AN OVERVIEW OF HUMAN PAPILLOMAVIRUSES AND CURRENT ...

10 Indian Journal of Medical Microbiology, (2007) 25 (1):10-7

Review Article

AN OVERVIEW OF HUMAN PAPILLOMAVIRUSES AND CURRENT VACCINE STRATEGIES M Gnanamony, A Peedicayil, *PAbraham

Abstract Cervical cancer is one of the most common cancers in women worldwide, particularly in developing countries. The viral origin of cervical cancer has been proven beyond any reasonable doubt. Persistent infection with certain subsets of human papillomaviruses is recognized as a necessary cause for the development of cervical cancer. Persistence of oncogenic HPVs, immunodeficiency, high HPV viral load and cofactors like smoking, multiple sex partners and poor nutrition predispose to cervical cancer. Prophylactic vaccines using HPV virus-like particles containing capsid protein L1 have shown protection against disease in animals and are currently undergoing clinical trials. Therapeutic vaccines using HPV E6 and E7 proteins are also being investigated for their ability to remove residual infection.

m rf o d ns a Epidemiological evidence has proved beyond doubt that to aid in cellular transformation and plays a significant role in o late tproteins o l i human papillomavirus (HPV) infection is the most important viral replication. The L1 and L2 make up the n risk factor for cervical intraepithelial neoplasia and invasive viral capsid. a w lic cervical cancer. HPVs infect the basal epithelium and are o E6 and play a major role in the malignant d E7 ofproteins grouped as cutaneous and mucosal types. Based on the b transformation cervical cells as can be seen in studies using u association with cervical cancer, genital HPVs are further e . P various cell lines. The E6 protein forms a complex with the e ) grouped into high risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, r f cell cycle regulator protein p53 using cellular ubiquitin ligase 56, 58, 59, 68, 73 and 82), probable high-risk types (26, 53 and r w m E6AP. This ubiquitination was found to lead to accelerated o o 66) and low risk types (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 o c n f degradation of p53. The E6 protein also down regulates p53 . and CP6108). Of the high risk types, HPV 16 is the most k e activity by targeting the co-activator CBP/p300. w l d oof p53 leads to loss of cell ofcyclep53,control prevalent type and is seen in at least 50% of invasive cervical b e Inactivation such as cancer patients globally followed by types 18, 45, 31, 33, 52 n a l cell cycle arrest and apoptosis. E6 protein associates M k proteins and Sp-1 and this complex binds towith i of HPV and 58. Various studies have looked at the prevalence d a Myc/Mak the y genotypes in Indian women and the most common v e b promoter region of the catalytic subunit of telomerase genotypes reported are HPV 16 and 18. a d .menzyme, hTERT. This leads to an increase in telomerase s i te w activity, thereby facilitating immortalization (Fig. 1). Basic Virology s wThey The E7 protein associates with retinoblastoma family of F o HPV are members of the family Papillomaviridae. D h (w proteins. The E7 protein binds to the phosphorylated are small, non-enveloped DNAPviruses, 55 nm in diameter. e of two structural retinoblastoma protein (Rb) and separates it from E2F/DP1 They have an outer icosahedral capsiditmade s i sof HPV is circular and complex. The E7 protein then mediates degradation of the proteins, L1 and L2. The ThThegenome approximately 8 Kb long. genome has eight open reading Rb protein through the ubiquitin-proteosome pathway. E7 a frames coding for six early proteins (E1, E2, E4, E5, E6 and E7) protein also binds to various other cellular proteins like cyclin Key words: Cervical cancer, human papilloma virus, vaccines

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and two late proteins (L1 and L2). E1 and E2 genes are involved in viral replication. 10 E2 protein also plays an important role as a repressor of E6 and E7 gene expression. E4 gene codes for protein E1-E4 that disrupt the cytoplasmic keratin network.11 The E4 protein is also found to contribute to regulation of host cell cycle control.12 E5 protein is known *Corresponding author (email: ) Department of Clinical Virology (MG, PA) and Department of Obstetrics and Gynaecology (AP), Christian Medical College, Vellore - 632 004, Tamil Nadu, India Received : 07-02-06 Accepted : 04-04-06

dependent kinase (cdk) inhibitor p21, possibly leading to loss of cell cycle control.25 The E7 protein also associates with another group of proteins called histone deacetylases (HDACs) independent of Rb protein and this results in the expression of E2F/DP1 inducible genes. E2F proteins activate expression of various genes necessary for cell cycle progression and DNA replication26 (Fig. 2). Thus the combined effect of these two viral oncoproteins and above mentioned cellular factors contribute to the malignant transformation of the cervical cells. Epidemiology Human papillomavirus is transmitted by sexual contact.

