1. TOWARD A UNIVERSAL INFLUENZA VIRUS VACCINE Icahn School of Medicine at Mount Sinai PETER PALESE Department of Microbiology Munich, May 30, 2013

2. >1 in 5 hospitalizations in the U.S. influenza-related (Jan. 2013) https://login.medscape.com/login/sso/getloginhttps://login.medscape.com/login/sso/getlogin? urlCache=aHR0cDovL3d3dy5tZWRzY2FwZS5jb20 vdmlld2FydGljbGUvNzc4MjMyX3ByaW50&ac=401

3. Influenza virus vaccine formulations (2000 – 2010) Vaccine Recommendations H1N1H3N2B 2000 – 2001A/NEW CALEDONIA/20/99A/MOSCOW/10/99B/BEIJING/184/93 2001 – 2002A/NEW CALEDONIA/20/99A/MOSCOW/10/99B/SICHUAN/379/99 2002 – 2003A/NEW CALEDONIA/20/99A/MOSCOW/10/99B/HONG KONG/330/2001 2003 – 2004A/NEW CALEDONIA/20/99A/MOSCOW/10/99B/HONG KONG/330/2001 2004 – 2005A/NEW CALEDONIA/20/99A/FUJIAN/411/2002B/SHANGHAI/361/2002 2005 – 2006A/NEW CALEDONIA/20/99A/CALIFORNIA/7/2004B/SHANGHAI/361/2002 2006 – 2007A/NEW CALEDONIA/20/99A/WISCONSIN/67/2005B/MALAYSIA/2506/2004 2007 – 2008A/SOLOMON ISLANDS/3/2006A/WISCONSIN/67/2005B/MALAYSIA/2506/2004 2008 – 2009A/BRISBANE/59/2007A/BRISBANE/10/2007B/FLORIDA/4/2006 2009 – 2010A/BRISBANE/59/2007A/BRISBANE/10/2007B/BRISBANE/60/2008

4. Source: CDC ILI and Vaccine Distribution Data Percentage of Visits for ILI and H1N1 Vaccine Distribution, Sep 2009 – May 2010

5. HOW CAN WE DO BETTER?

6. HEMAGGLUTININ STALK- SPECIFIC ANTIBODIES HUMAN STALK-SPECIFIC MONOCLONAL ANTIBODIES HAVE BEEN IDENTIFIED WE CAN GENERATE MONOCLONAL ANTIBODIES OF SUCH SPECIFICITIES IN THE MOUSE NATURE USES THIS MECHANISM TO ELIMINATE “EARLIER” STRAINS – EXTINCTION OF sH1N1 BY pH1N1 VIRUSES

7. Sui, J.,Hwang, W. C., Perez, S., Wei, G., Aird, D., Chen, L. M., Santelli, E., Stec, B., Cadwell, G. Ali, M., Wan, H., Murakami, A., Yammanuru, A., Han, T., Cox, N. J., Bankston, L. A., Donis, R. O., Liddington, R. C., Marasco, W. A. (2009) Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16: 265-273. CROSS-REACTIVE ANTIBODY BINDS TO STALK REGION OF HEMAGGLUTININ

8. Strategy for boosting the antibody response against the conserved regions (grey) of the influenza virus hemagglutinin Wang et al., Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathogens 2010

9. Seasonal H1N1 (sH1N1/pre-2009) sH1N1/pre-2009 Pandemic H1N1 (pH1N1/2009) Elimination of seasonal H1N1 viruses by anti-stalk antibodies

10. ASSAY FOR STALK SPECIFIC ANTIBODIES USING CHIMERIC HEMAGGLUTININS

11. REASSORTANT VIRUSES EXPRESS CHIMERIC HEMAGGLUTININ cH9/1 Rong Hai

12. PATIENTS INFECTED WITH pH1N1 HAVE HIGHER TITERS OF ANTIBODIES THAT NEUTRALIZE cHA-EXPRESSING VIRUS Natalie Pica and Dirk Eggink cH9/1N3 virus

