[Frontiers In Bioscience, Elite, 10, 15-73, January 1, 2018]

Therapeutic startegies for human papillomavirus infection and associated cancers

Alok Chandra Bharti1,2, Tejveer Singh1, Anjali Bhat1, Deepti Pande1, Mohit Jadli1

1 Molecular Oncology Laboratory, Department of Zoology, University of Delhi, Delhi 110007, India, 2Division of Molecular Oncology, National Institute of Cancer Prevention and Research (ICMR), Noida, Uttar Pradesh, India

TABLE OF CONTENTS

1. Abstract 2. Introduction 3. Spectrum of HPV-associated diseases and magnitude of the problem 4. Molecular characteristics and Infection cycle of HPV 5. Conventional therapies for management of benign and malignant cervical lesions 6. Emerging therapeutics 6.1. Immune checkpoint inhibitors and repurposed anti-cancer drugs 6.2. Therapeutic HPV vaccines 6.3. HPV genome targeting strategies 6.3.1. Silencing HPV RNA by antisense oligonucleotides 6.3.2. Silencing HPV RNA by catalytic oligonucleotides 6.3.3. Silencing HPV RNA by RNA interference 6.3.4. HPV Genome targeting approaches 6.4. Emerging anti-HPV pharmaceuticals from nature 6.5. Anti-HPV leads in alternative medicines 7. Conclusion 8. Acknowledgements 9. References

1. ABSTRACT

Human papillomavirus (HPV) infection is linked to development of cancer of cervix, vagina, vulva, penis, ano-genital and non-genital oro-pharyngeal sites. HPV being a sexually transmitted virus infects both genders equally but with higher chances of pathological outcome in women. In the absence of organized screening programs, women report HPV-infected lesions at relatively advanced stages where they are subjected to standard treatments that are not HPV-specific. HPV infection-driven lesions usually take 10-20 years for malignant progression and are preceded by well-characterized pre-cancer stages. Despite availability of window for pharmacological intervention, therapeutic that could eradicate HPV from infected lesions is currently lacking. A variety of experimental approaches have been made to address this lacuna and there has been significant progress in a number of lead molecules which are in different stages of clinical and pre-clinical development. Present review provides a brief overview of the magnitude of the problem and current status of research on promising lead molecules, formulations and therapeutic strategies that showed potential to translate to clinically-viable HPV therapeutics to counteract this reproductive health challenge.

2. INTRODUCTION

Strong awareness drives around the globe against sexually transmitted infections and organized periodic cervical screenings using a simple cytology smear test devised by Papanicolaou have brought down cervical cancer from its topmost killer position to the fourth leading cause of cancer death in women (1, 2). An estimated 527,600 new cervical cancer cases, 265,700 deaths were reported worldwide in 2012 (3). With a due credit to the organized cervical screening, the incidence of invasive cervical cancer is now restricted significantly in the developed countries. In contrast, a disproportionately high disease burden, exceeding 90% of total, is contributed solely by the less developed countries. Cervical cancer, which is usually reported late and in advanced stage of the disease in most of the developing nations, represents a formidable reproductive health challenge (4). Clinico-epidemiological and molecular studies have established causal link between persistent infection of human papillomavirus (HPV) with neoplastic growth of uterine cervix and other parts of the reproductive tract (5, 6). HPV-induced tumors can be either benign or malignant depending upon the genotype (s) of the infecting virus (7). These infection-induced tumors are frequently detected in cervix and to a lesser extent in vagina, vulva, penis, ano-genital and non-genital sites (8), though the non-cervical tumors are showing an increasing trend in last two decades (6, 9). The earliest evidence of sexual route of transmission of these cancers came with historical notions (10) and later supported by epidemiological studies that women with multiple sex partners or having spouses with multiple sexual contacts were at an increased risk of contracting these cancers (11, 12). Harald zur Hausen, a German virologist while researching on cancer of the cervix, discovered HPV as human carcinogen and a causative factor for onset and progression of genital neoplasia (13), for which he received the Nobel Prize in Physiology or Medicine 2008. With over 200 different genotypes having a varied genetic and oncogenic potential identified till date (14), this class of viruses is known to infect cutaneous and mucosal epithelium of both men and women worldwide. About 80% of women and likely the same number of men are estimated to get this infection sometime in their lifetime (15). Most of these incident infections are transient and clear spontaneously, more than 90% within one to two years; however, about 10% persist and can cause neoplastic transformation (16-19). Spontaneous clearance without any clinical intervention is common till 30-35 years of age; following which the clearance rate decreases with increasing age and decreasing immune status (16, 17). Discovery of infectious etiology of cancer catalyzed growth in understanding the basic biology and molecular diagnosis of the virus, formulation of cervical screening programs and development of various vaccines and therapeutics to control HPV infection in order to prevent or treat cervical and other HPV-associated neoplasias. Till date, prophylactic vaccines developed against the most prevalent oncogenic HPV that are given at an early adolescent age prevent incident infection and have become a clinical reality (20, 21). However, interventions against already infected lesions are still in different phases of pre-clinical clinical development and evaluation. Such therapeutic entities are, however, much needed in resource constraint under-developed nations that cannot support and implement nation-wide periodic screening or vaccination drives. As per estimates from World Health Organization’s (WHO) Catala Institute of Oncology, 453 million women (female population aged ? 15 years) are at risk for cervical cancer, whereas only 3.1.% fraction of these women are covered by opportunistic cervical cancer screening (22). Present review is, therefore, aimed to provide a brief overview of the magnitude of the problem of HPV-associated malignancies with a particular emphasis on cervical cancer, basic biology of the disease and status of research on lead molecules, formulations and therapeutic strategies that showed potential in recent time to translate to a clinically viable HPV therapeutic that could meet this reproductive health challenge.

3. SPECTRUM OF HPV-ASSOCIATED DISEASES AND MAGNITUDE OF THE PROBLEM

HPV infection is causally associated with cervical cancer; whereas in other genital cancers including anal, penile, vulva, and vaginal cancers, HPV infection may act as co-factor (6).Compared to cervical cancer, other HPV-associated ano-genital cancers are relatively less frequent (15, 23-25) (Figure 1). Apart from frank malignancies, HPV can cause various types of “genital” or “venereal” warts. Depending upon the frequency of association of a particular HPV genotype with malignant or benign lesion, HPVs are stratified as high-risk (HR) or low-risk (LR) types (5, 26).The established HR-HPV include HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 59; and LR-HPV includeHPV6, 11, 40, 42, 43, 44, 54, 61, 72, and 81. Recently some of the types have been recognized as intermediate or probable high risk (pHR) which includes HPV26, 30, 34, 53, 66, 67, 68, 69, 70, 73, and 82. Among all these types HR-HPV type 16 and HPV18 in cervical cancer and LR-HPV type 6 and 11 in genital warts are the most common HPV types (15). HPV16 is the most virulent strain accounting for more than 50% overall disease burden and usually lead to squamous cell carcinoma, whereas HPV18 is primarily associated with a less common adenocarcinoma of the cervix. Population prevalence of HPV in asymptomatic, reproductively-active women is highly variable (15). The meta-analysis shows HPV positivity reported in this group range from as low as 0.1.-100% in different studies. Such variations are ascribed to the test used and the population of women sampled. A median range of HPV prevalence is about11.7% where HPV16 constitutes the highest proportion of 2.8% (15, 27). The proportions and spectrum of HPV types change with severity of the lesions (Figure 1).

Natural history of HPV infection and cervical cancer has revealed cytologically (low grade and high grade) and histopathologically (Cervical Intraepithelial Neoplasia or CIN1, CIN2, CIN3) well-defined pre-cancer stages that precede cervical cancer (28, 29) (Figure 2). The process of tumorigenic transformation from entry of the virus to invasive carcinoma takes 10-20 years and thus provides a desirable window period to treat this disease at an early stage. Early infection is primarily latent, subclinical, and opportunistic (30). Genital HPV infection is highly prevalent in sexually active young women. Furthermore, sexual contact with an HPV infected individual results in an increased probability of contracting the virus (11, 29, 31). Some additional risk factors include use of oral contraceptives, a history of sexually transmitted infections, smoking, and immunosuppression (32). Various factors depending upon their role in carcinogenic progression of the disease can be classified into exposure-related, host susceptibility-related, factors that affect tumor microenvironment, and somatic/epigenetic. Among factors that strongly influence HPV transmission and pathogenicity are prevalence of different HPV types in a particular population, duration of infectivity, patterns of sexual contacts and pathogenic behavior of the infecting virus (28, 29) (Figure 3). Since HPV is a sexually transmitted virus primarily it is transmitted through heterosexual intercourse, however, oro-genital and anogenital transmission is also possible resulting in its presence in extra-genital sites that can occur due to varied sexual practices like oral and anal sex (33) (Figure 4). However, oral to oral transmission is still debatable. The basis of transmission is always a direct skin-to-skin contact. In the same corollary, condom provides protection but not 100% safe to prevent transmission. Interestingly, circumcision is found protective in reducing transmission thus highlighting its importance in prevention of viral transmission (34). Friction and micro-abrasions play a key role in exposure of dividing cells of the basal layer that host a productive viral infection (29). Spontaneous HPV clearance is another attribute of HPV infection among females. Majority of the cases regress spontaneously without any need of intervention. In a 12-year follow-up study of women with two positive tests for HPV16 (at baseline and 2 years later) the risk of CIN3+ lesions estimated was 47.4.% (95% confidence interval 34.9.–57.5.%). Several long-term natural history studies revealed a reduced risk of CIN3+ lesions in women who crossed the peak age of HPV acquisition (e.g., 30 years or older) who remain HPV-negative or could clear their HPV. CIN2 is considered as an intermediary step between CIN1 (HPV infection) to CIN3 (direct cancer precursor). Recent data have shown that regression rates of CIN1 among young women (defined as less than 25 years of age) are quite high (up to 90%) which fall as the lesion progresses (29, 30).

HPV is necessary but not sufficient to cause cervical cancer (35). Several other sexually transmitted infections such as Candida, Trichomonas vaginalis, Chlmydia trachomatis, and HSV-2 may participate as cofactors (35-37). It is likely that these infections result in localized chronic inflammation that provides a suitable niche for persistent HPV infection (Figure 5). A recent study evaluated association between bacterial vaginosis (BV) and inflammatory response (IR) with the severity of cervical neoplasia in HPV-infected women and have revealed vaginal infections irrespective of their nature (bacterial, fungal, yeast) increased the susceptibility, effects the clearance of HPV infection and eventually contributed towards HPV-induced cervical or genital carcinogenesis (38). These infections alter the microenvironment in the genital areas thereby causing severe inflammation, which en routes the development of cervical neoplasia. The key transcription factors that mediate inflammatory response, i.e. Nuclear Factor-kappa B (NF- B), Signal Transducer and Activator of Transcription (STAT)-3, and Activator Protein (AP)-1 (39), are also important regulators of HPV infection (40-42) and have showed an aberrant expression and constitutive activity in cervical precancer and cancer tissues which increase with disease severity (43-45).These observations suggest potential utility of anti-inflammatory anti-cancer drugs in control of HPV infection and cervical cancer.

