HPV Vaccines to Prevent Cervical Cancer

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1 HPV Vaccines to Prevent Cervical Cancer John Schiller, National Cancer Institute, NIH, USA HPV and Cancer Current Vaccine 2nd Generation Vaccines Other Interventions to Prevent HPV Infections

2 Incidence and Distribution of Cancers Attributable to HPV Cervix Oropharnyx Anus Oral cavity Larynx Vulva Penis HPV cases Total cases 0 100, , , , ,000 Annual number of cases

3 Distribution of HPV Types in Cervical Cancer by Geographical Region % of cervical cancer are HPV DNA positive. 15 types are thought to cause cervical cancer. Worldwide, 70% caused by HPV16 or HPV18 Africa Latin America South-East Asia Europe North America HPV 45 HPV 31 HPV 18 HPV 16

4 Precursor lesions for cervical cancer From Lowy & Schiller, J Clin Invest, 116: , 2006

5 Time Line of Cervical HPV Infections And Progession to Cancer Rate HPV Precancer 15 y.o. 30 y.o. 45 y.o. Cancer Age Lifetime incident of genital HPV infection >80% in U.S.

6 HPVs Cause A Variety of Proliferative Diseases foot warts EV cutaneous, mostly asymptomatic HPV16 mucosal, cancer- associated hand warts HPV18 HPV6 HPV11 genital warts

7 Therapeutic vs Prophylactic HPV Vaccines Therapeutic vaccine attractive since Cervical CA develops slowly from well defined and routinely identified viral lesions. E6 and E7 expressed. Approved vaccines against other viral infections are preventive (based on neutralizing antibodies), not therapeutic (based on cell mediated immune responses). We initially concentrated on Prophylactic Vaccines

8 Live Attenuated Viruses Are Not Suitable For an HPV Prophylactic Vaccine Papillomavirus cannot be efficiently grown in cultured cells The viral genomes contain oncogenes Virion protein-based subunit vaccines are preferable, if they could efficiently induce neutralizing antibodies.

9 Papillomaviruses Encode Two Structural Proteins Papillomavirus Particle L1: the major structural protein. Each viral particle has 360 copies in 72 pentamers. L1 L1 L1 L1 L2 L1 L2: the minor structural protein. Up to 72 copies per particle.

HPV Virion Prophyactic HPV Vaccines Are L1 Virus Like Particles (VLPs( VLPs) L1 coding region L1 Insertion in Baculovirus Expression Vector Production in Insect Cells

10 HPV Virion Prophyactic HPV Vaccines Are L1 Virus Like Particles (VLPs( VLPs) L1 coding region L1 Insertion in Baculovirus Expression Vector Production in Insect Cells HPV16 L1 VLPs Spontaneous assembly of L1 into VLPs Induce high titers of virion neutralizing antibodies Non-infectious, Non-oncogenic Reinhard Kirnbauer et al. PNAS 1992

11 VLP Vaccination in Animal PV Models Implicate Antibodies as Immune Effectors (Cottontail Rabbit PV, Bovine PV 4, Canine Oral PV) VLPs induce high titers of type-restricted virion neutralizing antibodies, all of which recognize conformation-dependent epitopes* Vaccination with VLPs of animal PVs** induces type-specific protection from experimental infection with high dose virus Protection can be passively transferred** in serum antibodies No regression of established lesions** No HPV or sexual transmission model * Kirnbauer PNAS 1992 **Breitburd J.Virol. 1995

12 Two Distinct HPV VLP Vaccines Are Under Commercial Development GlaxoSmithKline: HPV16 Cervarix HPV18 70% of Cervical Ca ASO4 Adjuvant (Aluminum + MPL) Made in insect cells Merck: Gardasil HPV16 70% of Cervical Ca HPV18 HPV6 90% of Genital Warts HPV11 Aluminum Adjuvant Made in yeast IM Injections at 0, 1 or 2, and 6 months

13 Consistency of Antibody Response to VLPs Percent of Women Serocoverting to Individual HPV VLPs in Merck Quadravalent VLP Vaccine Gardasil* HPV6 99.8% HPV % HPV % HPV % *4666 women vaccined 3 times by intramuscular injection

14 Effect of GSK s Adjuvant on VLP Antibody Response In vitro Pseudovirus Neutralization* AS04 Al(OH) 3 * Significant Difference (p<0.05) between Ab titers Giannini et al. Vaccine, 2006 * Pastrana et al. Virology, 2005

