Review Article

Indian J Med Res 130, September 2009, pp 296-310

Anti-human papillomavirus therapeutics: Facts & future

Alok C. Bharti, Shirish Shukla, Sutapa Mahata, Suresh Hedau & Bhudev C. Das*

Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, India

Received February 16, 2009

Even after 25 years of establishing Human Papillomavirus (HPV) as the causative agent for cervical cancer,
effective treatment of HPV infection still unavailable. Comprehensive efforts especially for targeting HPV
infection have been made only in recent years. Conventional physical ablation of HPV-induced lesions
such as cryo-therapy, photo-therapy, LEEP, laser cone-biopsy and localized radiotherapy are shown to
be effective to some extent in treating localized lesions where the removal of diseased tissue is associated
with removal of transforming keratinocytes harboring HPV. Apart from currently available prophylactic
vaccines which prevent the viral entry and should be given prior to viral exposure, several attempts
are being made to develop therapeutic vaccines that could treat prevailing HPV infection. In addition,
immunomodulators like interferons and imiquimod that have been shown to elicit cytokine milieu to
enhance host immune response against HPV infection. Also, antiviral approaches such as RNA interference
(RNAi) nucleotide analogs, antioxidants and herbal derivatives have shown effective therapeutic potential
against HPV infection. These leads are being tested in pre-clinical and clinical studies. Present article
provides a brief overview of conventional therapies for HPV-associated diseases. Potential of non-ablative
anti-HPV treatment modalities that could prove useful for either elimination of HPV in early stages of
infection when the virus is not integrated into the host cell genome or suppression of the expression of viral
oncogenes that dysregulate the host cell cycle following transformation is discussed.

Key words cervical cancer - herbal therapeutics - human papillomavirus - immunotherapy - RNA interference - therapeutic vaccines -
transcriptional regulation

Introduction of different organ sites and have been associated with

The plurality of HPV genotypes is associated
with its ability to cause a wide spectrum of epithelial
proliferative lesions1,2. The global burden of HPV
associated diseases is quite high, and is estimated to
be about 5.17 per cent of total cancer burden. Among
the HPV-associated diseases, cervical cancer which is
the second most common cancer among women world
over tops the list. Papillomaviruses exhibit a high
degree of cellular tropism for squamous epithelial cells
various clinical manifestations ranging from benign
hyperplastic epithelial proliferative innocuous lesions
(warts, papillomas) to invasive carcinomas (Table I).
On the basis of their association with disease types,
papillomaviruses are classified into high-risk (HR) and
low-risk (LR) types. HR-HPV types (HPV 16, 18, 31,
35, 39, 45, 51, 52, 56, 59, 66, 68, 69 and 73) are often
associated with high grade lesions and invasive cancer,
whereas the LR-HPV types (HPV 6, 11, 40, 42, 43,