4. MOLECULAR CHARACTERISTICS AND INFECTION CYCLE OF HPV

HPV is an epitheliotropic, double stranded DNA virus and belongs to the family of papillomaviridae. HPV genome of about 8 kb consists of eight open reading frames, encoding two structural proteins namely L1 and L2, which constitute the outer capsid of 55nm and 6 regulatory/functional proteins namely E1, E2, E4, E5, E6, E7 (46, 47). L1 protein has been characterized as highly immunogenic, and is found to be conserved across different HPV types. Due to this fact, the protein has been well exploited for the development of different forms of DNA diagnostics and prophylactic vaccines, and in the serological assays. This protein is also important as it self assembles to form small virus like particles (VLP) which can elicit an active immune response. The L2 protein on the other hand, constitutes the minor capsid protein. Other proteins encoded by HPV include E1 and E2. E1 has helicase activity and participate in viral replication, genome partitioning, and transcription. Proteins coded by E6, E7 and E5 HPV genes have key oncogenic role and disturb the normal cell cycle regulation leading to tumorigenic transformation. The oncogenic activities of E6 and E7 are well characterized and the proteins seem to have different host cell targets. The tumor suppressor protein p53, retinoblastoma protein (pRB) and epidermal growth factor receptor (EGFR) are targeted by E6, E7 and E5 respectively. Recent developments in bioinformatics suggest a more complex multiple interaction of viral oncoproteins with other key components of cellular machinery. These interactions collectively and synergistically promote continuous cell proliferation, DNA amplification, and impairment of DNA repair mechanisms leading to genome instability, and anti-apoptosis (47-49). E2 plays a strong regulatory role by promoting viral replication thereby promoting productive viral life cycle and preventing expression of E6 and E7 oncogenes from early promoter. Epigenetic silencing or inactivation of E2 due to integration of the virus DNA in host genome via E2 coding sequences results in loss of E2 expression which leads to (over)expression of E6 and E7 through an early promoter (p97). The viral genome can stably exist in both episomal and integrated forms. However, integration makes tumorigenic transformation an irreversible event. Viral gene expression is highly orchestrated that align with epithelial keratinization (49). Sequential expression of genes is controlled through a 800bp region named as upstream regulatory region (URR) that contains sequences for binding of (A) host transcription factors like NF- B, STAT3 and AP-1; and (B) HPV’s transcription regulator E2 (40-42). Constitutive expression of E6 and E7 is a necessary prerequisite for growth and survival of cervical cancer cells as well as initiation and maintenance of malignant transformation (47). Recent investigations from our group demonstrated novel role of HPVE6 in maintenance of stemness and chemoresistance in cervical cancer cells (50, 51) by regulation of key signaling mediators like STAT3, Notch/hes and Gli (45, 50-53). These leads make HPV oncoproteins and their upstream regulatory mechanism a suitable target to design effective anti-HPV therapeutics.

5. CONVENTIONAL THERAPIES FOR MANAGEMENT OF BENIGN AND MALIGNANT CERVICAL LESIONS

Current primary and secondary prevention measures and clinical management of HPV infected lesions have been periodically reviewed (30, 54) and schematically summarized in Figure 6. Primary prevention of infection with high-risk HPV types remains the most efficient and logistically feasible preventive measure for cervical cancer. Regulatory authorities initially approved two vaccines to prevent HPV infection i.e. Gardasil (HPV16, 18, 6, & 11) (20), Cervarix (HPV16 & 18) (21, 55, 56) and later in 2014 Gardasil 9 which targeted 5 additional HPV types (HPV31, 33, 45, 52, 58) apart from the earlier version of the vaccine (57). These HPV vaccines, if administered before sexual activity, can reduce the risk of infection of the HPV types targeted by the vaccine. Although the vaccines are safe and effective in preventing HPV16 and HPV18 persistent infections and associated CIN2/3 lesions in young women who had no HPV infection at the time of first vaccination (58, 59), neither of the two vaccines was able to clear existing HPV16/HPV18 infection, nor could prevent their progress to CIN2/3 (60, 61).

As a secondary prevention measure, screening of women by a regular gynaecological examination that include either a standard Pap smear test, visual inspection with acetic acid (VIA) or Lugol’s iodine (VILI) is recommended. These are low cost screening tools that examine the presence of cytological or clinical lesions. Hybrid Capture II (HCII) and APTIMA are most popular United States Food and Drug administration (US-FDA) approved molecular tests based on detection of HPV DNA and RNA respectively with very high sensitivity, specificity and clinical relevance. Emerging techniques like OncoE6, CINTECP16 can detect HPV and its types. Cervista, first US-FDA-approved genotyping test can detect 14 high-risk HPV types (62) making it easier to know the HPV types involved in the disease. Detailed description of various other molecular tests and their targeted HPV genotypes has been made earlier (63). Nevertheless, HPV testing is gradually replacing cytology-based cervical screening owing to greater reassurance when the test is negative. Despite significant progress in both primary and secondary prevention strategies, the effective implementation of HPV vaccination and screening particularly in low resource settings remain a challenge.

Treatment modalities depend upon the type and location of manifestation i.e. genital warts, pre-cancer and finally cervical cancer. Genital warts can be treated by topical treatment (Imiquimod, Podofilox), interferons, cryotherapy surgical removal, trichloro acetic acid, carbon dioxide laser therapy [Reviewed in (64)]. Apart from topical treatments, all other interventions are provided by trained healthcare professionals. Interferon which by their nature are generic anti-viral agent, have been used for the treatment of laryngeal papillomas as well as cutaneous and anogenital warts (65). Partial and total remissions have been achieved with topical, intra-lesion and systemic administration of the interferon. While IFN-α is clinically approved for the treatment of genital warts and used as an essential part of treatment, it is generally not recommended, as the doses required for clinical effects are not tolerable by patients (66). In some reports, lack of efficacy of IFN-α therapy in recurrent, advanced cervical cancer was described. It shows minimal activity against advanced, recurrent cervical cancer (67).

Among patient applied therapies Imiquimod (Aldara) is a potent adjuvant for the topical treatment of HPV lesions and is an immunomodulator. Imiquimod works by stimulating the immune system to release cytokines, which are important in fighting viruses and destroying cancer cells. Several randomized controlled trials were conducted to estimate the efficacy of a treatment with imiquimod in patients with CIN 2-3 (68). Though, it is well tolerated, irritation usually develops on treated skin and it is sometimes not effective for all patients. Moreover, its long treatment time (that extends beyond 4 months) and cost is a major concern. Podophyllotoxin is another patient applied therapy which is widely available in a various formulations apart from imiquimod. In general, podophyllotoxin is cheaper than imiquimod, whereas imiquimod 5% is associated with lower recurrence rates than podophyllotoxin (69). However, none of these methods are HPV-specific nor uniformly successful. Recurrence rates vary tremendously, from 5% to 65% depending on treatment modality (70).

Methods commonly used to treat precancerous cervical changes could be ablative and excisional, and include cryosurgery, loop electrosugical excision procedure (LEEP), surgical conization and laser vaporization conisation which may require local or general anaesthesia. LEEP remains an important classic technique for obtaining an excellent diagnostic and therapeutic specimen. For settings where LEEP cannot be performed, cryotherapy is used as an alternative treatment for eligible VIA positive lesions. Cryotherapy is the only option available outside of surgical settings and more popular treatment with a widespread use in many countries because of its ease of use. However, a special attention and certainty is warranted in the diagnosis and visualization of the lesion as the technique does not provide specimen for confirmatory histopathology (30).

Other techniques such as cone biopsy along with laser surgery can also be used. The treatment of choice in early cervical cancer is surgery which can either be radical or total hysterectomy depending on the location and size of the tumors (71). Radiotherapy is the treatment of choice for cervical cancer. For women with locally-advanced cervical cancer, external beam radiation therapy (EBRT) was the standard of care earlier. With advancements in radiotherapy, EBRT has been coupled with brachytherapy, or combined with brachytherapy and concurrent chemotherapy (72). Chemotherapy alone is generally ineffective for cervical cancer although it can complement other treatment regime in late stages (73). Chemotherapy when given with radiation can be an effective treatment of choice and largely accepted as the standard of care for patients with stage II to stage III cervical cancer. For metastatic cervical cancers (stage IVB), and recurrent cervical cancer patients who cannot be effectively treated using radiation or surgery, chemotherapy is the primary treatment (74). However, in spite of the compliance to these therapeutic regimes, up to 70 % of the cancer patients experience pelvic recurrence of tumor, distant metastases, or both along with an increasing chemoresistance that ultimately culminates into death of the patient (75).

6. EMERGING THERAPEUTICS

The major challenge for emerging immune- or antiviral-based therapies for HPV-associated conditions is to safely provide a clear advantage over any existing treatment; particularly for late precancer lesions like CIN2/3 where treatment efficacy is 90–95%. Keeping in view spontaneous regression of CIN2/3 lesions non-surgical methods are always preferred over unnecessary surgical interventions that lead to patient anxiety and risk for premature delivery in case of a reproductively active woman. Apart from effective utilization of clinically available treatment modalities, there has been an extensive research for the treatment and management of advanced cervical cancer and HPV infection using immunomodulators, viral-derived proteins or small molecule inhibitors which can be broadly and interchangeably classified as immunotherapeutics, anti-viral therapeutics, or anti-cancer therapeutics without discrete boundaries.

6.1. Immune checkpoint inhibitors and repurposed anti-cancer drugs

Use of T cell immune checkpoint inhibitors is emerging as a promising approach in clinical research for treatment of cervical cancer. These inhibitors target molecules that regulate T cell immune responses. Several different checkpoints inhibitors particularly the monoclonal antibodies that target specific antigens on tumors, are currently in different developmental phases like Ipilimumab [(anti-CTLA-4 antibody) NCT01711515], Pembrolizumab [(PD-1 antibody) NCT02628067], and Durvalumab [programmed death ligand-1 (PD-L1) NCT01975831]. Bevacizumab that targets vascular endothelial growth factor (VEGF) prevents angiogenesis, is USFDA approved for the treatment of late-stage or recurrent cervical cancer. Other similar inhibitors in clinical trials are HuMax®-TF-ADC. This is drug-conjugated antibody targeting tissue factor-specific cells, and is being evaluated in clinical trials in patients with cervical or other advanced cancers (NCT02001623, NCT02552121). Another drug-conjugate antibody, IMMU-132 that target human trophoblast cell-surface antigen TROP-2 ectopically expressed in a variety of cancers, is being evaluated in a phase I/II clinical trial in patients with advanced cancer, including cervical cancer (76). Several T cell based-immunotherapy approaches are being examined in clinical trials for cervical cancers and have shown promise (76). Again, most of these therapeutic approaches are not specific to HPV infection.

In parallel to these attempts, HPV-specific treatment strategies are being developed that can be broadly classified into therapeutic vaccines that boost anti-viral immune mechanisms or curtail viral addiction of host cell by targeting its oncogenes by gene silencing using RNA-based strategies or more radical newly emerging genome editing approaches, as well as by use of herbal derivatives that come as pure compounds, extracts, polyherbals or as formulations. Research carried out in areas has been summarized in the following sections.

6.2. Therapeutic HPV vaccines

As specific anti-HPV approaches, therapeutic vaccines are being examined to treat HPV-associated pre-cancers and cancer lesions where HPV infection is already established. As opposed to prophylactic vaccines that induce neutralizing antibodies against L1 capsid proteins, therapeutic vaccines are targeted against viral E6 and E7oncoproteins that are expressed throughout the life cycle of the virus, as other viral proteins like L1/L2 are either not expressed or deleted/inactivated due to viral integration (E1 and E2) in transformed tumor cells. Therapeutic vaccines can be broadly classified as (a) live bacterial or viral vector based (77, 78); (b) peptide or protein based (79, 80); nucleic acid based (81, 82); and whole cell based (83, 84) [reviewed in (85-87)]. Although the list of therapeutic vaccines is rapidly increasing as more than 20 trials are in on-going phase I/II stage whereas some interventions have already completed phase II/III. HPV therapeutic vaccines recently evaluated in stage II or III of clinical evaluation have been listed in Table 1. Among these MVA E2 targeted against HPV16E2 has successfully completed Phase III trial with 90% to 100% remission of anogenital intraepithelial neoplasia (AGIN) lesions in women and men respectively, and HPV clearance was observed in 83% cases in total patients (88). Earlier this vaccine showed 94% efficacy in treating CIN1/2/3 lesions (89), whereas in high grade lesions (CIN2/3) its complete remission was in 56% and partial remission in 32% (90). The vaccine was also useful in treatment of flat condylomas in men leading to eradication of lesion and HPV in 93% of vaccine administered cases (91). Interestingly, MVA E2 is a vaccinia virus Ankara (MVA) containing the bovine papillomavirus E2 protein, but not the expected oncoproteins E6, E7 or E5 of human counterpart of the vaccine strain. As mentioned earlier, expression of E6 and E7 oncoproteins is controlled by E2 that exerts a negative regulatory function on early promoter that transcribes these oncogenes. Therefore, reintroduction of E2 suppressed the expression of E6 and E7 transcripts in HPV-infected cells, and subsequently reduced the transforming ability of the infected, malignant HPV-associated tumor cells (87).

