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The Development of a Human Papillomavirus Vaccine

The Development of a Human Papillomavirus Vaccine. Mark F. Doerner, M.D. Resident Grand Rounds March 24, 2004. QUESTIONS. Does it work? Who should get it? When will it be available for my patients?. OVERVIEW. Epidemiology of Cervical Cancer Cervical Cancer Screening

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The Development of a Human Papillomavirus Vaccine

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  1. The Development of a Human Papillomavirus Vaccine Mark F. Doerner, M.D. Resident Grand Rounds March 24, 2004

  2. QUESTIONS • Does it work? • Who should get it? • When will it be available for my patients?

  3. OVERVIEW • Epidemiology of Cervical Cancer • Cervical Cancer Screening • HPV and Cervical Cancer • Early Steps in HPV Vaccine Development • Animal Studies • Human Studies • Studies in Progress • Screening and Vaccination • Conclusions

  4. Epidemiology • Cervical cancer second most common cancer among women worldwide • 15.8 cases per 100,000 in less developed countries • 15.0 cases per 100,000 in more developed countries • 379,000 new cases in the year 2000 in less developed countries • 5-year prevalence 1.4 million cases worldwide Ferlay J, et al., editors. IARC Cancer-Base No. 5. International Agency for Research on Cancer (IARC Press); 2001.

  5. Screening Costs Efficiency Curve Goldie SJ. Journal of the National Cancer Institute Monographs No. 31, 2003: 102-110.

  6. Current Cervical Cancer Screening Guidelines • American Cancer Society • Start 3 years after onset of vaginal intercourse • Start no later than age 21 • Annual screening with conventional cervical cytology smear • Every 2 years with liquid-based cytology • Can increase interval to every 2 to 3 years in women over the age of 29 who have had 3 or more consecutive normal PAPs

  7. Current Cervical Cancer Screening Guidelines • American College of Obstetricians and Gynecologists • Annual cytology screening for women under age 30 • Can extend interval to every 2 to 3 years for women over the age of 29 who have had 3 consecutive negative PAPs • The United States Preventive Services Task Force • Can extend interval to every 2 to 3 years for all women who have had 2 consecutive negative PAPs, regardless of age

  8. HPV and Cervical Cancer • Numerous studies have confirmed causal relationship between high-risk HPV types and cervical cancer since development of technology to test for HPV DNA in early 1980s. • Bosch et al looked at tumor samples from 932 women in 22 countries. • The samples were tested for 25 different HPV types by PCR. Bosch, et al. Prevalence of human papillomavirus in cervical cancer. Journal of the National Cancer Institute 1995.

  9. Bosch, Manos, et al. Prevalence of human papillomavirus in cervical cancer. Journal of the National Cancer Institute 1995. • Overall prevalence 92.9% (CI 91.1-94.5) • HPV type 16 comprised 53.7% of specimens positive for HPV. • HPV-16 most prevalent in all regions studied • Types 16 and 18 together comprised 68.5% of specimens positive for HPV. • Types 16, 18, 31, and 45 comprised 83.1% of specimens positive for HPV.

  10. Bosch FX, Lorinca A, Muñoz N, Meijer CJLM, Shag KV, The causal relation between human papillomavirus and cervical cancer. Journal of Clinical Pathology 2002; 55: 244-265.) • Pooled data from 11 case-controlled studies dating from 1985-1997 • Overall odds ratio for cervical cancer associated with HPV DNA positivity = 158.2

  11. Cervical Cancer and HPV Types • High-Risk Types (in decreasing order of prevalence) = 16, 18, 45, 31, 52, 58, 59, 35, 33, 51, 33, 56, 73, 68, 39, and 82 • Probable High-Risk Types = 26, 53, 66 • Low-Risk Types = 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 Muñoz N, Bosch FX, et al. The New England Journal of Medicine 2003;348:518-27.

  12. Papillomavirus Structure • A 7904 base-pair molecule of double-stranded DNA within a spherical protein capsid consisting of 72 capsomeres. • The capsid is comprised of two virally encoded proteins. • The major protein is L1, which forms most of the capsid by forming 72 pentamers • The minor protein in L2, which functions primarily in the process of encapsidation of the viral genome.

  13. HPV Genomic Map

  14. HPV Protein Capsid

  15. The Process of HPV Infection Fields Virology 2001.

  16. Oncogenic Potential • The E6 and E7 proteins of high-risk HPV types are capable of extending the lifespan of human keratinocytes and forming cells resistant to terminal differentiation. • The E6 oncoprotein can complex with and inactivate the tumor suppressor gene product p53. • The E7 oncoprotein complexes with and inactivates the retinoblastoma protein, a protein which normally has the ability to inhibit cell cycle progression, thereby allowing the cell to progress into the S phase of the cell cycle, inducing DNA synthesis and cellular proliferation.

  17. Time From HPV Infection to Development of Cervical Cancer • Most infections are acquired shortly after sexual activity begins and are cleared within 1 to 2 years. • It has been estimated that the average time between HPV infection and the onset of precancer is about 7-10 years. • Estimates of the time between HPV infection and development of cervical cancer range between 10 and 19 years.

