Recombinant EBOV/Makona Glycoprotein (GP) Nanoparticle Vaccine Produced in Sf9 Insect Cells

Transcription

1 Recombinant EBOV/Makona Glycoprotein (GP) Nanoparticle Vaccine Produced in Sf9 Insect Cells Gale Smith, PhD Novavax, Rockville, MD ISBioTech 5 th Spring Meeting March 9 11, 2015 Washington, DC EBOV/Makona GP Nanoparticles

2 Presentation Overview Ebola glycoprotein (GP) brief background Recombinant EBOV/Makona GP nanoparticle vaccine cloning, expression, purification, characterization EBOV/Makona GP vaccine non-clinical evaluation mice NHP Phase 1 human trial 2

3 Ebolavirus glycoprotein (GP) Ebolavirus GP Filoviruses express a single protein on their envelope surface, glycoprotein GP, GP attachment to, and entry of, host cells, GP forms a trimer on the viral surface, GP monomers S-S linked GP1 and GP2, GP1 contains a receptor-binding core topped by a glycan cap and a heavily glycosylated mucin-like domain, GP2 contains two heptad repeats and a transmembrane domain, Filoiruses initially enter cells via macropinocytosis In the endosome, GP is cleaved by host cathepsin, Protease removes the mucin-like domains and glycan cap and renders GP competent to bind to the receptor, and GP2 subunit unwinds from its GP1 clamp and rearranges irreversibly into a six-helix bundle to drive fusion of virus and host membranes. Pre-fusion EBOV GP Lee et al Nature. 454:

4 Nonsynonymous AA between 2014 EBOV/Makona and 1976 EBOV/Mayinga GP 20 AA changes EBOV vaccines in NHP glycoprotein (GP): Necessary Sufficient 1 Gire, et al, Science 12 Sept

5 Nonsynonymous changes in EBOV/Makona GP to protective monoclonal antibodies epitopes EBOV/Makona 1995 EBOV/Kikwik 1976 EBOV/Mayinga 17 AA changes (red) 20 AA changes (red) Neutralizing/protective EBOV mabs 1 Kugelman, et al. Mbio. 6(1); 20 Jan 2015, Nonsynonymous (red), synonymous (blue), noncoding (green) 5

6 Novavax EBOV/Mak GP recombinant nanoparticle vaccine Recombinant EBOV/Makona Glycoprotein (GP) GenBank #AIG96283 EBOV [H.sapiens -wt/sle/2014/makona-g3798] Full length, unmodified GP gene Synthetic, codon optimized Cloned into a baculovirus vector rbv-gp GP CLONING Baculovirus vector rbv-gp rbv-gp EXPRESSION rbv-gp infected Sf9 cells PURIFICATION EBOV GP nanoparticles EBOV GP 30-40nm Sf9 cells 6

7 Purification process recombinant EBOV/Makona GP expressed in Sf9 cell membranes (MOI = 0.1; 64hr harvest) Non-ionic detergent lysis Sf9 cells: GP trimers Cation exchange chromatography Load Eluate Centrifugation/filtration Anion exchange GP0 GP1 BV gp64 Affinity Cation exchange: 2 9 GP trimers/nanoparticle GP2 7

8 MS/peptide mapping and terminal sequencing EBOV/Makona GP Signal peptide GP1 N-terminus GP0 GP1 GP1 1 mgvtgilqlp rdrfkrtsff lwviilfqrt fsiplgvihn stlqvsdvdk lvcrdklsst 61 nqlrsvglnl egngvatdvp svtkrwgfrs gvppkvvnye agewaencyn leikkpdgse 121 clpaapdgir gfprcryvhk vsgtgpcagd fafhkegaff lydrlastvi yrgttfaegv 181 vaflilpqak kdffsshplr epvnatedps sgyysttiry qatgfgtnet eylfevdnlt 241 yvqlesrftp qfllqlneti yasgkrsntt gkliwkvnpe idttigewaf wetkknltrk 301 irseelsfta vsngpknisg qspartssdp etnttnedhk imasenssam vqvhsqgrka 361 avshlttlat istspqpptt ktgpdnsthn tpvykldise atqvgqhhrr adndstasdt 421 ppattaagpl kaentntsks adsldlattt spqnysetag nnnthhqdtg eesassgklg 481 litntiagva glitggrrtr revivnaqpk cnpnlhywtt qdegaaigla wipyfgpaae 541 giyteglmhn qdglicglrq lanettqalq lflrattelr tfsilnrkai dfllqrwggt 601 chilgpdcci ephdwtknit dkidqiihdf vdktlpdqgd ndnwwtgwrq wipagigvtg 661 viiavialfc ickfvf GP2 Furin cleavage site GP2 N-terminus EBOV/Makona GP 16Dec14 8