Other vaccines that completed phase II clinical trial are DNA-based VXG3100 (81, 92), protein-based HPV16 E6/E7 Synthetic Long Peptide (79, 80, 93-95), bacterial vector-based GLBL1010c (77), fusion protein-based TA-CIN (96) and TA-CIN+TA-HPV vaccines (97). Most of these vaccines except orally-administered GLBL101c, have local mild to moderate side effects (Table 1). Further, HPV16 SLP vaccine tested in HPV16 positive advanced recurrent gynaecological carcinoma, 9 out of 16 tested patients induced HPV16-specific proliferative response (79). No regression of tumors was observed among the 12 evaluable patients. On the other hand, whole cell based dendritic cell vaccines which hold a lot of promise (98), have proven to be weakly immunogenic in two of the completed phase I studies (83, 84). Apart from these, T-cell based vaccines, involving increase of tumor reactivity of tumor infiltrating lymphocytes using defined antigens in different phases of clinical development pipeline [listed in (87)], and leaving one study (99) the results of these on-going clinical trials are yet to become public. Most of these vaccines showed an efficacy associated with the induction of a strong HPV-specific CD4+ and CD8+ T cell activity. Careful analysis of patients with unresponsive lesions showed reduced systemic vaccine responses but an increased numbers of lesion associated immune suppressive T regulatory cells (Tregs) (93, 100). The failure to cure some premalignant lesions and the cancers appears to result from an unfavorable balance in effector T cells and Tregs. The increased understanding of the role of immune regulation in prevention of effective anti-tumor responses, particularly optimal use of adjuvants in vaccines leading to adequate infiltration of lesion by immune effector cells, may be an adjunct strategy to improve the clinical outcome of the immunotherapeutic regimens in future.

6.3. HPV genome targeting strategies

With emergence of genetic engineering and evolution of gene manipulation technologies, strategies involving nucleic acid (NA) – based targeting of specific HPV gene(s) are now increasingly gaining recognition in both experimental and clinical research. Early NA-based anti-viral agents included antisense oligonucleotides (AS-ODN), ribozymes (Rz) & DNAzymes (Dz), short interfering RNA (siRNA), and short hairpin RNA (shRNA). These therapeutic sequences were designed to target specifically the E6 and E7 regions of HR-HPV genome or their mRNAs, leading to specific, strong and effective down regulation of viral oncogene expression. Recent evolution of gene specific editing technologies utilizing zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat-associated nuclease (CRISPR/Cas9) RNA-guided endonuclease has resulted in further expansion of specific, robust and precise targeting of HPV genome of infected cells and could provide a long lasting solution particularly in advanced stages where the HPV genome is already integrated in host cell. Clinical implementation of these therapies, however, demands a parallel progress in suitable vectors and intracellular targeted delivery of the NA-based therapeutics to the infected cells. The most lucrative aspect that derives this segment of research is high probability to generate revenues as the reagents created are custom-made and may lead to generation of intellectual property. Specific HPV genome targeting approaches employed in different studies are summarized in Table 2. Each of these endeavors has consumed nearly three decades of scientific effort, but still it is very much a work-in-progress.

6.3.1. Silencing HPV RNA by antisense oligonucleotides (AS-ODN)

Use of antisense approach started with testing of native antisense oligonucleotides (AS-ODN) (101). Because of the intracellular stability issues, AS-ODNs were subsequently modified in their phosphate group to phosphorothioate, which made them resistant to intracellular exonucleases (102-105). These AS-ODNs worked efficiently and could inhibit HPV genes by translational silencing, target degradation with the help of endogenous (nuclear) RNaseH, or potentially by exon skipping. Unique feature of these molecules was that they did not require any specific delivery system. However, these early approaches required large quantities of ODNs. Alternatively, different expression vector constructs were employed using plasmids (106-108), adeno- (109, 110) and retro-viral vectors (111, 112). This considerably improved duration of antisense DNA/RNA mediated silencing and helped in avoiding repeat applications in in vivo settings. These approaches were effective in in vitro and to some extent in in vivo experimental setups but essentially required additional delivery methods that are generally not suitable to implement in patients. Further apart, the off target effects were also frequently noted while working with these reagents. Incidentally, use of AS-ODNs which is otherwise very promising molecular therapy remains untouched for clinical evaluation due to parallel advancements in other gene silencing technologies (i.e. siRNA and shRNAs). A wide array of strategic questions pertaining to the medicinal chemistry of oligonucleotides, pharmacokinetics, toxicology, as well as the molecular pharmacology of AS-OSN, remain unaddressed.

6.3.2. Silencing HPV RNA by catalytic oligonucleotides

Another equally promising experimental approach to target HPV transcripts has been using specifically engineered oligonucleotides with trans-acting catalytic properties (113). Commonly referred to as ribozymes and DNAzymes based on their ribose or deoxyribose sugar backbone, both lead to site-specific cleavage of target mRNAs but considerably differ in origin of their catalytic core, mechanism of action and stability within the system (114). Both, engineered ribozymes and recently the DNAzymes have been examined against HPV (Table 2). Among these, about 60 nucleotide long hammerhead ribozymes were designed first and showed activity in cell free system or in vitro cultured cells lines (115-118). Due to size and other experimental variables, research shifted to hairpin ribozymes that were significantly smaller in size (30 nucleotides) (119). A number of ribozyme targets were discovered in viral transcripts, among them R434 and R309 were the most effective target sites reported in HPV16 and HPV18, respectively in some studies (117-119). Ribozyme targeting HPV16 E6E7 transcripts (Rz170) suppressed cell growth and sensitized cervical cancer cells to chemotherapy and radiotherapy (120). To counteract size related problems due to cloning in plasmid vectors, the construct was modified to triplex ribozyme expression system that released processed ribozymes as a single transcript by a self-processing mechanism (121, 122). However, these modifications did not increase ribozyme mediated inhibition more than 30% (122), and thus this approach required further improvement to make ribozymes a therapeutic moiety. Further, the catalytic activity the ribozymes found to dramatically drop within the cellular environment due to Mg++ availability, nuclease action protein binding and with reduced probability of co-localization with the target. Because of deleterious off-target effects in intracellular milieu of an otherwise specific and proven catalytic molecule against HPV in biochemical assays, the pursuit to make this class of molecules as potential therapeutics faded considerably. To overcome problems encountered with ribozymes, alternative molecules particularly the DNAzymes were explored (123, 124). DNAzymes combined the efficiency of ribozymes and intracellular stability and simplicity of AS-ODNs (125). Out of the two commonly used DNAzymes, a widely used and well-characterized 10-23 motif has been engineered to destroy HPV oncogenes (123, 124). DNAzyme Dz1023-434 showed efficient cleavage against HPV-16 E6/E7 mRNA within bona fide antisense window at nt 410-445 even in low (Mg++) condition cell free systems. The stability of these molecules was further improved by locked nucleic acid (LNA)-modification with specific cleavage of HPV transcripts in cell cultures. Despite these improvements, current significance of ribozymes does not look very promising in the light of the emergence of new powerful, simple and easy to use anti-mRNA strategies, particularly the RNA interference (RNAi).

6.3.3. Silencing HPV RNA by RNA interference

This technology chiefly relies on perfect (or nearly perfect) matching of specific 2-8nt RNA sequences known as ‘seed’ regions that are naturally present on 22nt ( 4nt) long guide RNA in RISC complex ubiquitously present in all eukaryotic cells. These small interfering RNAs (siRNA) recognize and guide degradation of the target mRNA, or lead to its translational arrest depending upon the degree of complementarity (126). By exogenously supplementing synthetic siRNA, the naturally existing RNAi machinery can be manipulated to silence a desired mRNA with precision (127). The efficiency of the process is several orders of magnitude more than antisense or ribozyme treatment (128), though its utility is also restricted by its dependence on acceptable delivery methods that are rapidly evolving (129). SiRNA technology has evolved extensively over last decade leading to its evaluation in several clinical trials including some cancers, but targeting HPV or cervical cancer by siRNA is yet to be attempted in clinical setting and it is expected anytime. There have been periodic reviews that evaluated the therapeutic potential of siRNA in treating HPV-associated malignant disease as well as evaluation of the progress in its delivery methods (130-132), and only salient finding of the studies are being discussed.

Efforts to develop siRNA-based anti-HPV therapeutics have been entirely focused around E6 and E7 transcripts of the high risk HPV16 and HPV18 (50, 51, 53, 133-158) (Table 2). A superior silencing was observed if the E6 specific monoclonal antibodies were co-delivered with siRNA in intra-cytoplasmic compartment using liposomes (143). These studies have exhaustively utilized HPV16- (SiHa, CaSki) and HPV18-positive cells (HeLa, C4-I) or murine TC-1 cells (lung epithelial cells transformed with HPV16 E6/7 and ras oncogenes) both in vitro and in vivo as xenografts in nude mice. Despite nucleotide modifications that improved the intracellular stability and off target effects (159) targeting of E6 or E7 by siRNA has been a transient phenomenon and limits its use in vivo. The challenge was mitigated by utilizing shRNA approach (138, 141, 142, 145, 149, 157) or by making double-stranded RNA–DNA chimera (dsRNADNA) (151). Though theoretical off target effects of siRNA seed sequences are high which raised concern, experimental studies show lack of any correlation between predicted and actual off-target effects of siRNAs targeting the HPV16 E7 oncogene (160). Further, expression of shRNA in cells is stable and induces robust silencing for a long period ranging beyond 4 months in vitro (141). However, this strategy typically requires delivery through viral (142, 149) or bacterial expression vectors (134, 141, 145, 150, 157) which can pose safety concerns (161).

Targeting E6 or E7 by siRNA and snRNA invariably resulted in concomitant reemergence of p53 and phospho-pRB pools that were accompanied by a variable extent of a spectrum of anti-cancer effects characterized by (i) reduced cell growth in monolayers and in anchorage-independent conditions, (ii) arrested cell cycle and DNA synthesis, (iii) reduced colony forming ability, (iv) induction of senescence and apoptosis (v) reduced invasion and expression of epithelial mesenchymal transition markers, and (vi) loss of stemness and associated signaling in cultured cells (Table 2). Similarly, when these siRNA were administered in vivo in xenografted HPV-positive cells that form tumors in nude mice, tumorigenecity of treated cells declined and a reduced tumor formation was invariably noticed along with reduced angiogenesis and metastatic potential but to a variable extent. These variations were largely dependent on dose of siRNA, delivery methods, vectors, route of administration, or the transcript region against which the siRNA were raised.

Parallel to the development in siRNA technology, studies were carried out to integrate this promising RNAi approach with conventional and targeted anti-cancer therapy against cervical cancer (137, 148, 152-155). Combination studies performed in in vitro cultures showed a variable wide-ranging response from synergistic to additive to sub-additive within different studies. However, most of the combinations failed in animal models. As siRNAs are polyanions that do not readily cross the cell membrane in vivo coupled with immune-hostile tumor microenvironment and physiological barriers of the circulatory system, susceptibility to ribonuclease degradation, rapid renal excretion and nonspecific uptake by the reticulo-endothelial system, delivery of siRNAs remains a major obstacle to their clinical or even in vivo use (162). These bottlenecks are currently guiding research to develop safe, stable, and efficient nano-engineered targeted delivery systems some of which are currently under clinical testing (132).

