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