Journal of General Virology (2012), 93, 681–691 DOI 10.1099/vir.0.

039677-0

Review Genital human papillomavirus infections: current

and prospective therapies
Margaret A. Stanley
Correspondence Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK
Margaret A. Stanley
mas1001@cam.ac.uk
Infection with human papillomaviruses (HPVs) is very common and associated with benign and
malignant epithelial proliferations of skin and internal squamous mucosae. A subset of the
mucosal HPVs are oncogenic and associated with 5 % of all cancers in men and women. There
are two licensed prophylactic vaccines, both target HPV 16 and 18, the two most pathogenic,
oncogenic types and one, additionally, targets HPV 6 and 11 the cause of genital warts. The
approach of deliberate immunization with oncogenic HPV E6 and/or E7 proteins and the
generation of antigen-specific cytotoxic T-cells as an immunotherapy for HPV-associated cancer
and their high-grade pre-cancers has been tested with a wide array of potential vaccine delivery
systems in Phase I/II trials with varying success. Understanding local viral and tumour immune
evasion strategies is a prerequisite for the rational design of therapeutic vaccines for HPV-
associated infection and disease, progress in this is discussed. There are no antiviral drugs for the
treatment of HPV infection and disease. Current therapies are not targeted antiviral therapies, but
either attempt physical removal of the lesion or induce inflammation and a bystander immune
response. There has been recent progress in the identification and characterization of molecular
targets for small molecule antagonists of the HPV proteins E1, E2 and E6 or their interactions with
their cellular targets. Lead compounds that could disrupt E1–E2 protein–protein interactions have
been discovered as have inhibitors of E6–E6-AP-binding interactions. Some of these compounds
showed nanomolar affinities and high specificities and demonstrate the feasibility of this approach
for HPV infections. These studies are, however, at an early phase and it is unlikely that any specific
anti-HPV chemotherapeutic will be in the clinic within the next 10–20 years.

Introduction genotypes on the basis of DNA sequence and, since in vitro

growth of these viruses is problematic, HPV infection is
As recently as 1970 it was assumed that there was only one
determined by detecting HPV DNA. The viruses have a
human papillomavirus (HPV) and that infection with this
predilection for either cutaneous or mucosal epithelial
was the cause of the various warty lesions that decorated
surfaces and fall into two groups – low-risk types that
different tissue sites. HPV was seen, except in rare instances
predominantly cause benign warts or high-risk types
(Jablonska et al., 1972), as causing unsightly but essen-
associated with malignant disease. This risk stratification
tially trivial excrescences that, given time, would regress
is shown clearly in the ano-genital tract where about 30–40
spontaneously. The advent of recombinant DNA technol-
HPVs regularly or sporadically infect the mucosal epithe-
ogy overturned this simple view of the HPV world, and it
lium of men and women. The two most common low-risk
became clear within a decade that there were multiple
mucosal HPVs are HPV 6 and 11, which together cause
HPVs and that the warts on different tissue sites were
about 90 % of genital warts and almost all recurrent
caused by different HPV types with a tropism for mucosal
respiratory papillomas (RRP) (Lacey et al., 2006) as well
or cutaneous squamous surfaces (Orth et al., 1978). It also
as a proportion of low-grade, cervical intra-epithelial
became evident that HPV did not cause trivial disease but
neoplasms (CIN 1), vulval and vaginal intra-epithelial
that some members of the HPV family, particularly a
neoplasms grade 1 (VIN 1 and VAIN 1) and anal intra-
subset infecting the ano-genital tract, were true human
epithelial neoplasia grade 1 (AIN 1) (Moscicki et al., 2006).
carcinogens and were the cause of carcinoma of the cervix
There are about 15 high-risk mucosal HPV types of which
(zur Hausen et al., 1981), the second most common cancer
the most important are HPV 16 and 18, which together
in women worldwide (Arbyn et al., 2011).
cause more than 70 % of cervical carcinomas in women
HPVs are a very large family of dsDNA viruses comprising (Arbyn, et al., 2011). Cancers attributable to HPV are not
more than 180 types, numbered sequentially, that have confined to the cervix. In approximately 60 % of cancers of
been cloned from various clinical lesions (Bernard et al., the vagina, 40 % of vulva and penis, 80 % of anus (Parkin &
2010). These viruses are not classified as serotypes but as Bray, 2006) and more than 60 % of tonsil and base of

