Prospect of Universal Influenza Vaccine Designed by Broadly Neutralizing Monoclonal Antibodies (1) Yoshinobu Okuno

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1 Vaccine World East Asia 2015 Seoul, January 26, 2015 Prospect of Universal Influenza Vaccine Designed by Broadly Neutralizing Monoclonal Antibodies (1) Yoshinobu Okuno The Research Foundation for Microbial Diseases of Osaka University, Japan

2 Schematic diagram of influenza virus particle Fields Virology (6th edition)

3 Influenza viral proteins and their immunological function in host animals Influenza viral gene Protein Major immunological function Antigenic variability HA Hemagglutinin (HA) Antibody (head) + (stem) NA Neuraminidase Antibody M M1 CTL - M2 (M2e) Antibody + NP Nucleoprotein (NP) CTL - NS Nonstractural (NS) CTL - PA Polymerase acidic (PA) CTL - PB1 Polymerase basic 1 (PB1) CTL - PB2 Polymerase basic 2 (PB2) CTL -

4 Receptor binding site Three-dimensional structure of HA Globular head region Stem (Stalk) region HA 1 HA 2 Fusion peptide

5 Antigenic diversity of influenza A virus hemagglutinin

6 HA stalk reactive monoclonal antibodies Name of Publication Cross-reactivity MoAb among HA subtypes C (Okuno Y, et al; JVI) Group 1 A (Kashyap AK, et al; PNAS USA) Group 1 CR (Throsby M, et al; PLos One) Group 1 F (Sui J, et al; Nature Struct Mol Biol) Group 1 CR (Ekiert DC, et al; Science) Group 2 CR (Friesen RH, et al; PNAS USA) Group 2 F (Corti D, et al; Science) Group 1 and G (Li GM, et al: PNAS USA) Group 1 and 2 CR (Dreyfus C, et al; Science) Influenza A and B 5A (Yasugi M, et al; PLos Pathog) Influenza B

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8 Site A Site B Receptor binding site Site D Site E Site C Common neutralizing epitope (recognized by the MoAb, C179) Fusion peptide

9 Biological activity of C179 measured by neutralization and staining tests Virus strains Neutralization test Staining test H1N1 A/PR/8/ A/FM/1/ A/Bangkok/10/ A/Yamagata/120/ A/Osaka/930/ A/Suita/1/ H2N2 A/Okuda/ A/Adachi/2/ A/Kumamoto/1/ A/Kaizuka/2/ A/Izumi/5/ H3N2 A/Aichi/2/68 A/Fukuoka/C29/85 A/Ibaraki/1/90 H3N8 A/Budgerigar/Aichi/1/77 H4N6 A/Duck/Czechoslovakia/1/56 H5N9 A/Turkey/Ontario/7732/ H5N2 A/Duck/Hong Kong/342/ H5N3 A/Duck/Hong Kong/820/ H5N3 A/Whistling swan/shimane/476/ H6N5 A/Shearwater/Australia/1/72 H7N7 A/Tufted duck/shimane/124r/80 H8N4 A/Turkey/Ontario/6118/68 H9N2 A/Turkey/Wisconsin/ H9N2 A/Duck/Hong Kong/448/ H9N5 A/Duck/Hong Kong/702/ H10N7 A/Chicken/Germany/N/49 H11N6 A/Duck/England/56 H12N5 A/Duck/Alberta/60/76 H13N6 A/Gull/Maryland/704/77 H14N5 A/Mallard/Astrakhan/263/82 H15N8 A/Duck/Australia/341/83

10 -C179 +C179 H1N1 H2N2 H3N2

11 Throsby M et al. PlosOne 3 e3942 (2008) Sui J et al. Nature Structural & Molecular Biology 16: (2009)

12 Journal of Virology, 87: , 2014

13 HA stalk reactive antibodies as prophylactic and/or therapeutic measures for influenza virus infections Name of Publication Test animal MoAb C (Okuno Y, et al; JVI) Mice CR (Ekiert DC, et al ; Science) Mice CR (Friesen RH, et al ; PLos One) Ferrets CR6261 Human clinical trial 6F (Tan GS, et al; JVI) Mice 6F (Krammer F, et al; JVI) Ferrets CR (Ekiert DC, et al; Science) Mice CR8020 Human clinical trial Many MoAbs 2012 (Thomson CA, et al; Front Immunol) Mice CR (Dreyfus C, et al; Science) Mice