6.3.4. HPV Genome Targeting approaches

Targeting DNA through triplex forming oligonucleotides (TFO) was the initial and oldest attempt for transcriptional interference of HPV (163). Subsequently, several natural triplex forming sites were reported in HPV genome (164). Though DNA triplexes exhibited remarkable sequence specificity (165) and stability (166), the approach did not affect HPV replication or transcription appreciably. Among highly popular and easy to use RNA interference approaches, there has been emergence of a revolutionary research in specific targeted DNA manipulation techniques that led to HPV genome editing. These include ZFNs, TALENs, and the CRISPR/Cas9RNA-guided endonuclease system. Each of these technologies specifically recognize and locate a target sequence with homologous binding specific protein domains or the guide RNA (gRNA) and catalyze a DNA double stranded break by attached restriction enzyme and reviewed extensively in recent past (167). Because of the precision of programmable nucleases these technologies are proposed as a powerful antiviral therapy for persistent viral infections (168, 169). Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials (NCT01455389, NCT00595088, and NCT01118052).

ZFN were the first among engineer customized zinc-finger proteins created by fusing DNA-binding zinc finger proteins to the FokI DNA-cleavage domain (170) and are protected by patent to Sangamo Biosciences that resulted prohibitive costs in negotiating multiple use (171). Two custom-designed ZFNs catalyze cleavage in both strands upon binding to DNA in dimeric form that activates FokI nuclease. Double-strand breaks (DSB) created by ZFNs are repaired primarily by non-homologous end joining pathway in the absence of any other similar (donor) fragment. In case these enzymes are co-delivered with a donor template that directs gene replacement, the two DSBs are repaired by the homology-directed repair pathway. Early ZFN targeted against HPV were engineered for HPV18 E2 region which prevented replication of the virus in the cell (172, 173). Subsequently, engineered FokI-based constructs were developed against HPV16 and HPV18 E7 (174). These ZFNs disrupted HPV E7 oncogenes in both HPV16/18–positive cervical cancer cells, led to cell growth inhibition and induced apoptosis of corresponding HPV16- and HPV18-positive cervical cancer cell lines. ZFNs also repressed xenograft formation in vivo. Further work in this direction is less due to patent restrictions associated with this valuable technology.

Like ZFN, TALENs contain two domains out of which one depend upon repeat variable diresidues (RVD) that are arranged in 33~35 amino acid tandem repeat arrays which determine the specificity of the protein domain and can be engineered to recognize specific target sequence, and the other is FokI nuclease. Compared to ZFNs, TALENs possess several advantages including lower cytotoxicity, greater design flexibility, and simple engineering (175). Recently, TALEN-mediated targeting of HPV oncogenes was found to ameliorate HPV-related cervical malignancy (176). TALENs disrupted E6 and E7 oncogenes, successfully triggered specific apoptosis and growth inhibition and reduced tumorigenecity of HPV-infected cells in vitro, and mutated the E7 oncogene, reduced viral DNA load, and restored RB1 function and downstream E2F1 and CDK2, and reversed the malignant phenotype in K14 HPV16 transgenic female mice when administered in vivo directly at cervix. TALENs have their own limitations. The numbers of exogenous sequences that can be targeted are limited by the use of a thymidine at 4th position (177). Further, methylcytosine is not cleaved by the conventional TALENs (178). Methylcytosine is indistinguishable from thymidine in major groove which reduces target specificity. Further, TALEN repeats are difficult to amplify in PCR due to repeats and are susceptible to recombination.

The CRISPR/Cas system consists of two components: guide gRNA and nucleases (Cas). This originally constitutes the immune system of some bacteria and used for defending against foreign nucleic acids. The most commonly used type is CRISPR/Cas9 including a conjugation of the tracrRNA (trans-activating crRNA)—crRNA (CRISPR RNA) to a single guide RNA and Cas9 (175). Unlike ZFNs and TALENs that involve engineering protein domains, this system utilizes a simple guide RNA that directs DSB in DNA and the specificity can be changed by simply altering the guide RNA in the complex. Recently the CRISPR/Cas9 system has emerged as a promising candidate against HPV (179-183) (Table 2). Both high risk HPV16 and HPV18 as well as low risk HPV6 and HPV11 have been targeted with reasonable efficiency. These genome editing experiments also resulted in loss of correspondingly targeted genes and associated downstream events. Interestingly, recent study showed targeting of a large HPV genome fragment can result in restoration of gene functions of integration-targeted cellular genes thus facilitating reversal of the HPV integration event (183).

Even though HPV genome editing is a promising approach, properties, like negative charge, large size, low membrane penetration, low endosomal escape, and weak serum tolerance, genome editing by these nucleases prevent successful implementation of the technology unless an effective in vivo delivery system is developed for these programmable nucleases. Potential delivery strategies under clinical testing and commonly used vectors available and suitable for each class of nucleases are discussed elsewhere (175) and are equally applicable in the context of HPV genome editing. Further, the obstacles being faced in testing of gene editing-based antiviral therapeutics in animal models and transition towards human application have discussed in detail elsewhere (169). Even if these operational problems could be resolved, a major impediment to the widespread application of gene editing and gene manipulation based antivirals will be the potential cost to the populations at risk and will push development of alternate low cost therapeutics for treatment of target population which usually belongs to low socio-economic strata and cannot afford this sophisticated technology.

6.4. Emerging anti-HPV pharmaceuticals from nature

As per the survey conducted by National Cancer Institute (USA), of 175 approved anti-tumor drugs 131 are non-synthetic and are either natural products, their mimics or directly derived from them (184, 185). Even the standard anti-cancer therapeutics like vincristine, vinblastine, paclitaxel and camptothecin as well as podophyllotoxin, which are the established treatments for genital warts, are of plant origin (186). Therefore, in addition to developing immunotherapeutics, another major area of active research for developing treatments against cervical cancer is to explore potential pharmaceutical agents from natural resources especially plants. Ever since the cultures of HeLa cells were developed in 1952 (187, 188), the most practiced approach that prevailed prior to the knowledge of viral etiology of cervical cancer, was to test potential drugs or phytochemicals on this established cervical cancer cell line. Many natural products have frequently been tested and were found to possess potentially useful anti-cancer activities that have been summarized in Table 3. However, majority of these investigational entities displayed generic anti-cancer effects and the investigations remained restricted to testing them in the cell lines. These leads should be screened further to explore if they have any HPV-specific effects and their potential utility in clinical settings if feasible need to be established. Nevertheless, a sizable effort has been directed towards specific pharmacological targeting of HPV-mediated events during cervical carcinogenesis that can be broadly grouped together as (a) prevention of virus entry into the host cell; (b) to eliminate virally-infected cervical cells or to reduce their viral load in precancer stage; (c) to prevent virus-induced tumorigenic transformation; (d) abrogate carcinogenic progression by reducing expression of high risk oncoproteins E6/E7 or rendering them non-functional, (e) targeting host factors that are essentially required for expression of viral oncogenes or elimination of oxidative stress leading to apoptotic cell death of cervical cancer cells; (f) boosting immune surveillance or inhibiting co-infections that work as cofactors, and/or (f) to improve the efficacy of standard chemo-radiation therapies.

Natural products demonstrated to have anti-HPV activities and can emerge as potential treatments for cervical cancer are listed in Table 4. These natural products can be broadly classified as (a) phytochemicals which are mostly available as cataloged purified and characterized chemical entities, (b) isolated active compounds from crude or fractionated extracts, or (c) the extracts themselves that are used as such for treatment of cervical cells in vitro and are poorly defined and hard to reproduce, or (d) polyherbals having different constituent herbal derivatives with pharmacologically approved excipients that contain a combination of pure compounds or extracts and are the most complex formulations among all. Individual anti-viral and anticancer activities and drug interaction between components of these formulations are poorly defined. Keeping in view the space constraints, only those natural products that showed specific anti-HPV effects have been discussed in detail (Table 4).

Among phytochemicals, curcumin (diferulylmethane) derived from Curucuma longa Linn. displayed strong anti-HPV activity by targeting the expression of viral oncogenes (44, 45, 189). Curcumin is a known potent anti-inflammatory and anti-cancer small molecule inhibitor with proven pharmacological safety (190). The mechanistic studies revealed the inhibitory effect on oncogenes transcription was through downregulation of essentially required transcription factors AP-1, NF- B and STAT3 activity by curcumin (44, 45, 189). Blocking of HPV transcription was accompanied with induction of apoptotic cell death in cervical cancer cells. The effect was observed in HPV-positive cancer cells irrespective of the infecting high risk HPV types. Subsequent studies that evaluate anti-cancer activities of curcumin in cervical cancer cells also validated these findings (191, 192). A preliminary testing of oral administration of curcumin upto 12gm a day for 3 months in 4 CIN cases demonstrated clinical safety of the phytochemical with histological improvements in 1 out of 4 cases (193). Later, phase I/II double-blind clinical trial were conducted using intra-vaginal application of curcumin-containing gelatin passaries in women infected with HPV and without any high grade CIN (194). The study showed higher clearance of cervical HPV infection (81.3 %) compared to the control arm (73.3 %) (195). The formulation was found safe as no serious adverse events were noted with topical application of curcumin. The major difference among the two studies (193, 194) was the route of administration. Since premalignant tumors in cervix are accessible to topical applications, any therapy directly applied to the affected tissue appeared to be more effective and avoided the problem of bioavailability that is frequently encountered in working with majority of the phytochemicals when they are administered orally.

Similarly, berberine isolated from perennial herb Berberis displayed anti-HPV activity and suppressed the expression of viral oncoproteins E6 and E7 (52). Berberine possesses anti-inflammatory and anti-cancer properties with no known toxicity. Berberine exerted its inhibitory action through inhibition of host transcription factor AP-1 in a dose- and time-dependent manner and altered the composition of AP-1 DNA-binding complex. Berberine specifically downregulated expression of oncogenic c-Fos which was also absent in the AP-1 binding complex. Loss of E6 and E7 levels was associated with concomitant increase in p53 and pRb expression, loss of telomerase protein, hTERT, and resulted in growth inhibition of cervical cancer cells. Further, berberine altered epigenetic modifications, disrupted microtubule network by targeting p53 in cervical cancer cells (196). Thus, berberine proved its potential as a promising chemotherapeutic agent in cervical cancer. Though berberine is yet to be tested clinically in cervical cancer it is already being examined in clinical settings for prevention of colorectal adenomas recurrence (NCT02226185). It is also an important component of botanical medical practice including Goldenseal (Hydrastis canadensis), Berberis (Berberis vulgaris), Oregon grape (Berberis aquifolium), Barberry and Chinese Goldthread (Coptis chinensis). Phellodendron chinense and Phellodendron amurense are other two berberine-containing plants that are extensively utilized in Chinese medicine.

Another interesting phytochemical described with anti-HPV activity is Jaceosidin that inhibited the function of E6 and E7 oncogenes by physical interaction with oncoproteins that renders them non-functional (197). Other phytochemicals like Nor-dihydro-guaiaretic acid (NGDA) from plant lignin, and woginin, an anti-cancer flavonoid compound extracted from Scutellaria baicalensis, downregulated HPV E6 and E7 expressions and promoted intrinsic apoptosis in human cervical cancer cells through suppression of HPV transcription (198-200). Some other natural products like heparin (201), carrageenan a polysaccharide extracted from marine red algae (seaweed) (202), sulindac (non-steroidal anti-inflammatory drug) and sulfated K5 E. coli polysaccharide derivatives (203) reportedly exert anti-viral action against HPV primarily by preventing the entry of the virus to its target cells. Apart from the purified chemical entities, some crude and partially fractionated extracts of medicinal plants like rhizome extract of Pinellia pedatisecta Schott, Fractionated leaf extract of Bryophyllum pinnata rich in Bryophyllin A, crude fruit extract of Phyllanthus emblica Linn. and Brucea javanica oil emulsion has shown potent anti-HPV activities that are mediated through their inhibitory action on host factors like AP-1 and STAT3. These transcription factors promote cervical carcinogenesis and specifically downregulated expression of viral oncogenes E6 and E7 (204-207) (Table 4).