  18. Early Steps in HPV Vaccine Development

  19. Difficulties Associated with Early HPV Vaccine Development • No reliable source for intact papillomaviruses • Presence of viral oncogenes • Papillomaviruses are highly species specific.

  20. Virus-Like Particles • Zhou et al found, in an article published in 1991, that L1 and L2, the two capsid proteins, were capable of self assembly into so-called virus-like particles (VLPs) when expressed in insect cells via a baculovirus vector. Zhou J, et al.Journal of Virology 1991; 185: 251-257

  21. VLPs are morphologically similar to native virions L1-L2 BPV-4 VLP Native BPV-4 virus

  22. Kirnbauer R, et al. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proceedings of the National Academy of Science 1992; 89: 12180-12184. • Kirnbauer et al went on to show that L1 alone was able to self-assemble into VLPs. • They also showed that the L1 VLPs were able to induce high titers of neutralizing rabbit antisera similar to that of infectious virions. • These discoveries led to the initiation of animal trials with species-specific VLP vaccines.

  23. ANIMAL STUDIES

  24. Breitburd F, Kirnbauer R, et al. Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection. Journal of Virology 1995; 69 (6): 3959-396. • New Zealand White rabbits were divided into 7 groups of 10 • 1 control group received adjuvant only • 1 group received CRPV L1-L2 with Freund’s adjuvant • 1 group received CRPV L1-L2 with alum as the adjuvant • 1 group received CRPV L1 with Freund’s adjuvant • 1 group received denatured CRPV L1-L2 with Freund’s adjuvant • 1 group received BPV L1-L2 with Freund’s adjuvant • 1 group received denatured BPV L1-L2 with Freund’s adjuvant

  25. Breitburd F, Kirnbauer R, et al.Journal of Virology 1995. • The initial inoculation was with 50μg of the vaccine or adjuvant alone injected subcutaneously. Booster injections were given at 2 and 4 weeks. • Two weeks after the last booster the animals were challenged with infectious CRPV virions in both high and low doses. • Virions were applied on areas of shaved skin abraded with sandpaper, a low dose to one flank and a high dose to the other flank. • The animals were examined for a total of one year for the development of papillomas – weekly for the first 24 weeks and monthly thereafter.

  26. Breitburd F, Kirnbauer R, et al.Journal of Virology 1995. • Sera from the immunized animals were tested with a standard ELISA . • The mean titer of sera prior to immunization was less than 5. • After immunization with CRPV L1 or L1-L2 VLPs, mean titers ranged from 5,000 to 10,000 (depending on the advujant used) one week after the second booster.

  27. Breitburd F, Kirnbauer R, et al.Journal of Virology 1995. • Passive transfer of serum and IgG was also carried out to help determine if humoral or cellular immunity was required to confer immunity. • Of the 4 rabbits inoculated with hyperimmune sera or IgG, three developed no papillomas, while one of the animals who had received hyperimmune IgG developed three papillomas at the high-dose side and none at the low-dose side. • This suggests that the protective effect seen in those rabbits inoculated with CRPV VLPs came from neutralizing antibodies.

  28. Christensen ND, et al. Immunization with virus-like particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus. Journal of Virology 1996; 70 (2): 960-965. • A second study on rabbits published the following year looked at long-term protection. • Each group received three injections, a primary at day 0, and boosters at day 21 and day 35. • After the full immunization schedule had been administered, rabbits from each group were challenged with infectious CRPV at either 2 weeks, 6 months, or 12 months.

  29. Christensen ND, et al.Journal of Virology 1996. • The groups challenged at 2 weeks after immunization developed no papillomas. • Those challenged at 6 months showed a high level of protection, with 2 of 4 rabbits developing small papillomas at the strongest challenge dose. • Those challenged at 12 months also showed excellent protection -- 2 rabbits developed small papillomas at one site in response to the strongest challenge dose.

  30. Christensen ND, et al.Journal of Virology 1996. Challenge with CRPV infection 12 months after vaccination. Control group is on the left.

  31. Suzich JA, et al. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc. Natl. Acad. Sci. 1995; 92: 11553-11557. • The papillomas induced in the rabbits were cutaneous lesions, whereas those of greatest concern in humans are mucosal infections. • A study published in 1995 looked at the efficacy of VLP vaccines against mucosal papillomas in dogs.

  32. Suzich JA, et al.Proc. Natl. Acad. Sci. 1995. • Fourteen eight-week old beagles were vaccinated with COPV L1 VLPs by intradermal injection at week 0 and week 2. • Two weeks after their last vaccine dose, the dogs were infected with COPV on oral mucosa and followed for a total of 13 additional weeks. • None of the dogs receiving COPV L1 VLPs developed papillomas, whereas all of the controls developed them.