039677 G 2012 SGM Printed in Great Britain 681

M. A. Stanley
tongue (D’Souza et al., 2007) and the high-grade intra-
epithelial precursor lesions of these cancers, HPV DNA and
HPV early gene expression, principally HPV 16, can be
detected. Overall, current estimates are that 5.2 % of all
cancers in men and women are attributable to HPV and
this, together with the intra-epithelial lesions of all grades
and the warts, constitutes a huge disease burden.
Prophylaxis
There are two commercially available prophylactic HPV
vaccines, a bivalent HPV 16.18 virus-like particle (VLP)
vaccine from Glaxo Smith Kline Biologicals and a quad-
rivalent HPV 6, 11, 16, 18 VLP (qHPV) vaccine from MSD
Merck. Both vaccines have shown very high efficacy
against HPV 16/18 caused high-grade CIN 2/3 the end-
point accepted as the ethically acceptable proxy for
vaccine efficacy against cervical cancer (Kjaer et al., 2009;
Paavonen et al., 2009). The qHPV has shown greater than
98 % efficacy against HPV 6/11-associated genital warts
(women and men), HPV 6/11/16/18-associated VIN, VAIN
(women) and AIN (men) (Dillner et al., 2010). In the long-
term 30–50 years, and with high vaccine coverage, the
prophylactic vaccines will dramatically reduce the incidence
of disease associated with the vaccine HPV types. However,
the vaccines target only HPV 16 and 18 and in the case of the
qHPV vaccine HPV 6 and 11; there is some cross-reactivity
but this is partial (Paavonen et al., 2009). Despite the high
efficacy of the vaccines only 70 % reduction in cervical
cancers could be expected. Further, unless populations are
vaccinated and coverage exceeds 50 %, a large unvaccinated
population will remain with a huge burden of ano-genital
disease requiring treatment. The global burden of genital
HPV-associated disease is estimated as more than 60 million
cases per annum (Koutsky, 1997). Cutaneous HPV infec-
tions are common, treatments are unsatisfactory and there is
no prophylaxis. Effective, preferably virus-specific therapies
remain a priority.
Infectious cycle of HPV
Progress in the development of effective therapies for HPV
infection and disease has been slow, largely due to the
difficulties in studying the biology and pathogenesis of
these viruses that have a unique and complex replication
cycle. HPVs are exclusively intra-epithelial pathogens with
a replication cycle which is both time and differentiation
dependent. The viral replication cycle is one in which viral
infection is targeted to basal keratinocytes, but high level
expression of viral proteins and viral assembly occur only
in differentiating keratinocytes in the stratum spinosum
and granulosum of squamous epithelium (Doorbar, 2005).
The life cycle can only be reproduced experimentally in
complex, technically demanding in vitro culture systems
that generate small amounts of infectious virus (Meyers
et al., 1997). There are no easily manipulable animal
models for HPV and a mouse papillomavirus infection is at
a very early phase of analysis (Ingle et al., 2011). The viral
682
genome is small – 8 kb of dsDNA – and encodes a
maximum of eight genes, six of which encode non-
structural or early proteins E1, E2, E4, E5, E6 and E7 and
two of which encode structural or late proteins L1 and L2
(Fig. 1). Viral genes are differentially expressed both
temporally and spatially throughout the infectious cycle.
(Fig. 2). Despite the small genome the number of gene
products is much larger because of the complex use of
splice sites within the genome (Schwartz, 2008)
A further complication of these infections is the phenom-
enon of latency. After infection, HPV DNA can remain latent
within cells even though others have entered the productive
cycle. Spontaneous wart regression is immune mediated, but
this does not result in virus clearance and viral genomes can
be detected in apparently normal epithelium (Abramson
et al., 2004) many months and years after wart regression
(Moore et al., 2002). However the strong, local, cell-
mediated immunity that engenders regression of HPV-
infected lesions probably controls latent infection and, in
healthy immunocompetent individuals, recurrence of dis-
ease is unlikely. In contrast, immunosuppression predisposes
to reactivation as is demonstrated by the high levels of
genital HPV infection and neoplasia seen in immuno-
supressed organ transplant recipients and those with human
immunodeficiency virus (HIV) infection (Palefsky et al.,
2006). Antiviral chemotherapies are essential for such
patients.
Ano-genital HPV-associated disease
HPV infection of the ano-genital skin and mucosae results in
lesions with two morphologies – ano-genital warts (con-
dyloma acuminata) and squamous intra-epithelial lesions.
Condylomata are associated predominantly, but not exclu-
sively, with infection by low-risk types – HPV 6 and it’s
relatives. These are frank, polyploid growths that generate
infectious virus and have a low to negligible risk of malignant
progression. Squamous intra-epithelial lesions are classified
histologically and form a distinct spectrum of histological
atypia. In the cervix these are graded on the degree to which
they have lost cytoplasmic maturation and exhibit cytologi-
cal atypia. In Europe three grades of cervical intra-epithelial
neoplasia (CIN) are recognized: CIN 1, mild; CIN 2,
moderate and CIN 3, severe. In the vagina, vulva, penis
and anus a similar but not identical spectrum of changes can
be identified, VAIN, VIN, PIN and AIN. There is con-
siderable confusion about the terminology used to define
these lesions and to ensure clarity in this review the term
high-grade intra-epithelial neoplasia (HGIN) includes: CIN
2/3, AIN 3, PIN 3, VIN 3 and VAIN 3; low-grade squamous
intra-epithelial neoplasia (LGIN) encompasses CIN 1 and
the equivalent lesions in other sites.
LGIN at any site can be associated with both high- and
low-risk HPV types, although low-risk types predominate
(Moscicki, et al., 2006). The majority of lesions maintain
the virus as an episome, support a complete virus replication
cycle and viral gene expression is tightly regulated. Late
Journal of General Virology 93
 HPV therapy