www.ijmm.org 10 CMYK

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Binds to Myc, Mak

Gnanamony et al - HPV: Overview and Vaccine Strategies

HPV E6 Oncoprotein

(B)

p53 (A)

Ubiquitin ligase Myc, Sp-1 Ubiquitinized p53

Cellular protease

Transcriptional activation of hTERT

Degraded p53

Reconstitution of telomerase activity

Immortalization of HPV infected cell

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risk factors. Impaired cell mediated immunity is a major risk factor for cervical cancer. Evidence for this is derived from studies showing a high incidence of HPV infection in human immunodeficiency virus seropositive women.28 High HPV viral load is a risk factor associated with high stages of cervical disease.29 Persistence of high-risk types of HPV is another major risk factor in the development of cervical cancer. A higher risk of acquiring HPV was attributed to decreasing age of first sexual intercourse, multiple sexual partners and clinical history of other venereal diseases.30 High parity was found to increase the risk of infection.31 Long-term use of oral contraceptives32 is also a significant cofactor in the development of cervical cancer. Most Indian studies have reported HPV 16 and 18 as the major genotypes. There is evidence that there are other oncogenic HPV types circulating in our population. In our center, the predominant HPV types detected were HPV 16 and 18 followed by 52, 33, 58, 35, 31, 45, 51 and 56 (unpublished data).

m o fr d ns a lo tio HPV variants are gaining More recently,nnaturally occurring aof epidemiology and pathogenesis. importance as w viral markers c i Based on sequence L2 and long control l of theintoE6,fiveL1,major do 16uisanalysis bgrouped region, HPV phylogenetic e clusters. They are European (E), Asian (As), Asian-American . P and) African-2 (Af2). A study from our eAfrican-1(Af1) (AA), r f m prevelance (92%) of European (E) center has w shown a higher r o o c 16 in Indian population. fo variantknclass of .HPV le ePathogenesis d ow b n infect the basal layers of the cervical squamous HPVs la M epithelium k i mild abrasions or trauma. HPV is thought d the through a by toeenter v cell after attaching to cell surface receptor heparan a d .msulphate. The virus life cycle depends on the differentiation is te w of the host squamous epithelium. In the basal layers of the s w epithelium, the virus genome is episomal and establishes itself F o copy numbers. The early protein E1 in association with PD te h (w atE2lowis thought to trigger the replication process. At this stage, si si the expression of the viral oncoproteins E6 and E7 are kept in check by the E2 protein, which acts as a transcriptional Th a repressor. In the terminal layers, the virus switches from theta

Figure 1: E6 dependent modulation of cell cycle

(A) HPV E6 oncoprotein associates with Myc/Mak and Sp-1 proteins and these complexes induce expression of hTERT gene leading to an increase in the telomerase activity. (B) HPV E6 oncoprotein binds to p53 protein using ubiquitin ligase E6AP and this complex is degraded by cellular proteases leading to a loss of cell control.

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Figure 2: E7 Dependent modulation of cell cycle (A) HPV E7 oncoprotein forms a complex with cyclin dependent kinase inhibitor p21 leading to increased expression of cyclin dependent kinase (CDK). (B) HPV E7 oncoprotein binds to phosphorylated retinoblastoma protein (Rb) causing separation of Rb from elongation factor (E2F) complex. The Rb protein is then degraded through the ubiquitin proteosome pathway. (C) HPV E7 oncoprotein associates with histone deacetylases (HDACs) resulting in an increased expression of transcription factors E2F/DP1 inducible genes.

