A Recombinant Virus Like Particle (VLP) Vaccine for Influenza ?· A Recombinant Virus Like Particle…

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<ul><li><p>A Recombinant Virus Like Particle (VLP) Vaccine for Influenza</p><p>Rahul SinghviPresident &amp; CEO </p><p>Novavax, Inc. , Rockville MD</p><p>WCBP, 2010January 26, 2010</p></li><li><p>Recombinant Virus-Like Particle (VLP) Vaccines</p><p>Non-enveloped Hepatitis B vaccines (recombinant)- Recombivax HB (Merck)- Engerix B (GSK)</p><p>Human Papilloma Virus Vaccines (recombinant)- Gardasil (Merck)- Cervarix (GSK)</p><p>- Made in insect (Lepidoptera) cells- Recently licensed in the U.S.</p><p>EnvelopedSeasonal and Pandemic Influenza- Novavax HA-NA-M1 VLPs</p></li><li><p>Recombinant Influenza VLPs:Pleomorphic Spherical Particles</p><p>HA and NASpikes</p><p>Lipid bilayer</p><p>M1 helicalmatrix</p><p>120nm</p></li><li><p>Potential Immunologic Advantages of Novavax VLP Influenza Vaccine Candidates</p><p> Particulate nature and repeated display of HA protein on VLP surface</p><p> Processed through the MHC-I and MHC-II pathways</p><p> Generate antibody and cell-mediated immune responses to tomultiple influenza proteins leading to enhanced efficacy</p></li><li><p> Select proteins important for inducing virus neutralizing antibody and CMI Surface hemagglutinin (HA) Neuraminidase (NA) Matrix (M1)</p><p>Development of Novavax VLPs</p><p>Proteins (HA, NA, M1)spontaneously form</p><p>VLPs</p><p>Genes coding forthe HA, NA, and Mproteins are put into baculovirus</p><p>rBaculovirus </p><p>Infect cell culture(Sf9) with baculovirus</p><p>Baculovirus-infected Sf9 Cells</p></li><li><p>Manufacturing Process Influenza VLPs</p><p>Sf9 cells: VLPs secreted 100L Wave bioreactor</p><p>Ultrafiltration500K</p><p>AnionExchange</p><p>BVInactivation</p><p>0.2 filtration FillingSEC</p><p>Cross flow0.65 micron</p><p>TFF Filtration</p><p>Baculovirus HA/NA/M1infect Sf9 cells</p><p>Harvest day 3ConcentrationBV/NA removal</p><p>FinalPurification</p></li><li><p>7</p><p>VLP Flu Vaccines are Safe &amp; Immunogenic</p><p>Vaccine Candidate</p><p>Phase I Phase II Phase III Approval</p><p>H1N1</p><p>(2009 pandemic) Not required</p><p> Two part pivotal trial (n=4,000) </p><p> Part 1: 1000 subjects, safe &amp; immunogenic</p><p> Part 2: Enrollment ongoing</p><p>Approval / launch (Mexico) in 2010</p><p>Seasonal (trivalent)</p><p> Two Phase II studies (n = 571) </p><p> Well tolerated and immunogenic </p><p> VLPs for 6 flu strains tested</p><p> Head to head study ongoing in elderly</p><p> Upcoming in 2010</p><p>H5N1</p><p>(Indonesia) Phase I/II study (n =230)</p><p> Well tolerated and immunogenic</p></li><li><p>HA and NA Composition of Influenza VLP Vaccines in Human Clinical Trials</p><p>Antigen Composition (mcg)</p><p>Trivalent VLP Monovalent VLP </p><p>2005 2006 </p><p>15 mcg</p><p>dose</p><p>HA</p><p>NA</p><p>2008 2009</p><p>2009 2010 H5N1 2009 H1N1</p><p>45 45 45 15</p><p>5.4</p><p>15</p><p>11.8 13.6 12.2 3.0</p><p>HANA</p><p>M1</p><p>A/California/04/09 (H1N1) VLPs</p><p>SDS-PAGE HA Blot</p></li><li><p>SRID HA Potency Assay</p><p>SRID is the only assay currently approved by FDA for testing and standardization of hemagglutinin (HA) antigen concentration of influenza virus vaccines</p><p>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.</p><p>SRID measures antigenically active material and specificity of antiserum used is critical for accurate results.