Based on safety of herbal ingredients and their general anti-microbicidal action, a few poly-herbal formulations or mixtures based on herbal derivatives have been evaluated in clinical studies. Praneem, a combination of leaf extract of Azadirachta indica, saponins, purified extracts of Emblica officinalis, Mentha citrata oil, and extracts of Aloe vera gel was examined in HPV infected women in a placebo controlled clinical study (208). One course of thirty day intra-vaginal application of Praneem polyherbal tablet in women with early cervical intraepithelial lesions resulted in elimination of HPV16 infection in 60% of the women (6/10). The patients that could not clear HPV infection were subjected to another round of Praneem treatment and HPV clearance was observed in 50% of individuals resulting in total of 80% HPV clearance rate (208). Though the mechanism of action of Praneem or its individual components is not known, but a general microbicidal activity of the formulation against reproductive tract infections that can promote HPV-mediated cervical carcinogenesis as co-factors had been reported earlier (209). Later, following discovery of curcumin’s specific action on HPV transcription (44), another polyherbal cream formulation was developed and named ‘Basant’ for its saffron color that was imparted by chief ingredient curcumin. The formulation apart from curcumin, contained purified saponins, extracts of Emblica officinalis, Mentha citrata oil and extracts of Aloe vera gel that were common with Praneem (210). Although Basant showed a broad spectrum anti-microbicidal activity against drug resistant bacterial and fungal strains, it had a strong virucidal activity against HPV as well as HIV in in vitro analysis. Two of its ingredients, Aloe and Amla, which also formed part of Praneem, inhibited the transduction of HPV16 pseudovirus in HeLa cells at concentrations far below those that are cytotoxic and those used in the formulation. Therefore, the mechanism of these two components may be different and complementary to curcumin which target viral transcription (44, 45). Though Basant was found to be totally safe according to pre-clinical toxicology carried out on rabbit vagina by applying the formulation for 7 consecutive days or twice daily for 3 weeks (210), in clinical studies a mild to moderate vaginal irritation and itching was reported after Basant application (195). In this double blind randomized clinical evaluation study, women showed a statistically significant clearance of HPV infection following Basant application (87.7 % vs. placebo arms -73.3 %) (195). In another recent clinical study, Basant application in 11 women infected with HPV with low grade cervical abnormalities resulted in clearance of HPV16 infection in all the women recruited (11/11) (211). Further large scale clinical evaluation of these promising polyherbals is warranted to make them a useful, effective, cheap and clinically available therapeutic.

Apart from herbal derivatives, other natural sources have been explored for potential anti-HPV activity. The most prominent among them was carrageenan which was found to be is a potent inhibitor of HPV infection (202). Carrageenan extracted from marine red algae is composed of a sulfated polysaccharides of D-galactose and 3, 6-anhydro-D-galactose, and present in some vaginal lubricants (184). Carrageenan prevented the entry of the virus into the host cell both in vitro (202) and in animal models (212). A novel intra-vaginal ring of core-matrix having combination microbicide containing carrageenan as constituent to prevent HPV infection have been developed (213). Further clinical trials are needed to determine the efficacy of carrageenan-based products against genital HPVs in humans. Similarly, alginic acid and fucoidan derived from red algae were found to possess anti-HPV infection activities in high-throughput in vitro screens based on high-titer HPV pseudoviruses to identify HPV infection inhibitors (202). Other marine derivatives like gliotoxin, neoechinulin A, physcion, epoxydon and heterofucan also showed generic anti-cervical cancer activity and have been reviewed separately (184). Further studies are needed to examine their anti-HPV activities.

In view of the emerging anticancer activities, the herbal derivatives are being examined for their used as adjuvant to mainstream chemo- and radiotherapies for treatment of cervical cancer. Preliminary investigations demonstrate potential role of phytochemicals as chemosensitizers in cervical cancer. Phytochemicals like curcumin sensitized cervical cancer cells to taxol-induced or cisplatin-induced apoptosis by down-regulation of NF- B (214-216). Similarly, other phytochemicals like quercitin, saikasaponins, wogonin, tea polyphenol epigallocatechin galate (EGCG) and apigenin also sensitized cervical cancer cells to cisplatin (217-220), resveratrol, trihydroxyisoflavone and formononetin were found to sensitize cervical cancer cells to different anthracyclines (217-220). Further, dietary flavonoids sensitized cervical cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (221). TRAIL is another promising candidate as cancer therapeutics that preferentially induces apoptosis in cancer cells. Flavone, apigenin genistein, decursin and decursinol markedly augmented TRAIL-mediated cytotoxicity (221, 222). Many of these phytochemicals mediate their action through reactive oxygen species (ROS) (218-221, 223-226) or targeting different drug resistance mechanisms upregulated by viral oncogenes (51, 158).

A few phytochemicals have shown potent radiosensitizing effects. Resveratrol (227), genestein (228-230), curcumin (223, 231), ferulic acid (232) and quercitin (233) have shown synergistic anti-cancer properties and potentiation of apoptotic effects of ionizing radiation on cervical cancer cells primarily in vitro and in some studies in vivo (233). A ROS-dependent mechanism has again been implicated in radiosensitivity mediated by at least some of the phytochemicals (223)

With evolution of sophisticated molecular modeling, in silico approaches have been utilized to screen phytochemicals for their anti-HPV activity by specifically targeting molecular interaction of viral oncogenes with their respective cellular targets, thus rendering the viral oncogenes non-functional (234-237). In silico docking of Carrageenan, Daphnoretin, Withaferin A and Artemisinin against HPV16 and HPV18 E6 revealed nearly 20X stronger inhibitory effect as compared to curcumin and tea polyphenol ECGC or jaceosidin found to functionally interfere with E6 (234-236). Further studies are needed to validate these findings in experimental settings. Nevertheless, the approach is extremely powerful tool to examine potentially useful phytochemicals and developing them into a rationally-designed drug in a very short period.

Pure herbal derivatives, even though show remarkable anti-cervical cancer or anti-HPV activity in in vitro studies, they have poor bioavailability due to factors that vary with each derivative. Solubility of herbal derivatives in water has been a great challenge which prevents their intravenous administration. Therefore, majority of the herbal derivatives are administered orally, and it is the foremost reason of reduced bioavailability which is attributed to poor absorption, and metabolism while in gut or in liver followed by rapid excretion through kidney. These plant products are metabolized by hydrolysis, glucuronidation or glycosidation by gut flora [reviewed in (238)]. Such modifications severely impact the activity of those plant products where the metabolites are inactive. It is, therefore, important that these herbal derivatives be used in combination with biomolecules like piperine that is known to prevent glucuronidation (239-241). Further, mixing of two or more herbal products essentially requires optimization of mixtures which further increases magnitude of in vivo experimentation. Alternatively, nano-formulations and chemically-modified herbal derivatives have been examined in cervical cancer cells and have shown superior anti-cancer effects compared to their native counterparts at an equal strength in some cases. During last decade, there has been a significant progress in nano-formulations and derivatization of phytochemicals to improve their efficacy and bioavailability. Experimental combination of natural products/phytochemicals with silver nano- or liposomal formulations have been attempted to improve the anti-cancer activity (242-245) or to improve the chemosensitization of cervical cancer cells (246, 247). Phytochemicals derivatized to enhance activities against cervical cancer cells include lignin, chrysin, nitroflavone, propyl gallate, bromofascaplysins, butylated hydroxyanisole, flavonoid and curcumin (248-255). Treatment with most of the chemically-modified derivatives resulted in reduced proliferation and enhanced apoptosis and showed targeting of different but complementary anti-cancer mechanisms such as inhibiting telomerase and activation of c-myc and caspases by substituted methylenedioxy lignan (254), or cell cycle arrest G2/M by nitroflavone (249) and in G1 phase by propyl gallate (250), or GSH level depletion by butylated hydroxyanisole (252) or recently by downregulation of HR-HPV E6 and E7 (255).

Apart from chemically-defined entities or their nano-formulations, all other plant derivatives suffer from common drawbacks of batch to batch variation. Nevertheless, the herbal derivatives’ group as a whole shares many advantages include abundance, ease of isolation and their pharmacological safety that make them low cost, effective therapeutic entities. Despite these advantages, highly encouraging leads obtained from herbal or natural sources are not being put forward in large scale Phase II/III/IV clinical trials and preventing these formulations to become a clinical reality. Because of prevailing stringent regulations, execution of interventional clinical trials is now becoming more and more expensive, labor-intensive and demands an exhaustive infrastructure. Unfortunately, due to low commercial gains associated with these commonly-used, widely-available traditional and herbal remedies, testing these entities in large-scale clinical trials have become prohibitive in itself thus creating a major bottleneck and a roadblock to introduction of anti-HPV molecules/formulations from herbal sources in mainstream medicine.

6.5. Anti-HPV leads in alternative medicines

Interestingly, there are several parallel and concurrent systems of medicine that are operating along with mainstream medicine which extensively utilize same plants as source of their medicines. Among them, Indian, Chinese, Japanese and many other traditional and folklore medicines are popularly being practiced along with mainstream allopathic medicine all over the word. Nevertheless, Homeopathy is one of the most accepted 2nd line clinical management systems operating globally. Several formulations are well-standardized, and commercially available. Formulations are available for the treatment of symptoms associated with genital warts and cervical cancer these include Iodum, Kreosotum, Natrum Carb, Carbo Animalis, Calcaria Fluor, Hydrocotyle, Sabina, Calcaria Carb, Kali Iodide, Conium, Hydrastis, Sanguinaria, and Lachesis (256). Despite claimed efficacy, the mechanism of action of these homeopathic formulations/drugs, particularly on HPV and its oncogenes, is not known. Only a few reports describe the anti-cancer effect of homeopathic remedies and their mechanism of action in experimental cancers and cell cultures (257-260). On the other hand, Lycopodine from Lycopodium clavatum extract has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease, was found to inhibit proliferation of HeLa cells and induced caspase-3 mediated apoptosis (261). Similarly, Conium maculatum extract which is used as a traditional medicine for cervix carcinoma including homeopathy, induced apoptosis in cervical cells through ROS generation (262). Drug-DNA interaction was implicated in this phenomenon. Interestingly, recent studies showed existence of nanoparticle-like structures in some homeopathy formulations (239, 263-265). In this regard, it would be relevant to investigate anti-HPV effects and mechanism of action of these homeopathic drugs. Alternatively, as a second approach, the homeopathic remedies that utilize herbal derivatives outlines above (curcumin, berberine, Bryophyllum, Phyllanthus, spindus, Azadirachta etc.) may be explored for potential anti-HPV activities. These research objectives should be currently the area of active investigation for discovery and development of low-cost anti-HPV molecule (s).

7. CONCLUSION

It is a matter of prime importance to develop anti-HPV molecules/formulations that could eliminate HPV infection and are safe. Since HPV and related malignancies are multifactorial with long incubation period of the virus, combination of approaches may be used for effective control of virus-driven oncogenic progression. Targeting HPV persistence and specific downregulation of viral oncoproteins by any HPV-specific approach whether using therapeutic vaccines or genome silencing or genome editing are expected to provide a useful therapy. However, in practice such approaches have never been foolproof and rewarding and the reason could be that some of the malignant lesions despite HPV positivity behave like an HPV-negative tumors at molecular level. Recent TCGA (The Cancer Genome Atlas) study examined genome features of cervical cancers and identified novel genomic and molecular characteristics of HPV negative endometrial-like cancers (266). Therefore, it is important that investigational drugs under development not only target HPV or its oncogenes but also additionally target factors that influence carcinogenic progression; which could also curtail local inflammation by eradicating co-infectors and dampening of carcinogenic pro-inflammatory transcription factors that additionally activate HPV transcription. Incidentally, phytochemicals and some of the plant derivatives have been shown to target such carcinogenic transcription factors (43-45, 50-52, 158, 189, 205, 207, 208, 267) and this area should be explored further more vigorously. Further, developing active principles and leads from alternate medicine that already have well-defined safety and efficacy profile, as potential anti-HPV therapeutics against cervical cancer in mainstream medicine will be economic, easily translatable in clinic without undergoing expansive phase II/III/IV clinical trials and will help in creation of a fusion medicine.

