Schiller Extending Duration

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Presented by Dr. John Schiller at the Alliance for Contraception in Cats & Dogs’ 4th International Symposium on Non-Surgical Contraceptive Methods of Pet Population Control, April 8-10, 2010, in Dallas, Texas, U.S.

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Slide 1: 

Contraceptive Vaccines Based on Virus-Like Particles: Lessons from HPV Vaccines John Schiller National Cancer Institute Bethesda MD USA

Slide 2: 

What are Virus-like Particles? VLPs are composed of viral coat proteins that, when over expressed, spontaneously self-assemble into particles that are morphologically similar to infectious virus. However, they do not contain viral nucleic acid and are, therefore, non-infectious. Commercial VLP-based vaccines: Hepatitis B Vaccine (HBV) FDA approved - 1986. Human Papillomavirus (HPV) FDA approved - 2006.

Slide 3: 

HBsAg Particles Are S Proteins Floating in a Yeast Lipid Membrane MaAleeer et al. Nature 1984; 307: 178-80

Slide 4: 

Gardasil (Merck) Cervarix (GlaxoSmithKline) 20-40 micrograms each VLP Three injections over 6 months HPV VLPs Are Non-Enveloped Icosohedrons Composed of the L1 Major Capsid Protein 70% of Cervix Cancer 90% of Genital Warts

Slide 5: 

VLPs Are Consistently Immunogenic In Humans Percent of Women Serocoverting to Individual HPV VLPs in Merck Quadravalent VLP Vaccine Gardasil* HPV6 99.8% HPV11 99.8% HPV16 99.8% HPV18 99.5% *4666 women vaccined 3 times by intramuscular injection

Slide 6: 

HPV VLP Induced Antibody Responses Are Durable in Humans Month: HPV16 VLP ELISA Natural Infection 0 7 12 18 25 33 39 45 51 57 63 69 75 Peak Plateau The GSK Vaccine HPV-007 Study Group Lancet 374:301-14, 2009 Antibody levels stable in years 2 to 6 post vaccination Critical Value Geometric Mean Titer (95% C.I.) 10 100 1000 10000 1

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VLPs are Effective At Preventing Disease Merck Quadravalent VLP Vaccine Gardasil Vaccine Placebo Efficacy Number Cases* Number Cases* CIN2/3# 8487 0 8460 53 100% Genital 7897 1 7899 91 98.9% Warts *Analysis limited to cases by the types targeted by the vaccine # CIN2/3 = moderate and high grade precancer cervical lesions

Slide 8: 

Why are VLPs So Immunogenic? Because they are particulate Because they display surface epitopes in a dense repetitive array

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Size Range of Pathogens and Particulate Delivery Systems VLP Jennings and Bachmann Curr Mol Med 2007

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Particulate Nature VLPs have a particle size that greatly facilitates uptake by dendritic cells and macrophages. Particles 20-200 nm diffuse freely to lymph nodes, particles 500-2000 nm don’t Hence, VLPs are targeted in vivo to relevant APC for optimal induction of immune responses.

Slide 11: 

B cells specifically recognize particulate antigens with epitope spacing of 50-100Å as foreign. This epitope spacing is commonly found on microbial surfaces, e.g. viruses or bacterial pili. Protein complexes with this spacing rarely occur in vertebrate animals. So BCRs have evolved as antigen specific pattern recognition receptors. The Bachmann-Zinkernagel Hypothesis Highly Repetitive Antigen Display Bachman et al. Science 1993; 262: 1448

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Streptavidin-TNF-a Fusion Protein (Tetramers) Truncated streptavidin (core) aa1 159 16 133 3 22 SA SA-TNF-a (3-22) Full length streptavidin TNF-a Biotinylated VLP TNF-a peptide Streptavidin (foreign) VLP 4 TNF / biotin X 1.5 Biotin/L1 X 5 L1 / pentamer X 72 Pentamer/VLP = 2160 TNF / VLP Virus-Like Display Preferentially Augments Antibody Responses To Self Antigens Express in bacteria, purify TNF-a conjugated VLP

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Vaccinated w/ Adjuvant SA-TNF-a None SA-TNF-a Freund’s VLP:SA-TNF-a None VLP:SA-TNF-a Freund’s <10 Breaking of B Cell Tolerance to Self Antigens By Virus-Like Display Mice (3-8 per group) were given a total of 3 doses of 15 µg SA-TNF-a conjugated to 5 µg VLPs biweekly. Ratio SA/TNF-a N/A 133 6 1.4 TNF-a SA Chackerian et al. 2001; J Clin Invest 108:415

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+ µg per injection VLP SA-TNF-a SA The Density of Self-antigen on VLP Surfaces Effects Autoantibody Responses TNF-a SA 1 2 0 2 2 2 6 2 8 10 2 18 1 2 0 + 9 0 18 100% 50% 10% 20% 100% 0% Spacing: 60Å 85Å 140Å 190Å 106 Chackerian et al, 2002; J Immunol 169:6120

Slide 15: 

Strong Signals Proliferation/Survival Fate of Auto-Reactive B Cells After Encountering Conjugated VLPs Weak Signals Peripheral Tolerance Oligomerization of BCR/Ag Signaling Complexes Monomeric BCR/ Self Ag Complexes

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same : 

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same Baker et al. Microbiol Mol Biol Rev 1999; 63:862

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same : 

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same Mammalian VLPs Avian VLPs

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same : 

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same Plant VLPs

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same : 

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same Phage VLPs

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same : 

Naked Icosahedral Virions Vary in Size, But the Distance Between Subunits Stays the Same Yeast VLPs

Criteria For Choosing a Virus-Like Display Platform : 

Criteria For Choosing a Virus-Like Display Platform Immunogenicity Potential Antigen Display Potential Production Potential Intellectual Property Issues

Immunogenicity Issues : 

Immunogenicity Issues Particulate: uptake by APCs for CMI - Common Feature High density display: BCR activation - Common Feature Rigid Conformation vs Freely Diffusible in Membrane? Naked Icosohedral VLPs vs HBs Ag particles/liposomes Usefulness of Exogenous Adjuvant? Encapsidatation of Nucleic Acid as TLR agonist? Are preexisting Abs to the platform an issue?

