E.J. Remarque & G. Koopman. Confiden'al 2

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1 EDUcate influenza VACcine A Combinatorial immunization strategy to educate the immune system towards cross recognition and coverage against antigenic drift in seasonal influenza virus exposure Confiden'al 1

2 EDUcate influenza VACcine E.J. Remarque & G. Koopman Confiden'al 2

3 Presentation Outline AMA1 findings, covering polymorphism Influenza background EduFluVac project: Scientific background for broad flu coverage

4 AMA1 Polymorphism Database at 1778 valid entries Allelic variation at 103 polymorphic AA positions in DI, II and III (Ectodomain 521 AA) Prevalence Domain all 99% 95% 90% 85% 80% 75% Pro DI DII DIII Cytoplasmic Total

5 Crystal structure of AMA1

6 Three Allele Vaccine Kusi et al, PLoS One 2009

7 Three allele vaccine Multivalent equals responses of single allele vaccine when tested on homologous strain Multivalent covers all constituents and is broadened Indication of broadening (e.g. CAMP response) Differs from pooling monovalent immunisation IgGs

8 Conclusions AMA1 Each of the vaccine antigens in mix yields homologous titres (IgG and functional) similar to monovalent (despite 1/3 rd Ag dose). Broadening due to dilution of strain-specific epitopes, response focuses on cross-reactive epitopes (Kusi 2009 PLoS One) Simultaneous or sequential immunisation yields similar patterns of Ab breadth. (Kusi 2011 Mal J)

9 Influenza Virus

10 Influenza Background HA and NA are major surface antigens: HA: attachment, entry and membrane fusion NA: viral release from host cell M2: Ion channel (infected host cell), ph control, virus replication HA and NA are extremely variable HA has 16 subtypes H1, H2, H3, H5, H7 and H9 have caused disease in humans. H1, H2 and H3 have caused pandemics NA has 9 subtypes B type has two lineages: B/Yamagata and B/Victoria

11 Broadening of responses to flu Huber et al 2009, H3N2: DNA prime, LAIV boost. Mixed immunisation (DNA & LAIV) yields broadened response to H3N2 viruses. H3N2 strains from 3 alternating clusters as defined by Smith (Science 305, ) Carter et al 2013, H1N1: 3 sequential infections with H1 viruses induces Ab reactive with and protects against H1N1pdm challenge (not contained in infections) Prakbaran H5N1 mixture of 3 strains induces broadened response VLP induce broader response than whole virus vaccine

12 EduFluVac Approach Analogous to AMA1 approach, use mixture of variable antigens Investigate whether a mixture of 3 5 HA s from a (sub)type yields broadened responses (H1, H3 and B strains for seasonal flu vaccine) Investigate whether a mixture of 3 NA s from the N1 subtype yields broadened responses Investigate whether broad Group 2 HA neutralisation can be obtained by vaccination with 6 Group 2 HA s (Pandemic vaccine): H3, H4, H7, H10, H14, H15

13 Antigens in EduFluVac Haemagglutinin: H1, H3 and B. Five sequences per (sub)type selected Group 2 HA: H3, H4, H7, H10, H14 & H15 Neuraminidase N1. Three sequences. M1 protein included (also T cell help) Total 31 VLP products:

14 Project Phases Standard generation Exploratory Studies (regime, dose) Seasonal candidate selection (exclude Ag competition subtypes and HA/ NA VLP). Group 2 HA study Lead Candidate Selection Proof of Concept studies (Ferret & NHP)

15 Selection of lead candidate Q lead candidate selection, i.e. seasonal vaccine (H1, H3 & B) yielding the broadest response as determined by VNA. Based on mouse data. Contribution of adjuvant established Group 2 HA vaccine will move forward to ferret challenge model The seasonal vaccine is expected to contain H1N1 and this is the strain that will be used for the Ferret / NHP challenge studies ± N1 VLP

16 Proof of Concept studies Ferret (Q1 2016): Confirmation of dose and Ab response after 3 immunisations Heterologous challenge with H1N1 or H3N2 Heterologous challenge with Group2 HA strain (e.g. H7N7) Rhesus (Q2 2016): Heterologous challenge with H1N1 virus. Analysis of humoral (HI, VNA, ELISA) and cellular responses (ICS, ELISpot), both central (blood) and mucosal (BAL)

17 Partners: European Vaccine Initiative Biomedical primate Research Center Central Veterinary Institute ETNA Biotech Instituto de Biologia Experimental e Tecnologica National Institute for Biological Standard and Control Redbiotec Confiden'al 17

18 This presenta'on reflects only the author s views and the European Union is not liable for any use that may be made of the informa:on contained therein Confiden'al 18

19 Confiden'al 19

20 Step 1 Standard generation Groups of 4 mice immunised with 1.5 µg antigen in Montanide ISA 51, 3 x s.c. injections (0, 28 & 56). Sera collected and IgGt quantified for: H1 (N = 5), H3 (N = 5), B (N = 5), N1 (N = 3) and Group 2 HA (H3, H4, H7, H10, H14 and H15). Also HI and VN characterisation.