15 Three Phase III Trials Are in Progress Sponsor VLP Types Trial Sites Enrolled NCI: HPV16, 18 Costa Rica 7,500 GSK: HPV16, 18 Multicentric 18,000 Merck: HPV16, 18, 6, 11 Multicentric 25,000 Women followed for several years Virologic Endpoint: Persistent Cervical HPV DNA Clinical Endpoint: Intermediate and High Graded Cervical Dysplasia (CIN 2/3)

16 Design, Safety and Immunogenicity Design: double blind; placebo controlled Young women, yrs old, often fewer than 4 (or 6) lifetime sex partners Primary analyses for types in the vaccine and exclude women HPV DNA or seropositive at entry Tolerability: Slightly more injection site pain than Alum; No effect on drop out rates No vaccine-related SAEs Immunogencity: >99% Seroconversion; GMTs fold higher than natural infection

17 Gardasil: : Phase 3 Prophylactic Efficacy Results Vaccine Placebo n Cases n Cases Efficacy C.I. *HPV16 or 18 CIN 2/3 or AIS % **HPV16 or 18 VIN2/3 or VaIN2/ % *HPV6, 11, 16, 18 Genital warts % Results in HPV DNA and seronegatives at baseline after three doses (*) or after at least one dose (**), as reported in Gardasil package insert. Average Duration of Follow-up: 1.5 Years After the Last Vaccination

Exudated Abs at Sites of Trauma Y Y Y Y Y Y Y Y Y Y Basement Membrane Y Y Y Dermis Y *

18 How Could IM Injection of a VLP Vaccine Prevent Mucosal Infection at the Cervix? *Transudated Abs in Mucus Y YY Y Y Y Y Y Y Y Y Cervical Mucus Y Y Y Y Cervical Epidermis Y Exudated Abs at Sites of Trauma Y Y Y Y Y Y Y Y Y Y Basement Membrane Y Y Y Dermis Y * VLP-specific IgG in cervical mucus ~10-fold lower than in the serum after IM vaccination Denise Nardelli et al. JNCI, 2003

19 Protection After Plateau of VLP Antibody Titers Suggests Long Term Protection Geometric Mean Titer (mmu/ml) Vaccination HPV 16 L1 VLP Vaccinees Placebo recipients who were HPV 16 Sero(+)/PCR( (+)/PCR(-)) at Day Month Since Enrollment Merck data, Mao et al. 2006

20 Partial Cross-Protection Against Incident Infection by Other High Risk HPV Types GSK HPV16/18 Vaccine ITT Analysis; ca. 500 per arm: 3.5 yr follow-up HPV Type # Vaccine # Placebo Efficacy (95%CI) (71-99) From Harper et al, Lancet 367: , (66-100) 94 (63-100) 54 (11-78) ( ) ( ) ( ) X X 52 X High-risk HPV types

21 PERCENTAGES OF CERVICAL CANCER CASES ATTRIBUTED TO THE MOST FREQUENT HPV TYPES IN ALL WORLD REGIONS HPV-16 HPV-18 X HPV-33 HPV-45 HPV-31 X HPV-58 X HPV-52 HPV-35 HPV-59 HPV-56 HPV-51 HPV-39 HPV-73 HPV-68 HPV-82 META-ANALYSIS OF 14,500 CASES CUMULATIVE % Estimates adapted from Smith JS, et al. Br J Cancer, Submitted. Vaccine, Vol 24 Supplement 3, Elsevier Limited. All rights reserved. Chapter 03, Figure 1

22 Will the Vaccines Influence Prevalent Infection? NCI/Costa Rican HPV16/18 VLP Vaccine Trial : Rate of HPV-16/18 DNA Clearance by Treatment Arm 60 Controls Arm (184/56) HPV Arm (155/34) % Clearance VE = 5.8% VE = -5.4% Mo (p=0.44) 12-Mo (p=0.82) Time Conclusion: Vaccination does not increase rate of clearance of HPV infection Thanks to Allan Hidesheim, NCI

23 T Cell Responses To L1 Unlikely to Induce Regression: Virion Protein Expression is Lost During Progression CIN 1 CIN 2 CIN 3 Decreasing Epithelial Differentiation Virus Producing Non-productive Non-productive Doorbar, J Clin Virol 32:7-15, 2005

24 Overall Efficacy in Preventing HPV Cervical Disease GSK HPV16/18 Vaccine ATP analysis; 3.5 Yr Follow-Up; ca. 500 per arm* HPV Types Endpoint # Vaccine # Placebo Efficacy (95%CI) 16/18 CIN (42-100) 16/18 CIN2/ ( ) Any Type CIN ( ) Any Type CIN2/ ( ) *Harper et al. Lancet 2006

25 Potential Impact of the Vaccines in the US HPV infection Spontaneous regression Subclinical HPV infection US Cases 10,000,000 Spontaneous regression ~30% HPV 16/18 Low-grade dysplasia 1,400,000 Pap screen- surgical treatment ~70% HPV 16/18 High-grade grade dysplasia Cancer 300,000 11,000 Many years (20+) *The number of US cases and percentages attributed to each disease category are estimates.

26 Managing Expectations If VLP vaccine protection is predominantly type specific: It will prevent most high-grade cervical dysplasias, and cervical cancer, but cancers rates will not decrease for many years It will not prevent many of the low-grade cervical abnormalities that appear soon after infection and are routinely detected in Pap screens Women, and healthcare providers, need to know that the vaccine may be working, even if many vaccinated women develop lowgrade Pap smear abnormalities or HPV DNA positivity

27 Conclusions: HPV VLP Vaccine Efficacy VLP vaccines are highly effective at protection against a spectrum of cervical HPV endpoints from incident infection to high grade precancer. A VLP vaccine is also highly protective against lesions of the external genitalia in women. Protection is type-restricted/type-specific, consistent with protection being antibody mediated. Duration of protection is unknown, but strong protection at 3.5 years, after antibody levels have reached a plateau, is very encouraging.

28 Regulatory Approval of HPV VLP Vaccines Merck s Gardasil approved in 2006 in the U.S., EU, Australia, Canada, Mexico, New Zealand, Brazil, and recently in Switzerland. GSK s Cervarix was filed with EU s EMEA in Mar. 06, decision expected within one year. U.S. filing expected before end of the year.

29 Gardasil Approval Specifics US FDA approved for 9-26 yr old females in June 06 European Union also approved for 9-26 yr old females Australia approved for:9-26 yr old females 9-15 yr old boys Approval in 9-15 yr old girls, and boys of all ages, is based on immunogenicity bridging study not efficacy

30 GARDASIL Phase III Adolescent Immunogenicity Bridging Study: Anti-HPV GMTs * at Month Anti-HPV 6 (HPV 6 mmu/ml) 0 Anti-HPV 11 (HPV 11 mmu/ml) Anti-HPV 16 (HPV 16 mmu/ml) 0 Anti-HPV 18 (HPV 18 mmu/ml) Females Years of Age Males Years of Age Females Years of Age *GMT = geometric mean titers Thanks to Eliav Barr, Merck for this slide

31 Who Should Be Vaccinated? In descending order of importance: year old girls - the ultimate target group since they have not yet been exposed to these sexually transmitted viruses Sexually active women - since many may not yet been exposed to at least one of the vaccine types; may reduce transmission Adolescent boys and men - the vaccines have yet not shown to prevent infection in males, only 10% of HPV cancer in men; may be small impact on herd immunity if coverage of women high

32 Outstanding Implementation Issues High Cost of the Vaccine: Gardasil is $120 per dose What ages to start vaccinating What age is too old for catch up vaccination Delivery of 3 IM doses to early adolescents Acceptance of an STI vs Cevical Cancer vaccine Effects of vaccine on Cervical Ca screening recommendations and compliance

33 We Can t Give Up Screening The vaccine won t help women with established infections/lesions. If type restricted, the current VLP vaccines could not prevent ~20-30% of cervical cancers. Need to convince vaccinated women to comply with existing screening programs.

34 A Shift to an HPV-Based Cervical Cancer Prevention Strategy HPV Precancer 15 y.o. 30 y.o. 45 y.o. Cancer Currently: Pap Smears/Cytology Future?: Vaccinate HPV DNA Test 1 HPV DNA Test 2 HPV DNA Test 3 Thanks to Mark Schiffman and Phil Castle, NCI

35 2nd Generation Prophylactic Vaccines Might Overcome Some of the Limitations of VLPs High cost of production Type restricted protection Three injections of adolescents

36 L2 Induces Cross-Neutralizing Antibodies Papillomavirus Particle Richard Roden (NCI, now JHU) 10 5 HPV 6 HPV 16 HPV Immunogen: L1/L2 VLPs Neutralization titier L1 L1 L1 L1 L2 L2 L Neutralization assay: Immunogen: L2 protein Neutralization titier HPV 6 HPV 16 HPV Neutralization assay:

37 Papillomaviruses Neutralized by BPV1 L2 peptide HPV16 HPV31 HPV6 HPV45 HPV18 HPV5 Neutralizing antibodies induced by BPV L peptide Neutralization Assay Antibody Titer* for BPV1 L2 peptide BPV1 HPV5 HPV16 HPV18 HPV31 HPV45 HPV6 CRPV CRPV BPV1 Diana Pastrana et al, Virology 337: , 2005

38 Conclusion: L2 Minor Vaccines L2 induces a broadly cross-neutralizing antibody response so possibility for a monovalent vaccine against cutaneous and mucosal types. Delivered as part of infant vaccination? Production in bacteria could make it relatively inexpensive. Disadvantage: Neutralizing titers lower than induced by VLPs. Working to increase titers and initiate early phase clinical trials, with JHU and Shanta Biotechnics.

39 Mucosal Vaccination with L1-Salmonella Live Vaccine: Murine Model* Anti-HPV16 VLP IgG in serum HPV16 PsV Neutralizing Titer Log 10 Titers Ty21a Kan 3 -L1S Log 10 Titers Weeks 0 Intranasal Delivery at 0 and 4 wks Ty21a is an attenuated and widely used typhoid vaccine strain * Codon Modified HPV16 L1 expressed inside the bacteria Baud et al., J. Virol. 78: , 2004, Denise Nardelli-Haefliger, unpublished

40 Live Bacterial Vector Expressing VLP Advantages: Inexpensive production and delivery if oral, perhaps fewer doses Potential Disadvantages: trials expensive w/ GM organisms, variability in response? Approach hasn t been tested clinically, but trials with Indian Immunologicals possible within the next year

41 Looking to the Future Both the Merck and GSK VLP vaccines will be approved for sales in many countries over the next two years Who to vaccinate, how to maximize uptake, and how to pay for the vaccination programs will dominate the agenda The valency of both vaccines will likely increase; but don t wait to vaccinate because they won t be therapeutic Several 2nd generation vaccines will likely be tested in clinical trials, but it will be difficult to equal the performance of the current vaccines

42 Other Interventions to Prevent Genital HPV Infection

43 Need for Anti-HPV Topical Microbicides In contrast to other STDs, studies indicate that condoms afford at best limited protection against sexual HPV transmission, at least under normal patterns of use. HPV VLP vaccines appear to be highly effective, but are type-restricted and expensive. Topically applied HPV inhibitors could serve as an adjunct to, or intermediate substitute for, VLP vaccination, particularly if they were inexpensive and had broad spectrum activity.

+ L2 helper plasmid (12 kb) SV40 Ori Transfect SV40 Ori 10 10

44 The Availability of HPV Pseudoviruses Permits Rapid Screening for Infection Inhibitors L1 L2 GFP Target (6-8 kb) GFP codon-modified L1 + L2 helper plasmid (12 kb) SV40 Ori Transfect SV40 Ori infectious units/ ml T Ag 293TT Buck (2004) JV 78:751 Buck (2005) JV 79:2839

45 Model Types Available foot warts cutaneous, mostly asymptomatic HPV16 mucosal, cancer- associated hand warts genital warts

46 heparin - Rationale for Examining Anionic Polysaccharides Heparan Sulfate Proteoglycans are primary attachment factors for papillomaviruses. Attachment is blocked by Heparin, a highly sulfated form of HS.

47 Carrageenan Is An Exceptionally Potent Infection Inhibitor IC 50 = 2 ng/ml (~10 pm) (!!)

48 Carrageenans Carrageenans are unbranched, sulfated galactan polysaccharides. Extracted from cell walls of red algae (seaweed) Widespread use as food thickeners

49 Susceptibility To Carrageenan Varies By Genus completely resistant HPV5 cutaneous less susceptible BPV1 CRPV less susceptible HPV31 HPV16 mucosal, cancer- associated HPV6&11 HPV18 HPV45 genital warts highly susceptible

Consumer Products Carrageenan is frequently

agent Found lubricants containing: carrageenan

50 Consumer Products Carrageenan is frequently used in commercial products Hypothesis: existing sexual lubricant products might already incorporate carrageenan as a thickening agent Found lubricants containing: carrageenan kelp extract carrageenan carrageenan no sulfated polysaccharides

51 Anti-HPV16 Activity of Sexual Lubricants Lubricant products that use carrageenan as a primary gelling agent behave like ~1% carrageenan * * fifth ingredient IC 50 = 5 million-fold dilution (!!)