However a non-sexual mode of transmission through fomites has also been proposed.27 Epidemiological evidence also points to an association between cervical cancer and various

replication to rolling circle mode of replication, resulting in a high copy number of viral DNA.35 At this stage, the virus manages to integrate its DNA into the host chromosome. This disrupts the E2 open reading frame leading to excess synthesis of the E6 and E7 oncoproteins. This eventually is thought to lead to malignant transformation of the epithelial cells. Studies from our centre have shown that HPV 16 E6 and E7 transcripts are seen in all cases of invasive cancer but not in cases of cervical intraepithelial neoplasia (CIN) I and II. The presence of episomal E2 DNA in high propotion (52.4%) of patients with invasive carcinoma and the presence of significant variations in the E2 gene suggests that there are alternate mechanisms of E6 and E7 gene expression.36,37 Finally, capsid proteins L1 and L2 are synthesized and mature virions are produced. The


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release of HPV virions is thought to be facilitated by the E1 E4 protein. Laboratory Diagnosis The high incidence of cervical cancer in developing countries emphasizes the need for proper screening to reduce this global burden. The primary tools for diagnosis of HPV infection are cytology, histology and recently, detection of HPV DNA. Cytology and histology The Papanicolou (Pap) smear, introduced by Papanicolau and Traut in 1943, identifies changes in cells of the transformation zone of cervix caused by HPV infection.38 Abnormal cells are vacuolated with a pyknotic nucleus surrounded by a halo and are termed as “koilocytes”. The current interpretation of Pap smear is based on the Bethesda system.39 However, cytology has its limitations. Inadequate sampling, poor sensitivity with false negative results, contaminants in the sample, have been reported.9 Automated cytologic tests using PapNet (Neuromedical systems, Suffern, NY) and Autopap 300 QC (Neopath, Redmond, Wash) have been approved by the Food and Drug Administration, USA for screening smears to identify false negative smears. Fluid based technology also reduces false negative smear results. The specimen is collected in a preservative solution, debris is removed thereby aiding clear visualization of the cells.40 Colposcopy and colposcopy directed biopsies are done in patients with abnormal pap smears. Visual inspection is done with the naked eye after application of 3% acetic acid solution (VIA).9 Dysplastic cells appear as acetowhite lesions. Visual inspection with Lugol’s iodine (VILI) is another approach where the cervix is viewed with the naked eye after application of iodine solution. Normal squamous epithelial cells appear brown or almost black in colour whereas abnormal cells appear colourless, pale or mustard yellow in colour.41 Cervical biopsy is then done to conform malignancy. Cervical biopsy is considered the gold standard for the detection of cervical neoplasia and HPV infection.

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and uses chemiluminescence to detect the presence of HPV.43 Recently a prototype version of Hybrid Capture 3 has been released. The hybrid capture 3 was found to be more sensitive and equally specific like its predecessor when compared to the PCR based assays.44 But this method does not distinguish between the different HPV genotypes. Most PCR protocols for the detection of genital HPVs use consensus primers GP5/6,45 degenerate primers MY09/11 and its modified version PGMY09/11,46 amplifying a wide spectrum of genital HPVs. HPV genotyping is done by nucleotide sequencing, restriction fragment length polymorphism,47 reverse hybridization line probe assay48 and line blot assay.49 Quantification of HPV viral load has been done by real time PCR assays.50

m rf o d ns a o tio l n Immunology a w c i o HPV infection is ltransient and 70% of women clear the b suggesting the role of an effective infectiond in one year u e immune system clearing virus. Immune response to Pin weak ).andthestudies rf eis however, HPV using HPV virus-like w m particles (VLPs) have shown that antibodies are detected r o eight omonths after incident HPV infection. The c n fo approximately . kstudy also shows that women who seroconverted were e same w l d 5.7 times more likely to progress to HPV associated squamous o b eintraepithelial n neoplasia (SIL) than women who did not a l M seroconvert. k i These results suggest that in natural infection, d to HPV are not protective a by antibodies and serve as a marker of v e a d .mdisease progression. Another study showed a seroprevalence is te w of16.46% in sexually active college women infected with HPV s w F o importance of cell mediated immunity in controlling PD te h (w HPVTheinfection comes from studies showing increased s si i prevalence of HPV in HIV seropositive women. Cytotoxic T h a HPV DNA detection T lymphocyte (CTL) response to HPV E6 and E7 proteins were detected more commonly in HPV 16 positive patients without HPV cannot be easily cultured in the laboratory. Therefore, molecular methods to detect HPV DNA are been used to confirm the presence to HPV in clinical specimens.