HPV genome organization

URR
E6 E7
Promoter and enhancer URR
elements
Viral ORI
AL Early genes
L1
E1 _ replication
HPV 16 E2 _ replication and
E1
transcription
Late genes E4 _ viral release

L1 _ major capsid E5 _ immune evasion

AE
protein E4 E6 _ binds p53
_
L2 minor capsid L2
E7 _ binds pRB
protein E2
E5

Fig. 1. This cartoon demonstrates the genomic organization of HPV 16. The genome contains early and late regions that relate
to the position of the genes within the genome and their timing of expression during the viral life cycle. The early region encodes
a number of genes that function at the level of viral replication and transcription: E2, E1, E6 and E7 genes. E2 encodes a protein
that has an auxiliary role in viral replication and also functions at the level of transcriptional regulation of the viral early genes.
Many studies have shown that it is a negative regulator of viral gene expression. The E6 and E7 genes encode the major
transforming proteins of the oncogenic HPVs, but it should be noted that although they are transforming proteins they also have
important roles in the normal viral life cycle. The late region encodes viral structural proteins, with L1 the major capsid protein
and L2 the minor capsid protein. Control of early gene transcription and replication is conferred by the upstream regulatory
region, which contains promoter and enhancer elements, as well as the viral origin of replication. Here, the transcriptional
apparatus assembles and generates polycistronic transcripts that utilize the ‘early’ polyadenylation signal.

genes are expressed and virus particles generated. Warts and
low-grade squamous intra-epithelial lesion at all sites are
likely to be amenable to the same therapeutic strategies.
HGIN are associated almost exclusively with high-risk
types. In general because of the defects in cellular
differentiation that characterize these lesions, they do not
support a complete viral infectious cycle. Late gene
expression is either lost or significantly reduced, the viral
DNA sequences may be integrated into the host genome
and E6–E7 oncogene expression is deregulated. The genetic
instability of these high-grade lesions and the changes in
viral gene expression such as the loss of E2 and E1
expression that occur in association with this, means that
they are unlikely to be amenable to the same therapeutic
strategies as low-grade lesions.
Current therapeutic options
Ablative/excisional therapies. Current therapies for
ano-genital HPV infections are mostly ablative and/or
cytodestructive (von Krogh et al., 2000). Ablative therapies
for genital warts include cryotherapy, scissor excision,
laser therapy and electrosurgery (Sonnex & Lacey, 2001).
Physically ablative therapies such as cryotherapy are often
highly effective in the short term, with clearances of 70–80 %,
but recurrence rates can be as high as 25–39 % despite
multiple treatments (Maw, 2004).
CIN 3 arises almost exclusively at the squamo–columnar
junction. Disease therefore is localized even though infection
may be regional and excisional therapies such as loop
excision of the transformation zone are highly effective
(Prendiville, 1995). However, AIN, PIN, VAIN and VIN are
often multi-focal, both disease and infection are regional
and ablation may not be feasible or, if attempted, ineffective
(Jones, 2001).
Non-surgical treatments
Cytotoxic agents. Cytotoxic agents are widely used in the
treatment of genital warts (for review see Viera et al., 2010).
They are topical preparations that kill cells on contact,
irrespective of HPV status, by anti-proliferative or chemo-
destructive modes of action.
Podophyllotoxin. Podophyllotoxin, as a cream (Europe) or a
gel (USA) that can be self applied by the patient, is the first
line treatment for genital warts (Lacey, 2005) achieving
50 % clearance, but with recurrence rates of 25–30 %
(Maw, 2004). The mechanism of action is thought to be
due to the binding of lignans to microtubule proteins with
cell cycle arrest at metaphase. Podophyllotoxin is contra-
indicated in pregnancy.
Trichloracetic acid (TCA). TCA is a clinic-based topical
therapy that has a local caustic action effectively generating a
chemical burn of the wart. It is as effective as podophyllotoxin

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M. A. Stanley

Infectious cycle of high-risk HPVs

Virus-laden cells ready for _
Virus particles assembled desquamation and infection of 6 12 weeks
naive individual L1, L2
Differentiated cells L1, L2, E4
E and L viral Viral genomes at 1000’s per cell
genes expressed E6, E7, E1, E2, E5
Viral DNA amplification in
Dividing cells
non-dividing cells
only E genes expressed
very low levels of Viral and cell replicate together
protein made E1, E2
0 week
E1, E2 ? E5, E6, E7
Virus infects a primitive
basal keratinocyte