52 Carrageenans As Microbicides Carrageenans are currently under investigation as topical microbicides against HIV and HSV. HPV16 is 1,000-fold more susceptible: IC 50 ng/ml vs mg/ml for HIV and HSV The Population Council (non-profit) is currently running a phase III clinical trial of Caraguard in South Africa. Focus of the trial is efficacy against HIV. An NCI sponsored trial for prevention of genital HPV infection in women is in the planning stage

53 Caveat Cells in a dish may not be the same as cells resident in the genital epithelium

54 Development of a murine model for the genital transmission of human papillomavirus Jeff Roberts Chris Buck, Cindy Thompson, Rhonda Kines, Marcelino Bernardo, Peter Choyke, Doug Lowy, John Schiller National Cancer Institute Bethesda, MD

55 HPV genital challenge model general protocol Day 1 Day 4 Day 7 Progesterone pretreatment: Depo-Provera 3mg SQ Physical or chemical disruption of genital tract mucosa challenge with pseudovirus (~10 7 IU s) Sacrifice mouse; dissect out genital tissues Histological analysis Section tissue with cryostat Identify reporter gene expression by confocal microscopy Quantitative analysis Fluorescence imaging of entire genital tract analyze signal indicating overall level of infection

56 HPV16 RFP PsV infects the murine genital epithelium vaginal lumen Blue = DAPI staining of nuclei Red = RFP expression indicating infection epithelium basement membrane stroma

57 Quantitation of Infection: Multispectral Imaging of the Murine Reproductive Tract Uterine horns cervix A. Spectral unmixing for RFP B. Conversion to grey scale; region of interest (ROI) drawn Area: ROI 3.3X10e7 C. ImageJ analysis vagina Mean signal per pixel: 8.1 -psv challenge Uterine horns cervix Area: ROI 3.5X10e7 Mean signal per pixel: 42.7 vagina +psv challenge

58 The Intact Cervicovaginal Mucosa Is Resistant to HPV Infection - PsV challenge: + + Pretreatment: cytobrush -

59 Nonoxynol 9 (N-9) Potentiates HPV16 Infection Nonoxynol 9 Pretreatment 6 hr Prior to virus challenge A detergent Solubilizes sperm cell membrane An over the counter spermacide containing 4% N-9 Disrubts cervicovaginal epithelium - PsV challenge: + + Pretreatment: cytobrush 3%CMC + 4%N-9 in 3%CMC + Conceptrol

60 It would be difficult to recommend discontinuing use of spermicides Unwanted pregnancy is more of a problem than HPV infection for most women Preferable to eliminate the HPV infection promoting activity of the spermacides

61 Carrageenan prevents HPV infection in vivo Pretreatment: <Intravaginal, 6hrs before challenge> Conceptrol 4% N-9 with CMC 4% N-9 with Carr PsV challenge: PsV inoculum premixed with: Carr = carrageenan CMC = carboxymethylcellulose CMC* Carr* Divine #8 Bioglide - - Mix Microbicide with Virus Mix Microbicide with N-9

62 Conclusions: Genital HPV Infection Potentiators/Inhibitors Use of spermacides containing Nonoxynol-9 may be an unappreciated risk factor gential HPV infection. Carrageenan is an attractive candidate for a safe, broad spectrum, and inexpensive genital HPV topical microbicide, but clinical trials are needed before this application can be promoted. Reformulation of N-9 containing spermacides in a carrageenan-based gel should be considered if above are demonstrated in future studies.

63 Key Collaborators National Cancer Institute Doug Lowy Past Members of the Lab: Reinhard Kirnbauer Richard Roden Diana Pastrana Present Members of the Lab Chris Buck Susana Pang Patricia Day Cindy Thompson Jeff Roberts Outside Clayton Harro: Johns Hopkins University- Baltimore Denise Nardelli: Universitaire Vaudois - Lausanne

64 Thanks For Your Attention

65 VLP and L2 Vaccines Prevent Infection in the Murine Challenge Model All mice challenged with HPV16-RFP PsV challenge: vaccine Polyoma VLP HPV16L1 VLP BPVL1 VLP BPVL peptide

HPV Life Cycle & Vaccination: Possible Relevance for HSV Vaccines

HPV Life Cycle & Vaccination: Possible Relevance for HSV Vaccines Douglas R. Lowy Center for Cancer Research, NCI Deputy Director, NCI National Institutes of Health Herpes Virus Infection & Immunity Meeting