Slide 23: 

Virion Capsid Protein Target sequence Overexpression Approaches For Virus-Like Display Genetic Fusion Conjugate to Pre-formed Capsids Activated Biotin Biotinylated Target Ag Conjugated SA-Target + Wild-type SA Tetramer Biotin Binding Site Streptavidin Bridging Chemical Conjugation + Target Peptide Sulfo-MBS (cross-linker) Cys HBcAg Parvovirus Qbeta HPV VLPs

Slide 24: 

Antigen Display Potential Conjugation to Preformed VLPs: Flexible with regard to antigen More complex manufacturing Genetic fusion w/ major capsid protein Limited to short peptides Inserts are often incompatible with assembly Less complex manufacturing

Slide 25: 

A Partial List of VLPs Used as Vaccine Platforms VLP Type Linking Method Target Antigen Animal Viruses Hepadnavirus Core Genetic, Chemical malaria, flu, FMDV, HCV (HBV, WHV, DHV) hantavirus, others HPV Genetic, Chemical HIV, flu, HPV E2 and E7, P1A tumor Ag Parvovirus Genetic dengue virus, anthrax (PPV, B19) LCMV Mouse Polymavirus Genetic Her2/Neu Plant Viruses CPMV Genetic, Chemical HIV, rinovirus, MEV, Pseuomonas TMV Genetic, Chemical HPV L2, melanoma Ags Bacteriophage Q Chemical nicotine, Der p1 allergen LCMV MS2 Genetic HIV Yeast Ty particles Genetic HIV, flu B Chackerian Expert Rev Vaccines 2007; 6:381-90

Slide 26: 

Production Potential Efficient and low cost vaccine manufacture and delivery is important if wide spread distribution in low resource settings is a goal. Comparative studies of vaccine yields and stability are rarely conducted. Difficult to project industrial scale GMP yields based upon results in academic laboratories. Industry rarely reveals cost of vaccine production.

Advantages of VLP Vaccines : 

Advantages of VLP Vaccines Best subunit vaccine for Ab responses: Low dose Consistent seroconversion High titers Durable response No or Conventional Adjuvant Proven safety and effectiveness in humans

GnRH As an Antigen for a VLP Vaccine : 

Gonadotropin-Releasing Hormone Hypothalamus-pituitary-gonade axis GnRH action Release of FSH and LH Androstenone, testosterone and sperm production in male Follicle maturation and progesterone and oestrogen production in female GnRH As an Antigen for a VLP Vaccine

A Candidate GnRH VLP VaccineCytos Biotechnology : 

Principle of vaccine production VLP-Carrier Chemical Linker + + carrier Antigen antigen SMPH Succinimidyl-6[(β-maleimidopropionamido)hexanoate] Mr.: 379.36 CGGEHWSYGLRPG (GnRH with NH2-terminal cys linker) A Candidate GnRH VLP VaccineCytos Biotechnology Thanks to Gary Jennings, Cytos, for these slides

GnRH VLP Vaccine Cytos Biotechnology : 

Proof of principle experiments C57Bl/6 mice - male or female - 2 x 50μg Qb-GnRH (+/- Alum) sera collected periodically: aGNRH-titers and testosterone (EIA) determined immunized animals mated with healthy mice for fertility test d0 d14 d21 d28 d42 d56 d70 GnRH VLP Vaccine Cytos Biotechnology fertility

GnRH VLP Vaccine Cytos Biotechnology : 

aGNRH IgG induced in immunized mice - 2 doses male mice 50 ug Qb-CGG-GnRH +/- Alum d0 d28 d70 GnRH VLP Vaccine Cytos Biotechnology

GnRH VLP Vaccine Cytos Biotechnology : 

Reduced serum testosterone in GnRH-VLP immunized mice GnRH VLP Vaccine Cytos Biotechnology

GnRH VLP Vaccine Cytos Biotechnology : 

GnRH VLP Vaccine Cytos Biotechnology Reduced fertility in immunized male and female mice Female fertility 100% reduced, Male fertility 70% reduced Fertility correlates with low a-GnRH titer Unfortunately many animals regained fertility by 160 days!

Will A GnRH VLP Vaccine Workin Cats and Dogs? : 

Will A GnRH VLP Vaccine Workin Cats and Dogs? There are no assurances at this point. I wouldn’t give up on the idea of an effective immuno-contraceptive until VLP-based vaccines have been evaluated in fertility trials. Possible improvements: Stronger adjuvants Slow or delayed release formulations Don’t throw out the good in pursuit of the ideal!

The Big Question: Will Plateau AntibodyLevels Be Above or Below That Needed For Contraception? : 

The Big Question: Will Plateau AntibodyLevels Be Above or Below That Needed For Contraception? Ab Conc. Years Protective Level Good Vaccine Poor Vaccine

Will A GnRH VLP Vaccine Workin Cats and Dogs? : 

Will A GnRH VLP Vaccine Workin Cats and Dogs? There are no assurances at this point. I wouldn’t give up on the idea of an effective immuno-contraceptive until VLP-based vaccines have been evaluated in fertility trials. Possible improvements: Stronger adjuvants Slow or delayed release formulations Don’t throw out the good in pursuit of the ideal!

Thanks For Your Attention : 

Thanks For Your Attention

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