Fig. 2. Papillomaviruses are absolutely species-specific and tissue-specific. HPV will only infect and replicate in a fully
differentiated squamous epithelial cell. The virus infectious cycle is rather complex and can explain the duration of an HPV
infection. It involves both temporal and spatial separation of viral protein expression. The virus first infects a keratinocyte in the
basal layer of the epithelium as a consequence of microtrauma, i.e. an abrasion of the epithelium that exposes the basement
membrane and basal cells. In the proliferative compartments of the epithelium, there is a phase of plasmid maintenance and the
virus, the cells replicate together and viral copy number is maintained at around 50–100 in the daughter cells. For the oncogenic
viruses in particular, viral gene expression is very tightly controlled during this phase. As long as the cell is dividing, the high-risk
HPVs control the expression of their viral proteins very tightly. The oncogenes E6 and E7 are thus expressed at very low levels.
When the host cell stops dividing and begins to differentiate into a mature keratinocyte, this provides a signal to the virus to
activate all its genes to increase viral genome copy number to the thousands. In the case of incipient malignancy, control of E6
and E7 expression is lost and gene expression in the cell becomes deregulated. In the top layers of the epithelium, all the viral
genes, including those encoding the L1 and L2 proteins, are expressed and many thousands of viral genomes are encapsidated.
They exit the cell as infectious virus particles. The time taken from infection to the generation of infectious virus is at least 3
months. HPV thus has a very long infectious cycle, has no blood-borne phase and does not cause cell death.

but can result in ulceration, dermal scarring and
secondary infections if inappropriately applied. It has,
however, no systemic toxicity and can be used during
pregnancy.
Immunomodulators.
Imiquimod. Imiquimod formulated as the self-applied topical
therapy Aldara is a pharmacological agent that can modulate
innate immune responses. Imiquimod is an agonist for Toll-
like receptor 7, ligation of which activates dendritic cells,
macrophages and keratinocytes to release type I interferons
(IFNs) and other pro-inflammatory cytokines (Stanley,
2002). Randomized clinical trials (RCTs) with Imiquimod
5 % cream applied topically to genital warts have shown
efficacy and safety and a reduced recurrence rate (12 %)
compared with placebo (30 %) (Beutner et al., 1998). It is
feasible that Imiquimod will have a therapeutic effect on
intra-epithelial disease and small trials on VIN (Terlou et al.,
2011) and AIN (Fox et al., 2010) have demonstrated efficacy,
but the drug is not licensed for this use. The inflammatory
side effects of Imiquimod (erythema, oedema, itching and
pain) have limited its use on mucosal surfaces.
Immunomodulators such as Imiquimod that induce the
secretion of type I IFNs should be used with caution on
intra-epithelial lesions infected with high-risk HPV types.
Experimental studies in vitro have shown that both
exogenously or endogenously derived IFN-a and IFN-b are
highly effective in cells containing episomal HPV DNA,
inducing growth arrest and episomal loss, but have no
growth inhibitory effects on cells containing integrated HPV
DNA species and thus select for cells with integrated genomes
(Herdman et al., 2006; Pett et al., 2006). Repeated and
protracted use of preparations that generate locally high
concentrations of type I IFNs could result in the selection of
cells with integrated HPV, thus driving progression to high-
grade intra-epithelial and/or malignant disease.
Polyphenon E. Polyphenon E is a standardized extract of
green tea leaves from Camellia sinensis that, self adminis-
tered as a topical ointment, has shown efficacy against
genital warts in RCTs with lower recurrence rates and
greater clearance of warts in the treated (54 %) compared
with placebo groups (35 %) (Tatti et al., 2010). The main
catechin in Polyphenol E is (–) epigallocatechin gallate
(ECGC), a molecule that impacts on multiple signalling
pathways, inducing cell cycle arrest or apoptosis via caspase
activation, altered Bcl-2 family member expression and
inhibition of telomerase activity (Beltz et al., 2006; Yang
et al., 1998). ECGC has been shown to have antioxidant
properties, blocking nitric oxide production by suppres-
sing inducible nitric oxide synthase via blocking nuclear
translocation of the transcription factor nuclear factor-kB
as a result of decreased IkB kinase activity (Ahmad et al.,
2000; Khan et al., 2006).