</p></li><li><p>Source of Reagents For SRID HA Potency Assay</p><p>Reagent source</p><p>CBER/NIBSC Novavax</p><p>Antigen used for immunization</p><p>Bromelian cleaved HA from flu virus grown in eggs</p><p>Recombinant HA purified from BV infected insect cells</p><p>Antiserum sheep sheep</p><p>Reference antigen Whole inactivated virus grown in eggs</p><p>Viral like particle purified from BV infected insect cells</p></li><li><p>SRID NVAX and CBER Reagents are Exchangeable when Testing Matched Influenza Strains</p><p>AB/Antigen AC BVS Average % Bias vs NVAX</p><p>CBER/CBER 124.2 130.3 127.3 5.25NVAX/NVAX 122.3 119.5 120.9</p><p>CBER/CBER 120.1 104 112.1 -13.87NVAX/NVAX 134.8 125.3 130.1</p><p>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</p><p>H3N2 Brisbane</p><p>SRID values 2008/2009 NVAX VLP's 75508013-1 120g HA/ml</p><p>H1N1</p><p>B/Florida</p><p>AB/Antigen AC BVS Average % Bias vs CBER</p><p>CBER/CBER 39.6 39.4 39.5NVAX/NVAX 31.3 32 31.7 -19.87</p><p>CBER/CBER 28.2 30.6 29.4NVAX/NVAX 34.3 37 35.7 21.26</p><p>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</p><p>H3N2 Uruguay</p><p>SRID values 2008/2009 Fluzone </p><p>H1N1</p><p>B/Florida</p></li><li><p>VLP Can be Used Instead of Virus to Measure HA Antibody in HAI</p><p>0</p><p>2</p><p>4</p><p>6</p><p>8</p><p>10</p><p>12</p><p>Serum 1 Serum 2 Serum 3 Serum 4</p><p>Rabbit Tox Serum</p><p>HA</p><p>Itite</p><p>r(lo</p><p>g2)</p><p>HAI using H1N1 VirusHAI using H1N1 VLP</p><p>0</p><p>2</p><p>4</p><p>6</p><p>8</p><p>10</p><p>12</p><p>14</p><p>Serum 1 Serum 2 Serum 3 Serum 4</p><p>Rabbit Tox Serum</p><p>HA</p><p>ITite</p><p>r(lo</p><p>g2)</p><p>HAI using H3N2 VirusHAI using H3N2 VLP</p><p>1% turkey RBC, </p><p>cell-derived virus.</p></li><li><p>VLP Associated NA Attributes are Similar to NA in Influenza Virus</p><p> Enzymatic Properties Vmax (maximal velocity) and Km (substrate concentration required to achieve maximal velocity) for VLP and viral NA are similar</p><p> VLP NA is inhibited by Oseltamivir Phosphate (Tamiflu) at a concentration similar to the corresponding virus</p><p> VLP can be formulated to maintain stable NA activity for at least 6 months</p></li><li><p>Human Sera with NA Inhibiting Antibody give Similar Titers Against VLP and Virus NA</p><p>Correlation between NA inhibition (Log2NIT25) in B/FL VLP and live virus for pre- and post immunized serums with </p><p>15-60 mcg HA of VLP vaccine</p><p>y = 1.0057x + 1.9717R2 = 0.8407</p><p>0</p><p>2</p><p>4</p><p>6</p><p>8</p><p>10</p><p>12</p><p>14</p><p>0 2 4 6 8 10 12</p><p>Log2NIT in B/FL VLP</p><p>Log2</p><p>NIT</p><p> in B</p><p>/FL </p><p>live </p><p>viru</p><p>s</p></li><li><p>VLP Characterization</p><p> Carbohydrate AnalysisIs there an Immune Response to Carbohydrate?</p><p> Sf9 and Baculovirus ProteinsIs there a clinical Immune response</p><p> Fatty Acids and Lipids</p><p> Aggregation</p></li><li><p>Carbohydrate Analysis 2008-2009 Trivalent VLP Vaccine</p><p>gF Map gA Map</p><p>Oligosaccharides</p><p>Consistent with the presence of truncated complex type and/or high Mannose structures expected from insect cells</p><p>Performed under contract by MScan, Inc.</p><p>Possible Oligosaccharide Assignment</p><p>Hex5</p><p>Hex3HexNAc2</p><p>Hex6</p><p>Hex3HexNAc2DeoxyHex1</p><p>Hex3HexNAc3</p><p>Hex7</p><p>Hex5HexNAc2</p><p>Hex3HexNAc3DeoxyHex1</p><p>Hex8</p><p>Hex5HexNAc2</p><p>Hex3HexNAc4DeoxyHex1</p><p>Hex9</p><p>Hex7HexNAc2</p><p>Hex8HexNAc2</p><p>Hex9HexNAc2</p><p>Possible Oligosaccharide Assignment</p><p>Hex3HexNAc2</p><p>Hex3HexNAc2DeoxyHex1</p><p>Hex3HexNAc3</p><p>Hex5HexNAc2</p><p>Hex6HexNAc2</p><p>Hex7HexNAc2</p><p>Hex8HexNAc2</p><p>Hex9HexNAc2</p></li><li><p>Potential Insect Cell Glycoallergens</p><p>Alpha 1 3 fucose Plants and some insects </p><p>glycoproteins Very low or absent in Sf9 </p><p>(S. frugiperda) cells</p><p>Galactose-alpha-1,3-galactose Food allergen Significant levels High5 (T. ni) cells Very low level in Sf9 cells</p><p>No Evidence of potential glycoallergensalpha 1,3 fucose and alpha 1,3 galactose linkages in H5N1, 2005-2006 or 2008-2009 VLPs</p></li><li><p>Are the Sf9 Derived Carbohydrate Structures Immunogenic?