*
Present address: Dr B.R. Ambedkar Center for Biomedical Research, University of Delhi (North Campus), Delhi 110 007, India
e-mail: bcdas48@hotmail.com
296
 Bharti et al: Anti-human papillomavirus therapeutics: Facts & future 297
Table I. Association of various HPV types with different diseases outcomea
Disease
Benign and pre-cancerous Lesions
Plantar warts
Common warts
Flat warts
Other cutaneous lesions (e.g., epidermoid cysts)
Epidermodysplasia verruciformis
Recurrent respiratory papillomatosis
Laryngeal papillomatosis
Focal epithelial hyperplasia of Heck
Conjunctival papillomas
Condyloma Acuminate (genital warts)
Cancerous lesions
Cervical carcinoma
Oral carcinoma
Esophageal carcinoma
Laryngeal carcinoma
Conjunctival carcinoma
Anal carcinoma
Vaginal carcinoma
Vulvar carcinoma
Lung carcinoma
Bladder carcinoma
Penile carcinoma
Non-melanoma skin cancer
a
Data from3,4
b
Order indicates relative frequency; bold type indicates most frequent association
44, 54, 61, 70, 72, 81, CP6108) are mainly found in
low grade lesions, genital or skin warts and condyloma
accuminata. There are about 15 high-risk HPV types
have been described, which contribute to about 100 per
cent to the HPV-attributable cancer burden worldwide.
Among them, HPV 16 and HPV 18 are the most
prevalent oncogenic genotypes as two together are
responsible for more than 80 per cent of total HPV-
associated cancerous lesions.
Persistent infection of specific types of HR-HPVs
particularly the type 16 and 18 is a prerequisite for
the development of cervical intraepithelial neoplasia
(CIN), and cervical cancer5,6. However, HPV infection
is a transient phenomenon. Following infection, the
virus may persist over time, leading to development of
low grade cervical lesions where the virus is present in
non-integrated form, progressing in course of time to
cancer in a well-characterized process that takes 15-
20 years. Though the natural history of HPV infection
demonstrates spontaneous clearance of HPV infection
in a large number of cases7-9, persistence of high risk
HPV infection for a year or more confers an increased
risk of progression to cancer10. It has also been shown
that it takes about two years to clear type-specific HPV
infection11. Although HPV genome, which is a ~ 8Kb
HPV type(s) associated
1, 2, 4, 63
2, 1, 7, 4, 26, 27, 29, 41, 57, 65, 77, 1, 3, 4, 10, 28
3, 10, 26, 27, 28, 38, 41, 49, 75, 76
6, 11, 16, 30, 33, 36, 37, 38, 41, 48, 60, 72, 73
2, 3, 10, 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 50
6, 11
16,11,6,10
13, 32
6, 11, 16
6, 11, 30, 42, 43, 45, 51, 54, 55, 70
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
16, 18
16, 18
6, 11, 16, 18
6, 11, 16, 18
16, 18, 31, 33
16, 18, 31, 33
16, 18, 31, 33
18
16
16, 18, 31, 33
5, 8
circular DNA, codes for about 6 early (E1, E2, E4, E5,
E6 and E7) and two late genes (L1 and L2), expression
of E6 and E7 of the HR-HPVs can immortalize human
squamous epithelial cells in vitro, the discovery that
lead to establishment of the role of these viruses
in development of cervical carcinoma12,13. Virus-
encoded E6 and E7 oncoproteins interact specifically
with important cell cycle regulatory proteins. The
E7 protein of the genital tract HPVs, similar to the
adenovirus ElA proteins and the large tumour antigens
of the polyomaviruses, can complex with the product
of the retinoblastoma tumour suppressor gene, pRB,
and disrupts the association of E2F transcription factor
with pRB14,15. The E7 proteins of the “high risk” HPVs,
such as HPV-16 and HPV-18, bind pRB with ‘10-fold
higher affinity than do the E7 proteins of the “low risk”
HPV types 6 and 11, and this difference in binding
affinity correlates with the transforming potential
of the different E7 proteins15. Like simian virus 40
(SV40) large tumour antigen and adenovirus E1B,
the E6 protein of the “high risk” HPVs can complex
with the p53 protein16. Primary role of E6 interaction
with p53 is formation of a ternary complex alongwith
a ubiquitin ligase called E6AP17. This ternary complex
formation leads to the ubiquitation and degradation of
298 INDIAN J MED RES, September 2009
p53 by action of 26S proteasome which reduces the
half life of p53 to less than 20 min in HPV infected
keratinocytes18,19. Establishment of E6 and E7’s
oncogenic properties has made these viral genes the two
most sought after targets for anti-HPV therapeutics.
The HPV life cycle comprises of four important
steps necessary for its pathological consequences and
are considered as potential targets for any anti-HPV
therapeutic strategies. The viral entry is the first step
in HPV pathogenesis followed by its persistence in
epithelial cells. These two steps establish the viral
genome into the host cell and has been the subject of
therapeutic interventions through prophylactic and
therapeutic vaccination. Most of the lesions maintain
the virus as an episome and support complete virus
replication cycle with tight regulation of viral gene
expression. The third and most important step of HPV
life cycle is its integration into the human genome
which is frequently observed in CIN3 or higher
lesions. The exact mechanism of this integration is not
clearly understood, but loss of E2 ORF and release of
inhibition on E6 and E7 expression and transformation/
immortalization of keratinocytes follows this integration
event. It is also not clear whether this integration is
reversible or not. The integration of HPV genome is
the key point which facilitates the progression towards
Fig. Conventional therapies for treatment of cervical pre-cancers and cancers.
malignancy. The fourth important step of viral life
cycle is expression/upregulation of viral oncogene
transcription in differentiating epithelium. As HPV gene
expression is associated with epithelial differentiation
programme and it has been controlled by specific
cellular factors. Several host cell transcription factor
binding sites and keratinocytes specific enhancers like
AP-1, Sp1, NF-1, TEF-1, TEF-2, Oct-1, AP-2, KRF-1,
YY1, STAT3 and glucocorticoid responsive elements
have been identified in the upstream regulatory region
(URR) of HPV, which determine the cell-type-specific
viral gene expression and contribute to the tissue
tropism of HPV20,21. All these four steps provide means
of interference with HPV life cycle and thus represent
targets for development of anti-HPV therapeutics.
Current treatment modalities
A) Conventional therapies:
Even after establishment of causal relationship
between HPV and cervical cancer, currently, presence
or absence of HPV does not have any impact on
deciding the treatment; the strategies are primarily
anti-cancer and not anti-viral. Several therapies are
available for treatment of HPV-associated diseases
(Fig.). These treatment strategies are ablative in nature
and aim to remove the lesion rather than specifically
 Bharti et al: Anti-human papillomavirus therapeutics: Facts & future 299
targeting the HPV infection. These treatments though
effective are applicable only when the lesion is visible
and does not necessarily eliminate HPV infection.
Some of these procedure are also associated with
significant morbidity such as profuse watery discharge,
bleeding, cervical stenosis (narrowing) and in some
cases cervical incompetence and pelvic infection.
Even though these treatment modalities are effective,
all of them are associated with considerable degree of
recurrence which ranges upto 10 per cent of the cases.
Studies aiming to correlate the status of HPV infection
with efficacy of conization treatment for CIN 3 lesions
revealed persistent HPV infection in all the cases
which showed recurrence22,23 probably due to positive
resection margins24 . Thus, the studies emphasized that
patients with persistent HPV infection after conization
for CIN 3 should be followed because they are at
increased risk of developing disease recurrence.
Before the advent of colposcopy, hysterectomy
was considered necessary for CIN lesions. However,
the natural history of CIN has clearly shown that in a
large majority of cases, CIN lesion does not progress
to invasive cancer, thus, making hysterectomy an
unnecessary and over-treatment procedure for CIN. At
the same time, management of invasive carcinomas has
little development as standard radiation or chemotherapy
is applied along with surgical removal of primary
tumour in advanced cancers. On the other hand, it is
recommended that the earliest lesions, CIN1, should not
be treated and should be followed up and treated only
if they progress to higher lesion. In such cases, HPV
tests are being recommended as adjunct to cytology for
identification of women at risk of disease progression
and selective ablation of their lesions25. In such cases,
anti-HPV therapeutics may have the greatest impact.
B) Alternate therapies:
Cytotoxic agents: Though scissor excision is the
most preferred methods for genital warts, topical
preparations of cytotoxic compounds like Podophyllin
or Trichloroacetic acid are also utilized in USA and
Europe26. Similarly 5-Fluorouracil is also available
but with very limited use for external genital lesions
as it generates strong inflammatory reaction27. Use of
Podophyllin and 5-Fluorouracil is contra-indicated
in pregnancy. Most of these therapies show no or
inconsistent antiviral dose response against HPV28.
Recently, two most common anti-cancer agents for
treatment of many cancers including cervical cancer,
arsenic trioxide (As2O3) and carboplatin have been
shown to target two most important transcription
factors, AP-1 and NF-κB respectively that play a
critical role in expression of HPV oncoprotiens E6 and
E7 though activation of HPV URR29,30 for which their
anti-viral role is being reinvestigated.
Photodynamic therapy (PDT): PDT is a new treatment
for a wide variety of malignancies and premalignant
dysplasias, as well as some non-cancer indications.
Therapeutic response to PTD is achieved through
the activation of non-toxic photosensitiser located
within neoplastic tissue, using visible light tuned to
the appropriate absorption band of the photosensitiser
molecule. This produces cytotoxic free radicals
such as singlet oxygen, which result in local photo-
oxidation, cell damage and destruction of the tumour
cells that may induce bystander effect and activate
host immunity. Topical administration of PDT
though considered most appropriate for cervical and
valval intraepithelial lesions, the clinical trials
with 5-aminolevulinic acid has revealed variable
results31-33. Though PDT is not equally efficacious for
all subgroups, PDT for condyloma and intraepithelial
neoplasia appears to be as effective as conventional
treatments like cold-knife conization34. In contrast
to cold-knife conization, PDT causes no substantial
cervical stroma destruction and hence reduces the
risk of a possible subsequent incompetent cervix, but
the effect on HPV eradication was found to be the
same as for other ablative therapies35. Subsequent
study however showed that PDT could be a promising
therapeutic modality as the HPV infection found in
follow up studies could be a result of reinfection36.
Immunotherapy: Interferons (IFNs) are the only anti-
viral drugs approved for the therapy of benign HPV-
related lesions. While IFN-α, IFN-β and IFN-γ have
all been tested against condyloma acuminata, most
information is available on IFN-α which appears
efficacious via a number of routes of administration,
schedules and dosages with an acceptable safety
profile. Success with IFN-α therapy, in terms of
reduced recurrence rates of condylomas was reported
from studies in which all visible lesions were
surgically removed with subsequent administration
of subcutaneous local IFN-α37 . Limited data are
available on the efficacy of IFNs in the treatment of
HPV-related dysplasia and carcinoma. Combination
therapy of IFN-α with retinoids appears promising for
cervical carcinoma38,39. Though, along with retinoids,
IFN-α showed synergistic anti-angiogenic effect
and upregulation of HLA class I and cell adhesion
300 INDIAN J MED RES, September 2009
molecule, ICAM-1 in HPV-harboring tumour-cell line,
the anti-viral effects of the combination have not been
explored. In another study, IFNα showed no effect on
HPV11 replication28. The treatment is expensive, has
limited efficacy and not recommended for routine
clinical practice in the treatment of high-grade HPV-
associated lesions37.
Apart from directly injecting IFNs,
immunomodulators, such as imidazoquinolones that
induce production of inflammatory cytokines are being
used for potentiation of innate immune mechanism
mediated through macrophages and dendritic cells.
Imiquimod, one of the imidazoquinolone compounds
has shown efficacy and safety in clinical trials for the
treatment of external HPV-infected genital warts40.
This class of compounds generates a cytokine milieu
that activates dendritic cell migration and leads to a
Th1 type of response. Recent study showed presence of
HPV-specific CD4+ regulatory T cells isolated in lymph
nodes of cervical cancer patients that could suppress
proliferation and IFN γ & IL-2 cytokine production by
responder T cells41. It is likely that these compounds
might have antagonizing role on these regulatory
T cells.
Prospective therapeutics
The most significant events in the HPV biology have
been successful clinical trial with potent prophylactic
anti-HPV vaccines, Gardasil from Merck, USA and
Cervarix from GlaxoSmithKline, Belgium. However,
there are a number of limitations like cost of the
vaccine, efficacy in target population, sex preference
and age of immunization that prevent their effective
mass scale implementation in near future. In view
of these bottlenecks, it becomes essential to explore
alternative methods for control of HPV infection and
effective management of cervical and other HPV-
related pre-malignant and malignant lesions. Several
therapeutic strategies are being explored which can
effectively target at various phases of viral infection
such as viral entry, viral latency, viral replication, and
its oncogenic expression. Since low grade and high
grade lesions differ with respect to the viral activity,
the treatment approaches also differ and are specific to
the grade of the lesions to be treated. Low grade lesions
are usually homogeneous, genetically stable with
permissive viral replication; in a immunocompetent
individual, therapeutics are aimed to resolve these
lesions and to prevent entry, replication and latency
of the virus without any recurrence. In contrast, high
grade disease is heterogeneous and genetically unstable
with less active viral replication but with expression
of oncogenic proteins. Therefore, targeting expression
of these viral oncogenes either by promoting anti-
HPV immunity or RNA interference or transcriptional
inactivation is the norm. Prospective therapeutics can
be classified as therapeutic vaccines, immunotherapies,
RNA interference-based therapies, anti-retrovirals and
natural/herbal derivatives. Many of these therapeutics
are at later stages of their clinical evaluation.
A) Therapeutic HPV vaccines:
Vaccination with virus-like particles (VLP) such
as Gardasil and Cervarix has demonstrated efficacy in
HPV prophylaxis but these vaccines lack therapeutic
potential. Considering the vital role of HPV16 in
carcinogenesis, most of the efforts for developing
therapeutic HPV vaccines have been directed towards
development of vaccines against HPV16 viral oncogenes
E6 and E742-52. Based upon the nature of immunogen
the therapeutic vaccines can be divided into two major
classes, (i) proteins/peptide-based vaccines, and (ii)
DNA-based vaccines. Majority of the studies conducted
as phase I clinical trials both in healthy individuals as
well as in patients having high grade lesions or frank
malignancies have used whole protein or peptides
derived from HPV16 or HPV18 E6 and E7 (Table II)
mainly because of their oncogenic potential and they
are invariably retained and expressed throughout HPV-
related disease progression and carcinogenesis.
These immunogens were found to be highly
effective in generating both humoral as well as
cytotoxic T cell response in majority of trial subjects,
but their effect on lesion clearance/regression or on
HPV positivity were determined in a very few studies.
Several animal studies have shown promising results
indicating that therapeutic HPV vaccine may regress
disease progression43,53 Phase I trials that recorded
these parameters indicate that despite effective anti-
HPV immunity, lesion regression as well as HPV
clearance was suboptimal and was only observed
in a subset of patients42-49. Perhaps this could be the
potential reason why these therapeutics could not enter
in Phase II and Phase III studies. Apart from these,
there are several other limitations that remain to be
overcome like subunit vaccines are costly to prepare
and require cold chain and the response is short term
and requires several boosters. To overcome some of
these limitations, second generation, less expensive
therapeutic DNA-based vaccines are being prepared
against HPV16 E750-52. These efforts are also directed
towards generation of effective immune response