684 Journal of General Virology 93


Cidofovir. Cidofovir is a monophosphate nucleotide ana-
logue of deoxycytidine (dCTP). After undergoing cellular
phosphorylation, it competitively inhibits the incorpora-
tion of dCTP into viral DNA by viral DNA polymerase.
Incorporation of the drug disrupts further chain elonga-
tion. Small trials in CIN have been reported (Van
Pachterbeke et al., 2009) with promising efficacy. Cido-
fovir is currently being used off-licence to treat different
viral infections, such as benign low-risk human HPV-
related RRP but concerns about safety have been raised
(Donne et al., 2009).
Future prospects
Antivirals for HPV proteins
At present there are no virus-specific therapies for
papillomavirus infection, but such therapies are necessary
for several reasons. Antiviral therapy has the potential to
treat both inapparent HPV infection as well as visible
clinical disease. There is a significant population of HPV-
infected, immunosuppressed individuals who, at the
present time, cannot be treated with immunotherapy –
drugs are their only option. Multifocal lesions such as VIN
and AIN are not amenable to ablation, may not respond to
immunotherapy but could be targeted by chemotherapy
and furthermore antivirals, unlike immunotherapies, may
not be HPV-type restricted in their efficacy.
Traditionally, antiviral therapies have targeted viral enzy-
mes. The papillomaviruses encode only one enzyme, the E1
helicase, rather limiting traditional approaches. The remain-
ing early genes E2, E4, E5, E6 and E7 function largely by
macromolecular interactions with host DNA and proteins
and these interactions have not been readily amenable to
drug design and the high-throughput screening necessary to
identify candidate molecules.
E1 and E2 proteins as antiviral targets. E1 and E2 are the
most highly conserved of the papillomavirus proteins and
are essential for viral DNA replication. E1 is the only viral
enzyme with ATPase and helicase activity. The protein is
divided into an N9 domain (Amin et al., 2000), a sequence-
specific DNA-binding domain (DBD) (Titolo et al., 2003)
and a C9 helicase domain (Seo et al., 1993; Titolo et al.,
2000). E2 is a sequence-specific DNA-binding protein with
roles in DNA replication, transcriptional regulation (Hegde,
2002) and partitioning of viral genomes to daughter cells
during mitosis (Ilves et al., 1999). The protein is organized
into two functional domains, an N9 transactivation domain
(TAD) involved in transcriptional regulation and direct
association with E1 and a C9 DBD/dimerization domain
(Hegde, 2002). Both domains are separated by a hinge
region whose function remains poorly characterized.
HPV genome replication requires both E1 and E2 (Chiang
et al., 1992; Yang et al., 1991). E1 monomers are specifically
recruited to the viral ori through interaction with the E1
DBD as well as protein–protein interaction with the E2
TAD bound also to the viral ori via the E2 DBD (Sanders
http://vir.sgmjournals.org
HPV therapy
& Stenlund, 1998). The E1 DBD dimerizes on the ori,
additional E1 monomers are recruited, a double hexamer
forms and in the process melts the duplex DNA, resulting
in the assembly of a hexamer around each strand of the ori
DNA (Fouts et al., 1999). ATP plays an important role in
these processes. ATP-binding enhances the E1–E2 inter-
action with the viral ori (Titolo et al., 1999), but also
changes the conformation of the E1–E2 protein interaction
(White et al., 2001) and as the E1 double hexamers are
formed E2 dissociates and DNA pol-a is recruited to the
viral ori (Masterson et al., 1998) via p70 and E1 (Lusky
et al., 1994), the full replication complex assembles and
viral DNA replication proceeds. In theory any one of these
interactions could be targeted by small molecule inhibitors.
Small molecule inhibitors targeting the ATPase activity of
HPV 6 E1 were identified by high-throughput screening of
a large collection of in house compounds by scientists
working for Boehringer–Ingelheim Canada (White et al.,
2011). The lead molecules from this screen were biphenyl-
sulfonacetic acid analogues (Faucher et al., 2004) whose
mode of action probably affected ATP binding by an
allosteric mechanism (White et al., 2005). Unfortunately
this class of inhibitors were not active in cell-based assays.
These workers also screened for inhibitors of helicase
activity but were unsuccessful, an outcome shared by many
research groups both in academia and industry.
Inhibitors of E1–E2 interactions. The conventional
wisdom is that small molecules will be unable to inhibit
protein–protein interactions because the interfaces are
large, relatively flat and without pockets into which
small molecules can bind (Fradet-Turcotte & Archambault,
2007). White and colleagues addressed these issues in a
series of detailed studies to identify inhibitors of the co-
operative assembly of HPV E1 and E2 on the ori, focussing
on HPV 6 and 11 (White et al., 2011). They identified two
series of inhibitors that bound to overlapping sites at the E1-
binding interface on the E2 TAD (Yoakim et al., 2003). Both
series of compounds, the indandiones and repaglinides, were
optimized for binding by medicinal chemistry approaches
and in both series the best compounds had nanomolar
activity against HPV 11 E1–E2 activity (Goudreau et al.,
2007). These studies demonstrate that druggable pockets can
be found at protein interfaces even though the apo crystal
structure may not appear favourable and, as the authors of
these studies comment, ‘binding of a ligand can be necessary
to induce formation of a pocket’ (White et al., 2011).
Unfortunately when the high efficacy of the prophylactic
vaccines in randomized control trials was demonstrated,
these studies on E1–E2 inhibitors were halted by the
pharmaceutical company.
Inhibitors of E2 binding to cellular proteins. Other targets
for small molecule inhibitors of E2 are in the frame.
The bromodomain and ET domain (BET) proteins that
bind acetylated histones are of considerable contemporary
interest as potential anticancer and antiviral targets (Smith
685
M. A. Stanley
et al., 2010). Brd4, one of the most studied of the BET
