed the hypothesis (7) and subsequent demonstration in
the 1980s by zur Hausen and colleagues (8, 9) of a PV DNA
in cervical cancers that was related to the PV DNA in genital
warts, but was not identical. Molecular typing of many can-
cers worldwide has subsequently established that .99% of
cervical cancer is associated with 1 of 10 genotypes of a-clade
HPV (10) termed “high risk” genital PVs. The commonest
association is with HPV16, as first identified by zur Hausen
and colleagues, which is associated with .50% of cervical
cancer worldwide. Subsequent epidemiological studies have
established that genital tract infections with cervical cancer–
associated PVs are extremely common, as .50% of young
women and men are infected with HPV16 within 3 years of
onset of sexual activity (11). Such infections are generally
benign, associated with neither symptoms nor cellular ab-
normalities (12), and are relatively short-lived, with 50% of
genital infection cleared within a year (11). However, per-
sisting PV infection of the cervix with a high-risk HPV is
associated with increasing risk of cervical dysplasia, and
eventually malignancy ensues in ∼30% of women with per-
sisting dysplasia (13). Epidemiological studies have also ex-
tended the catalog of human PVs to .200 (14), of which
most are b-clade viruses and appear to be benign skin sapro-
phytes, although ∼5 b-clade types are associated with skin cancer
in patients with a rare genetic disorder, epidermodysplasia ver-
ruciformis (15). The a-clade infects the genital tract, with
two (HPV6 and HPV11) genotypes responsible for most
genital warts, and ∼10 (including HPV16 and HPV18) as-
sociated with risk of cervical cancer, whereas several other
genotypes produce no evident disease. It is also now ac-
cepted that ∼50% of other anogenital cancers (vulvar, pe-
nile, and anal) and ∼50% of oropharyngeal cancers are
Abbreviations used in this article: HPV, human papillomavirus; PV, papillomavirus;
VLP, virus-like particle.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
4008
attributable to an initiating infection with high-risk a-clade
PVs (16).
The immunological challenge: translating knowledge into outcomes
Vaccines are the most effective public health measure for re-
ducing disease burden, after safe food and water. Although it
was not clear in the late 1980s whether HPV infections were
rare and commonly initiated cancers or, as turned out to be the
case, common with cancer a rare consequence of persisting
infection, the argument for developing a prophylactic vaccine
was strong. Protection of cattle against challenge with bovine
PV2 by a vaccine consisting of purified bovine PV2 virus (17)
and protection of dogs against canine oral PV by formalin-
inactivated virus (18) suggested that vaccination against human
PVs would be practical.
The first translational goal (Fig. 1) on the pathway to de-
veloping protection against HPV infection and cervical cancer
was to derive a source of Ag for HPVs. As PV could not be
grown in culture, the standard approaches to vaccination
through production of inactivated or attenuated virus were
not practical. The PV virion was well understood as non-
enveloped capsid comprising 72 pentamers of a major (L1)
capsid protein, and a variable amount of a minor (L2) capsid
protein. Some L1 genetic material cloned from tumor cells
was available in prokaryotic expression vectors, which pro-
duced an immunogenic L1 protein (19). However, this was
isolated from inclusion bodies, presumably in denatured
form, and it seemed likely that correct folding and assembly
of L1 into virion-like structures would be necessary to pro-
duce antigenic material that would express epitopes to induce
host-protective Ab responses. Several groups achieved expres-
sion of PV L1 and production of virus-like particles (VLPs),
visualized by electron microscopy, in the early 1990s using
a range of expression systems. This strategy had already
proven practical for a recombinant hepatitis B vaccine. Initial
research was undertaken with recombinant vaccinia expressed
in COS cells with L1 and L2 genes cloned from a clinical
lesion (20), and with recombinant baculovirus in insect cells
with L1 cloned from a tumor and, with better yields, using
the L1 gene from clinical material (21–24). In each case, L1
protein expressed in eukaryotic cells, which, for HPV16, re-
quired expression from the second translation initiation co-
don in the L1 open reading frame, self assembles into VLPs
morphologically resembling the naturally occurring virus.