21 Anti-PfAMA1 antibodies from single vs mixed allele immunisations in celisa FVO AMA1 capture Residual binding (%) Soluble an'gen (µg/ml) sag Minimum (95% CI) FVO 3.7 ( ) 3.0 ( ) HB ( ) 2.9 ( ) 3D ( ) 8.0 ( ) CAMP 31.6 ( ) 16.8 ( )

22 Most cross-reactive epitopes common to all alleles FVO AMA1 capture Residual binding (%) FVO Soluble an'gen (ug/ml)

23 Anti-PfAMA1 antibodies from single vs mixed allele immunisations in celisa 3D7 AMA1 capture Residual binding (%) Soluble an'gen (µg/ml) sag Minimum (95% CI) FVO 40.2 ( ) 17.8 ( ) HB ( ) 5.7 ( ) 3D7 3.3 ( ) 1.9 ( ) CAMP 40.0 ( ) 25.7 ( )

24 Most cross-reactive epitopes common to all alleles 3D7 AMA1 capture Residual binding (%) Soluble an'gen (ug/ml)

25 Influenza Serology Haemagglutination inhibition (HI): Virus agglutinates erythrocytes, if Ab blocks receptor function agglutination is inhibited. HI detects Head Antibodies, but not stalk antibodies Virus Neutralisation assay. VNA involves infection of target cells ± Ab and detects both head and stalk Ab.

26 Influenza A Subtypes history

27 Broadening of responses to flu Carter et al 2013 J. Virol.

28 H1 HA Sequences * * * * * * * * * *---- A/California/07/2009 DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYE A/Puerto_Rico/8/ I S------R-K-I---Q L------DP-LPVR N-E--I A/Fort_Monmouth/1/ I S------R-K-I---Q LSKR-----A---N-E A--- A/Denver/1/ I S------R-K-K---Q--N------V LSNR-----A---N-E A--- A/Texas/36/ I S------R-K-I---Q--N-SV K----FSKE-----A---NPE Y-A--- A/New_Caledonia/20/ I S------L-K-I---Q--N-SV L-ISKE NPE Y-A--- A/Brisbane/59/ I NS------L-K-I---Q--N-SV L-ISKE K-NPE H-A * * * * * * * * * *--- A/California/07/2009 ELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAY A/Puerto_Rico/8/ E------NTT S---KS---R--L--TE-EG-----KN--V-K N-K---NI---EN-- A/Fort_Monmouth/1/ ER---K-NITR------S---KS------L--TETDG V-NNE V----NIE--KT--RKEN-- A/Denver/1/ ER-----TTR RKS------V--TEANG---N--R--V-NQE V----NIEE-RA--RKDN-- A/Texas/36/ E------TVT----TS-S-N-KS---R--L--TE-NGL--N-----V-N-E V----NIR--RAI-HTEN-- A/New_Caledonia/20/ E------TVT.--S-S-S-N-KS---R--L--TG-NGL--N-----V-N-E V---PNIG--RA--HTEN-- A/Brisbane/59/ E------TVT.--S-S-S-N-ES---R--L--TG-NGL--N-----A-N-E V---PNIGN-KA--HTEN * * * * * * * * * *--- A/California/07/2009 FVGSSRYSKKFKPEIAIRPKVRDREGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPI A/Puerto_Rico/8/1934 S-VT-N-NRR-T----E------QA LK---T-I---N---IA------LS-GF-----T-NASM-E---K----L----S V A/Fort_Monmouth/1/1947 S-V--N-NRR-T----E-----GQA L----T-I---N---IA-WH---LS-GF-----T-NA-MDE-D-K----Q----S V A/Denver/1/1957 S-V--N-NRR-T----K------QS L----T-I IA-W----LS-GP-----T-NA-LDE-D-K----Q----S V A/Texas/36/1991 S-V--H--RR-T----K-----GQ---I------L----T-I---N---IA-W----LS-GF-----T-NASMDE-DAK----Q----S------V--V A/New_Caledonia/20/1999 S-V--H--RR-T----K------Q---I------L----T-I---N---IA-W----LS-GF-----T-NA-MDE-DAK----Q----S------V--V A/Brisbane/59/2007 S-V--H--R--T----K------Q---I------L----T-I---N---IA------LS-GF-----N-NA-MDK-DAK----Q----S------V--V * * * * * * * * * *--- A/California/07/2009 IGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFN A/Puerto_Rico/8/ E-----R-A---MV I Q NG I A/Fort_Monmouth/1/ E MV A/Denver/1/ E-----R-----MV V M Q NG A/Texas/36/ E-----R-----MV I Q NG A/New_Caledonia/20/ E-----R-A---MV Q NG A/Brisbane/59/ E-----R-A---MV Q NG # AA differences between influenza A H1N1 haemagglu'nin HA1 globular head (326 AA total) A/PR/8/34 A/FM/1/47 A/Den/1/57 A/Tex/91 A/NC/99 A/Bris/2007 A/California07/

29 Influenza Virus

30 Haemagglutinin Conserved Epitopes

31 Haemagglutinin Conserved Epitopes