proteins, binds to bovine papillomavirus E2 to tether the
viral episome to the mitotic spindle and permit equal
partitioning of viral genomes at mitosis (Abbate et al., 2006;
McBride et al., 2004). There is accumulating evidence that
Brd4 is a cellular-binding partner for the E2 protein of both
low and high-risk HPVs (Gagnon et al., 2009), and that these
interactions are important for the maintenance of viral
episomes and transcriptional regulation of viral oncogenes
(Smith et al., 2010). Inhibition of Brd4–E2 interactions are
potential drug targets; recently structure and function
studies of a small molecule competitive inhibitor of Brd4,
JQ1, that displaces Brd4 from chromatin were reported
(Filippakopoulos et al., 2010).
E6 and E7 as antiviral targets. The combined actions of
the high-risk E6 and E7 oncoproteins are essential for the
maintenance of the neoplastic phenotype and the evasion of
apoptosis (Moody & Laimins, 2010). Abrogation of either
E6 or E7 function (or both) in neoplastic cells by targeting
gene expression or protein–protein interactions should be
an effective strategy. They are therefore the targets of choice
for high-grade disease and a number of approaches have
been employed: siRNA, antisenseRNA, ribozymes and
peptide aptamers (Govan, 2005). In vitro data using HPV-
expressing cells suggest that targeting E6 and/or E7 is a
feasible approach, but to date, unfortunately, these proteins
have not been amenable to inhibition by small molecules,
although some progress has been made with E6.
The high-risk HPV E6 gene encodes a small protein of
approximately 150 aa. This protein deregulates the cell cycle
and one of the key molecular interactions by which this is
achieved is through inhibition of p53 function. E6 binds
to p53 to form a stable complex, which then undergoes
proteolytic degradation. This E6-mediated degradation
requires a third protein, E6-AP, which in combination with
E6 acts as a ubiquitin ligase (Talis et al., 1998). E6 mediates
degradation not only of p53 but also the PSD95/Dlg/ZO-1
domain-containing proteins that modulate signalling path-
ways deregulating differentiation and E6-AP appears to be
the primary partner in these E6 degradation functions
(Nominé et al., 2006). Inhibition of the E6–E6-AP complex
should reactivate p53 function in infected cells, resulting in
growth arrest and/or apoptosis. Small peptides that inhibit
E6–E6-AP binding by competing with E6-AP have been
designed (Be et al., 2001; Liu et al., 2004) and there are some
candidate inhibitors that have potential for optimization for
potency and selectivity, but these studies are at a very early
stage and at least a decade from the clinic.
Protecting p53 from E6-mediated degradation. Small
molecules that target p53 and protect it from E6-mediated
degradation rather than a direct inhibition of E6 may be
more tractable strategies. In this regard the small molecule
activation of p53 and induction of apoptosis (RITA) was
shown to suppress the growth of cervical cancer cells in
vitro and also of carcinoma xenografts in vivo (Zhao et al.,
686
2010). RITA blocks p53 ubiquitination by preventing
E6-AP binding to p53 with the consequent upregulation
of proapoptotic p53 targets Noxa, PUMA and BAX
and downregulation of pro-proliferative factors cyclin B1,
CDC2 and CDC25C.
Selective inhibition of the proteasome is a property of HIV
proteases, raising the possibility that the drugs that inhibit
this function might also affect the ability of E6 to mediate
proteasomal p53 degradation. This notion has been
explored by Hampson and colleagues in a series of studies
that have shown that Lopinavir, an anti-retroviral protease
used in a fixed-dose combination therapy (KALETRA) in
HIV infections, can stabilize p53 and induce apoptosis of
HPV-positive cell lines (Hampson et al., 2006; Kim et al.,
2010). They have shown further that the mechanism
of action of Lopinavir is related to its ability to block
viral proteasomal activation and induce upregulation of
RNASEL in HPV-containing cells (Batman et al., 2011).
These are interesting and promising data, but HIV patients
receiving Lopinavir do not show enhanced clearance of
HPV-associated lesions (De Vuyst et al., 2008). However,
the concentration of drug required to mediate the in vitro
growth inhibition is higher than that achieved by oral
administration (Hampson et al., 2006) and cervico-vaginal
concentrations in patients are probably sub-optimal. If a
topical preparation of Lopinavir can be formulated then
clinical trials to test the safety and efficacy of the drug
against both low- and high-grade intra-epithelial lesions
would be indicated. Since Lopinavir is a licensed drug a
successful outcome to such a trial could result in a topical
treatment for high-grade HPV-associated disease being in
the clinic within years rather than decades.
Inhibition of virus entry
Prevention of virus entry by microbicides or virocides is
an alternative approach to targeting viral proteins after
exposure. Virus entry for HPV 16, 18 and related types is
a complex process that requires epithelial microabrasion and
wound healing (Sapp & Bienkowska-Haba, 2009). The
microabrasion results in full thickness epithelial denudation
but retention of the basement membrane (BM) (Roberts
et al., 2007). HPV binds initially to heparin sulphate
proteoglycans (HSPGs) in the BM via the L1 protein before
attaching to and entering the wound keratinocyte (Kines
et al., 2009). Carrageenan, a sulphated polysachharide
extracted from red seaweed, is a potent inhibitor of the
attachment of the virus particle to HSPGs on the BM and the
cell surface. In an experimental cervico-vaginal challenge
model in the mouse, carrageenan was highly effective at
preventing HPV transmission to cervical epithelial cells
even in the presence of nonoxynol-9, a product that en-
hances HPV infection in this model (Roberts et al.,
2007). Carrageenan is a component of several gels used as
lubricants in vaginal examination and the use of carragee-
nan-based gels during digital vaginal examination and Pap
smear collection is advisable (Roberts et al., 2011).
Journal of General Virology 93