Self-assembly allowed for a scalable industrial process pro-
ducing replicas of the PV virion sufficiently accurate in
protein conformational structure to become the basis of a
potential vaccine.
FIGURE 1. The stages of translational research. Research drives the pro-
gression of an observation on health from initial basic science (T0) through to
successful adoption of a proven effective intervention (T4). Each stage involves
different people with different skills, needing different funding sources and
evaluation through different output measures.
TRANSLATING IMMUNOLOGY: HPV VACCINES
The second translational goal was to determine whether a
vaccine based on HPV VLPs would induce virus-neutralizing
Ab and host protection. Again, the inability to grow PV pre-
sented problems, and there were also the conceptual issues of
whether systemic immunization would provide genital tract
protection, and whether it was desirable to immunize with
material from a virus associated with cancer. The natural im-
mune response to HPV infection was not well studied. De-
velopment of Ab was slow and relatively weak after infection,
with only 50% of subjects naturally infected with HPV16
producing measurable virion-specific Ab within a year of in-
fection (25). Furthermore, past HPV infection does not ap-
pear to protect effectively against reinfection with the same
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genotype of the virus, as demonstrated by significant rates
of infection in placebo recipients in the pivotal vaccine trials
with serological evidence of past HPV infection. Therefore,
utilization of human sera from infected subjects as reference
standards for immunogenicity and protection was not feasi-
ble. However, a key study in dogs challenged with canine
oral papillomavirus after immunization with COPV L1
VLPs (26) showed that L1 VLPs administered as a vaccine
with or without adjuvant induced high-titer Ab and protective
immunity against challenge with live virus. Protection re-
quired only Ab as protection and could be passively transferred
by immune serum, and Ab had to be directed at conformational
determinants on the VLP, as denatured VLPs did not produce
protection. Studies examining the in vitro–neutralizing activity
of polyclonal antisera raised against individual VLP types in ani-
mals established that HPV genotypes are distinct serotypes, and
therefore that the vaccines would need to be multivalent. De-
velopment of mAbs specific for PV virions (27) and partial so-
lution of the crystal structure of the PV VLP (28) confirmed the
clinical and laboratory evidence that PV genotypes were also
distinct serotypes, and that neutralizing Ab was directed against
surface-conformational determinants on the external face of the
L1 molecule that were nonconserved between HPV genotypes.
Subsequent studies demonstrated that, following immunization,
serum IgG was effectively transudated or exudated into the
genital tract, and is likely to be the basis of host protection (29).
The third translational goal was to ensure a pathway, and the
necessary reagents, for commercial development of vaccines
based on the PV VLPs. Extensive epidemiological studies in the
1990s had defined the widespread nature of high-risk HPV
infections, their common acquisition shortly after onset of
sexual activity, and the HPV genotypes most commonly as-
sociated with cancer. Having established patentable technologies
for VLP production, two vaccine companies (Merck & Co. and
GSK) took on the risk of developing and testing potential clinical
vaccine products. Merck used yeast fermentation technology to
make a quadrivalent vaccine based on HPVs 6, 11, 16, and
18 that might potentially protect against genital warts and
∼70% of cervical cancer, while GSK used baculovirus/insect
cell fermentation to make a bivalent HPV16 and HPV18
vaccine that would potentially protect against 70% of cervical
cancer but not genital warts. Both vaccines use adjuvanted
VLPs: Merck uses a proprietary aluminum hydroxide for-
mulation, and GSK uses an aluminum hydroxide adjuvant
supplemented with monophosphoryl lipid A, a TLR4 agonist.