Table II. Anti-HPV therapeutic vaccines in various stages of clinical evaluation
Target Antigen Phase/study Disease model Response References
status Immunity Lesion HPV
A. Protein-based Vaccines
HPV16 and 18 E6 and Phase I/II trial Late Stage Cervical HPV-specific antibody response in 3 out ND ND 42
E7 (TA-HPV) in live Cancer of 8, presence of HPV-specific cytotoxic
vaccinia virus (n = 8) T lymphocytes
HPV16 L1/L2- E7 Pre-clinical C57BL/6 mice Agglutinate erythrocytes and elicit high ND ND 43
chimeric VLPs injected with TC-1 titers of neutralizing antibodies
expressing HPV16 Effect mediated through class I restricted
E7 cytotoxic T lymphocytes
HPV16 E7 peptides Phase I CIN/VIN II/III DC infiltration in 6/6 cases 3 cases had complete 12/18 scrapes showed 44
(amino acids 12-20  (n = 8) No positive delayed type hypersensitivity clearance of dysplasia clearance of virus but
and 86-93) for E7 increase in cytokine response & and CIN 6 had partial biopsy were positive for
E7-specific cytolysis in 10/16 cases regression HPV
HPV16 L2, E6 and E7 Phase I Healthy volunteers Induced IgG antibodies & enhanced T-cell ND ND 45
(n = 40) immunity in 8/11 cases
HPV16 E6E7 fusion Phase I CIN HPV-specific antibody response, delayed ND Loss of HPV16 DNA 46
protein (n = 31) type hypersensitivity, in vitro cytokine from the cervix in some
release, and CD8 T cell responses to E6 cases
and E7 proteins
HPV16 L1-E7 Phase I CIN II/III Induces with high antibody titers against Histological improvement 56% of the responders 47
chimeric virus-like (n = 39) L1 and low titers against E7 as well as to CIN 1 or normal were also HPV16 DNA-
particles (CVLP) cellular immune responses against E7 and was seen in 39% of the negative
L1 patients
HPV16/18 E7 Phase I Stage IB or IIA Enhanced CD4+ T-cell and antibody ND ND 48
cervical cancer responses,  E7-specific CD8+ T-cell 
(n = 10) response, DTH responses to intradermal
injections of HPV E7 antigen 
HPV16 E6 and E7 Phase I Cervical cancer Elevated CD4+ and CD8+ T cells response ND ND 49
synthetic long peptides patients (n = 6) against E6 and E7
B. DNA- based vaccines
HPV16 E7 Phase I Anal dysplasia 10/12 subjects showed increased immune Partial histological ND 50
(ZYC 101) (n = 12) response to E7 peptide responses
Bharti et al: Anti-human papillomavirus therapeutics: Facts & future