The currently used techniques for HPV DNA detection are the hybrid capture second generation (HC2) assay and polymerase chain reaction (PCR). The hybrid capture assay is the only commercial kit approved by the FDA for the detection of HPV. The hybrid capture assay detects 13 highrisk types (16,18,31,33,35,39,45,51,52,56,58,59 and 68) and 5 low risk types (6,11,42,43,44). Hybrid Capture 2 assay was found to be a good screening tool for the detection of cervical intraepithelial neoplasia III and invasive cervical cancer.42 The assay works on a hybridization/ signal amplification principle

Studies including those from this centre have shown an association between the presence of HPV DNA in plasma of patients with advanced stages of invasive cervical cancer and metastasis to distinct organs.51,52 HPV DNA in plasma can thus be used as a marker of poor disease progression.

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CIN than in HPV 16 positive patients with (CIN), suggesting a protective role of CTL’s in HPV infection.56 This CTL response was found to be mediated by both CD4-positive and CD8-positive T lymphocytes.57 Lymphoproliferative responses to a HPV 16 E7 peptide 37-54 were found to be associated with regression of disease and loss of HPV infection suggesting a protective role of cell mediated immune response.58 Treatment Treatment options for cervical neoplasia depend on the stage of the disease. Cervical non-invasive lesions are treated by ablative methods like cryotherapy, laser therapy, cold


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coagulation and diathermy or excisional methods like loop electrosurgical excision procedure (LEEP) and conization. In cryotherapy, abnormal tissue is frozen by an instrument and destroyed. Laser therapy uses a single laser beam to cut abnormal tissue. In loop LEEP, an electrically charged wire is used to excise the lesion. Early invasive cancers are treated with radical hysterectomy or internal/external radiation therapy. Advanced cancer patients are treated with internal/ external radiation therapy and chemotherapy.9 Additional therapies to treat HPV infection of the cervix have also been tried. Local application of antiviral agents like cidofovir has been shown to be partly effective against CIN III lesions. 59 Various immunomodulatory agents like recombinant interferon gamma,60 imiquinod 5%61 have also been shown to be efficacious in CIN treatment and management. In a preliminary study, antioxidant curcumin caused a down regulation of HPV 18 transcription in Hela cell line.62 Further studies will establish the clinical efficacy of curcumin in treatment of cervical cancer.

being developed for HPV infection, prophylactic vaccine and therapeutic vaccine. Some of the studies on vaccine trials and future candidate vaccines are summarized in Tables 1 and 2. Prophylactic vaccines prevent infection by inducing production of neutralizing antibodies. A good prophylactic vaccine should be safe and should induce long-lasting protection. Development of prophylactic vaccines for HPV is hindered by various factors. HPVs are difficult to grow in vitro. Moreover, HPVs are species specific, so vaccine evaluation in animal models is also not possible. Sub-unit vaccines using HPV VLPs, obtained by expressing capsid protein L1and L2 in both prokaryotic and eukaryotic cells are currently being used as candidate vaccines. In early animal models like rabbits63 and canines,64 these VLPs have shown to be highly immunogenic and also shown to elicit high titres of neutralizing antibodies. An early human trial using HPV 16 VLPs produced in insect cells showed high titres of neutralizing antibodies up to 40 times higher than in a natural infection.65 Another randomized control trial was conducted using HPV 16 VLPs consisting of HPV 16 L1 protein expressed in yeast. This study showed complete protection from disease in the vaccinated group compared to the unvaccinated group.66 This study has shown that HPV 16 VLPs used as prophylactic vaccines can reduce cervical cancer risk in women who are HPV 16 negative. Recently, a bivalent HPV 16/ 18 virus-like particle vaccine has been shown to be efficacious in preventing incident and persistent infections with HPV 16 and 18.67 Quadrivalent vaccines using VLPs made of HPV 6, 11, 16, 18 have been tested for their efficacy in chimpanzees. The study shows that this vaccine elicits both neutralizing antibodies as well as a transient CTL responses.68 Currently phase III trials are being undertaken to prove its

