The Development of Human Papillomavirus Vaccine by mikeholy


									The Development of a
Human Papillomavirus
     Mark F. Doerner, M.D.
    Resident Grand Rounds
        March 24, 2004
Does it work?
Who should get it?
When will it be available for
 my patients?
 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
Cervical cancer second most common
 cancer among women worldwide
15.8 cases per 100,000 in less developed
15.0 cases per 100,000 in more developed
379,000 new cases in the year 2000 in
 less developed countries
5-year prevalence 1.4 million cases
                 Ferlay J, et al., editors. IARC Cancer-Base No. 5. International
                 Agency for Research on Cancer (IARC Press); 2001.
Screening Costs

 Efficiency Curve
                   Goldie SJ. Journal of the National Cancer
                Institute Monographs No. 31, 2003: 102-110.
         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
          Current Cervical Cancer
           Screening Guidelines
American College of Obstetricians and
  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
      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
 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.
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.
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 =
 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.
       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.
HPV Genomic Map
HPV Protein Capsid
The Process of HPV Infection

                    Fields Virology 2001.
           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
 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.
     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
 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.
 Early Steps in HPV
Vaccine Development
  Difficulties Associated with Early
     HPV Vaccine Development

No reliable source for intact
Presence of viral oncogenes
Papillomaviruses are highly species
          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
VLPs are morphologically similar to
          native virions

    L1-L2 BPV-4 VLP   Native BPV-4 virus
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:

 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
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
   1 group received BPV L1-L2 with Freund’s adjuvant
   1 group received denatured BPV L1-L2 with Freund’s
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.
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.
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.
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):

 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.
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.
Christensen ND, et al. Journal of Virology 1996.

       Challenge with CRPV infection 12 months after
         vaccination. Control group is on the left.
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.
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.
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.
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.
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
 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.
Harro CD, et al. Journal of the National Cancer
Institute 2001.
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.
Harro CD, et al. Journal of the National Cancer
Institute 2001.
       Limitations of the Study

 Short duration of follow-up after the last
 Antibody levels were measured in serum, not
  at the site where infection would be likely to
 Inability to detect whether these serum
  antibody titers were sufficient to protect
  against mucosal infection
 Small sample size
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
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
 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.
Nardelli-Haefliger D, et al. Jounal of the National
Cancer Institute 2003.
Nardelli-Haefliger D, et al. Jounal of the National
Cancer Institute 2003.

    IgG titers remained relatively constant throughout the
                cycle in the contraceptive group
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.
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.
Emany RT, et al. Journal of Virology 2002.

          Immunization Schedule
      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.
T and B Cell Responses to Viral Antigen

                        Parslow TG, et al. Eds. Medical
                        Immunology 10th ed. 2001
     Emany RT, et al. Journal of Virology 2002.

 Titers of greater than 200 mMU/ml were
  found in most subjects after the third
 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.
Emany RT, et al. Journal of Virology 2002.
     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.
    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.
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
Pinto LA, et al. Journal of Infectious Diseases 2003.

               P = Placebo; V = Vaccine
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.
Koutsky LA, Ault KA, Wheeler CM, et al. A controlled trial of a
human papillomavirus type 16 vaccine. NEJM 2002; 347 (21):

 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.
Koutsky LA, et al. NEJM 2002.
            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.
              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.
        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.
                                 Koutsky LA, et al. NEJM 2002.

              Efficacy Analyses of HPV-16 L1 VLP Vaccine
Type of Analysis    End Point        HPV-16 Vaccine        Placebo          Observed
                                                                             (95% CI)     P
                                  No. of   Cases of    No. of   Cases of       %
                                  Women    Infection   Women    Infection

Primary per-        Persistent     768        0         765       41          100       <0.001
protocol efficacy   HPV-16
analysis            infection                                               (90-100)

Efficacy            Persistent     800        0         793       42          100        -----
analysis            HPV-16
including           infection                                               (90-100)
women with
general protocol
       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.
          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

                Billich A, HPV vaccine
                MedImmune/GlaxoSmithKline. Current Opinion
                in Investigational Drugs 2003; 4 (2): 210-213.
           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.
  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.
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.
          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
 Even with yearly screening, therefore, rapidly
  progressive cancers in younger women may
  not be detected until a relatively advanced
         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.
Does it work?
Who should get it?
When will it be available for
 my patients?
Does it work?
The Future
When will it be available for my patients?
More Studies
Are Needed
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
Who should get it?
Unclear at
this time.
Try again
 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.
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
Ideally, males should also receive the
 vaccine, as they are the primary vectors.
 Dr. Kevin High
 Dr. Raquel Watkins
 Dr. Wasil Khan
 My Mother-In Law
 Clara

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