HPV16 E7 (ZYC101)  Phase I  CIN II/III HPV-specific T-cell responses Partial histo-pathological ND 51
(n = 15) response
Response
HPV16 E7 Preclinical E7-expressing Higher E7-specific CD8+ T-cell immune Apoptotic tumour cell ND 52
tumour-bearing responses  death 
mice
301

CIN, cervical intraepithelial neoplasia; ND, not determined; VIN, valval intraepithelial neoplasia
302 INDIAN J MED RES, September 2009
against HPV by intra dermal administration of DNA
vaccines which represents a feasible strategy to deliver
DNA directly into the professional antigen-presenting
cells of the skin but the efficacy of this strategy for
regression or downstaging of the lesion is yet to be
proven. It is likely that additional targets are needed
to be identified to compensate dysregulated host cells
apart from targeting only the HPV oncoproteins. Recent
studies show that combining DNA vaccine with either
death receptor-5 antibody or silencing of fas ligand
by RNA interference can substantially enhance the
therapeutic potential of these vaccines54,55.
Apart from therapeutic HPV vaccines, there have
been attempts to pulse dendritic cells (DC) with tumour
lysate expressing HPV16 antigens56. Such DCs when
matured with non-toxic double stranded RNA were
capable of inducing class I and class II T cell immunity
in a pilot clinical study. This strategy is currently being
tested in a clinical trial in patients with cervical cancer48.
B) RNA interference-based therapies:
Following identification of target genes involved
in neoplastic transformation and tumour growth in
HPV-associated disease, target-specific therapeutic
approaches are being extensively investigated.
Last decade has witnessed a major advancement in
RNA interference technology specifically targeting
HPV oncogenes E6 and E7 without any damage to
normal cellular RNA pool, which is often lacking
in conventional therapeutics. E6 and E7 oncogenic
expression is detected in both upper epithelial layers
as well as during early stages in advanced stage of
disease and targeting these proteins will eventually
lead to the treatment of active lesions. Although, there
are a number of RNA interference-based therapies for
several oncogenes in Phase I-III clinical trials, they are
yet to be tested clinically for HPV-associated lesions.
Several in vitro and in vivo studies show effective
targetting of high-risk HPVs using antiviral ribozymes
and antisense molecules, siRNA and shRNA (Table
III).
RNA interference has been achieved by four
distinct approaches primarily against HPV16/18 E6
and E757-68. Most well established among these are anti-
sense oligonucleotides and ribozymes. Application of
antisense or ribozyme molecules has a favored position
because of their specificity and the requirement of
continued expression of the transforming genes E6 and
E7 for maintaining the malignant phenotype of cervical
cancer cells69,70. The effects of anti-sense plasmids or
oligonucleotides on the cell proliferation of cervical
cancer cell lines have already been demonstrated in
a variety of assays59,71-73. Inhibition of E6 and E7 by
complementary anti-sense transcripts led to reduced
growth rates, loss of the transformed phenotype
of cervical and oral carcinoma cell lines,57,71,72 and
inhibition of tumour formation in an animal model71,74.
Antisense E6 and E7 oligonucleotides also showed an
anti-proliferative effect in primary tumour explants73.
Treatment of HPV16-infected CaSki and SiHa cells
with phosphorothioate oligonucleotides against HPV16
E6 and E7 not only reduced cell proliferation in vitro
but also led to a reduction in the weight of tumours
derived from SiHa cells implanted into nude mice57,58.
Expression of antisense E6/E7 genes using adenoviral
gene transfer has resulted in a strong inhibition of
SiHa cell proliferation and a complete suppression of
tumour formation after ex vivo application75. However,
for therapeutic application of antisense genes, their
delivery is the major problem as a high percentage of
target cells has to be transduced, because no bystander
effect will help to spread the therapeutic principle.
In addition, the expression of anti-sense genes has to
be stable because the tumour cells are not eliminated
by treatment with a proliferation-inhibiting antisense
construct. To address this issue, Liposome-mediated
transfer of genes efficiently expressing anti-sense E7
has been tried alonwith co-delivery of interleukin-12
cytokine genes76 and this approach was found to induce
tumour cell apoptosis as well as an anti-tumoural
immune response which was further augmented.
On the other hand, ribozymes offer several
advantages over conventional anti-sense RNA. They
are capable of catalytic activity and do not require the
presence of an auxiliary enzyme such as RNase H.
Furthermore, one molecule of ribozyme can bind and
cleave several molecules of mRNA, giving rise to an
amplification of the net effect, and thus a desirable
reduction in the dosage required. In vitro studies
showed that Ribozymes can be used for effective
cleavage of HPV transcripts77 and can inhibit E6/E7-
mediated immortalization59. Recently, a hammerhead
ribozyme, Rz170, have been successfully designed
that can specifically target HPV16 E6E7 transcripts
and was found to be effective in inhibiting cell growth
and promoting apoptosis. Rz170 also appears to be
very efficient in inhibiting tumour growth in nude
mice78.
In contrast, achieving RNA interference using
small interfering RNA is more efficient than Anti-sense
 Bharti et al: Anti-human papillomavirus therapeutics: Facts & future 303