</p></li><li><p>Baculovirus ELISAA VLP Release Assay</p><p> Rabbit immunized with purified baculovirus to produce polyclonal antisera</p><p> IgG isolated used to coat microtiter plate</p><p> Baculovirus proteins captured</p><p> Biotin conjugated rabbit anti-baculovirus IgG used to detect bound protein</p><p> If the antibody reacts with the carbohydrate moieties on the baculovirus proteins the assay will overestimate the amount of baculovirus protein in samples</p></li><li><p>Experimental Approach</p><p> Use Carbo Release Kit from QA-bio to remove carbohydrate from baculovirus proteins</p><p>Removes N-linked using PNGaseF</p><p>Removes o-linked using O-Glycosidase, Sialidase, B-Galactosidase, Hexaminidase</p><p>Bovine Fetuin control 24h without any denaturation</p><p> Test reactivity of baculovirus antiserum with baculovirusproteins before and after deglycosylation by SDS-PAGE and Western Blot</p><p> Use Baculovirus ELISA to determine concentration of baculovirus proteins before and after deglycosylation</p></li><li><p>24 Hr Treatment</p><p>Lane123456</p><p>SampleMolecular Weight</p><p>BlankBV IEX (Treated)</p><p>BlankBV IEX (Untreated)</p><p>Blank</p><p>24 Hr BV IEX Degly 10Mar09</p><p>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</p><p>T U T U</p></li><li><p>ELISA</p><p>Dilution 1, gBV/ml</p><p>Dilution 2, gBV/ml</p><p>Average, gBV/ml %CV</p><p>BV IEX 24 hr Deglycosylation 50.1 50.9 50.5 1.12</p><p>BV IEX Untreated 64.7 51.0 57.9 16.75</p><p>24h Deglycosylation had no effect on antibody binding to BV antigen</p><p>Antibody does not react with carbohydrate</p></li><li><p>Are the Protein Impurities Immunogenic?</p></li><li><p>LC/Mass Spec Analysis 0f Proteins In B/Florida/4/06 VLPs</p><p>HA0gp64 BVNAM1 dimer/tubulin</p><p>p39 BV capsid</p><p>M1</p><p>Influenzatarget HA, NAM1</p><p>Baculovirusgp64 envelopep39 capsidubiquitinminor structural proteins</p><p>Sf9 host proteinsalpha-actin and tubulinHSP 70 (chaperon)several housekeeping proteins</p><p>Performed under contract by John Hopkins University</p></li><li><p>Lack of Response to BV Proteins in Humans Vaccinated with VLP</p><p>( Tested by ELISA against 2 ug/mL of BV protein )t test</p><p>Group GMT pre SD SE GMT post SD SE Ratio P value </p><p>Placebo 60.95 1.52 1.13 61.85 1.55 1.14 1.01 0.9261N = 12</p><p>15 mg 63.62 1.23 1.06 65.69 1.26 1.07 1.03 0.6737N = 1260 mgN = 12 68.38 1.23 1.06 81.14 1.31 1.08 1.19 0.0950</p></li><li><p>Lack of Response to Sf9 Proteins in Humans Vaccinated with VLP</p><p>( Tested by ELISA against 2 ug/mL of SF9 protein ) t testGroup GMT pre SD SE GMT post SD SE Ratio P value</p><p>Placebo 103.28 3.11 1.39 108.95 3.51 1.44 1.05 0.8588N = 12</p><p>15 mg 185.12 2.05 1.23 340.31 2.36 1.28 1.84 0.0877N = 1260 mgN = 12 104.05 1.84 1.19 173.74 1.91 1.21 1.67 0.0709</p></li><li><p>Fatty Acid and Lipid AnalysisInitial Results</p><p> 80% of fatty acids belong to 4 classesC16.0 Palmitate, C16.1 Palmitoleate, C18.0 Stearate, C18.1 n9 Oleate</p><p> Seasonal and H5n1 pandemic VLPs differ in their total percentage of saturated vs