9 AU AU 54.4 Purity capillary electrophroesis (CE) deglyosylated/reduced Ebola/Makona GP PDA - 220nm Ebola GP 1613 Red Degly Corrected Area Percent PDA - 220nm Ebola GP 1613 Reduced Corrected Area Percent p10kd marker GP PNGase F GP Minutes

10 EBOV GP dynamic light scattering (DLS) = 36.7 nm Z-Ave (d.nm) Mean DLS PdI Mean PdI

11 2014 Guinea EBOV Recombinant GP 36.7nm Particles 11

12 Guinea EBOV GP transmission EM imaging 2D Class averaging analysis (NanoImaging) Guinea EBOV GP Lot 15Sep14; 13.2 µg/ml Particle Selection >1,000 images Computer Particle Alignment and Classification: particles sorted into self-similar groups of classes Figure 1. Transmission Electron Microscopy Negative Stained Guinea EBOV GP. FEI Tecnai T12 electron microscope, operating at 120keV equipped with an FEI Eagle 4k x 4k CCD camera. Purified Guinea EBOV GP 1623 Lot 15sep14; 13.2 µg/ml stained uranyl formate then imaged at 67,000x and 110,000x (insert). 12

13 Guinea EBOV GP and RSV F 2D nanoimaging Ebola/Mak GP pre-fusion nanoparticles Particles with 2 to 9 GP trimer protrusions Arranged as concentric EBOV/Mak GP pre-fusion trimers 10 nm 10 nm RSV fusion (F) post-fusion nanoparticles >1,000 RSV F nanoparticles sorted into self-similar groups Particles with 2 to 9 RSV F trimer protrusions Arranged as apposing RSV F post-fusion trimers 13

14 Ebolavirus GP is heavily glycosylated the glycan cap and mucin-like domain (MLD) N-linked only N- and O-linked Filoviruses maintain glycoprotein glycosylation to protect against proteases and antibody neutralization at the expense of efficient entry. Lennemann, et al 2014 mbio 5(1) e

15 Recombinant EBOV/Mak GP N-linked glycosylated sites and peptides: MS/Trypic peptide mapping Number Site Glycopeptide Sequence Major Glycoform Occupied 1 Asn8 IPLGVIHNSTLQVSDVDK M3F 98% 2 Asn172 EPVNATEDPSSGYYSTTIR M3F 91% 3 & 4 Asn(196 & 206) YQATGFGTNETEYLFEVDNLTYVQLE SR 2 M3F 99% 5 Asn225 FTPQFLLQLNETIYASGK M3F, M6, M5 99% 6 Asn236 SNTTGK ND 7 Asn264 NLTR M3F 99% 8 Asn285 NISGQSPAR M3F 58% 9 Asn301 TSSDPETNTTNEDHK M3F 74% 10 Asn314 IMASENSSAMVQVHSQGR M3F 41% 11 Asn354 TGPDNSTHNTPVYK M3F, A1G0F 99% 12 & 13 Asn(381 & 404) 14 & 15 Asn(422 & 430) RADNDSTASDTPPATTAAGPLKAENT NTSK SADSLDLATTTSPQNYSETAGNNNTH HQDTGEESASSGK 2 M3F and M3F/ A1G0F 98% M3F/ A1G0Fand 2 M3F 16 Asn531 GP2 QLANETTQALQLFLR M8, M7,M6, M5 99% 17 Asn586 GP2 NITDKIDQIIHDFVDK M3F 84% 15

16 Binding kinetics purified recombinant EBOV/Mak GP to neutralizing/protective EBOV monoclonal antibodies mab KZ52 13C6 6D8 13F6 EBOV GP Epitope aa 42-43, 513, , 556 GP1/GP2 aa GP1 aa GP1 HNTPVYKLDISEATQVE aa GP1 ATQVEQHHRRTDNDSTA ATQVGQHHRRADNDSTA 1 Conformational Pre-fusion GP2 Neutralizing Conformational Neutralizing Protective In ZMapp Linear Neutralizing Protective Linear Neutralizing SPR Analysis K a (1/Msec) K d (1/sec) K D (nm) 2.90E E E E E E No binding No binding No binding 16