Table III. Molecular targeting of HPV gene expression using RNA interference technology
RNA Interference Target Study type Biological response References
approach
Anti-sense HPV16 & HPV18 E6 In vitro Growth inhibition of cervical and oral cancer cells 57,58
oligonucleotides and E7 harboring HPV18 or HPV16 but had little effect on
(AS-ODNs) cells lacking HPV
Inhibited formation of colonies in soft agar
In vivo in nude mice Substantially reduced tumour formation in nude 58
mice
Ribozymes (Rz) HPV16-E6/E7 HP In vitro Reduced the growth rate and prevented 59
ribozyme (R434) immortalization of E6/E7 transformed normal human
keratinocytes
HPV6b and HPV11 E1 In vitro Efficiently cleaved E1 gene RNA transcripts 60
genes
Short-interfering HPV16 & HPV18 E6 In vitro E6 silencing induced accumulation of cellular p53 61-65
RNAs (siRNA) and E7 protein, transactivation of the cell cycle control p21
gene and reduced cell growth.
E7 silencing induced hypo-phosphorylation of
retinoblastoma protein and apoptotic cell death.
Inhibited cellular DNA synthesis
Induced morphological and biochemical changes
characteristic of cellular senescence
HPV16 E6 and E7 in vivo in NOD/SCID Retarded tumour formation in nude mice 62,66
nude mice
Short hairpin RNA HPV16 & HPV18 E6 In vitro Accumulation of p53, p21, and hypophosphorylated 67
(sh-RNA) and E7 pRb proteins in cells 68
Lentiviral (LV) delivery of low dose sh-RNA
reduced cell growth and the induction of senescence,
high-dose infection resulted in specific cell death via
apoptosis
HPV18 E6 and E7 in vivo in NOD/SCID Transplant of HeLa cells infected with a low dose of LV- 68
nude mice shRNA into Rag-/- mice significantly reduced the tumour
weight, whereas transplant of cells infected with a high
dose resulted in a complete loss of tumour growth
Systemic delivery of LV-shRNA into mice with
established HeLa cell lung metastases led to a
significant reduction in the number of tumour nodules

oligos or ribozymes. Recent studies were demonstrated of p21 (WAF1/CIP1)64,66, hypophosphorylation of