13. SUMMARY We have developed analytical tools that allow us to detect and quantify antibodies that bind the hemagglutinin protein and neutralize influenza virus but do not have hemagglutination inhibition activity Influenza virus pH1N1 infected patients have higher titers of stalk-specific antibodies than uninfected controls Increased titers of stalk antibodies in the population infected with pH1N1 serve as an explanation of why sH1N1 viruses have disappeared from circulation

14. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

15. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Y Y Y Y Y Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

16. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Y Y Y Y Y Y Y Y Y Y Y Y Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

17. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

18. A/Netherlands/602/09 pH1N1 challenge positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA

19. A/Fort Monmouth/1/47 and A/Puerto Rico/8/34 H1N1 challenge positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA

20. H5N1 and H6N1 challenge positive control (matched inactivated) cH9/1 DNA + H1 protein/cH6/1 protein + cH5/1 protein/H1 protein cH9/1 DNA + BSA +BSA

21. A more realistic approach – mimicking pre-existing immunity as it is present in the human population A/cHA expressing influenza B virus as tool to mimic pre-existing immunity in the mouse model No background immunity against internal viral proteins and neuraminidase Control groups: BcH9/1+ BSA + BSA Bwt + BSA + BSA naïve (neg. contr.) matched vaccine (pos. contr.)

22. Mechanism of action

23. Alternative mechanisms of neutralization

24. CD8 T-cell depletion and PR8 challenge positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA

25. Targeting group 2 HAs

26. H7N9

27. Group 1-Group 2 cross-reactivity is not sufficient to protect from virus challenge Phil82 (H3N2) ELISA Phil82 (H3N2) challenge

28. Group 2 proof of concept cH4/3 DNAcH5/3 protein boost cH7/3 protein boost (H3 protein for the H7 challenge) ma Phil82 (H3) ma X31 (H3) Rhea (H7) challenge Control groups: cH4/3 DNA + BSA + BSA naïve (neg. contr.) matched vaccine (pos. contr.) 4 weeks 3 weeks

29. A/Philippines/2/82 H3N2 challenge ***, p=0.0008 N=1

30. X-31 H3N2 and H7N1 challenge ***, p=0.0001 **, p=0.0088

31. Conclusions from proof of principle studies A broadly protective immune response to the stalk domain can be induced by vaccine constructs in mice Chimeric HA constructs protect mice from challenge with heterologous and heterosubtypic virus strains The observed protection is antibody mediated Good protection against the new Chinese H7N9 strain is expected, data will be generated in the coming weeks

32. Ferrets (Mustela putorius furo)

33. Vaccination scheme pre-prime bleedpost-prime bleed post-boost bleed ~ 3 weeks ~ 5 weeks A/Netherlands/602/09 challenge AdV cH6/1 VSV cH5/1B cH9/1 infection Control groups: B wild type infection VSV-GFP Adenovirus-GFP Prime: 10 7 PFU/ferret Challenge: 10 4 PFU/ferret

34. Challenge virus titers A/Netherlands/602/09 (pH1N1) virus

35. Conclusions Immunization with chimeric hemagglutinins induces broadly protective antibodies directed against the stalk domain Vaccinated animals are protected against homologous, heterologous as well as heterosubtypic challenge Natalie Pica, Florian Krammer, Matthew Miller, Adolfo García-Sastre Taia Wang, Rong Hai, Irina Margine, Peter Palese Randy Albrecht

37. Induction of a broadly neutralizing immune response against influenza HA by novel universal influenza virus vaccine constructs Y Y Y Y Y Y Y Y Y Y Globular head domain (immuno-dominant) Previous exposure Pre-existing memory Annual vaccination with similar strain Chimeric HA Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Vaccination with universal vaccine constructs Stalk domain Boost of anti-globular head antibodies Boost of anti-stalk antibodies Y

38. A proof of principle: Protective levels of stalk-reactive antibodies can be induced by chimeric HA constructs in mice cH9/1 DNA cH6/1 protein cH5/1 protein PR/8/34 H1N1 FM/1/47 H1N1 or pH1N1 (2009) challenge Controls: cH9/1 DNA plus 2x BSA (neg. contr.) inactivated challenge virus (pos. contr.)

