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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 µ 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 120µg 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 ½ 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, µgBV/ml
Dilution 2, µgBV/ml
Average, µgBV/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 value
Placebo 60.95 1.52 1.13 61.85 1.55 1.14 1.01 0.9261N = 12
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
Lack of Response to Sf9 Proteins in Humans Vaccinated with VLP
( Tested by ELISA against 2 ug/mL of SF9 protein ) t testGroup GMT pre SD SE GMT post SD SE Ratio P value
Placebo 103.28 3.11 1.39 108.95 3.51 1.44 1.05 0.8588N = 12
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
Fatty Acid and Lipid AnalysisInitial Results
• 80% of fatty acids belong to 4 classesC16.0 Palmitate, C16.1 Palmitoleate, C18.0 Stearate, C18.1 n9 Oleate
• Seasonal and H5n1 pandemic VLPs differ in their total percentage of saturated vs unsaturated fatty acids
Seasonal longer chainsHigher saturated/unsaturated ratio in seasonal suggesting greater lateral segregation
• VLP fatty acid lower content of saturated fatty acids than baculovirus and may bud from different membrane regions
• Seasonal VLP 4-fold higher content choline lipids than pandemic VLP
Fatty Acid Composition
05
10152025303540
C14:0
MYRIS
TATE
C16:0
PALM
ITATE
C16:1
PALM
ITO
LEATE
C18:0
STE
ARAT
EC18
:1n9
OLE
ATE
C18:
1n7
VACCE
NATE
C20:0
EIC
OSANOATE
C20:1
11-
EICOSEN
OATEC22
:0 B
EHENATE
Perc
enta
ge
VLPSF9 CellsBV
0
10
20
30
40
50
60
70
saturated monounsaturated
Perc
ent,
%
VLPBV
There are detectable differences in the fatty acid content of VLP, Sf9 cells and BV.
VLP AggregationWyatt technology
Field Flow Fractionation online static and dynamic LS detection– No aggregation
Different trivalent seasonal or monovalent VLPs similar size anddistribution
RMS Radius (root mean square)/Rh (radius of hydration) Radius ratio ~ 1:1– Spherical shells with open center
Cryo EM of H5N1 VLP Vaccine
Particle sizing Malvern Zetasizer
No evidence aggregation in VLP samples
After acid treatment can detect aggregates
Sample Particle Size (nm)
pH 7.2 pH 4
H5N1 VLP’s, Lot#: 11508-1 (Stage B)180µg HA/mL
178 1435
Seasonal Trivalent VLP’s, Lot#: 75508008-2A (05/06 strains) 30µg HA/mL/ strain
160 268
Seasonal Trivalent VLP’s, Lot#: 75508013-1 (08/09 strains) 120µg HA/mL/ strain
180 284
Conclusions
• VLP is a promising vaccine technology for Influenza
• HA and NA antigens on recombinant VLPs have high fidelity to natural virus
• HA and NA in VLPs elicit functional antibodies in humans
• Traditional assays for potency and immunogenicity can be reliably used for VLP vaccines
• VLPs can be used as antigen reagents in potency and immunogenicty assays
• Carbohydrate and protein impurities are not immunogencic in VLP preparations