that using siRNA is more rational as these molecules
can induce selective silencing of exogenous viral
genes in mammalian cells, and the process does not
interfere with the recovery of cellular regulatory
systems previously inhibited by viral gene expression61.
Moreover, because E6 and E7 genes are not present in
normal cells, RNAi-based therapies would not affect
them. Several studies have shown that HPV E6 and E7
can be efficiently targeted by small interfering RNA,
resulting in suppressed monolayer and anchorage-
independent growth and induce apoptosis and
replicative senescence of SiHa cells61-64,66,68,79. The most
interesting feature of E6 and E7 targeting by siRNA is
the stabilization of E6 target protein p53 and its nuclear
accumulation which eventually leads to the expression
retinoblastoma protein (pRb 105) and activation of
apoptotic pathways in HPV-infected target cells.
Moreover, delivery of E6/E7 siRNA into nude mice has
shown significant reduction in the number of tumour
nodules and retarded tumour growth of HPV16+ cells
in NOD/SCID mice64,66.
Despite being a totally rational antiviral strategy,
RNA interference operates only at post-transcriptional
level to suppress gene expression of viral oncogenes and
does not have any impact on viral latency. Moreover,
due to lack of bystander effect and requirement
of large amount of therapeutic principle, the cells
with targeted oncogenic RNA tend to loose their
proliferative capacity (as desired) and get diluted out
in fast expanding pool of non-targeted or inefficiently
304 INDIAN J MED RES, September 2009
targeted cells expressing viral oncogenes. Therefore,
in view of these major shortcomings RNA interference
may not be useful as stand alone therapy. Keeping
these inherent lacunae, a few more rational approaches
have been developed by combining RNAi against
HPV E6 and E7 with the current forms of treatment
for cervical cancer. In a recent study, targeting HPV
oncogene by siRNA and reactivation of p53 pathway
was found to increase the sensitivity of tumour cells
to cisplastin80. This strategy also reduced the cisplastin
dose requirement and the prevented occurrence of drug
resistance and resulted apoptotic death in cancer cells.
In another study, the use of photocross-linking reagent,
4,5’,8-trimethylpsoralen, conjugated oligo (nucleoside
phosphorothioate) (Ps-S-Oligo) for photodynamic
anti-sense regulation of the anti-apoptotic HPV18 E6
expression alongwith low concentration of cisplastin,
up regulated more p53 activity and induced massive
apoptotic death in comparison to that induced by Ps-S-
Oligo81 alone.
As with the other technologies, in vitro testing of
siRNAs against cervical cancer has shown promising
result. However, there are issues that have held up
the clinical development of ribozymes and antisense
molecules are target selection, specificity and delivery
along with low efficiency, short-period of maintenance,
and high cost82. In addition, transfection with siRNA
can activate stress response genes (e.g., GADD and
p38), and there is evidence that in some case, siRNA
might degrade some off-target mRNAs in human
cells83,84. This non-specific effects may explain, in
part, why cytotoxicity can occur after transfection with
seemingly harmless siRNAs and thus underline the
importance of siRNA optimization.
C) Natural/herbal derivatives:
In this age of targeted therapy, the failure of most
current drug –discovery efforts to yield safe, effective,
and inexpensive drugs has generated widespread
concern. Successful drug development has been
obstructed by a general focus on target selection rather
than clinical safety and efficacy. The very process of
validating the targets themselves is inefficient and in
many cases leads to drugs having poor efficacy and
undesirable side effects not only for HPV-related
diseases but cancer in general. Since any given cancer
carries an estimated 300 gene alterations, this raises
an important question about how effective these
targeted therapies can ever be against cancer. Thus,
it has become necessary to rethink drug development
strategies and emphasis is being given to natural and
herbal derivatives that are safe and possess multi-hit
capability not only to target against the viral oncogenes
but also to inhibit the co-operative signaling and
dysregulated gene expression of the host cells.
Since productive life cycle of HPV is tightly
linked with the differentiation programme of infected
epithelial cells, various leads are being tested for their
activity at different stages of HPV’s life cycle in the host
cell which include virus binding and entry, presence of
viral DNA in episomal form and its replication in host
cells as well as host cell-dependent expression of viral
oncogenes following integration of its DNA into the
host cells. The noncoding upstream regulatory region
(URR) of HPV consists of myriad of transcription
factor binding sites like AP-1, SP1, NFκB, NF-1,
TEF-1, TEF-2, Oct-1, AP-2, KRF-1, YY-1, STAT-3
and glucocorticoid responsive elements85. During
differentiation, these upregulated cellular transcription
factors bind to the HPV URR and facilitate the
expression of viral oncoproteins and late structural
proteins. As these cellular transcription factors serve as
a major link between oncogenic host cell transcription
and also the viral gene expression, transcription factors
provides a unique target for development of anti-HPV
therapeutics86. Theoretically, preventing binding of
these transcription factors will eventually leads to the
suppression of HPV oncogenic gene expression and
stop assembly of virion particles. In past few years,
several herbal compounds, derivatives, antioxidant and
small polyphenol plant compounds have been used for
selective suppression of host cell transcription factors
like AP-1, and NF-κB in HPV infected cells87-91. The
selective suppression and alteration in composition
of these factors has been shown to be associated with
downregulation of HPV gene expression and induction
of apoptosis in infected cells91. Here, in this section we
have described some of the potent anti-HPV activities
which are in process of development for being used as
anti-HPV therapeutics (Table IV).
Curcumin: Curcumin is a potent antioxidative agent
(diferuloymethane) and an active compound of the
perennial herb which also exhibits anti-inflammatory
and antitumour activity110. Recent studies using this
curcumin on HPV positive human cervical cancer cell
lines have demonstrated that curcumin downregulates
the AP-1 and NF-κB expression in a dose and time
dependent manner89,91. The AP-1 plays a crucial role
during the development of cervical cancer by binding
to its potential cognate binding sites within HPV URR
 Bharti et al: Anti-human papillomavirus therapeutics: Facts & future 305

Table IV. Molecular targeting of HPV and Host cellular factors interaction using natural and herbal derivatives
Natural/ Herbal derivative Anti-viral action References
Curcumin and curcumin-based poly-herbal Inhibition of oncogenic and viral transcription through inhibition of AP-1 89-92
formulation, Basant and NF-κB
Block viral entry in cervical cells
Praneem poly herbal formulation Clearance of viral DNA from precancer lesions 93
Epigallocatechin gallate (EGCG) Inhibition of proliferation with suppression of pKi-67 and telomerase 94-98
activity and induction of apoptosis
Nordihydroguaiaretic acid (NGDA) from Inhibition of HPV16 transciption by inhibition SP1-mediated 99
plant lignan transcription 100
Stabilization of p53
Silymarin Apoptosis by no-mitochondrial pathway mediated through p38/ JNK 101
MAPKs
Jaceosidin ( 4’,5,7-trihydroxy-3’,6- Inhibit binding of E6 with p53 and E7 with pRb 102
dimethoxyflavone) Suppress growth of cervical cancer cells
Heparin Inhibition of AP-1 activity and inhibition of HPV LCR and trascription of 103, 104
E6 & E7
Supression of cell proliferation
Carrageenan Acts primarily by preventing the binding of HPV virions to cells 105
Sulindac (NSAID) Degradation of E7 by the proteasomal pathway 106
Inhibition of host cell cycle and suppression of cyclin E and A,
inactivation of cdk2,
Solanum nigrum Phytoglycoprotein Reduces the binding activities of NF-kB and AP-1 and declines the level 107
of Nitric Oxide (NO) production
Soyasaponins Apoptosis by mitochondrial pathway 108
Sulfated K5 E. coli polysaccharide Prevented the interaction between HPV-16 pseudovirions and 109
derivatives immobilized heparin
Inhibited HPV-16, HPV-18, and HPV-6 pseudovirion infection