39. pH1N1(2009) challenge positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + ch5/1 protein cH9/1 DNA + BSA +BSA challenge dose: 10 LD50

40. positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + ch5/1 protein cH9/1 DNA + BSA +BSA FM/1/47 and PR/8/34 challenge CD8 T-cell depletion and PR/8/34 challenge challenge dose: 5 LD50

41. Naïve Positive control WT fluB +BSA+BSA fluB cH9/1 + cH6/1 + cH5/1 (or PR8) fluB cH9/1 + BSA + BSA pH1N1(2009) and H5N1 challenge challenge dose: 250 (pH1N1) and 5 LD50 (H5N1) Passive transfer and PR/8/34 challenge passive transfer challenge dose: 5 LD50

42. What about H3/group 2 HA vaccines in mice? cH4/3 DNAcH5/3 protein boost cH7/3 protein boost (PerthH3 protein for the H7 challenge) 5 LD50 ma Phil82 (H3) 5 LD50 ma X31 (H3) 5 LD50 Rhea (H7) challenge Control groups: cH4/3 DNA + BSA + BSA naïve (neg. contr.) matched vaccine (pos. contr.) 4 weeks 3 weeks

43. Phil82 H3N2 challenge (5 LD50) Day post infection % weight loss % survival N=1 SurvivalWeight loss

44. X31(1968) H3N2 challenge (5 LD50) Day post infection % survival A/Rhea/NC/39482/93 H7N1 challenge (5 LD50) Day post infection % survival

45. cH5/3 N1 virus challenge (100 LD50) Day post infection % weight loss % survival Weight lossSurvival

46. Cross-protection in vivo by passive transfer of anti-H3 stalk mAbs Mice were given 30mg/kg mAb 12D1, 39A4 or isotype control by intraperitoneal injection 1 hour prior to infection with A/Georgia/1981. Data represent lung titers from groups of 5 mice, 2 days post infection. Taia Wang

47. ACEACE BDFBDF Prophylactic treatment of mice with anti-H3 mAbs diminishes lung damage associated with viral pneumonia Lung tissue taken 4 days post infection with the A/HK/68 reassortant virus. A,B: Untreated C,D: Mice treated with mAb 39A4 E,F: Mice treated with mAb 12D1 Taia Wang

48. Mechanism of action: Red blood cell fusion assay 1) Incubate virus + mAb. 2) Add chicken red blood cells. 3) Add low pH buffer. 4) Evaluate supernatant for presence of NADPH (340nm). HK/68 virus Taia Wang

49. Reactivity of group 2 HA stalk vaccinated mice to Perth09 H3 and Shanghai13 H7 HA protein Florian Krammer and Irina Margine 5/1/13

50. Proteins used as substrate have different trimerization domains and purification tags than proteins used for vaccination. Substrate: Perth/09 H3 protein Substrate: Shanghai/13 H7 protein

51. Conclusions Immunization with chimeric hemagglutinins induces broadly protective antibodies directed against the stalk domain Vaccinated animals are protected against homologous, heterologous as well as heterosubtypic challenge Florian Krammer, Irina Margine, Rong Hai, Adolfo García-Sastre Matthew Miller, Taia Wang, Natalie Pica, Peter Palese

52. FUTURE Precise mechanism of how stalk-specific anti- hemagglutin antibodies work Hemagglutinin-specific binding antibodies and effector functions such as phagocytosis, complement activation and antibody- dependent cellular cytotoxicity (ADCC) Efficay of chimeric hemagglutinin-based vaccines in humans

