1. transgene 1 Title Human papillomavirus vaccines : The knowns and the (still) unknowns FEAM Conference, Lisboa, 15 December 2007 Marc P Girard Lyon, France

2. transgene 2 Papillomaviruses Papillomaviruses are animal viruses which affect man and animals. They typically are the agents of warts Among animal viruses, one finds : The Shope cotton tail rabbit virus The agent of canine oral papillomatosis The agent of oesophagal papillomatosis of calves

3. transgene 3 Human Papillomaviruses More than 100 HPV types have been identified that affect humans. They all are small icosahedral viruses, with a circular double-stranded DNA genome and a strong epithelial tropism. About 40 HPV genotypes spread through sexual contact and infect the ano-genital mucosae. HPV infection is one of the most, if not the most frequent sexually transmitted infection (STI) in humans.

4. transgene 4 Six early genes: E1, E2, E4, E5, E6 and E7 E1 & E2 proteins: viral replication E6 & E7 oncoproteins: responsible for transformation of infected cells (induction of premalignant alterations) and directly contribute to malignant progression Two late genes: L1 and L2 L1 & L2 : capsid proteins The HPV Genome HPV genomic organization E7 L1 E5 E6 L2

5. transgene 5 Prevalence of genital HPV infections Prevalence of genital HPV in the female population varies from 2%- 44% in different studies. In a study on 474 young female students in the USA, the cumulative risk of HPV infection after onset of sexual activity was 45% over 2 years Prevalence of genital HPV infection among female college students in the USA was estimated to be 20%-30%.

6. transgene 6 Infection can be multiple Multiple genital infections (several types of HPV together) can also be observed. Thus, of 3578 female volunteers recruited for a vaccine study in the USA: 23.7% were infected by one HPV of genotype 16, 18, 6 or 11; 4.8% were infected by two of the 4 genotypes 1% were infected by three of the four genotypes And 0.1% carried the four genotypes together

7. transgene 7 Fifteen HPV genotypes (high-risk HPVs) are recognized as the major cause of cervical cancer. Of these, HPV 16 represents 57% of cases, and HPV 18 14% Altogether, five specific high-risk HPV ( HPV 16, 18, 31, 33 and 45 ) cause more than 80% of the cervical cancers diagnosed worldwide. Over 99% of cervical cancer biopsies contain HPV DNA Bosch et al. J Clin Pathol. 2002 Apr;55(4):244-65 Walboomers at al. J Pathol.199 sep;189(1):12-19 HPVs: at the Root of Cervical Cancer

8. transgene 8 Burden of HPV-related cancers High-risk HPVs, especially HPV 16 and 18, were estimated to cause 100% of the almost 260,000 deaths from cervical cancer worldwide in 2005. They also caused 60-65% of cancers of the vagina, 20-50% of cancers of the vulva, 85%-95% of cancers of the anus and 12%-36% of cancers of the oropharynx. Altogether, cancer cases attributed to HPVs in 2002 were estimated to number about 450,000 in developing countries and 110,000 in industrialized countries.

9. transgene 9 Disease burden Cervical cancer is the second most frequent cancer in women worldwide In developing countries, cervical cancer is the leading cause of cancer deaths in women

10. transgene 10 Low-risk HPVs « Low-risk » genital HPVs are responsible for genital warts (condyloma acuminata) which grow on the cervix, vagina, vulva or anus in women and penis, scrotum or anus in men. HPV genotypes most frequently responsible for genital warts are HPV 6 and HPV 11. HPVs also cause epithelial growths over the vocal cords of children and adults that require surgical intervention (recurrent juvenile respiratory papillomatosis)

11. transgene 11 The outcome of HPV infection Most genital HPV infections are asymptomatic and more than 90% of detected infections are spontaneously cleared within two years. Persisting infection leads to integration of the viral genome into human DNA, which translates into lesions called squamous intraepithelial lesions (SIL) or cervical intraepithelial lesions (CIN) of low then high grade. If untreated, moderate (CIN2) or severe (CIN3) lesions (often grouped together as CIN 2/3) lead to cancer.

12. transgene 12 CIN2/3: Screening / Diagnostic Suspected on cytology : HSIL on screening Pap smear Visualized by colposcopy directed biopsy of acidophil lesion CIN2/3 diagnosis based on histology : epithelial hyperplasia, atypical mitoses, marked nuclear abnormalities, etc.

13. transgene 13 Treatment of CIN2/3 CIN2/3: indication for the removal of the entire extent of the suspicious area - Outpatient technique known as the loop electrosurgical excision procedure (LEEP). - More aggressive procedure called conization (cone biopsy). Treatment efficacy >90% Relapse after conization (disease present more than 6 months after treatment) can reach a rate of 15% Safety: conization is associated with a low rate of significant hemorrhage and may have consequences in terms of fertility

14. transgene 14 The need for preventive vaccines Routine screening of 25-65 years-old women has been estimated to prevent the occurrence of 90% of cervical cancers (Europ Union Recommendations, Vienna 1999). However, neither routine screening for precancerous lesions nor their eventual treatment are done in developing countries. The need for a preventive vaccine is therefore both obvious and urgent

15. transgene 15 Size of the problem The population of less than 15 years-old girls worldwide is about 80 million today. It is projected to be 300 million by 2040. The 493,000 cases of cervical cancer in 2002 is projected to become 702,000 by 2020

16. transgene 16 Vaccine Positioning HPV Infection CIN 2/3 Invasive cancer Persistent Infection Prevention 4045 35 302520 15 105 years Average age = 35 years GlaxoSmithKline - Cervarix Merck - Gardasil Therapeutic Vaccine

17. transgene 17 HPV VLPs HPV vaccines are prepared from empty viral protein shells called pseudo-virions, or « virus-like particles »(VLPs), that spontaneously assemble from the L1 viral capsid protein produced by recombinant technology in yeast or insect cells. They do not contain any DNA, so they are not infectious. They elicit the production of high-titer neutralizing antibodies (IgG) that diffuse by transudation into the mucosal ano-genital tissues where they can block incoming HPV particles of the cognate genotype and prevent infection

18. transgene 18 The two HPV vaccines Two HPV vaccines have been developed: Gardasil TM, a quadrivalent vaccine made of VLPs produced in yeast and corresponding to HPV 16, 18, 6 and 11 + Merck aluminium adjuvant Cervarix TM, a bivalent vaccine made of VLPs produced in insect cells and corresponding to HPV 16 and 18 + GSK AS04 adjuvant (oil-in-water emulsion with Al(OH) 3 and monophosphoryl lipid A) One month after the third dose of vaccine, nearly 100% of women in trials of either of the vaccines developed neutralizing antibodies to each of the HPV genotype in the vaccine at titers 10-100 fold higher than in natural infection.

19. transgene 19 Major properties of VLP vaccines VLP vaccines are prophylactic vaccines that work remarkably well only if administered prior to infection : ~ 100% efficacy proven over 36 months against HPV vaccine types infection in perprotocol phase III study of Gardasil TM ; But only 45% efficacy in intention-to-treat protocol (women who were infected at time of first vaccination). Protection is HPV genotype-specific, although evidence has been gathered for partial cross-protection against HPV 45 and 31. They induce a long-term neutralizing antibody response (>5 years) in vaccinated women.

20. transgene 20 The unknowns -1- Duration of protection : Persistence of neutralizing antibodies after 5 years? Minimum protective antibody threshold for disease proptection? Need for eventual booster injections? Target age group : pre-adolescent girls (age 9-12 years) Effectivness of the vaccine given at an earlier age?(school-age) Problem of limited efficacy in already infected young women Safety and efficacy in developing countries ? Especially in populations of high HIV prevalence Gender effect : efficacy in men?

21. transgene 21 The unknowns -2- HPV vaccination will reduce, but not eliminate the risk of cancer. Screening programmes will therefore remain necessary for cervical cancer prevention even after the vaccines are introduced. A combination of HPV vaccination and screening 1-3 times per lifetime might turned out to be cost-effective But much will depend on the cost of the vaccine in the different settings

22. transgene 22 A complementary approach: Therapeutic vaccination There is a high rate of spontaneous regression involving immune mechanisms A therapeutic vaccination that would improve HPV antigens presentation & immunogenicity should further enhance this mechanism CIN2/3 are lesions which slowly evolves into invasion Although conization leads to immediate cure of the CIN lesions, there is time, without raising safety concern, to implement an immune response through therapeutic vaccination E6 and E7 HPV antigen involvement in cervical cells transformation is clearly defined They are the ideal candidate vaccine antigens for a therapeutic vaccination

23. transgene 23 Therapeutic vaccination in CIN2/3: medical rationale In the context of CIN2/3 pathology and compared to current treatment methods, therapeutic vaccines would present several advantages: Non-invasive, safe procedure avoiding surgery and its complications Easy procedure: vaccination during visit at gynecologist, no need for out- patient hospitalization No relapse, due to eradication of the viral cause. The vaccine should induce an HPV-specific immune response able not only to cure CIN2/3 lesions but also, on a long-term basis, to reduce the rate of recurrent disease, even though booster immunizations may be needed

24. transgene 24 Therapeutic Vaccines: postulated mechanism of action www.cancer-info.com Migration T cells MVA-Antigen Antigen- specific CD8 T cells killing a cancer cell Dendritic cells