and is absolutely indispensable for efficient HPV
oncogene expression87. AP-1 has been shown to be
regulating the transcription of almost all HPV types
investigated so far111. Curcumin treatment of HPV 18
positive cervical cancer cells selectively suppresses the
HPV 18 transcription as well as abolished AP-1 binding
to the viral URR89. In subsequent studies, curcumin has
also been shown to suppress activation of transcription
factor NFκB. Curcumin also selectively blockes
IκBα phosphorylation and degradation and lead to
abrogation of NFκB activation and downregulation
of NF-κB dependent COX-2 expression91. These
activities of curcumin are primarily linked to its strong
anti-oxidant activity. Based on these leads a polyherbal
cream, Basant, has been formulated using curcumin
and other herbal components92. Basant was found to
not only inhibit HPV entry into the cervical cells in
vitro but also inhibited Neisseria gonorrhoeae, various
species of Candida and HIV-1in cultures92. Currently,
this formulation is being tested clinically for its safety
and efficacy against HPV infection in humans in a
Phase II multicentric clinical trial in women having
HPV positive pre-cancer (CIN1/LSIL) lesions112.
Praneem: Similar to Basant, Praneem also is a
polyherbal formulation based on purified extracts
of Neem (Azadirachta indica) leaves and other
ingredients113, strongly inhibited the growth of N.
gonorrhea, multi-drug resistant E. coli and various
species of Candida and has virucidal action against
HIV-1113-115. Praneem was shown to clear and prevent
in vivo transmission of HSV2 and Chlamydia
trachomatis infection114. Praneem dispensed as
pessary has completed Phase-I safety and Phase-II
efficacy studies for treatment of abnormal vaginal
discharge due to a variety of reproductive tract
infections (RTIs)116-118. In a pilot Phase II clinical trial
of Praneem in women positive for HPV 16, a 30 day
application of the Praneem polyherbal tablet resulted
in elimination of highly carcinogenic HPV type 16
from the uterine cervix in an overall 80 per cent of
cases93. The elimination of HPV DNA was found to
be accompanied by marked improvements in clinical
symptoms as well as cytological abnormalities in
Praneem-treated subjects. Though the mechanism(s)
of action of Praneem is not known, it appears to act
through immunomodulatory pathways.
306 INDIAN J MED RES, September 2009
Epigallocatechin gallate (EGCG): A constituent of
green tea, (-)-epigallocatechin-3-gallate (EGCG) is
known to possess anti-cancer and anti-proliferative
properties. Several studies conducted in recent years
on effect of EGCG on cervical cancer cell lines showed
inhibition of proliferation of cervical adenocarcinoma
cells which was associated with suppression of pKi-67
and telomerase activity and induction of apoptosis95-98 .
Treatment of EGCG in organotypic culture of cervical
cancer cell lines which mimics cervical cancer lesion
in vitro, also showed decreased thickness of epithelial
multi-layers and induction of apoptosis. EGCG was
also tested in clinical trial along with polyphenol E
of green tea in fifty-one patients with cervical lesions
(chronic cervicitis, mild dysplasia, moderate dysplasia
and severe dysplasia); 69 per cent response rate (35/51)
was noted as compared to 10 per cent response rate
(4/39) in control group94. Overall these observations
suggest that green tea polyphenols are effective against
HPV infection and for treating cervical lesions and can
be a potential drug as anti-HPV therapeutics.
Potential leads from herbal derivatives: With the
development of newer in vitro drug screening assays,
it is now easier to screen various agents that may have
anti-viral activities against HPV. Recent in vitro studies
have revealed a number of herbal anti-oxidants such
as plant lignan, nordihydroguaiaretic acid (NDGA)119,
silymarin101 , sulindac106 and jaceosidin102 or naturally
occurring polysaccharides and glycoproteins
like heparin104, carrageenan105, Solanum nigrum
glycoproteins107, soyasaponins108 and Sulphated E. coli
polysaccharide derivatives109 to possess anti-cervical
cancer and anti-HPV activities. These leads can be
further tested in clinical settings, and considering the
natural origin with multi hit capability against HPV
and the host cell, it is expected that these formulations
will be safe and effective.
Conclusion
Antiviral therapies based on natural/herbal
derivatives have the potential to treat both
inapparent HPV infection as well as visible clinical
disease. A substantial number of HPV-infected,
immunosuppressed individuals cannot be treated
with immunotherapies or high-cost RNA-interference
technology. In such patients anti-viral and anti-cancer
drugs are the only option. Furthermore, antiviral
agents, unlike immuno-therapies or RNA-interference,
may not be HPV type restricted in their efficacy. All
these reasons make development of natural/herbal
derivative-based anti-HPV therapies a high priority
as cheap and effective anti-viral therapies.
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