Immunotherapy
Prophylactic HPV VLP vaccines are highly effective and in
the long-term should control HPV infection and disease
against the HPV types in the vaccine. In the interim,
however, therapeutic intervention to enhance or induce
effective immune targeting and clearance of established
infections is an attractive strategy. Such therapies have the
potential for treating inapparent infection and/or disease in
addition to clinically visible lesions. A Th1 biased cell-
mediated immune response is critical for regression of
HPV-induced disease (Stanley, 2006). Agents, therefore,
that enhance or induce strong cell-mediated immune
responses would be predicted to be effective HPV thera-
pies. In HPV-associated cancers and HGIN, oncogenic
viral gene expression is deregulated and the E6 and E7
genes are constitutively expressed. The continued expres-
sion of these oncogenes is essential for progression to,
and maintenance of, the malignant phenotype. In effect,
therefore, there are only two possible antigenic targets, E6
and E7, since these are the only viral proteins that will be
expressed in all cancers and HGIN.
The approach of deliberate immunization with E6 and/or
E7 of HPV 16 and 18 predominantly, and the generation of
antigen-specific CTL as an immunotherapy for HPV-
associated cancer has been tested with a wide array of
potential vaccine delivery systems in transplantable rodent
tumour models (reviewed by Su et al., 2010). It turns out
from these studies that HPV-expressing cancers in mice are
relatively easy to cure, but human HPV-induced cancers
and HGIN have been, to date, largely refractory to the
approaches successful in rodents. All the vaccines tested in
clinical trials in humans have been safe and well tolerated,
they have induced vaccine-specific T-cell responses of
varying magnitude, but these did not necessarily correlate
with clinical responses (Trimble & Frazer, 2009).
Successful therapeutic HPV vaccines – what is
needed
Recent trials in patients with VIN (Daayana et al., 2010; Kenter
et al., 2009) provide reasons for cautious optimism and there
is now the outline of a blueprint that could lead to successful
therapeutic immunization of HPV-associated disease.
Immunogenicity
Firstly, vaccines must be highly immunogenic. Adjuvants
will be central to enhancing the immunogenicity of HPV
therapeutic vaccines. Early innate immune responses shape
subsequent immune responses and the development of
adjuvants that exploit innate signals specifically focusing
and biasing the adaptive response to cytotoxic effector Th1
type will be critical for effective HPV therapeutic vaccines.
The importance of robust immunogenicity and adjuvantation
has been demonstrated in a recently reported Phase II trial
(Kenter et al., 2009) in which patients with longstanding and
refractory VIN 3 were immunized with a highly immunogenic
http://vir.sgmjournals.org
HPV therapy
vaccine comprising HPV 16 E6 and E7 overlapping synthetic
long peptides with an oil in water adjuvant: 47 % showed a
durable and complete regression histologically and clinically.
Importantly, responders had small lesions and regression was
associated with a strong and broad HPV-specific Th1 type
CD4+ and CD8+ systemic T-cell response that peaked after
the first vaccination; non-responders on average had larger
lesions and mounted a vaccine-induced HPV-specific regu-
latory T-cell response (Welters et al., 2010).
Manipulation of the local, lesional, immune milieu
Secondly, the local mucosal immune milieu must be
manipulated. The many published studies show that
systemic T-cell responses to HPV infections are weak and
transient, but the presence of an intense T-cell infiltrate in
regressing lesions implies that modulating the local immune
micro-environment will be critical for successful immuno-
therapy. The cellular effectors in these local responses are
still not unequivocally identified, but regression of CIN 1 in
longitudinal studies has been shown to be correlated with
the presence at study entry of functional cytotoxic CD8+
cells producing Granzyme B (Woo et al., 2010). Intra-
epithelial CD8+ T-cells in the cervix express the intra-
epithelial homing receptor a4/b7 and, significantly, in a
retrospective analysis, CIN regression correlated with the
expression on the stromal microvascular endothelium in
dysplastic lesions of the mucosal addressin cell adhesion
molecule 1, the ligand for a4/b7 (Trimble et al., 2010). This
suggests that with increasing disease severity cytotoxic
effector lymphocytes could not exit the lymphatics and
vessels and enter the local lesion. Generating large numbers
of circulating vaccine-induced HPV-specific cytotoxic
effectors and antigen-specific CTL will be ineffective if their
ingress into the lesion is prevented and/or they are disabled
