The Development of a Human Papillomavirus Vaccine

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 2003348518-27.
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 Freunds
  adjuvant
• 1 group received CRPV L1-L2 with alum as the
  adjuvant
• 1 group received CRPV L1 with Freunds adjuvant
• 1 group received denatured CRPV L1-L2 with
  Freunds adjuvant
• 1 group received BPV L1-L2 with Freunds adjuvant
• 1 group received denatured BPV L1-L2 with
  Freunds 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.
51
Emany RT, et al. Journal of Virology 2002.
52
Emany RT, et al. Journal of Virology 2002.

• The predominant type of immunoglobulin at month
  7, one month after the third vaccine, was IgG,
  primarily IgG1, IgG3, and IgG4.
• IgA was also present in 25 of 30 subjects
• IgM was present in only 5 of 30 subjects.

53
Emany RT, et al. Journal of Virology 2002.
All subjects who received vaccine showed a
significant T-cell response, and the response
remained fairly stable after the first
immunization.
54
Pinto LA, et al. Cellular immune responses to
human papillomavirus (HPV)-16 L1 in healthy
volunteers immunized with reocmbinant HPV-16 L1
virus-like particles. Journal of Infectious
Diseases 2003 188 327-338.

• Preliminary results of a phase II trial published
  in 2003 also looked at cellular immune responses
  to the HPV-16 L1 VLP vaccine
• The results showed a statistically significant
  lymphoproliferative response of both CD4 and
  CD8 cells when compared with placebo, with a
  peak response at month 7.
• Similar responses were seen with cytokine
  production.

55
Pinto LA, et al. Journal of Infectious Diseases
2003.
P Placebo V Vaccine
56
Summary of Lymphoproliferative Studies

• The role of a lymphoproliferative response in a
  prophylactic vaccine remains unclear
• It may function to clear those cells that are
  infected despite an adequate antibody response,
  or it could simply play a role in potentiating
  the B cell response.
• It is unknown based on these in vitro studies if
  a significant lymphoproliferative response occurs
  at the site of infection in the genital tract,
  and whether this response would be targeted
  against HPV-16 infected cells.

57
Koutsky LA, Ault KA, Wheeler CM, et al. A
controlled trial of a human papillomavirus type
16 vaccine. NEJM 2002 347 (21) 1645-1651.

• A phase II clinical trial of an HPV-16 VLP
  vaccine was published in 2002.
• This was a double-blind, multicenter, randomized
  trial.
• Over 2000 women aged 16 to 23 from 16 centers in
  the U.S.A. were recruited.
• Some women were excluded from the primary
  analysis, most commonly because of HPV-16
  infection at enrollment.

58
Koutsky LA, et al. NEJM 2002.
59
Koutsky LA, et al. NEJM 2002.

• The vaccine was comprised of 40 µg of HPV-16 L1
  VLPs of 97 purity adsorbed to 225 µg of aluminum
  hydroxyphosphate sulfate adjuvant.
• Intramuscular injections were administered at day
  0, month 2, and month 6.
• Testing for PAP smears, HPV-16 DNA, and HPV-16
  antibody (by competitive radioimmunoassay) was
  done at enrollment, at month 7, month 12, and
  every 6 months thereafter until the conclusion of
  the study at month 48.

60
Koutsky LA, et al. NEJM 2002.

• The primary endpoint was persistent HPV-16
  infection, defined as follows
• negative HPV-16 infection on enrollment and at
  month 7, but HPV-16 subsequently detected by DNA
  PCR on 2 or more consecutive visits 4 or more
  months apart or
• cervical biopsy showing CIN or cervical cancer,
  and HPV-16 DNA detected in the biopsy tissue,
  swab or lavage sample collected at the preceding
  or following visit or
• HPV-16 DNA detected in a sample prior to the
  subject being lost to follow-up.

61
Koutsky LA, et al. NEJM 2002.

• The vaccine was very well tolerated, with no
  serious adverse events.
• The most common adverse event was pain at the
  injection site.