17 Human mab KZ52 binds to pre-fusion EBOV GP Ebola KZ52 Human Monoclonal Antibody Isolated from a human survivor of 1995 outbreak in Kikwit, Zaire Binds residues at the N terminus of GP1, and and at the N terminus of GP2 Requires an epitope seen only in the GP2 pre-fusion conformation Protection from lethal EBOV challenge in rodents; minimal protection of non-human primates Lee et al, Nature (7201):

18 EBOV GP potency assay 13C6 mab ELISA for product release 4F3 mab (linear) capture EBOV/Mak GP 13C6 mab (conformational) to quantify Sample EBPV/Mak DP Lot B531P12 13C6 ELISA (µg/ml) A280 O.D. (µg/ml)

19 EBOV/Makona GP Nanoparticle Vaccine Immunogenicity and Challenge in Mice

20 Study # : EBOV/Makona GP vaccine immunogenicity and protection in mice STUDY DESIGN Group N GP Dose (µg) Adjuvant (µg) Immunization days Blood draw days Challenge day none 0, 14, 28 0, 21, Matrix M (5) 0, 14, 28 0, 21, AlPO 4 (50) 0, 14, 28 0, 21, none - 0, 21,

21 EBOV/Makona GP vaccine protected mice against lethal challenge with Ebola virus Mice were challenged on day 42 with an intraperitoneal injection of 1,000 pfu mouse adapted EBOV/Mayinga 1976 strain 1 One animal with an unrelated abdominal abscess and was dropped from the study. 21

22 EBOV/Makona GP vaccine ELISA IgG (EC50) and neutralizing antibody responses in mice (day 28) 100% 100% 10% 0% 10% 0% CONFIDENTIAL EBOV/Mayinga PsVNA: Jay Hooper, USAMRIID 22

23 Review of ebolavirus vaccines protective in rodents Marzi and Feldmann Ebola virus vaccines: an overview of current approaches. Exp Rev Vac 13(4);521 23

24 Review ebolavirus vaccine protective NHP Until now no ebolavirus GP subunit or WIV vaccine reported to be protective in NHP Marzi and Feldmann Ebola virus vaccines: an overview of current approaches. Exp Rev Vac 13(4);

25 Novavax EBOV/Makona GP Nanoparticle Vaccine Immunogenicity in Olive Baboons Robert Welliver, MD Oklahoma Baboon Research Resource, University of Oklahoma Health Sciences Center

26 Baboon immunogenicity 2014 EBOV/Makona GP nanoparticle vaccine study design Group N Ebola GP Vaccine Adjuvant Day of Immunization Day for Blood Draw µg No Adjuvant µg 800µg AlPO µg 50µg Matrix-M 4 3 5µg 50µg Matrix-M 0, 21 0, 21, 31, 42, 60, 90, 120, 150,

27 E L IS A E C 9 0 T ite r E L IS A E C 9 0 T ite r Anti-EBOV/Makona GP ELISA EC90 responses in baboons DAY 21 Day EBOV GP 60 µg 60 µg 60 µg 5 µg Adjuvant None AlPO 4 Matrix Matrix EBOV GP 60 µg 60 µg 60 µg 5 µg Adjuvant None AlPO 4 Matrix Matrix ELISA Antigen: Makona GP Mayinga GP 27

28 Ebola biotinylated 13C6 mab competitive ELISA assay mab13c6 competition ELISA biotin-13c6 (0.3 mg/ml) % reduction Concentration (ug/ml) 4-P Fit: y = (A - D)/( 1 + (x/c)^b ) + D: A B C D R^2 13C6 (S#04: Dilution vs Adjusted %) Four parameter fit analysis: 50% reduction of biotin-13c6 mab binding to EBOV/Mak GP = 0.35 µg/ml 13C6- competing antibody Weighting: Fixed 0.35 Competing mab 13C6 (mg/ml) 28

29 Baboon Immunogenicity Study: anti-ebov/mak GP ELISA and competition ELISA with 13C6 mab Group Vaccine Day 0 Day 21 1 dose Day 31 2 doses EC90 EC90 EC90 13C6 mab 1 60µg EBOV GP < ,517 <4 µg/ml µg EBOV GP 800µg AlPO4 60µg EBOV GP 50µg Matrix 5µg EBOV GP 50µg Matrix <100 19, , µg/ml <100 13,115 6,870, µg/ml <100 3,242 11,302, µg/ml CONFIDENTIAL 29

30 EBOV/Mak GP vaccine IFNγ-Elispot response in baboons day 31 IFNγ-Elispot responses to 5ug EBOV GP + Matrix-M vaccine 30