53. Toward a universal influenza virus vaccine Project 1: Design of new immunogens based on conserved epitopes in the influenza virus hemagglutinin PI Peter Palese 1 st Scientific Advisory Board meeting 5/24/13

54. Conclusions from proof of principle studies A broadly protective immune response to the stalk domain can be induced by vaccine constructs in mice Chimeric HA constructs protect mice from challenge with heterologous and heterosubtypic virus strains The observed protection is antibody mediated Good protection against the new Chinese H7N9 strain is expected, data will be generated in the coming weeks

55. Toward a universal influenza virus vaccine Project 1: Design of new immunogens based on conserved epitopes in the influenza virus hemagglutinin PI Peter Palese 1 st Scientific Advisory Board meeting 5/24/13

56. Aims 1)Characterization of broadly neutralizing antibodies 2) Development of novel broad spectrum influenza virus vaccines

57. A universal influenza virus vaccine based on chimeric hemagglutinin constructs Florian Krammer, Irina Margine, Natalie Pica, Rong Hai and Peter Palese 5/24/13

58. Influenza A virus hemagglutinin adapted from Krammer and Grabherr, 2010, Trends Mol Med. Globular head domain: mediates binding to host receptors Stalk domain: mediates fusion of viral and endosomal membranes

59. Antibodies against the influenza virus HA stalk domain Ekiert et al., 2009, Science Rare and not induced/boosted upon regular seasonal vaccination Have been isolated from humans and mice Cross-reactive between HAs of different subtypes Have mostly conformational epitopes Broad neutralizing activity in vitro in passive transfer studies in animals (ferrets, mice) HI negative!! Immunodominant head domain Stalk domain

60. Alternative mechanisms of neutralization

61. The HA stalk is conserved among group 1 and among group 2 HAs Shaw and Palese, 2011, Field’s Virology

62. Chimeric hemagglutinins (cHAs) Conserved stalk domain Globular head domain H6 H1 chimeric H6/1 Hai et al., 2012, JVI

63. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

64. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Y Y Y Y Y Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

65. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Y Y Y Y Y Y Y Y Y Y Y Y Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

66. cH9/1 DNA cH6/1 protein (replace by H1 full length protein for H6N1 challenge) cH5/1 protein (replaced by H1 full length protein for H5N1 challenge) PR8 H1N1 FM1 H1N1 or pH1N1 H5N1 H6N1 challenge Control groups: cH9/1 DNA + BSA + BSA matched vaccine (pos. contr.) Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice

67. A/Netherlands/602/09 pH1N1 challenge positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA

68. A/Fort Monmouth/1/47 and A/Puerto Rico/8/34 H1N1 challenge positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA

69. H5N1 and H6N1 challenge positive control (matched inactivated) cH9/1 DNA + H1 protein/cH6/1 protein + cH5/1 protein/H1 protein cH9/1 DNA + BSA +BSA

70. How does pre-existing immunity to the HA stalk impact on the vaccine regimen?

71. A more realistic approach – mimicking pre-existing immunity as it is present in the human population A/cHA expressing influenza B virus as tool to mimic pre-existing immunity in the mouse model No background immunity against internal viral proteins and neuraminidase Control groups: BcH9/1+ BSA + BSA Bwt + BSA + BSA naïve (neg. contr.) matched vaccine (pos. contr.)

72. A/Puerto Rico/8/34 H1N1 challenge Naïve Positive control WT fluB +BSA+BSA fluB cH9/1 + cH6/1 + cH5/1 fluB cH9/1 + BSA + BSA

73. A/Fort Monmouth/1/47 and A/Netherlands/602/09 H1N1 challenge Naïve Positive control WT fluB +BSA+BSA fluB cH9/1 + cH6/1 + cH5/1 (or PR8) fluB cH9/1 + BSA + BSA H5N1 challenge

74. Mechanism of action

75. Binding to PR8 (H1N1) virusBinding to Cal09 (pH1N1) protein ELISA reactivity to H1 HA and H1N1 virus cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA naïve serum

