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  • A Recombinant Virus Like Particle (VLP) Vaccine for Influenza

    Rahul SinghviPresident & CEO

    Novavax, Inc. , Rockville MD

    WCBP, 2010January 26, 2010

  • Recombinant Virus-Like Particle (VLP) Vaccines

    Non-enveloped Hepatitis B vaccines (recombinant)- Recombivax HB (Merck)- Engerix B (GSK)

    Human Papilloma Virus Vaccines (recombinant)- Gardasil (Merck)- Cervarix (GSK)

    - Made in insect (Lepidoptera) cells- Recently licensed in the U.S.

    EnvelopedSeasonal and Pandemic Influenza- Novavax HA-NA-M1 VLPs

  • Recombinant Influenza VLPs:Pleomorphic Spherical Particles

    HA and NASpikes

    Lipid bilayer

    M1 helicalmatrix

    120nm

  • Potential Immunologic Advantages of Novavax VLP Influenza Vaccine Candidates

    Particulate nature and repeated display of HA protein on VLP surface

    Processed through the MHC-I and MHC-II pathways

    Generate antibody and cell-mediated immune responses to tomultiple influenza proteins leading to enhanced efficacy

  • Select proteins important for inducing virus neutralizing antibody and CMI Surface hemagglutinin (HA) Neuraminidase (NA) Matrix (M1)

    Development of Novavax VLPs

    Proteins (HA, NA, M1)spontaneously form

    VLPs

    Genes coding forthe HA, NA, and Mproteins are put into baculovirus

    rBaculovirus

    Infect cell culture(Sf9) with baculovirus

    Baculovirus-infected Sf9 Cells

  • Manufacturing Process Influenza VLPs

    Sf9 cells: VLPs secreted 100L Wave bioreactor

    Ultrafiltration500K

    AnionExchange

    BVInactivation

    0.2 filtration FillingSEC

    Cross flow0.65 micron

    TFF Filtration

    Baculovirus HA/NA/M1infect Sf9 cells

    Harvest day 3ConcentrationBV/NA removal

    FinalPurification

  • 7

    VLP Flu Vaccines are Safe & Immunogenic

    Vaccine Candidate

    Phase I Phase II Phase III Approval

    H1N1

    (2009 pandemic) Not required

    Two part pivotal trial (n=4,000)

    Part 1: 1000 subjects, safe & immunogenic

    Part 2: Enrollment ongoing

    Approval / launch (Mexico) in 2010

    Seasonal (trivalent)

    Two Phase II studies (n = 571)

    Well tolerated and immunogenic

    VLPs for 6 flu strains tested

    Head to head study ongoing in elderly

    Upcoming in 2010

    H5N1

    (Indonesia) Phase I/II study (n =230)

    Well tolerated and immunogenic

  • HA and NA Composition of Influenza VLP Vaccines in Human Clinical Trials

    Antigen Composition (mcg)

    Trivalent VLP Monovalent VLP

    2005 2006

    15 mcg

    dose

    HA

    NA

    2008 2009

    2009 2010 H5N1 2009 H1N1

    45 45 45 15

    5.4

    15

    11.8 13.6 12.2 3.0

    HANA

    M1

    A/California/04/09 (H1N1) VLPs

    SDS-PAGE HA Blot

  • SRID HA Potency Assay

    SRID is the only assay currently approved by FDA for testing and standardization of hemagglutinin (HA) antigen concentration of influenza virus vaccines

    Single Radial Immunodiffusion (SRID) calculates relative potency to qualified standard reference and carried out by comparing dilution series of test sample to a dilution series of the reference.

    SRID measures antigenically active material and specificity of antiserum used is critical for accurate results.

  • Source of Reagents For SRID HA Potency Assay

    Reagent source

    CBER/NIBSC Novavax

    Antigen used for immunization

    Bromelian cleaved HA from flu virus grown in eggs

    Recombinant HA purified from BV infected insect cells

    Antiserum sheep sheep

    Reference antigen Whole inactivated virus grown in eggs

    Viral like particle purified from BV infected insect cells

  • SRID NVAX and CBER Reagents are Exchangeable when Testing Matched Influenza Strains

    AB/Antigen AC BVS Average % Bias vs NVAX

    CBER/CBER 124.2 130.3 127.3 5.25NVAX/NVAX 122.3 119.5 120.9

    CBER/CBER 120.1 104 112.1 -13.87NVAX/NVAX 134.8 125.3 130.1

    CBER/CBER 240.2 195.7 218.0 73.11NVAX/NVAX 123.9 127.8 125.9CBER/NVAX 113.6 115 114.3 -9.21

    H3N2 Brisbane

    SRID values 2008/2009 NVAX VLP's 75508013-1 120g HA/ml

    H1N1

    B/Florida

    AB/Antigen AC BVS Average % Bias vs CBER

    CBER/CBER 39.6 39.4 39.5NVAX/NVAX 31.3 32 31.7 -19.87

    CBER/CBER 28.2 30.6 29.4NVAX/NVAX 34.3 37 35.7 21.26

    CBER/CBER 41.2 44.2 42.7NVAX/NVAX 70.8 62.2 66.5 55.74NVAX/CBER 41.8 31 36.4 -14.75

    H3N2 Uruguay

    SRID values 2008/2009 Fluzone

    H1N1

    B/Florida

  • VLP Can be Used Instead of Virus to Measure HA Antibody in HAI

    0

    2

    4

    6

    8

    10

    12

    Serum 1 Serum 2 Serum 3 Serum 4

    Rabbit Tox Serum

    HA

    Itite

    r(lo

    g2)

    HAI using H1N1 VirusHAI using H1N1 VLP

    0

    2

    4

    6

    8

    10

    12

    14

    Serum 1 Serum 2 Serum 3 Serum 4

    Rabbit Tox Serum

    HA

    ITite

    r(lo

    g2)

    HAI using H3N2 VirusHAI using H3N2 VLP

    1% turkey RBC,

    cell-derived virus.