m o fr d ns a o tio l Prevention n a w c HPV screening i o l d ub Screening programs have greatly reduced the incidence of e . cervical cancer in the developed world. Prevention programs P e ) r should comprise of health education and periodic pelvic f w m examinations, which may include direct visual inspection r o no .co (DVI), VIA, VILI, pap smear and HPV DNA testing. However,f k w large scale routine screening and treatment for cervical cancer e l d is hard to achieve in a developing country like India. A bcost-to e no a effective vaccine is therefore needed as an alternative l i y M dk screening and treatment. There are two typesaof vaccines v b e a m papillomavirus vaccines d for human Table 1:sClinical trials . e i t wStudy group Type of vaccine Antigen used Deliveryssystem Outcome F w o Prophylactic HPV 16 L1 protectivity in w Healthy volunteers 100% h particles ( PD tVirus-like protein (VLPs) study group e particles Healthy volunteers Increased si si Virus-like Prophylactic HPV 16 L1 systemic proteinh (VLPs) CMI response in patient T a group Prophylactic

HPV 16 L1 protein

Virus-like particles (VLPs)

Healthy volunteers

Prophylactic

HPV 16/18 L1 protein

Virus-like particles (VLPs)

Therapeutic

HPV 16 E7 peptide Microparticles (ZYC101) HPV 16/18 Recombinant E6/ E7 gene vaccinia virus HPV 16 E7 -

I: HPV negative volunteers II: cytologically confirmed LSIL, HSIL patients CIN II/ III

Highly immunogenic with 40 fold higher antibody titre than natural infection 91.6% efficient against incident infection and 100% efficient against persistent infection 5/11 regression, E2 specific immunity

Late stage cervical cancer CIN/VIN II/III

Mild increase in antibody titre and CTL response 3/17 had complete regression

Therapeutic Therapeutic

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Table 2: Future vaccine candidates/ vaccine chimeras Type of vaccine

Antigen used

Delivery system

Status of study

Ref

Prophylactic Therapeutic and Prophylactic Therapeutic

HPV 16 and HPV 18 HPV 16 L1/ E7

Virus like particles Virus like particles

Undergoing phase II trial Protection shown in mice

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HPV 16 E6 and E7 oncoprotein Cottontail rabbit papilloma virus E2 protein

Recombinant semliki Forest virus (SFC) Recombinant adenovirus vector

Shown to eradicate tumors in mice Shows clearance of the virus

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Therapeutic

80

HPV - Human papillomavirus

efficacy. Another human trial demonstrated the ability of HPV VLP vaccines to induce both humoral as well as cell-mediated immune responses in healthy individuals as shown by increased neutralizing antibodies as well as cytokine response.69

epidemiological evidence. J Clin Virol 2000;19:1-5. 3.

Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518-27.

m o r f Future vaccine candidates in our country should d ns incorporate the important prevalent high-risk HPV types. a lo tio Therapeutic vaccines n w lica Therapeutic vaccines are being developed to treat already o established infections. As the early oncoproteins E6 and E7 d ub are expressed through out the lifecycle of the virus and their e . P e presence necessary to maintain the transformed state in cell ) r lines, they are potential targets as therapeutic vaccines. A r f ow om phase I trial using E7 peptide vaccine showed regression of o n .c disease in the study population. Vaccination with longf k peptides of E7 protein of HPV 16 elicited a robust CD4-positive le ed ow b T helper cell response as well as a CD8-positive CTL response n a in mice. DNA vaccines comprising the HPV 16 E6ilgene wereM k d shown to elicit protective cell mediated immunearesponseyin v e b mice. Chimeric VLPs have also been developed by fusing a d These .m non-structural proteins E7 or E2 intosL2 protein. i te as wellw chimeric VLPs elicit both humoral immune responses s wfor as cell mediated immune responses F and may be relevant o a population that has established HPV h cervical(wdisease. PD related s site Conclusions i hbeen reported HPV infection has T a to be a frequently occurring sexually transmitted disease. It has also been 4.

Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: A meta-analysis. Br J Cancer 2003;88:63-73.

5. Chatterjee R, Roy A, Basu S. Detection of type specific human papillomavirus (HPV) DNA in cervical cancers of Indian women. Indian J Pathol Microbiol 1995;38:33-42.

6. Menon MM, Sinha MR, Doctor VM. Detection of human papillomavirus (HPV) types in precancerous and cancerous lesions of cervix in Indian women: A preliminary report. Indian J Cancer 1995;32:154-9.

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Das BC, Sharma JK, Gopalkrishna V, Das DK, Singh V, Gissmann L, et al. A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by southern blot hybridization and polymerase chain reaction. J Med Virol 1992;36:239-45.

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implicated in causing cervical cancer, which is the most frequently occurring cancer in Indian rural women. India carries a fourth of world’s burden of cervical cancer.75 It is important that health authorities place cervical cancer screening and HPV detection as a high priority in future cancer prevention health strategies. References 1. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12-9. 2. Munoz N. Human papillomavirus and cancer: The

10. Chiang CM, Ustav M, Stenlund A, Ho TF, Broker TR, Chow LT. Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins. Proc Natl Acad Sci USA 1992;89:5799-803. 11. Doorbar J, Ely S, Sterling J, McLean C, Crawford L. Specific interaction between HPV 16 E1-E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network. Nature 1991;352:824-7. 12. Nakahara T, Nishimura A, Tanaka M, Ueno T, Ishimoto A, Sakai H. Modulation of the cell division cycle by human papillomavirus type 18 E4. J Virol 2002;76:10914-20. 13. Genther SM, Sterling S, Duensing S, Munger K, Sattler C, Lambert PF. Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle. J Virol 2003;77:2832-42.


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Wright TC Jr. Human papillomavirus infection in women infected with the human immunodeficiency virus. N Engl J Med 1997;337:1343-9.

15. Hudson JB, Bedell MA, McCance DJ, Laimins LA. Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J Virol 1990;64:519-26.

29. Swan DC, Tucker RA, Tortolero-Luna G, Mitchell MF, Wideroff L, Unger ER, et al. Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type. J Clin Microbiol 1999;37:1030-4.

16. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990;248:76-9.

30. La Vecchia C, Franceschi S, Decarli A, Fasoli M, Gentile A, Parazzini F, et al. Sexual factors, venereal diseases and the risk of intraepithelial and invasive cervical neoplasia. Cancer 1986;58:935-41.

17. Cooper B, Schneider S, Bohl J, Jiang Y, Beaudet A, Vande Pol S. Requirement of E6AP and the features of human papillomavirus E6 necessary to support degradation of p53. Virology 2003;306:87-99.

31. Munoz N, Franceschi S, Bosetti C, Moreno V, Herrero R, Smith JS, et al. Role of parity and human papillomavirus in cervical cancer: The IARC multicentric case-control study. Lancet 2002;359:1093-101.

m o fr d ns a o tio l n a w c i o l d ub e rf e w P m). r o o o f kn .c le ed ow b la M dkn i a by e v a d .m is te w s w F o PD te h (w is si h T a

18. Huibregtse JM, Scheffner M, Howley PM. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol Cell Biol 1993;13:775-84. 19. Hubbert NL, Sedman SA, Schiller JT. Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes. J Virol 1992;66:6237-41. 20. Zimmermann H, Degenkolbe R, Bernard HU, O’Connor MJ. The human papillomavirus type 16 E6 oncoprotein can downregulate p53 activity by targeting the transcriptional coactivator CBP/p300. J Virol 1999;73:6209-19.

32. Moreno V, Bosch FX, Munoz N, Meijer CJ, Shah KV, Walboomers JM, et al. Effect of oral contraceptives on risk of cervical cancer in women with human papillomavirus infection: The IARC multicentric case-control study. Lancet 2002;359:1085-92. 33. Sathish N, Abraham P, Peedicayil A, Sridharan G, Chandy C. HPV 16 E6 sequence variations in Indian patients with cervical neoplasia. Cancer lett 2005;229:93-9. 34. Giroglou T, Florin L, Schafer F, Streeck RE, Sapp M. Human papillomavirus infection requires cell surface heparan sulfate. J Virol 2001;75:1565-70.

21. Veldman T, Horikawa I, Barrett JC, Schlegel R. Transcriptional activation of the telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein. J Virol 2001;75:446772.

35. Flores ER, Lambert PF. Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle. J Virol 1997;71:7167-79.

22. Veldman T, Liu X, Yuan H, Schlegel R. Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse transcriptase promoter. Proc Natl Acad Sci USA 2003;100:8211-6.

36. Sathish N, Abraham P, Peedicayil A, Sridharan G, John S, Chandy G. Human papillomavirus 16 E6/E7 transcript and E2 gene status in patients with cervical neoplasia. Mol Diagn 2004;8:57-64.

23. Oh ST, Kyo S, Laimins LA. Telomerase activation by human papillomavirus type 16 E6 protein: Induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites. J Virol 2001;75:5559-66.

37. Sathish N, Abraham P, Peedicayil A, Sridharan G, Shaji RV, Chandy G. E2 sequence variations of HPV 16 among patients with cervical neoplasia seen in the Indian subcontinent. Gynecol Oncol 2004;95:363-9.

24. Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. Embo J 1989;20:4099-105.

38. Shingleton HM, Orr JW Jr, editors. Carcinoma of the cervix: Historical aspects, epidemiology and screening, Chapter 1. In: Cancer of the cervix: Diagnosis and treatment. Churchill Livingston: New York; 1987.p. 1-15.

25. Funk JO, Waga S, Harry JB, Espling E, Stillman B, Galloway DA. Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein. Genes Dev 1997;11:2090-100.

39. Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M, et al. The 2001 Bethesda system. Terminology for reporting results of cervical cytology. JAMA 2002;287:2114-9.

26. DeGregori J, Leone G, Miron A, Jakoi L, Nevins JR. Distinct roles for E2F proteins in cell growth control and apoptosis. Proc Natl Acad Sci USA 1997;94:7245-50.

40. Spitzer M. Cervical screening adjuncts: Recent advances. Am J Obstet Gynecol 1998;179:544-56.

27. Roden RB, Lowy DR, Schiller JT. Papillomavirus is resistant to desiccation. J Infect Dis 1997;176:1076-9.

41. Sankaranarayanan R, Nene BM, Dinshaw K, Rajkumar R, Shastri S, Wesley R, et al. Early detection of cervical cancer with visual inspection methods: A summary of completed and on-going studies in India. Salud Publica Mex 2003;45:S399-407.

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42. Sherman ME, Lorincz AT, Scott DR, Wacholder S, Castle PE,



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72. Zwaveling S, Mota SC, Nouta J, Johnson M, Lipford GB, Offringa R, et al. Established human papillomavirus type 16expressing tumors are effectively eradicated following vaccination with long peptides. J Immunol 2002;169:350-8.

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78. Billich A. HPV vaccine MedImmune/ GlaxoSmith Kline. Curr Opin Investig Drugs 2003;4:210-3. 79. Daemen T, Riezebos-Brilman A, Regts J, Dontje B, van der Zee A, Wilschut J. Superior therapeutic efficacy of alphavirus mediated immunization against human papilloma virus type 16 antigens in a murine tumour model: Effects of the route of immunization. Antivir Ther 2004;9:733-42. 80. Brandsma JL, Shlyankevich M, Zhang L, Slade MD, Goodwin EC, Peh W, et al. Vaccination of rabbits with an adenovirus vector expressing the papillomavirus E2 protein leads to clearance of papillomas and infection. J Virol 2004;78:116-23. Source of Support: Nil, Conflict of Interest: None declared.

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IMARD: The age old structure for scientific papers Attracting readers: The importance of title and abstract Tables and Figures: Making figures worth 1000 words The ‘structured discussion’: The evidence based discussion Standardized reporting guidelines: The helping hands Reporting statistics: The common errors editors won’t miss to pickup The peer review process: Easier to pass through when you know how it works ‘I wish I had written that paper’: The publishable paper Authors vs. contributors: The importance of names in byline Ethical conduct of research: When and how of IRBs and more... For details: www.jpgmonline.com/writecon.asp www.ijmm.org 17 CMYK