A series of HPV genotype-specific Ab assays were developed to
assess vaccine immunogenicity, including ELISA with VLP
substrate (30), competitive inhibition assays using labeled
The Journal of Immunology
VLP-specific mAbs (31), and neutralization assays utilizing
VLP pseudovirions that could infect a reporter cell line with
a reporter gene (32). These assays have subsequently been
facilitated by Ab standards, which have been developed and
validated through the World Health Organization (33). The
efficacy endpoint of the phase 3 clinical trials of the HPV
VLPs was selected as prevention of premalignancy (cervical
intraepithelial neoplasia grade 2 or 3) attributable to the
HPV genotypes incorporated in the vaccines, and, for the
Merck vaccine, also prevention of genital warts. The vaccines
proved extremely effective in preventing cervical and other
premalignancy in large phase 3 clinical trials of women aged
15–26 who had no evidence of prior infection with HPVs of
the relevant genotypes (34). However, no protection against
development of premalignant disease was seen for women
already infected, but without evidence of disease at the time of
recruitment. Data on comparable Ab production then allowed
prediction of protection for younger women and men prior to
onset of sexual activity, as these are the preferred target pop-
ulation for publicly funded immunization programs. Registra-
tion of the vaccines for use to assist in prevention of cervical
cancer and, for the quadrivalent vaccine, for prevention of
genital warts, followed in most countries of the world between
2006 and 2010. Ab data have also been compared between
the two available vaccines. The GSK vaccine, adjuvanted with
a stronger adjuvant, produces higher titer Ab (35). However,
both vaccines proved to be essentially 100% effective in the
phase 3 clinical trials (36), and there is currently no evidence
to suggest that better initial Ab response translates into better
long-term protection. Both vaccines induce stable long-term
Ab titers, without evidence of breakthrough infection even in
those subjects with low initial Ab levels following vaccination,
precluding establishment of a surrogate marker for protection,
and both have proven safe in use, with only the usual local
and mild systemic reactions to vaccines commonly reported
(37), while the frequency of severe allergic reactions (38, 39)
is very low and comparable to other vaccines.
The fourth translational goal is to ensure effective deployment
of the HPV vaccines. For the developed world, cervical cancer
prevention prior to vaccine development was largely achievable
through effective implementation of screening programs based on
cervical cytology assessment and treatment of premalignancy by
surgical means. Vaccines are therefore an additional measure, with
the rationale for introduction based on analysis of cost effec-
tiveness and public wishes and acceptability. Some countries,
including Australia and the United Kingdom, were early adopters
of universal publicly funded immunization of young women
before the onset of sexual activity, and, from 2013, the Australian
program has been extended to young men. The success of the
program in Australia can be measured by a .80% uptake rate
of vaccination among 12- to 14-y-old girls, and by the virtual
disappearance of genital warts during the last 5 years, not only
among immunized younger women, but also among unimmu-
nized younger men (40), presumably as a result of the protective
effects of herd immunity. Significant reductions in the preva-
lence of HPV16 and HPV18 DNA have been similarly observed
in cervical smear samples from 18- to 24-y-old women (41).
Whereas herd immunity may help protect men from HPV-
associated anogenital and oropharyngeal cancers, the low level
of coverage of the female population in many countries (42) is
likely currently insufficient to provide good herd immunity, a
4009
justification for immunization of young men as a public health
measure where cost-benefit analysis permits.
Introduction of the HPV vaccines on a global basis have
encountered some significant issues (43), including an anti-
vaccine lobby that has tried to use misinterpreted adverse
events in vaccine recipients unrelated to vaccination, as well as
adverse events in placebo recipients, to suggest that the vac-
cines are not safe. This has had significant consequences in
India where vaccine programs were halted (44), and in in the
United States where uptake has been slow (∼33% for all three
doses in girls in 2012) (42). Vaccine costs have also impacted
on uptake. The initial programs sponsored by GSK and
Merck for the developing world, and the recent announce-
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ment that the vaccines will be made available to the Global
Alliance for Vaccines Initiative eligible countries at $5 U.S.