  • VLP Associated NA Attributes are Similar to NA in Influenza Virus

    Enzymatic Properties Vmax (maximal velocity) and Km (substrate concentration required to achieve maximal velocity) for VLP and viral NA are similar

    VLP NA is inhibited by Oseltamivir Phosphate (Tamiflu) at a concentration similar to the corresponding virus

    VLP can be formulated to maintain stable NA activity for at least 6 months

  • Human Sera with NA Inhibiting Antibody give Similar Titers Against VLP and Virus NA

    Correlation between NA inhibition (Log2NIT25) in B/FL VLP and live virus for pre- and post immunized serums with

    15-60 mcg HA of VLP vaccine

    y = 1.0057x + 1.9717R2 = 0.8407

    0

    2

    4

    6

    8

    10

    12

    14

    0 2 4 6 8 10 12

    Log2NIT in B/FL VLP

    Log2

    NIT

    in B

    /FL

    live

    viru

    s

  • VLP Characterization

    Carbohydrate AnalysisIs there an Immune Response to Carbohydrate?

    Sf9 and Baculovirus ProteinsIs there a clinical Immune response

    Fatty Acids and Lipids

    Aggregation

  • Carbohydrate Analysis 2008-2009 Trivalent VLP Vaccine

    gF Map gA Map

    Oligosaccharides

    Consistent with the presence of truncated complex type and/or high Mannose structures expected from insect cells

    Performed under contract by MScan, Inc.

    Possible Oligosaccharide Assignment

    Hex5

    Hex3HexNAc2

    Hex6

    Hex3HexNAc2DeoxyHex1

    Hex3HexNAc3

    Hex7

    Hex5HexNAc2

    Hex3HexNAc3DeoxyHex1

    Hex8

    Hex5HexNAc2

    Hex3HexNAc4DeoxyHex1

    Hex9

    Hex7HexNAc2

    Hex8HexNAc2

    Hex9HexNAc2

    Possible Oligosaccharide Assignment

    Hex3HexNAc2

    Hex3HexNAc2DeoxyHex1

    Hex3HexNAc3

    Hex5HexNAc2

    Hex6HexNAc2

    Hex7HexNAc2

    Hex8HexNAc2

    Hex9HexNAc2

  • Potential Insect Cell Glycoallergens

    Alpha 1 3 fucose Plants and some insects

    glycoproteins Very low or absent in Sf9

    (S. frugiperda) cells

    Galactose-alpha-1,3-galactose Food allergen Significant levels High5 (T. ni) cells Very low level in Sf9 cells

    No Evidence of potential glycoallergensalpha 1,3 fucose and alpha 1,3 galactose linkages in H5N1, 2005-2006 or 2008-2009 VLPs

  • Are the Sf9 Derived Carbohydrate Structures Immunogenic?

  • Baculovirus ELISAA VLP Release Assay

    Rabbit immunized with purified baculovirus to produce polyclonal antisera

    IgG isolated used to coat microtiter plate

    Baculovirus proteins captured

    Biotin conjugated rabbit anti-baculovirus IgG used to detect bound protein

    If the antibody reacts with the carbohydrate moieties on the baculovirus proteins the assay will overestimate the amount of baculovirus protein in samples

  • Experimental Approach

    Use Carbo Release Kit from QA-bio to remove carbohydrate from baculovirus proteins

    Removes N-linked using PNGaseF

    Removes o-linked using O-Glycosidase, Sialidase, B-Galactosidase, Hexaminidase

    Bovine Fetuin control 24h without any denaturation

    Test reactivity of baculovirus antiserum with baculovirusproteins before and after deglycosylation by SDS-PAGE and Western Blot

    Use Baculovirus ELISA to determine concentration of baculovirus proteins before and after deglycosylation

  • 24 Hr Treatment

    Lane123456

    SampleMolecular Weight

    BlankBV IEX (Treated)

    BlankBV IEX (Untreated)

    Blank

    24 Hr BV IEX Degly 10Mar09

    Lane Sample name1 Molecular Weight2 Blank3 Fetuin (TR)4 Blank5 Fetuin (UT)6 Blank7 Blank8 Blank9 BV IEX (TR)10 Blank11 BV IEX (UT)12 Blank

    T U T U

  • ELISA

    Dilution 1, gBV/ml

    Dilution 2, gBV/ml

    Average, gBV/ml %CV

    BV IEX 24 hr Deglycosylation 50.1 50.9 50.5 1.12

    BV IEX Untreated 64.7 51.0 57.9 16.75

    24h Deglycosylation had no effect on antibody binding to BV antigen

    Antibody does not react with carbohydrate

  • Are the Protein Impurities Immunogenic?

  • LC/Mass Spec Analysis 0f Proteins In B/Florida/4/06 VLPs

    HA0gp64 BVNAM1 dimer/tubulin

    p39 BV capsid

    M1

    Influenzatarget HA, NAM1

    Baculovirusgp64 envelopep39 capsidubiquitinminor structural proteins

    Sf9 host proteinsalpha-actin and tubulinHSP 70 (chaperon)several housekeeping proteins

    Performed under contract by John Hopkins University

  • Lack of Response to BV Proteins in Humans Vaccinated with VLP

    ( Tested by ELISA against 2 ug/mL of BV protein )t test

    Group GMT pre SD SE GMT post SD SE Ratio P

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