62
Koutsky LA, et al. NEJM 2002.Efficacy Analyses
of HPV-16 L1 VLP Vaccine
63
Limitations of the Study

• Median follow-up time after completion of
  vaccination was only 17.4 months the results
  from the full 4 years of follow-up have not yet
  been published.
• Larger study group needed to prove that clinical
  disease is prevented by vaccination.
• The study included only women, whereas men are
  the primary vectors and would likely need to be
  included in a comprehensive vaccination program.

64
Studies in Progress

• Phase II trials of a multivalent vaccine
  combining HPV-16 and HPV-18 VLPs were initiated
  in January of 2000.
• This vaccine, called MEDI-517, was developed by
  MedImmune and GlaxoSmithKline.

Billich A, HPV vaccine MedImmune/GlaxoSmithKline.
Current Opinion in Investigational Drugs 2003 4
(2) 210-213.
65
Studies in Progress

• Phase III clinical trials are currently underway
  for a quadrivalent HPV VLP vaccine.
• Developed by Merck against types 6, 11, 16, and
  18.

Jansen KU, Shaw AR. Human papillomavirus
vaccines and prevention of cervical cancer.
Annual Review of Medicine 2004 55 319-331.
66
Would a Successful Vaccine Lead to Changes in
Current Screening Practices?

• In less developed countries the hope would be
  that cervical cancer incidence could be reduced
  by administering the vaccine to a large
  proportion of the population, and any screening
  would continue to be available to a relatively
  small percentage of the population.

67
Kulasingam SL and Myers ER. Potential health and
economic impact of adding a human papillomavirus
vaccine to screening programs. JAMA 2003 290
(6) 781-789.

• The authors of this 2003 article used
  mathematical modeling to derive an optimal
  combination of vaccination and screening.
• The optimal strategy according to this model was
  vaccination plus screening every other year
  beginning at age 24, with a lifetime cost of
  834.

68
Further Considerations in Screening versus
Vaccine

• Many of the cervical cancers affecting younger
  women are rapidly progressive.
• Sensitivity of conventional cytology is highly
  variable, with estimates ranging from 50 to 90.
• Even with yearly screening, therefore, rapidly
  progressive cancers in younger women may not be
  detected until a relatively advanced stage.

69
Further Considerations in Screening versus
Vaccine

• Once more experience with the vaccine is gained,
  and more HPV types are included in the vaccine,
  the recommended age for screening onset can be
  expected to increase, and the recommended
  screening interval can be expected to lengthen.

70
QUESTIONS

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

71
Does it work?
72
The Future Looks Promising
73
When will it be available for my patients?
74
More Studies Are Needed
75

• If the phase III trials of the Merck and
  MedImmune HPV vaccines show that they are safe
  and effective in larger study groups, then it is
  likely that a commercially available HPV vaccine
  will be available in the next 5 to 10 years.
• Much like the Hepatitis B vaccine, however, it is
  likely to be relatively expensive, probably
  around 200 for a series of 3 injections

76
Who should get it?
77
Unclear at this time. Try again later.
78

• Given that HPV infection tends to occur in young
  women within the first one to two years after
  sexual activity is initiated, the vaccine should
  be administered prior to this time.
• A study of sexual behavior among American
  adolescents conducted in 1990 showed that only 5
  of boys at age 12 had had sex, while 67 at age
  17 reported having had sex. Of girls 0 had had
  sex at age 12 and 56 at age 17.

Leigh BC, et al. Sexual behavior of American
adolescents Results from a U.S. national survey.
Journal of Adolescent Health 1994 15 117-125.
79

• In developing countries, an infant vaccine would
  likely be most effective, as access to healthcare
  is limited.
• However, it is unknown if an infant vaccine would
  be effective into adolescence and adulthood.
• Ideally, males should also receive the vaccine,
  as they are the primary vectors.

80
THANKS TO

• Dr. Kevin High
• Dr. Raquel Watkins
• Dr. Wasil Khan
• My Mother-In Law
• Clara