unsaturated fatty acids </p><p>Seasonal longer chainsHigher saturated/unsaturated ratio in seasonal suggesting greater lateral segregation</p><p> VLP fatty acid lower content of saturated fatty acids than baculovirus and may bud from different membrane regions</p><p> Seasonal VLP 4-fold higher content choline lipids than pandemic VLP</p></li><li><p>Fatty Acid Composition </p><p>05</p><p>10152025303540</p><p>C14:</p><p>0 MYR</p><p>ISTA</p><p>TE</p><p>C16:</p><p>0 PAL</p><p>MIT</p><p>ATE</p><p>C16:</p><p>1 PAL</p><p>MIT</p><p>OLE</p><p>ATE</p><p>C18:</p><p>0 STE</p><p>ARAT</p><p>EC1</p><p>8:1n</p><p>9 OL</p><p>EATE</p><p>C18:</p><p>1n7 </p><p>VACC</p><p>ENAT</p><p>E</p><p>C20:</p><p>0 EIC</p><p>OSAN</p><p>OATE</p><p>C20:</p><p>1 11-</p><p>EICO</p><p>SENO</p><p>ATE</p><p>C22:</p><p>0 BEH</p><p>ENAT</p><p>E</p><p>Perc</p><p>enta</p><p>ge</p><p>VLPSF9 CellsBV</p><p>0</p><p>10</p><p>20</p><p>30</p><p>40</p><p>50</p><p>60</p><p>70</p><p>saturated monounsaturated</p><p>Perc</p><p>ent, </p><p>%</p><p>VLPBV</p><p>There are detectable differences in the fatty acid content of VLP, Sf9 cells and BV.</p></li><li><p>VLP AggregationWyatt technology</p><p>Field Flow Fractionation online static and dynamic LS detection No aggregationDifferent trivalent seasonal or monovalent VLPs similar size anddistribution</p><p>RMS Radius (root mean square)/Rh (radius of hydration) Radius ratio ~ 1:1 Spherical shells with open center</p></li><li><p>Cryo EM of H5N1 VLP Vaccine</p></li><li><p>Particle sizing Malvern Zetasizer</p><p>No evidence aggregation in VLP samples</p><p>After acid treatment can detect aggregates</p><p>Sample Particle Size (nm)</p><p>pH 7.2 pH 4</p><p>H5N1 VLPs, Lot#: 11508-1 (Stage B)180g HA/mL</p><p>178 1435</p><p>Seasonal Trivalent VLPs, Lot#: 75508008-2A (05/06 strains) 30g HA/mL/ strain </p><p>160 268</p><p>Seasonal Trivalent VLPs, Lot#: 75508013-1 (08/09 strains) 120g HA/mL/ strain </p><p>180 284</p></li><li><p>Conclusions</p><p> VLP is a promising vaccine technology for Influenza</p><p> HA and NA antigens on recombinant VLPs have high fidelity to natural virus</p><p> HA and NA in VLPs elicit functional antibodies in humans</p><p> Traditional assays for potency and immunogenicity can be reliably used for VLP vaccines</p><p> VLPs can be used as antigen reagents in potency and immunogenicty assays</p><p> Carbohydrate and protein impurities are not immunogencic in VLP preparations</p><p>A Recombinant Virus Like Particle (VLP) Vaccine for InfluenzaRecombinant Virus-Like Particle (VLP) VaccinesRecombinant Influenza VLPs:Pleomorphic Spherical ParticlesPotential Immunologic Advantages of Novavax VLP Influenza Vaccine CandidatesDevelopment of Novavax VLPsVLP Flu Vaccines are Safe &amp; ImmunogenicHA and NA Composition of Influenza VLP Vaccines in Human Clinical TrialsVLP Can be Used Instead of Virus to Measure HA Antibody in HAIVLP Associated NA Attributes are Similar to NA in Influenza VirusHuman Sera with NA Inhibiting Antibody give Similar Titers Against VLP and Virus NAVLP CharacterizationCarbohydrate Analysis 2008-2009 Trivalent VLP VaccineAre the Sf9 Derived Carbohydrate Structures Immunogenic? Baculovirus ELISAA VLP Release AssayExperimental Approach24 Hr TreatmentELISAAre the Protein Impurities Immunogenic? Lack of Response to BV Proteins in Humans Vaccinated with VLPLack of Response to Sf9 Proteins in Humans Vaccinated with VLPFatty Acid and Lipid AnalysisInitial ResultsVLP AggregationWyatt technologyCryo EM of H5N1 VLP VaccineParticle sizing Malvern ZetasizerConclusions</p></li></ul>