Hana Golding, Ph.D. Office of Vaccines Research and Review/CBER/FDA

1. Vaccines and Related Biological Products Advisory Committee MeetingNovember 14, 2012Vaccine Adjuvants & Mode of Action for GSK’s AS03 Adjuvanted Influenza A (H5N1) Virus Monovalent Vaccine Hana Golding, Ph.D. Office of Vaccines Research and Review/CBER/FDA

2. Vaccine Adjuvants Adjuvant – a substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used as part of vaccine formulation. Most adjuvants activate early innate immunity.

3. Immature dendritic cell Digests antigen from pathogen and displays pieces of it on cell surface Mature dendritic cell Interacts with T- and B- cells to launch a specific, adaptive immune response to pathogen Microbial products, cell-free genetic material from pathogens Antigen Lymph T cell, B cells Cytokine & Antibody production Peripheral sites (nose, muscle, skin, etc.) Lymphnode 1- Antigen uptake and Activation 2- Migration 3- Antigen Presentation to T cells and B cells. Most Adjuvants work at early time points of IR

4. Slide 3:Immune responses to vaccines: role for Dendritic cell maturation into antigen presenting cells (APC) • Vaccines are often delivered to the immune system in peripheral sites (such as muscle, skin or nose). • The vaccine antigen are picked up by immature dendritic cells (DC), which internalize and process it. DC are capable of expressing the “processed” antigen in the form of short peptides on their surface in close association with HLA molecules. • In order to activate pathogen-specific immune cells, the Ag-loaded DC must migrate from the site of vaccination to the local lymph nodes. • The immature DC must also undergo maturation, which include upregulation of surface HLA and co-stimulatory molecules, and secretion of soluble mediators (cytokines). They help to stimulate T and B cells in the lymph nodes that undergo maturation into effector cells. Such effector cells can fight pathogens and protect from disease. • Adjuvants act to increase the early stages of antigen uptake and DC maturation and migration to lymph nodes.

5. Dendritic cell Bacterial cell wall DNA genetic material virus, bacteria RNA geneticmaterial viruses Bacterial flagella (Flagellin protein) Some Adjuvants can activate dendritic cells by mimicking components of microbes that bind to Pattern Recognition Receptors (PRR) on APC (TLRs,NLRs,RLRs)

6. Slide 5: Dendritic cell express receptors that can sense different components of invading pathogens Pattern Recognition Receptors (PRR) on dendritic cells (such as TLRs, NLRs, RLRs) evolved to sense different components of inavding pathogens not found on self (host) cells: • Outer walls of gram negative or gram positive bacteria • Flagella used by bacteria for motility • Unique sequences in the genomic nucleic acids of bacteria and viruses Activation of these PRR result in DC maturation, migration to lymph nodes, and more efficient activation of antigen-specific B and T cells that can fight invading pathogens Adjuvants are designed to mimic the natural ligands of the PRR in dendritic cells and provide the signaling required for DC maturation into more effective antigen presenting Cells (APC) in the lymph nodes.

7. Current Regulatory State of Adjuvants:Adjuvant mode of action (MOA) is important but not always fully understood before licensure of a vaccine containing adjuvant.

8. Potential Benefits/Risks of Vaccine Adjuvants Benefits Risks Increase reactogenicity Local Systemic effects, e.g., inflammation, fever, myalgia Nonspecific immune activation Immune mediated diseases Organ specific Inflammatory diseases • Enhance / accelerate the immune response • Prolong the immune response • Focus the immune response (CMI vs. Ab, Th1/Th2) • Diversify the immune response • Increase antibody affinity • Improve long term memory • Special Patient Populations • Dose sparing

9. Oil-in-water Emulsion-Based adjuvant

10. AS03 Mode of Action (conducted by GSK) • No evidence for direct interaction between H5N1 antigen and AS03 adjuvant. Adjuvant and Ag need to be administered at the same site. • MOA studies in mice: AS03 modulates the local innate responses in the site of injection (muscle) and draining lymph nodes: - recruitment of monocytes, DC, and neutrophils to the muscle (transient chemokine/cytokine production) - enhanced antigen loading by APC (CD11b+, CD11c+); increase of co-stimulatory molecules (CD80, CD86, CD40) - NFkB activation in the muscle and draining LN but not in non- draining LN or liver. Innate responses were transient (24-48 hr). - antigen specific T cells: mixed TH1/Th2 cytokines - enhanced antibody responses (IgG1 > IgG2) - evidence for a-tocopherol contribution to immune response enhancement against Vaccine antigen • Strong evidence for dose sparing with AS03/H5N1 (Q-PAN)

11. AS03/H5N1 Vaccine in Ferret Challenge Model (conducted by ViroClinics) Protection in ferrets vaccinated with Q-PAN H5N1 vaccine against: • Homologous : A/Indonesia vaccine (clade 2.1) (N=40) • Heterologous :H5N1 A/Hong Kong/156/97 (clade 0) (N=48) • Heterologous :H5N1 A/Vietnam/1194/04 (clade 1) (N=48) All animals were evaluated: Lethality, fever, body weight, gross pathology (lungs), virus loads in URT / LRT (PCR and PFU in MDCK), virus shedding Immune responses: HI and neutralization titers against homologous/heterologous H5N1 strains

12. AS03/H5N1 Vaccine in Ferret Challenge Model (ViroClinics): Results • Animals vaccinated (x2) with the Q-PAN H5N1 vaccine (human dose) were protected from lethality following challenge with either homologous (A/Indonesia, clade 2.1) or heterologous (clade, 0, clade 1) strains. • Protected animals had much reduced lung pathology (both macroscopic and microscopic findings). • Vaccinated animals had lower viral loads in the nasal washes and the lungs: Virus replication in MDCK cells correlated with disease pathology and inversely correlated with vaccine dose (PCR data less correlative). • Immunogenicty data were supportive of the challenge outcome.

13. SummaryPreclinical studies with H5N1/AS03 Vaccine • AS03 Mode of Action at the cellular/receptor level is not fully understood. • Preclinical studies in mice (and human cells) demonstrated enhanced antigen uptake, APC function, and local cytokine release (IL-6, MCP-1,IL-1b,MIP-1a, RANTES, IL-8) • Innate immune activation was demonstrated to be transient and mostly localized to site of injection and draining lymph nodes. • Preclinical studies of AS03 in combination with TIV or H5N1 MIV demonstrated significant augmentation of HI titers and dose sparing. • Ferrets vaccinated with the Q-PAN H5N1 vaccine were protected from lethality following challenge with either homologous or heterologous H5N1 virus strains. Significant reduction in viral loads in URT/LRT and lung pathology • Preclinical studies supported the intended human dose of Q-PAN: 3.8 mg HA /dose + AS03 in 0.5 ml.