76. Binding to H2N2 virus Binding to H5N1 virus (altered vaccination regimen with H1 instead of cH5/1 HA) ELISA reactivity to H2N2 and H5N1 virus cH9/1 DNA + cH6/1 protein + cH5/1 protein/H1 protein cH9/1 DNA + BSA +BSA naïve serum

77. Passive transfer (PR8 H1N1 challenge) Naïve Positive control WT fluB +BSA+BSA fluB cH9/1 + cH6/1 + cH5/1 (or PR8) fluB cH9/1 + BSA + BSA IgG ug/ml positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA H2 pseudotyped particle neutralization assay Protection is mediated by humoral immunity

78. CD8 T-cell depletion and PR8 challenge positive control (matched inactivated) cH9/1 DNA + cH6/1 protein + cH5/1 protein cH9/1 DNA + BSA +BSA

79. Targeting group 2 HAs

80. H7N9

81. Group 1-Group 2 cross-reactivity is not sufficient to protect from virus challenge Phil82 (H3N2) ELISA Phil82 (H3N2) challenge

82. Group 2 proof of concept cH4/3 DNAcH5/3 protein boost cH7/3 protein boost (H3 protein for the H7 challenge) ma Phil82 (H3) ma X31 (H3) Rhea (H7) challenge Control groups: cH4/3 DNA + BSA + BSA naïve (neg. contr.) matched vaccine (pos. contr.) 4 weeks 3 weeks

83. A/Philippines/2/82 H3N2 challenge ***, p=0.0008 N=1

84. X-31 H3N2 and H7N1 challenge ***, p=0.0001 **, p=0.0088

85. Mimicking a pre-existing anti-H3 stalk response in the mouse model A/cH7/3 BcH7/3 exposure cH5/3 protein boost (H3 protein for the cH5/3 challenge) cH4/3 protein boost cH5/3N1 A/Philippines/82 (H3N2) X31 (H3N2) Control groups: Bwt+ BSA + BSA naïve (neg. contr.) matched vaccine (pos. contr.) BcH7/3 + BSA + BSA

86. cH5/3N1 challenge (A/Perth/16/09 H3 stalk) **, p=0.0082 N=2

87. A/Philippines/2/82 H3N2 and X-31 H3N2 challenge ****, p<0.0001

88. Lung titers

89. Mechanism of action

90. ELISA reactivity to H3N2 and H7N1 viruses and two HA proteins of H3N2 and H7N9 origin Substrate: A/rhea/NC/39482/93 virus (H7N1) virus Substrate: A/Victoria/361/11 (H3N2) virus Substrate: A/Perth/16/09 H3 protein Substrate: A/Shanghai/1/13 H7 protein

91. Mucosal IgA and Ig subtype distribution Reciprocal nasal wash dilution

92. Passive transfer and entry inhibition assay Passive transfer (Phil82 challenge) Pseudotyped particle entry assay (Vic11)

93. Conclusions from proof of principle studies A broadly protective immune response to the stalk domain can be induced by vaccine constructs in mice Chimeric HA constructs protect mice from challenge with heterologous and heterosubtypic virus strains The observed protection is antibody mediated Good protection against the new Chinese H7N9 strain is expected, data will be generated in the coming weeks

94. Ferrets (Mustela putorius furo)

95. Vaccination scheme pre-prime bleedpost-prime bleed post-boost bleed ~ 3 weeks ~ 5 weeks A/Netherlands/602/09 challenge AdV cH6/1 VSV cH5/1B cH9/1 infection Control groups: B wild type infection VSV-GFP Adenovirus-GFP Prime: 10 7 PFU/ferret Challenge: 10 4 PFU/ferret

96. ELISA data after vaccination

97. Challenge virus titers A/Netherlands/602/09 (pH1N1) virus

98. Influenza viruses circulating in the human population