8. ACKNOWLEDGEMENTS

The study was supported by extramural research grants from Department of Science and Technology (DST-PURSE Phase II/RC/2016/944), from Department of Biotechnology, Government of India (Grant Support BT/PR10347; & 102/IFD/SAN/PR-1612/2007; 6242-P34/RGCB/PMD/DBT/ALCB/2015) and intramural funding to ACB from University of Delhi and Indian Council of Medical Research (5/13/38/2014/NCD3), Department of Health Research, Ministry of Health and Family Welfare, Government of India, India. JRF to TS from UGC [2061430699 22/06/2014 (i) EU-V] and PDF to DP from UGC (E4-2/2006 (BSR)/BL/15-16/0328), SRF to MJ from ICMR (3/2/2/278/2014/NCD3).

9. REFERENCES

1. C. E. Hufford and E. L. Burns: Papanicolaou test in the early diagnosis of uterine cancer. Ohio State Med J, 44 (9), 900-3 (1948)

2. M. Safaeian, D. Solomon and P. E. Castle: Cervical cancer prevention--cervical screening: science in evolution. Obstet Gynecol Clin North Am, 34 (4), 739-60, ix (2007)

3. IARC: Globocan 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012. In: WHO, Lyon, France (2016)

4. S. K. Cesario: Global inequalities in the care of women with cancer. Nurs Womens Health, 16 (5), 372-85 (2012)

DOI:10.1111/j.1751-486X.2012.01761.x

PMid:23067282

5. IARC: Biological Agents: Volume 100B A Review of Human Carcinogens. IARC, Lyon (France) (2012)

6. M. Schiffman, J. Doorbar, N. Wentzensen, S. de Sanjose, C. Fakhry, B. J. Monk, M. A. Stanley and S. Franceschi: Carcinogenic human papillomavirus infection. Nat Rev Dis Primers, 2, 16086 (2016)

7. N. Munoz, X. Castellsague, A. B. de Gonzalez and L. Gissmann: Chapter 1: HPV in the etiology of human cancer. Vaccine, 24 Suppl 3, S3/1-10 (2006)

8. S. Shukla, A. C. Bharti, S. Mahata, S. Hussain, R. Kumar, S. Hedau and B. C. Das: Infection of human papillomaviruses in cancers of different human organ sites. Indian J Med Res, 130 (3), 222-33 (2009)

9. H. zur Hausen: Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer, 2 (5), 342-50 (2002)

DOI:10.1038/nrc798

PMid:12044010

10. M. Griffiths: 'Nuns, virgins, and spinsters'. Rigoni-Stern and cervical cancer revisited. Br J Obstet Gynaecol, 98 (8), 797-802 (1991)

DOI:10.1111/j.1471-0528.1991.tb13485.x

PMid:1911588

11. C. Ley, H. M. Bauer, A. Reingold, M. H. Schiffman, J. C. Chambers, C. J. Tashiro and M. M. Manos: Determinants of genital human papillomavirus infection in young women. J Natl Cancer Inst, 83 (14), 997-1003 (1991)

DOI:10.1093/jnci/83.14.997

PMid:1649312

12. A. B. Moscicki, N. Hills, S. Shiboski, K. Powell, N. Jay, E. Hanson, S. Miller, L. Clayton, S. Farhat, J. Broering, T. Darragh and J. Palefsky: Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females. JAMA, 285 (23), 2995-3002 (2001)

DOI:10.1001/jama.285.23.2995

PMid:11410098

13. L. Gissmann: Linking papillomaviruses to cervical cancer: a long and winding road In: Papillomavirus Research: From Natural History to Vaccines and Beyond. Ed M. S. Campo. Caister Academic Press, Norflok (2006)

14. D. Bzhalava, C. Eklund and J. Dillner: International standardization and classification of human papillomavirus types. Virology, 476, 341-4 (2015)

DOI:10.1016/j.virol.2014.12.028

PMid:25577151

15. L. Bruni, L. Barrionuevo-Rosas, G. Albero, B. Serrano, M. Mena, D. Gomez, J. Munoz, F. X. Bosch and S. de Sanjose: ICO Information Centre on HPV and Cancer, Summary report ,7 October 2016. Institu Catala d'Oncologia (2016)

16. M. Evander, K. Edlund, A. Gustafsson, M. Jonsson, R. Karlsson, E. Rylander and G. Wadell: Human papillomavirus infection is transient in young women: a population-based cohort study. J Infect Dis, 171 (4), 1026-30 (1995)

DOI:10.1093/infdis/171.4.1026

PMid:7706782

17. A. Hildesheim, M. H. Schiffman, P. E. Gravitt, A. G. Glass, C. E. Greer, T. Zhang, D. R. Scott, B. B. Rush, P. Lawler, M. E. Sherman and et al.: Persistence of type-specific human papillomavirus infection among cytologically normal women. J Infect Dis, 169 (2), 235-40 (1994)

DOI:10.1093/infdis/169.2.235

PMid:8106758

18. E. L. Franco, L. L. Villa, J. P. Sobrinho, J. M. Prado, M. C. Rousseau, M. Desy and T. E. Rohan: Epidemiology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer. J Infect Dis, 180 (5), 1415-23 (1999)

DOI:10.1086/315086

PMid:10515798

19. G. Y. Ho, R. Bierman, L. Beardsley, C. J. Chang and R. D. Burk: Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med, 338 (7), 423-8 (1998)

DOI:10.1056/NEJM199802123380703

PMid:9459645

20. USFDA: Approval Letter - Human Papillomavirus Quadrivalent (Types 6, 11, 16, 18) Vaccine, Recombinant. In: Vaccines, Blood and Biologics. Ed USFDA. USFDA, Rockville, MD (2006)

21. TGA: ARTG ID 126114 - CERVARIX human papillomavirus vaccine types 16 and 18 (recombinant, AS04 adjuvanted) suspension for injection pre-filled syringe (Approval date May 18, 2007). In: Therapeutic Goods Administration. Ed A. G. D. o. Health. TGA Australia, Symonston (Australia) (2007)

22. ICO: Human Papillomavirus and Related Diseases in India. 2014. In: Information Centre on HPV and cancer. Ed ICO. Institut Catala d'Oncologia (ICO), (2014)

23. C. de Martel, J. Ferlay, S. Franceschi, J. Vignat, F. Bray, D. Forman and M. Plummer: Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. Lancet Oncol, 13 (6), 607-15 (2012)

DOI:10.1016/S1470-2045(12)70137-7

24. H. De Vuyst, G. M. Clifford, M. C. Nascimento, M. M. Madeleine and S. Franceschi: Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis. Int J Cancer, 124 (7), 1626-36 (2009)

DOI:10.1002/ijc.24116

PMid:19115209

25. M. Plummer, C. de Martel, J. Vignat, J. Ferlay, F. Bray and S. Franceschi: Global burden of cancers attributable to infections in 2012: a synthetic analysis. Lancet Glob Health, 4 (9), e609-16 (2016)

DOI:10.1016/S2214-109X(16)30143-7

26. M. Schiffman, G. Clifford and F. M. Buonaguro: Classification of weakly carcinogenic human papillomavirus types: addressing the limits of epidemiology at the borderline. Infect Agent Cancer, 4, 8 (2009)

27. L. Bruni, M. Diaz, X. Castellsague, E. Ferrer, F. X. Bosch and S. de Sanjose: Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis, 202 (12), 1789-99 (2010)

DOI:10.1086/657321

PMid:21067372

28. B. C. Das, S. Hussain, V. Nasare and M. Bharadwaj: Prospects and prejudices of human papillomavirus vaccines in India. Vaccine, 26 (22), 2669-79 (2008)

DOI:10.1016/j.vaccine.2008.03.056

PMid:18455843

29. A. B. Moscicki, M. Schiffman, A. Burchell, G. Albero, A. R. Giuliano, M. T. Goodman, S. K. Kjaer and J. Palefsky: Updating the natural history of human papillomavirus and anogenital cancers. Vaccine, 30 Suppl 5, F24-33 (2012)

30. F. X. Bosch, T. R. Broker, D. Forman, A. B. Moscicki, M. L. Gillison, J. Doorbar, P. L. Stern, M. Stanley, M. Arbyn, M. Poljak, J. Cuzick, P. E. Castle, J. T. Schiller, L. E. Markowitz, W. A. Fisher, K. Canfell, L. A. Denny, E. L. Franco, M. Steben, M. A. Kane, M. Schiffman, C. J. Meijer, R. Sankaranarayanan, X. Castellsague, J. J. Kim, M. Brotons, L. Alemany, G. Albero, M. Diaz and S. de Sanjose: Comprehensive control of human papillomavirus infections and related diseases. Vaccine, 31 Suppl 7, H1-31 (2013)

31. J. T. Cox: Epidemiology of cervical intraepithelial neoplasia: the role of human papillomavirus. Baillieres Clin Obstet Gynaecol, 9 (1), 1-37 (1995)

DOI:10.1016/S0950-3552(05)80357-8

32. P. E. Castle: Beyond human papillomavirus: the cervix, exogenous secondary factors, and the development of cervical precancer and cancer. J Low Genit Tract Dis, 8 (3), 224-30 (2004)

DOI:10.1097/00128360-200407000-00011

PMid:15874868

33. K. R. Dahlstrom, A. N. Burchell, A. V. Ramanakumar, A. Rodrigues, P. P. Tellier, J. Hanley, F. Coutlee and E. L. Franco: Sexual transmission of oral human papillomavirus infection among men. Cancer Epidemiol Biomarkers Prev, 23 (12), 2959-64 (2014)

DOI:10.1158/1055-9965.EPI-14-0386

PMid:25392180

34. B. J. Morris, C. A. Hankins, A. A. Tobian, J. N. Krieger and J. D. Klausner: Does Male Circumcision Protect against Sexually Transmitted Infections? Arguments and Meta-Analyses to the Contrary Fail to Withstand Scrutiny. ISRN Urol, 2014, 684706 (2014)

35. H. zur Hausen and E. M. de Villiers: Cancer "causation" by infections--individual contributions and synergistic networks. Semin Oncol, 41 (6), 860-75 (2014)

DOI:10.1053/j.seminoncol.2014.10.003

PMid:25499643

36. M. Kyrgiou, A. Mitra and A. B. Moscicki: Does the vaginal microbiota play a role in the development of cervical cancer? Transl Res, 179, 168-182 (2017)

37. I. Ghosh, R. Mandal, P. Kundu and J. Biswas: Association of Genital Infections Other Than Human Papillomavirus with Pre-Invasive and Invasive Cervical Neoplasia. J Clin Diagn Res, 10 (2), XE01-XE06 (2016)

38. J. M. de Castro-Sobrinho, S. H. Rabelo-Santos, R. R. Fugueiredo-Alves, S. Derchain, L. O. Sarian, D. R. Pitta, E. A. Campos and L. C. Zeferino: Bacterial vaginosis and inflammatory response showed association with severity of cervical neoplasia in HPV-positive women. Diagn Cytopathol, 44 (2), 80-6 (2016)

DOI:10.1002/dc.23388

PMid:26644228

39. S. I. Grivennikov and M. Karin: Dangerous liaisons: STAT3 and NF-kappaB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev, 21 (1), 11-9 (2010)

DOI:10.1016/j.cytogfr.2009.11.005

PMid:20018552 PMCid:PMC2834864

40. I. Arany, K. G. Grattendick and S. K. Tyring: Interleukin-10 induces transcription of the early promoter of human papillomavirus type 16 (HPV16) through the 5'-segment of the upstream regulatory region (URR). Antiviral Res, 55 (2), 331-9 (2002)

DOI:10.1016/S0166-3542(02)00070-0

41. T. Chong, D. Apt, B. Gloss, M. Isa and H. U. Bernard: The enhancer of human papillomavirus type 16: binding sites for the ubiquitous transcription factors oct-1, NFA, TEF-2, NF1, and AP-1 participate in epithelial cell-specific transcription. J Virol, 65 (11), 5933-43 (1991)

42. V. Fontaine, E. van der Meijden, J. de Graaf, J. ter Schegget and L. Struyk: A functional NF-kappaB binding site in the human papillomavirus type 16 long control region. Virology, 272 (1), 40-9 (2000)

DOI:10.1006/viro.2000.0363

PMid:10873747

43. B. K. Prusty, S. A. Husain and B. C. Das: Constitutive activation of nuclear factor -kB: preferntial homodimerization of p50 subunits in cervical carcinoma. Front Biosci, 10, 1510-9 (2005)

DOI:10.2741/1635

PMid:15769641

44. B. K. Prusty and B. C. Das: Constitutive activation of transcription factor AP-1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP-1 activity in HeLa cells by curcumin. Int J Cancer, 113 (6), 951-60 (2005)

DOI:10.1002/ijc.20668

PMid:15514944

45. S. Shukla, S. Mahata, G. Shishodia, A. Pandey, A. Tyagi, K. Vishnoi, S. F. Basir, B. C. Das and A. C. Bharti: Functional regulatory role of STAT3 in HPV16-mediated cervical carcinogenesis. PLoS One, 8 (7), e67849 (2013)

DOI:10.1371/journal.pone.0067849

PMid:23874455 PMCid:PMC3715508

46. A. C. Bharti, S. Shukla, S. Mahata, S. Hedau and B. C. Das: Human papillomavirus and control of cervical cancer in India. Expert Rev. Obstet. Gynecol, 5 (3), 329–346 (2010)

47. J. Doorbar, N. Egawa, H. Griffin, C. Kranjec and I. Murakami: Human papillomavirus molecular biology and disease association. Rev Med Virol, 25 Suppl 1, 2-23 (2015)

DOI:10.1002/rmv.1822

PMid:25752814 PMCid:PMC5024016

48. B. A. Werness, A. J. Levine and P. M. Howley: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science, 248 (4951), 76-9 (1990)

DOI:10.1126/science.2157286

PMid:2157286

49. A. E. White, E. M. Livanos and T. D. Tlsty: Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins. Genes Dev, 8 (6), 666-77 (1994)

DOI:10.1101/gad.8.6.666

PMid:7926757

50. K. Vishnoi, S. Mahata, A. Tyagi, A. Pandey, G. Verma, M. Jadli, T. Singh, S. M. Singh and A. C. Bharti: Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells. Sci Rep, 6, 34377 (2016)

51. A. Tyagi, K. Vishnoi, S. Mahata, G. Verma, Y. Srivastava, S. Masaldan, B. G. Roy, A. C. Bharti and B. C. Das: Cervical Cancer Stem Cells Selectively Overexpress HPV Oncoprotein E6 that Controls Stemness and Self-Renewal through Upregulation of HES1. Clin Cancer Res (2016)

52. S. Mahata, A. C. Bharti, S. Shukla, A. Tyagi, S. A. Husain and B. C. Das: Berberine modulates AP-1 activity to suppress HPV transcription and downstream signaling to induce growth arrest and apoptosis in cervical cancer cells. Mol Cancer, 10, 39 (2011)

53. G. Shishodia, G. Verma, Y. Srivastava, R. Mehrotra, B. C. Das and A. C. Bharti: Deregulation of microRNAs Let-7a and miR-21 mediate aberrant STAT3 signaling during human papillomavirus-induced cervical carcinogenesis: role of E6 oncoprotein. BMC Cancer, 14 (1), 996 (2014)

DOI:10.1186/1471-2407-14-996

PMid:25539644 PMCid:PMC4364636

54. P. L. Stern, S. H. van der Burg, I. N. Hampson, T. R. Broker, A. Fiander, C. J. Lacey, H. C. Kitchener and M. H. Einstein: Therapy of human papillomavirus-related disease. Vaccine, 30 Suppl 5, F71-82 (2012)

55. P. S. Naud, C. M. Roteli-Martins, N. S. De Carvalho, J. C. Teixeira, P. C. de Borba, N. Sanchez, T. Zahaf, G. Catteau, B. Geeraerts and D. Descamps: Sustained efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine: final analysis of a long-term follow-up study up to 9.4. years post-vaccination. Hum Vaccin Immunother, 10 (8), 2147-62 (2014)

DOI:10.4161/hv.29532

PMid:25424918 PMCid:PMC4896780

56. USFDA: Approval Letter - Cervarix (October 16, 2009). In: Vaccines, Blood and Biologics. Ed USFDA. USFDA, Rockville, MD (2009)

57. USFDA: FDA approves Gardasil 9 for prevention of certain cancers caused by five additional types of HPV (December 10, 2014). In, (2014)

58. FUTURE-II-Study-Group: Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med, 356 (19), 1915-27 (2007)

DOI:10.1056/NEJMoa061741

PMid:17494925

59. J. Paavonen, P. Naud, J. Salmeron, C. M. Wheeler, S. N. Chow, D. Apter, H. Kitchener, X. Castellsague, J. C. Teixeira, S. R. Skinner, J. Hedrick, U. Jaisamrarn, G. Limson, S. Garland, A. Szarewski, B. Romanowski, F. Y. Aoki, T. F. Schwarz, W. A. Poppe, F. X. Bosch, D. Jenkins, K. Hardt, T. Zahaf, D. Descamps, F. Struyf, M. Lehtinen and G. Dubin: Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet, 374 (9686), 301-14 (2009)

DOI:10.1016/S0140-6736(09)61248-4

60. A. Hildesheim, R. Herrero, S. Wacholder, A. C. Rodriguez, D. Solomon, M. C. Bratti, J. T. Schiller, P. Gonzalez, G. Dubin, C. Porras, S. E. Jimenez and D. R. Lowy: Effect of human papillomavirus 16/18 L1 viruslike particle vaccine among young women with preexisting infection: a randomized trial. JAMA, 298 (7), 743-53 (2007)

DOI:10.1001/jama.298.7.743

PMid:17699008

61. S. M. Garland, M. Hernandez-Avila, C. M. Wheeler, G. Perez, D. M. Harper, S. Leodolter, G. W. Tang, D. G. Ferris, M. Steben, J. Bryan, F. J. Taddeo, R. Railkar, M. T. Esser, H. L. Sings, M. Nelson, J. Boslego, C. Sattler, E. Barr and L. A. Koutsky: Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. N Engl J Med, 356 (19), 1928-43 (2007)

DOI:10.1056/NEJMoa061760

PMid:17494926

62. L. R. Johnson, C. R. Starkey, J. Palmer, J. Taylor, S. Stout, S. Holt, R. Hendren, B. Bock, E. Waibel, G. Tyree and G. C. Miller: A comparison of two methods to determine the presence of high-risk HPV cervical infections. Am J Clin Pathol, 130 (3), 401-8 (2008)

DOI:10.1309/4DXEAFG2JXYF34N3

PMid:18701413

63. A. C. Bharti and G. Shishodia: Human Papillomavirus Detection Methods. In: Gynecological Cytopathology - Cervix - With Laboratory and Clinical Aspects: Third Edition. Ed S. Bhambhani. Mehta Publishers, New Delhi, India (2014)

64. V. R. Yanofsky, R. V. Patel and G. Goldenberg: Genital warts: a comprehensive review. J Clin Aesthet Dermatol, 5 (6), 25-36 (2012)

65. C. Penna, M. G. Fallani, R. Gordigiani, L. Sonni, G. L. Taddei and M. Marchionni: Intralesional beta-interferon treatment of cervical intraepithelial neoplasia associated with human papillomavirus infection. Tumori, 80 (2), 146-50 (1994)

66. N. R. Tirone, M. A. Michelin and E. F. Murta: Using cytokines to treat cervical intraepithelial and invasive neoplasia. Recent Pat Anticancer Drug Discov, 5 (2), 165-9 (2010)

DOI:10.2174/157489210790936225

PMid:19961435

67. S. Wadler, R. D. Burk, D. Neuberg, R. Rameau, C. D. Runowicz, G. Goldberg, F. McGill, R. Tachezy, R. Comis, J. Edmonson and et al.: Lack of efficacy of interferon-alpha therapy in recurrent, advanced cervical cancer. J Interferon Cytokine Res, 15 (12), 1011-6 (1995)

68. C. Grimm, S. Polterauer, C. Natter, J. Rahhal, L. Hefler, C. B. Tempfer, G. Heinze, G. Stary, A. Reinthaller and P. Speiser: Treatment of cervical intraepithelial neoplasia with topical imiquimod: a randomized controlled trial. Obstet Gynecol, 120 (1), 152-9 (2012)

DOI:10.1097/AOG.0b013e31825bc6e8

PMid:22914404

69. S. C. Woodhall, M. Jit, K. Soldan, G. Kinghorn, R. Gilson, M. Nathan, J. D. Ross and C. J. Lacey: The impact of genital warts: loss of quality of life and cost of treatment in eight sexual health clinics in the UK. Sex Transm Infect, 87 (6), 458-63 (2011)

DOI:10.1136/sextrans-2011-050073

PMid:21636616 PMCid:PMC3253069

70. C. M. Kodner and S. Nasraty: Management of genital warts. Am Fam Physician, 70 (12), 2335-42 (2004)

71. P. P. Martin-Hirsch, E. Paraskevaidis, A. Bryant, H. O. Dickinson and S. L. Keep: Surgery for cervical intraepithelial neoplasia. Cochrane Database Syst Rev (6), CD001318 (2010)

72. J. A. Green, J. M. Kirwan, J. F. Tierney, P. Symonds, L. Fresco, M. Collingwood and C. J. Williams: Survival and recurrence after concomitant chemotherapy and radiotherapy for cancer of the uterine cervix: a systematic review and meta-analysis. Lancet, 358 (9284), 781-6 (2001)

DOI:10.1016/S0140-6736(01)05965-7

73. K. Scatchard, J. L. Forrest, M. Flubacher, P. Cornes and C. Williams: Chemotherapy for metastatic and recurrent cervical cancer. Cochrane Database Syst Rev, 10, CD006469 (2012)

74. M. Candelaria, J. Chanona-Vilchis, L. Cetina, D. Flores-Estrada, C. Lopez-Graniel, A. Gonzalez-Enciso, D. Cantu, A. Poitevin, L. Rivera, J. Hinojosa, J. de la Garza and A. Duenas-Gonzalez: Prognostic significance of pathological response after neoadjuvant chemotherapy or chemoradiation for locally advanced cervical carcinoma. Int Semin Surg Oncol, 3, 3 (2006)

75. C. A. Perez, P. W. Grigsby, H. M. Camel, A. E. Galakatos, D. Mutch and M. A. Lockett: Irradiation alone or combined with surgery in stage IB, IIA, and IIB carcinoma of uterine cervix: update of a nonrandomized comparison. Int J Radiat Oncol Biol Phys, 31 (4), 703-16 (1995)