31 IFNγ-secreting I F N r e c r e g cells/10 c e n o./1 6 x PBMC c e lls EBOV/Makona GP vaccine IFNγ-Elispot response in baboons day u g G P 6 0 u g G P /A lu m 6 0 u g G P /M a trix -M 5 u g G P /M a trix -M N H P g r o u p s 31

32 IFNγ Ebola-GP/Matrix-M induced higher T cell responses in baboons 60 ug GP 60ug GP/Alum 60ug GP/Matrix-M 5ug GP/Matrix-M CD4+ T cells CD8+ T cells TNFα 32

33 % of cytokine producing cells % of cytokine producing cells Ebola-GP/Matrix-M induced multifunctional T cell response in baboons Single cytokine double cytokine triple cytokine single cytokines d o u b le c y to k in e s trip le c y to k in e s CD4+ T cells single cytokines CD8+ T cells u g G P 6 0 u g G P + A lu m 6 0 u g G P + M a trix -M 5 u g G P + M a trix -M 6 0 u g G P 6 0 u g G P + A lu m 6 0 u g G P + M a trix -M 5 u g G P + M a trix -M 6 0 u g G P 6 0 u g G P + A lu m 6 0 u g G P + M a trix -M 5 u g G P + M a trix -M Cytokines evaluated: interferon-, TNF-a and IL-2 33

34 E L IS A E C 9 0 G M T Baboons Durability of anti-ebov GP Response ELISA EC 90 GMT titers baboons (n=3/group) u g G P + M a trix 6 0 u g G P + M a trix 6 0 u g G P + A lp O u g G P D a y 34

35 Lethal Ebola virus challenge of Cynomolgus macaques Study Title: Efficacy screening of Novavax 2014 EBOV/Makona glycoprotein (GP) nanoparticle vaccine in Cynomolgus macaques. CRO: Texas Biomedical Research Institute, San Antonio, TX Ricardo Carrion, Jr., Ph.D. Study Director Study Protocol: Group Vaccine N 1 5 µg GP + Matrix Immunization Day Challenge 1 Day End of Study Day 3 0, control Challenge virus: 100 pfu wild-type EBOV/Kikwik

36 Anti-EBOV GP EC90 Titer (log10) EBOV/Makona 5µg GP + Matrix-M vaccine EC90 IgG day 28 response in Cynomolgus macaques ELISA EC 90 ELISA titers wk 4 (1 wk post boost) ELISA antigen 2014 Makona GP 1976 Mayinga GP Pre-Bleed Placebo EBOV GP + Matrix 36

37 IFNγ-secreting cells/10 6 PBMC IFNγ-secreting cells/10 6 PBMC IFNγ-secreting cells/10 6 PBMC EBOV/Makona GP vaccine IFNγ-Elispot response in macaques 350 Week Week placebo placebo Week placebo Immunization: Weeks 0, 3 Challenge: Week 7 Placebo: #33356 Vaccine: #33355, 33360, Ebola Zaire (EZ) peptide pools Pool 1: N-terminal sequence peptides Pool 2: C-terminal sequence peptides Con: Zaire/Sudan consensus peptides 37

38 Percent Survival EBOV/Makona GP vaccine 100% protective against a lethal Ebola virus challenge in macaques µg EBOV GP + Matrix-M Control PBS Days Post Challenge Macaques immunized on day 0 and 21 with 5µg EBOV/Makona GP + 50µg Matrix M. On Day 42 challenged by intramuscular inoculation of 100 pfu of Ebola/Kikwik 38

39 Recombinant EBOV/Makona GP + Matrix-M nanoparticle vaccine Recombinant GP Full length, unmodified, trimers assembled as stable nanoparticles Pre-fusion, binds to functional Ebola mab Highly purified with good stability 2 8 C Matrix-M adjuvant Dose sparring Induces multifunction CD4+ and CD8+ T cells Mice and NHP 5µg dose induces high levels of anti-ebov GP IgG High levels virus neutralizing antibodies 100% protection against Ebola virus Sustained anti-gp titers 4 months 39

40 Novavax EBOV-H-101: FSI February 10, 2015 Phase 1, blinded, controlled in 230 adults 18 to < 50 y.o. in Australia Dose ranging, 1 and 2 dose regimens w/wo Matrix-M adjuvant Immune responses thru 1 year Safety, GP ELISA, PRNT, anti-13c6 IgG, and T-cell responses 40

41 41 Ebola GP Vaccine Employee Acknowledgements