  33. Kirnbauer R, et al. Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization. Virology 1996; 219: 37-44. • A study of vaccines to mucosotropic bovine papillomavirus type 4 was carried out in a manner similar to that of the canine study. • Calves were vaccinated intramuscularly with 150 μg of L1 VLPs or 200 μg of L1-L2 VLPs at 0 and 4 weeks • Two weeks after the final dose of vaccine, the calves were infected with BPV-4 at 10 sites in the palate.

  34. Kirnbauer R, et al. Virology 1996. 21 weeks after challenge with BPV-4. Control calves on the left, calves vaccinated with L1 VLPs in the middle, and calves vaccinated with L1-L2 VLPs on the right.

  35. HUMAN STUDIES

  36. Harro CD, et al. Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine. Journal of the National Cancer Institute 2001; 93 (4): 284-292. • A double-blind, placebo-controlled, dose-escalation trial designed to assess the safety and immunogenicity in adults of an HPV-16 L1 VLP vaccine. • Study group included both men and women, ranging in age from 18 to 29 years old. • One group was randomized to receive three injections of vaccine at a dose of 10 µg or placebo, and another group was randomized to receive the vaccine at a dose of 50 µg or placebo.

  37. Harro CD, et al.Journal of the National Cancer Institute 2001.

  38. Harro CD, et al.Journal of the National Cancer Institute 2001. • The vaccine was very well tolerated, with the most common reported side effect being pain at the injection site. • All of those receiving vaccine showed significant serum IgG responses as measured by an ELISA assay, with the peak observed 1 month after the third vaccination at month 5.

  39. Harro CD, et al.Journal of the National Cancer Institute 2001.

  40. Limitations of the Study • Short duration of follow-up after the last vaccine • Antibody levels were measured in serum, not at the site where infection would be likely to occur • Inability to detect whether these serum antibody titers were sufficient to protect against mucosal infection • Small sample size

  41. Nardelli-Haefliger D, et al. Specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus 16 virus-like particles. Jounal of the National Cancer Institute 2003; 95(15): 1128-1137. • Evaluated antibody response at the cervix • 18 healthy adult women between the ages of 18 and 45 with normal PAP smears • Divided into 2 groups – those taking oral contraceptives and those not taking them • Subjects were administered HPV-16 L1 VLP vaccine.

  42. Nardelli-Haefliger D, et al.Jounal of the National Cancer Institute 2003. • All subjects had seroconverted by 4 weeks after the last immunization. • All subjects developed cervical anti-HPV 16 antibodies. • Wide variation in cervical IgG antibody titers during ovulatory cycles – they were highest during the proliferative phase, decreased ninefold around ovulation, and increased threefold during the luteal phase.

  43. Nardelli-Haefliger D, et al.Jounal of the National Cancer Institute 2003.

  44. Nardelli-Haefliger D, et al.Jounal of the National Cancer Institute 2003. IgG titers remained relatively constant throughout the cycle in the contraceptive group

  45. Nardelli-Haefliger D, et al.Jounal of the National Cancer Institute 2003. • In sum, it would appear from this small study that the HPV 16 L1 VLP vaccine does induce significant quantities of IgG antibody in the female genital tract. • IgG is known to be the predominant protective antibody in the female genital tract, not IgA as on other mucosal surfaces. • Whether this is a sufficient quantity to be protective against infection and how long the protection would last remains unclear.

  46. Emany RT, et al. Priming of human papillomavirus type 11-specific humoral and cellular immune responses in college-aged women with a virus-like particle vaccine. Journal of Virology 2002; 76 (15): 7832-7842. • Both humoral and cellular immune responses against an HPV 11 L1 VLP vaccine were assessed in this Phase I trial. • Study participants included 30 women aged 10 to 25 who were in general good health and showed no evidence of HPV-6 or HPV-11 infectivity.

  47. Emany RT, et al.Journal of Virology 2002. Immunization Schedule

  48. Emany RT, et al.Journal of Virology 2002. • Antibody titers were measured with a competitive radioimmunoassay, whereby antibodies in patient serum compete against a monoclonal antibody of established affinity for the target protein. • Lymphoproliferation was measured by incorporating titrated quantities of tagged thymidine into DNA. The more thymidine incorporated, the more DNA synthesized, which reflects cell proliferation. This was quantitated by counting the rads. • Lymphoproliferation results were expressed as a stimulation index (SI), calculated as the geometric mean of counts per minute of cells cultured in the presence of VLP divided by the mean count of cells cultured without VLPs. An SI of 5.0 or greater was considered positive.

  49. T and B Cell Responses to Viral Antigen Parslow TG, et al. Eds. Medical Immunology 10th ed. 2001

  50. Emany RT, et al.Journal of Virology 2002. • Titers of greater than 200 mMU/ml were found in most subjects after the third immunization. • An earlier study had shown that serum titers greater than 200 measured in the same manner were neutralizing against HPV-11 virions in 63 of 69 (91.3%) serum specimens using an athymic mouse xenograft model. Brown DR, et al.Journal of Infectious Disease 2001; 184: 1183-1186.

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