per dose, and that the Gates Foundation will meet this cost
for a period for approved programs (45), should enhance
more rapid deployment in the countries where cervical cancer
screening has not been implemented and most deaths from
cervical cancer occur. One clear lesson to date is that education
about the vaccine is key to its successful deployment, and that
education needs to be directed at health care professionals,
governments, recipients, parents, and schools to ensure effective
delivery programs (46). A recent program in Vanuatu, a de-
veloping Pacific island nation, established the high preva-
lence of high-risk HPV infection (47) and of cervical dysplasia
(48) in that country. This has enabled a joint program with
GSK to deliver vaccines through schools, which achieved
a 93% uptake rate of the full vaccine program across eligible
children on Efate Island. This program also established that
although there was a wide variation in nutritional status across
the immunized 10- to 12-y-old girls, the neutralizing Ab
responses after three immunizations in the quintile with the
lowest body mass index and skinfold thickness were not sig-
nificantly different from those in the other quintiles (Fig. 2).
The fifth translational goal is to improve the vaccine tech-
nology to achieve better adoption and better outcomes. The
recent announcement that a nine-valent vaccine based on the
current VLP technology and covering five further high-risk HPV
types has given good protection against each of these in HPV
naive recipients, without compromising the immune response to
the initial four types in previously unimmunized subjects (49),
suggests that vaccination should eventually replace screening
for cervical cancer as the primary means of cancer prevention.
Further studies will be needed to determine whether similar
broad-spectrum responses across all nine vaccine genotypes
can equally be obtained in individuals already immune to one
FIGURE 2. Ab to HPV VLPs as assessed by pseudovirion neutralization in
randomly selected HPV vaccine recipients in Vanuatu, stratified into quintiles
by body mass index.
4010
or more types through vaccination, or whether prior immunity
may divert immune responses toward already recognized geno-
types as predicted by the concept of “original antigenic sin” (50).
Although prior infection with one HPV genotype does not appear
to have influenced immunity to the others in the pivotal clinical
trials of the current vaccines, prior immunity generated by vac-
cination may be a more rigorous test of this concept. This will
possibly acquire increased significance if the current debate about
the role of b-PV in development of squamous skin cancer (51) is
resolved with the conclusion that these viruses do play a causative
role, and therefore that vaccination against b-PV is desirable.
A recent study has shown that two immunizations with the
GSK vaccine gave as good a durable Ab response as three
immunizations, with equal efficacy for disease prevention
over 4 years, whereas a single dose gave somewhat reduced
long-term Ab response but nevertheless with protection (52).
This observation may pave the way for cheaper and more
practical vaccine delivery strategies for the developing world,
although, as there are no surrogate markers of protection fol-
lowing vaccination based on Ab titer, this decision should be
based on clinical testing. Cheaper and more practical vaccine
delivery strategies may also allow programs aimed at preventing
other HPV-related cancers, including oropharyngeal cancer,
where men and women are equally affected, and where clin-
ical trials to provide an evidence base for protection are un-
feasible given the lengthy lag time between acquisition of in-
fection and clinical manifestation of disease.
This review has been focused on translation of knowledge of
the virology and immunology of HPV infection into practical
prophylactic vaccines, and has acknowledged that these vac-
cines have no efficacy as immunotherapeutics for existing
infection. Future research may enable development of effective
immunotherapy for HPV-associated cancers based on our
current knowledge of the potential target Ags and necessary
delivery strategies for such vaccines (53). This research is
justified by the estimated 25 million people already infected
with a high-risk HPV who will in consequence develop an
HPV-related cancer of the anogenital tract or oropharynx,
most of whom are in developing world countries where
effective treatment is not widely available.
Conclusions
A concerted effort from basic science and industry has enabled
the discovery of a connection between infection with HPV and
development of cervical cancer to be translated into a practical
means to prevent cervical cancer through immunization in
∼25 years. This success story demonstrates the benefits of
collaborative research activity between academia and industry
to translate scientific discoveries into public health benefits.
Disclosures
The University of Queensland derives royalties from HPV vaccines referred to
in this article. The author has accepted speaking engagements and expert ad-
visory panel consultancy payments from GSK and from Merck.
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