14 Survival rate (%) Survival rate (%) Effect of C179 against infection with A/FM/1/47(H1N1) in mice A Prophylactic effect B Therapeutic effect Days after infection 1000μg/mouse C μg/mouse 10μg/mouse PBS C179 Days after infection 1000μg/mouse 100μg/mouse PBS

15 HA stalk based universal influenza vaccines (Graves PN, et al; Virology 1983) (Wang TT, et al; PLos Pathog 2010) (Steel J, et al; MBio 2010) (Hai R, et al; JVI 2012) Quoted from figures of Krammer F and Palese P (Curr Opin Virol, 2010)

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17 HA head reactive broadly reactive monoclonal antibodies Name of Publication Recognition site MoAb F (Ohshima N, et al; JVI) Receptor-binding site CH (Whittle JR, et al; PNAS USA) Receptor-binding site C (Ekiert DC, et al; Nature) Receptor-binding site S139/ (Lee PS, et al; PNAS USA) Receptor-binding site B (Kubota-Koketsu, R et al; BBRC) a S139/ (Yoshida R, et al; PLos One) b 5J (Krause JC, et al; JVI) c CR (Dreyfus C, et al; Science) d CR (Dreyfus C, et al; Science) e a: underneath the receptor-binding site b: adjacent to the receptor-binding site c: between the receptor-binding site and the Ca 2 antigenic site d: near the receptor-binding site e: the base of the HA head distant from the receptor-binding site

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19 Summary M2e and HA stalk will be candidates for universal influenza vaccines. In particular, HA stalk induces broad neutralizing antibodies which will be useful for prevent influenza virus infection. Constructions based on HA stalk have been studied extensively and will be applied for clinical trials soon.

20 Vaccine World East Asia 2015 Seoul, January 26, 2015 Prospect of Universal Influenza Vaccine Designed by Broadly Neutralizing Monoclonal Antibodies (2) Production of Monoclonal Antibodies Derived from Human Donors, Epitope Analyses, and Vaccine Design Yuji Inoue The Research Foundation for Microbial Diseases of Osaka University, Japan

21 Production of Anti-Influenza Human Monoclonal Antibodies Vaccinees 1-4 wk Patients PBMCs Fusion partner cell line SPYMEG (MBL Co. Ltd.) Screening Cloning Human Monoclonal Antibodies (HuMAbs) Characterization in vivo Characterization in vitro

22 Sampling ~ Cloning Hybridomas Viral Titer Cross-reactive Abs (IgG) IgM IgG IgM IgG (Strain-specific Abs) Past Infection (or Vaccination) Present Infection (or Vaccination) Blood Collection Day 3 Day 7-9 Day Day Vaccinees(6 donors) 0 (0) 2 (2) 8 (5) 1 (1) 2011 Patients (2 donors) 0 (0) 26 (9) 8 (2) 2 (1) ( ) : neutralizing Abs

23 Isolated Human Monoclonal Antibodies Neutralizing Influenza Virus Universal Typespecific Lineagespecific Subtypespecific H1 H1N1pdm 5E4 Influenza A Group 1 1H11 2H5 5G2 H5 H9 Influenza Influenza B 5A7 Group 2 B-1 D-1 Yamagata lineage 3A2 10C4 Victoria lineage H3 H14 3G8 5D8 5E7 (Pan et al, 2014)

24 Human Monoclonal Antibodies 1H11, 2H5, and 5G2 Carries Neutralization Activities against H1N1, H5N1, H9N2 strains (Pan et al, 2014)

25 Epitope Analyses of HuMAbs 1H11, 2H5 and 5G2 Ab Ab-Sepharose HA Ag Wash Trypsin digestion Wash Elution Each HuMAb recognizes a similar region on HA stem. MALDI-MS analysis