DOI:10.1016/0360-3016(94)00523-0

76. CRI: Cancer Immunotherapy - Cervical Cancer. In: Ed CRI. (2017)

77. K. Kawana, K. Adachi, S. Kojima, A. Taguchi, K. Tomio, A. Yamashita, H. Nishida, K. Nagasaka, T. Arimoto, T. Yokoyama, O. Wada-Hiraike, K. Oda, T. Sewaki, Y. Osuga and T. Fujii: Oral vaccination against HPV E7 for treatment of cervical intraepithelial neoplasia grade 3 (CIN3) elicits E7-specific mucosal immunity in the cervix of CIN3 patients. Vaccine, 32 (47), 6233-9 (2014)

DOI:10.1016/j.vaccine.2014.09.020

PMid:25258102

78. A. M. Kaufmann, P. L. Stern, E. M. Rankin, H. Sommer, V. Nuessler, A. Schneider, M. Adams, T. S. Onon, T. Bauknecht, U. Wagner, K. Kroon, J. Hickling, C. M. Boswell, S. N. Stacey, H. C. Kitchener, J. Gillard, J. Wanders, J. S. Roberts and H. Zwierzina: Safety and immunogenicity of TA-HPV, a recombinant vaccinia virus expressing modified human papillomavirus (HPV)-16 and HPV-18 E6 and E7 genes, in women with progressive cervical cancer. Clin Cancer Res, 8 (12), 3676-85 (2002)

79. M. I. van Poelgeest, M. J. Welters, E. M. van Esch, L. F. Stynenbosch, G. Kerpershoek, E. L. van Persijn van Meerten, M. van den Hende, M. J. Lowik, D. M. Berends-van der Meer, L. M. Fathers, A. R. Valentijn, J. Oostendorp, G. J. Fleuren, C. J. Melief, G. G. Kenter and S. H. van der Burg: HPV16 synthetic long peptide (HPV16-SLP) vaccination therapy of patients with advanced or recurrent HPV16-induced gynecological carcinoma, a phase II trial. J Transl Med, 11, 88 (2013)

80. P. J. de Vos van Steenwijk, M. I. van Poelgeest, T. H. Ramwadhdoebe, M. J. Lowik, D. M. Berends-van der Meer, C. E. van der Minne, N. M. Loof, L. F. Stynenbosch, L. M. Fathers, A. R. Valentijn, J. Oostendorp, E. M. Osse, G. J. Fleuren, L. Nooij, M. J. Kagie, B. W. Hellebrekers, C. J. Melief, M. J. Welters, S. H. van der Burg and G. G. Kenter: The long-term immune response after HPV16 peptide vaccination in women with low-grade pre-malignant disorders of the uterine cervix: a placebo-controlled phase II study. Cancer Immunol Immunother, 63 (2), 147-60 (2014)

DOI:10.1007/s00262-013-1499-2

PMid:24233343

81. C. L. Trimble, M. P. Morrow, K. A. Kraynyak, X. Shen, M. Dallas, J. Yan, L. Edwards, R. L. Parker, L. Denny, M. Giffear, A. S. Brown, K. Marcozzi-Pierce, D. Shah, A. M. Slager, A. J. Sylvester, A. Khan, K. E. Broderick, R. J. Juba, T. A. Herring, J. Boyer, J. Lee, N. Y. Sardesai, D. B. Weiner and M. L. Bagarazzi: Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial. Lancet, 386 (10008), 2078-88 (2015)

DOI:10.1016/S0140-6736(15)00239-1

82. R. D. Alvarez, W. K. Huh, S. Bae, L. S. Lamb, Jr., M. G. Conner, J. Boyer, C. Wang, C. F. Hung, E. Sauter, M. Paradis, E. A. Adams, S. Hester, B. E. Jackson, T. C. Wu and C. L. Trimble: A pilot study of pNGVL4a-CRT/E7 (detox) for the treatment of patients with HPV16+ cervical intraepithelial neoplasia 2/3 (CIN2/3). Gynecol Oncol, 140 (2), 245-52 (2016)

DOI:10.1016/j.ygyno.2015.11.026

PMid:26616223 PMCid:PMC4724445

83. A. D. Santin, S. Bellone, M. Palmieri, A. Zanolini, A. Ravaggi, E. R. Siegel, J. J. Roman, S. Pecorelli and M. J. Cannon: Human papillomavirus type 16 and 18 E7-pulsed dendritic cell vaccination of stage IB or IIA cervical cancer patients: a phase I escalating-dose trial. J Virol, 82 (4), 1968-79 (2008)

DOI:10.1128/JVI.02343-07

PMid:18057249 PMCid:PMC2258728

84. P. Ramanathan, S. Ganeshrajah, R. K. Raghanvan, S. S. Singh and R. Thangarajan: Development and clinical evaluation of dendritic cell vaccines for HPV related cervical cancer--a feasibility study. Asian Pac J Cancer Prev, 15 (14), 5909-16 (2014)

DOI:10.7314/APJCP.2014.15.14.5909

PMid:25081721

85. P. Vici, L. Pizzuti, L. Mariani, G. Zampa, D. Santini, L. Di Lauro, T. Gamucci, C. Natoli, P. Marchetti, M. Barba, M. Maugeri-Sacca, D. Sergi, F. Tomao, E. Vizza, S. Di Filippo, F. Paolini, G. Curzio, G. Corrado, A. Michelotti, G. Sanguineti, A. Giordano, R. De Maria and A. Venuti: Targeting immune response with therapeutic vaccines in premalignant lesions and cervical cancer: hope or reality from clinical studies. Expert Rev Vaccines, 15 (10), 1327-36 (2016)

DOI:10.1080/14760584.2016.1176533

PMid:27063030 PMCid:PMC5152541

86. A. Yang, J. Jeang, K. Cheng, T. Cheng, B. Yang, T. C. Wu and C. F. Hung: Current state in the development of candidate therapeutic HPV vaccines. Expert Rev Vaccines, 15 (8), 989-1007 (2016)

DOI:10.1586/14760584.2016.1157477

PMid:26901118

87. A. Yang, E. Farmer, T. C. Wu and C. F. Hung: Perspectives for therapeutic HPV vaccine development. J Biomed Sci, 23 (1), 75 (2016)

DOI:10.1186/s12929-016-0293-9

PMid:27809842 PMCid:PMC5096309

88. R. Rosales, M. Lopez-Contreras, C. Rosales, J. R. Magallanes-Molina, R. Gonzalez-Vergara, J. M. Arroyo-Cazarez, A. Ricardez-Arenas, A. Del Follo-Valencia, S. Padilla-Arriaga, M. V. Guerrero, M. A. Pirez, C. Arellano-Fiore and F. Villarreal: Regression of human papillomavirus intraepithelial lesions is induced by MVA E2 therapeutic vaccine. Hum Gene Ther, 25 (12), 1035-49 (2014)

DOI:10.1089/hum.2014.024

PMid:25275724 PMCid:PMC4270165

89. C. M. Corona Gutierrez, A. Tinoco, T. Navarro, M. L. Contreras, R. R. Cortes, P. Calzado, L. Reyes, R. Posternak, G. Morosoli, M. L. Verde and R. Rosales: Therapeutic vaccination with MVA E2 can eliminate precancerous lesions (CIN 1, CIN 2, and CIN 3) associated with infection by oncogenic human papillomavirus. Hum Gene Ther, 15 (5), 421-31 (2004)

DOI:10.1089/10430340460745757

PMid:15144573

90. E. Garcia-Hernandez, J. L. Gonzalez-Sanchez, A. Andrade-Manzano, M. L. Contreras, S. Padilla, C. C. Guzman, R. Jimenez, L. Reyes, G. Morosoli, M. L. Verde and R. Rosales: Regression of papilloma high-grade lesions (CIN 2 and CIN 3) is stimulated by therapeutic vaccination with MVA E2 recombinant vaccine. Cancer Gene Ther, 13 (6), 592-7 (2006)

DOI:10.1038/sj.cgt.7700937

PMid:16456551

91. Y. C. A. Albarran, A. de la Garza, B. J. Cruz Quiroz, E. Vazquez Zea, I. Diaz Estrada, E. Mendez Fuentez, M. Lopez Contreras, A. Andrade-Manzano, S. Padilla, A. R. Varela and R. Rosales: MVA E2 recombinant vaccine in the treatment of human papillomavirus infection in men presenting intraurethral flat condyloma: a phase I/II study. BioDrugs, 21 (1), 47-59 (2007)

DOI:10.2165/00063030-200721010-00006

PMid:17263589

92. M. L. Bagarazzi, J. Yan, M. P. Morrow, X. Shen, R. L. Parker, J. C. Lee, M. Giffear, P. Pankhong, A. S. Khan, K. E. Broderick, C. Knott, F. Lin, J. D. Boyer, R. Draghia-Akli, C. J. White, J. J. Kim, D. B. Weiner and N. Y. Sardesai: Immunotherapy against HPV16/18 generates potent TH1 and cytotoxic cellular immune responses. Sci Transl Med, 4 (155), 155ra138 (2012)

93. G. G. Kenter, M. J. Welters, A. R. Valentijn, M. J. Lowik, D. M. Berends-van der Meer, A. P. Vloon, J. W. Drijfhout, A. R. Wafelman, J. Oostendorp, G. J. Fleuren, R. Offringa, S. H. van der Burg and C. J. Melief: Phase I immunotherapeutic trial with long peptides spanning the E6 and E7 sequences of high-risk human papillomavirus 16 in end-stage cervical cancer patients shows low toxicity and robust immunogenicity. Clin Cancer Res, 14 (1), 169-77 (2008)

DOI:10.1158/1078-0432.CCR-07-1881

PMid:18172268

94. P. J. de Vos van Steenwijk, T. H. Ramwadhdoebe, M. J. Lowik, C. E. van der Minne, D. M. Berends-van der Meer, L. M. Fathers, A. R. Valentijn, J. Oostendorp, G. J. Fleuren, B. W. Hellebrekers, M. J. Welters, M. I. van Poelgeest, C. J. Melief, G. G. Kenter and S. H. van der Burg: A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions. Cancer Immunol Immunother, 61 (9), 1485-92 (2012)

DOI:10.1007/s00262-012-1292-7

PMid:22684521 PMCid:PMC3427705

95. G. G. Kenter, M. J. Welters, A. R. Valentijn, M. J. Lowik, D. M. Berends-van der Meer, A. P. Vloon, F. Essahsah, L. M. Fathers, R. Offringa, J. W. Drijfhout, A. R. Wafelman, J. Oostendorp, G. J. Fleuren, S. H. van der Burg and C. J. Melief: Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med, 361 (19), 1838-47 (2009)

DOI:10.1056/NEJMoa0810097

PMid:19890126

96. S. Daayana, E. Elkord, U. Winters, M. Pawlita, R. Roden, P. L. Stern and H. C. Kitchener: Phase II trial of imiquimod and HPV therapeutic vaccination in patients with vulval intraepithelial neoplasia. Br J Cancer, 102 (7), 1129-36 (2010)

DOI:10.1038/sj.bjc.6605611

PMid:20234368 PMCid:PMC2853099

97. L. J. Smyth, M. I. Van Poelgeest, E. J. Davidson, K. M. Kwappenberg, D. Burt, P. Sehr, M. Pawlita, S. Man, J. K. Hickling, A. N. Fiander, A. Tristram, H. C. Kitchener, R. Offringa, P. L. Stern and S. H. Van Der Burg: Immunological responses in women with human papillomavirus type 16 (HPV-16)-associated anogenital intraepithelial neoplasia induced by heterologous prime-boost HPV-16 oncogene vaccination. Clin Cancer Res, 10 (9), 2954-61 (2004)

DOI:10.1158/1078-0432.CCR-03-0703

PMid:15131030

98. S. Bellone, S. Pecorelli, M. J. Cannon and A. D. Santin: Advances in dendritic-cell-based therapeutic vaccines for cervical cancer. Expert Rev Anticancer Ther, 7 (10), 1473-86 (2007)

DOI:10.1586/147