by regulatory T-cells when they arrive.
Immune trafficking and modulation of the local milieu
could be modified by mucosal immunization. The route of
administration significantly affects immunization outcome
and a mucosal route would be desirable for both practical
and target driven reasons, but immune induction at different
mucosal surfaces requires specific signals. Vaccines delivered
to mucosae will almost certainly require specific adjuvant
formulations to efficiently target the mucosal inductive sites
and induce strong cell-mediated Th1 type responses at these
sites. Recent studies give some support to the notion of
local immunomodulation. In a trial in patients with VIN
3 combining topical Imiquimod (functioning as a local
immunomodulator/adjuvant) with systemic immunization
with an HPV 16/18 E6/E7 protein delivered via vaccinia virus
(TA-CIN) resulted in 32 % of patients showing complete
regression of lesions (Daayana et al., 2010).
Regulating the regulators
Therapeutic vaccines cannot cure large established malig-
nancies or large persistent pre-cancerous lesions and the
687
M. A. Stanley
evidence to date is that this is due, at least in a large part, to
the dominance of T regs over cytotoxic effectors in the
lesions (van der Burg et al., 2007; Welters et al., 2010). If
therapeutic HPV vaccine success is to be improved then
either T regs should be depleted or the pool of cytotoxic
effectors increased, or both. T regs, probably because of
their lower intracellular ATP levels, are more sensitive to
cyclophosphamide than other T-cell subsets. Low-dose
cyclophosphamide treatment has been used to modify the
immune milieu in giant condyloma acuminata after laser
surgery, resulting in a reduction in Foxp3+T-cells and
absence of recurrence (Cao et al., 2010). Potentially one
could target the receptors and their ligands that regulate T-
cell function either by expanding the pool of effector T-
cells (Curran et al., 2010) or by reversing the T-cell anergy
and immunosuppression (Sharma et al., 2009) induced by
T regs. All of these strategies have been or are being tested
in mouse models and have shown their potential, but
transfer to the clinic remains to be achieved.
Conclusions
The fact that there are many therapies available for the
treatment of HPV-associated lesions is a reflection of the
reality that, on the whole, these are unsatisfactory. Current
therapies are not targeted antiviral therapies, but either
attempt physical removal of the lesion or induce inflam-
mation and a bystander immune response. There has been
recent progress in the identification and characterization of
molecular targets for small molecule antagonists of the
HPV proteins E1, E2 and E6 or their interactions with their
cellular targets. Lead compounds that could disrupt E1–
E2 protein–protein interactions have been discovered as
have inhibitors of E6–E6-AP-binding interactions. Some
of these compounds showed nanomolar affinities and
high specificities and demonstrate the feasibility of this
approach for HPV infections. These studies focussed on
the mucosal low-risk HPV 6 and 11 and on the major
oncogenic HPV 16 and 18 and their close relatives.
However, once the efficacy of the prophylactic vaccines
that target these HPV types was demonstrated in RCTs
further development of the small molecule programmes, at
least those in house in large Pharma, was halted in the
expectation that the market for antivirals would be very
significantly reduced.
The approach of deliberate immunization with HPV 16/18
E6 and/or E7 and the generation of antigen-specific CTL as
an immunotherapy for HPV-associated cancer and their
high-grade pre-cancers has been tested with a wide array of
potential vaccine delivery systems in Phase I/II trials with
varying success. Advances in our understanding of viral
and tumour immune evasion strategies has emphasized
that modulation of the local tumour immune micro-
environment combined with the induction of strong
systemic HPV E6 and E7 antigen-specific cytotoxic effector
responses are prerequisites for successful therapeutic
immunization.
688
The prophylactic vaccines are highly effective but focussed
on a few types, broadly cross-protective L2 vaccines are in
development pre-clinically, but have not entered clinical
trials to date. The reality is that for the next 20–40 years at
least there will remain a large unvaccinated population of
women and men for whom treatment, whether antiviral
drugs or immunotherapy or both, is needed. In addition,
there is a need for effective antiviral therapy for the large
population of immunosuppressed individuals and for the
neglected cutaneous infections for which prophylaxis may
not be effective or developed.
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