26 Isolated Human Monoclonal Antibodies Neutralizing Influenza Virus Universal Typespecific Lineagespecific Subtypespecific H1 Influenza A Group 1 1H11 2H5 5G2 H1N1pdm H5 H9 5E4 Influenza Group 2 B-1 D-1 H3 H14 3G8 5D8 5E7 (Yasugi et al, 2013) Influenza B 5A7 Yamagata lineage 3A2 10C4 Victoria lineage

27 Concentration of HuMAbs (mg/ml) Neutralization Test of Isolated MAbs against Influenza B Strains Yamagata lineage Victoria lineage Influenza B

28 5A7 Ab Neutralizes Influenza B Strains Belonging Both Yamagata and Victoria Lineages 4 h.p.i. HA1 HA2

29 Isolated Human Monoclonal Antibodies Neutralizing Influenza Virus Universal Typespecific Lineagespecific Subtypespecific H1 H1N1pdm 5E4 Influenza A Group 1 1H11 2H5 5G2 H5 H9 Influenza Group 2 B-1 D-1 H3 H14 3G8 5D8 5E7 (Koketsu et al, 2009) Influenza B 5A7 Yamagata lineage 3A2 10C4 Victoria lineage

30 B-1 and D-1 Commonly Recognizes a Novel Epitope Region on HA HA1 (A/H3N2) Ai/68 Gz/89 Wy/03 NY/04 Hi/05 Upper MP N N D N F D K L Y I WG V H H P S D K I E K V E K V E K G Location of B-1 epitope on HA molecule Upper Lower Ai/68 Gz/89 Wy/03 NY/04 Hi/ Lower G L S S R I S I Y WT I V K P G D V L V I I - L - D I I - L - D I P I - L - N I P I - L - (top view) (side view) Regions composing the epitope (namely Upper and Lower subregions) -are highly conserved. -form an anti-parallel β-sheet structure.

31 Antigen Design Targeting B-1 Epitope Structure (Inoue et al., 2013, J. Biol. Chem) HA(monomer) HA (monomer) EGFP β-sheet variant epitope (UL1) (Upper/Lower) Strategy: Carrier proteins exposing anti-parallel β-sheet part are selected. Epitope-derived residues are introduced onto the β-sheet portion of the carrier proteins. Carrier Proteins Carrier Proteins Carrier Proteins EGFP and mcherry

32 EGFP or mcherry Variants Exposing B-1 Epitope-Derived Structure Crystal Structure of an mcherry variant (UL1) The artificial epitope structure on mcherry variant compared with the original epitope structure on HA. EGFP U1 L1 UL1 UL2 mcherry U1 L1 UL1 UL2

33 Body Weight (%) Immunization and Virus Challenge EGFP UL1 Change of Body Weight 110 HA vaccine - positive control - PBS - negative control ELISA (Ag: HA vaccine soln.) #1 #2 #3 #4 # #1 #2 #3 #4 # Absorbance at 450 nm WT EGFP 110 #1 100 #2 #3 90 #4 #5 80 EGFP UL1 / mcherry / mcherry UL1 0.0 PBS WT EGFP PBS Wild-type EGFP Upper/Lower-like EGFP Immunogen EGFP UL d.p.i #1 #2 #3 #4 #

34 Summary A procedure to obtain human monoclonal antibodies (HuMAbs) derived from influenza patients or vaccinees was established. Several HuMAbs we isolated showed neutralizing activities against a broad range of influenza strains. Abs neutralizing group 1 influenza A strains (1H11, 2H5 and 5G2). Abs neutralizing influenza B strains (5A7, 10C4 and 3A2). Abs mainly neutralizing influenza A/H3N2 strains, including strains isolated more than 40 years (B-1 and D-1). Model vaccine antigens targeting the conformational epitope of B-1 MAb were tried to design.

35 Acknowledgements The Research Foundation for Microbial Diseases of Osaka University Yoshinobu Okuno Kazuyoshi Ikuta Ritsuko Koketsu Tadahiro Sasaki The Research Institute for Microbial Diseases, Osaka University Mayo Yasugi Yang Pan Mitsuhiro Nishimura Akifumi Yamashita Anariwa Du Japan Blood Products Organization Shoji Ideno Mikihiro Yunoki Molecular and Biological Laboratories, Co. Ltd. Ken-ichiro Ono Motoki Kuhara Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS)