Topics

1. Topics - Variation of receptor specificity of influenza viruses in different avian species - Receptor specificity and cell tropism of H1N1pdm and of its mutants associated with severe cases of infection

2. 2-3 2-3 2-3 2-6 2-3 2-3 2-6 2-3 2-6 2-6 2-3 2-3 3- and 6-linked Sia receptors in different species

3. Nikolai Bovin Institute of Bio-organic Chemistry, Moscow Characterization of receptor-binding specificity of avian influenza viruses, 2002  present Alexandra Gambaryan, Nikolai Bovin, Mikhail Matrosovich, Svetlana Yamnikova, Dmitri Lvov, Alexander Klimov, Robert Webster, Ilaria Capua, Jiovanni Cattoli, Ron Fouchier, Vincent Munster, Jill Banks et al., Alexandra Gambaryan M.P.Chumakov Institute of Poliomyelitis, Moscow

4. A panel of sialylglycopolymers (Nikolai Bovin’s lab)

5. Receptor specificity of avian viruses Fuc Fuc (H9 and H7)

6. 193 Fuc 193 Su 222 222 Ionic bond between sulfate (type II core) and conserved lysine/arginine in position 193 (H5, H7, some H13) Molecular features that determine binding of avian viruses to sulfated and fucosylated receptors Sia Fuc Sia No sterical conflicts between fucose and amino acid in position 222 (gull viruses, some poultry viruses) Crystal structure of H7 HA: Russell, Gamblin, Skehel et al.

7. - Receptor specificity of influenza viruses is not limited to recognition of the type of Sia-Gal linkage. • Receptor specificity of avian viruses is not uniform. Viruses of gulls and poultry are closer to each other than to duck viruses. This may facilitate virus exchange between gulls and poultry.

8. Expression of distinct 3-linked sequences varies depending on the animal species, tissue and cell type. Avian viruses may differ by their tissue and cell tropism in humans. • 6-linked Sia are present on epithelial cells in galliformes; evolution of viruses in these species may increase binding to receptors in humans.

9. Further studies are needed to characterize role of receptor specificity of avian viruses in their host-range, tissue tropism and pathogenicity

10. Alan Hay Ten Feizi

11. The neoglycolipid-based carbohydrate microarrays (Ten Feizi lab, Imperial College London, UK) Liu et al., Biol.Chem. 2009 • Sequence-defined natural and synthetic oligosaccharides are coupled to synthetic lipid to allow efficient immobilization on solid supports. • Neoglycolipids are spotted on nitrocellulose-coated glass slides. • The lipid-linked probes have some lateral mobility. Clustering of glycolipids allows their optimal multivalent presentation that mimics presentation at the cell surface in vivo.

12. Examples of binding profiles Virus 1 Virus 2 2-3 2-6 2-8 2-3 2-6 2-8 Examples of glycan probes 80 sialic acid-containing oligosaccharide probes with differing backbone types, chain lengths and branching patterns 2-3, 2-6, 2-8-linked sialyl epitopes and their combinations (depicted by different colors) 6 neutral probes (negative control; gray)

13. Binding profiles H1N1pdm (Hamburg/5/09) H1N1 seasonal (Memphis/14/96) H3N2 pandemic (Aichi/2/68) 2-3 2-6 2-8 2-3 2-6 2-8 2-3 2-6 2-8 • H1N1pdm, similar to 1918, 1957 and 1968 pandemic viruses, preferentially binds to 6-linked Sia • - H1N1pdm differs from seasonal H1N1 virus by a broader binding specificity and by low-avidity binding to 3-linked Sia

14. No binding of H1N1pdm to 2-3-probes in several assays Maines et al., Science 2009 Yang, Carney & Stevens, PLoS Curr Influenza 2010 3. Binding of soluble probes, 3- and 6-fetuin, to the solid-phase immobilized virus 1. 2. Blue – 2-3 probes, red: 2-6 probes Binding to the glycoarray of recombinant HA protein

15. Neoglycolipid-based arrays:Utilization of whole virus particles and clustered glycolipid probes could facilitate detection of low-affinity polyvalent interactions H1N1pdm H1N1 seasonal Biological significance of binding to 2-3 Sia ?

16. 6- and 3-linked Sia in human respiratory tract Binding: Lectins 2-6 (green), 2-3 (red) Viruses Nasal and tracheo-bronchial epithelial cells mainly express 6-linked Sia. Binding to 6-linked receptors seems to be essential for virus replication in the upper respiratory tract and efficient human-to-human transmission. 3-linked Sia are mainly present in bronchioles and alveoli. Binding to 3-linked receptors could facilitate virus replication in the lower respiratory tract and increase pathogenicity. Shinya et al. 2006 van Riel et al., 2007

17. Can low-avidity binding of H1N1pdm to 3-linked Sia contribute to viral capacity to causesevere disease in humans ?

18. HA polymorphism in position 222 (225, H3 numb.) • Major variant, 222D. Substitutions G,E,N. • 222G found in 7-10% of sequences in fatal and severe cases, but not in clinically mild cases. The mutants seem to occur sporadically with no evidence of sustained transmission. • 222E – No apparent correlation with disease severity; transmissible virus. • 222N – Not enough data.

19. Two of five HA sequences from the victims of the 1918 pandemic had mutation D222G

20. Amino acid 222 • Correlates with the virus host species. Avian viruses have 222G, human and swine viruses have 222D/E. Propagation of human viruses in hen’s eggs often leads to mutations D222G/N. • Mutation D222G increases binding to 3-linked receptors of human viruses(Gambaryan et al., 1997,1999; Glaser et al., 2005; Stevens et al., 2006),including H1N1pdm(Yang, Carney & Stevens, PLoS Curr Influenza 2010). • Mutation D222G decreased airborne transmission of the 1918 virus in ferrets(Tumpey et al., 2007).

21. Can mutations D222G/E in H1N1pdm change viral cell tropism and replication efficiency in human respiratory tract ?

22. Immuno-staining of cilia in fixed culture Primary airway epithelial cells grow on membrane supports at air-liquid interface Cross-section of 6-wk-old culture Beating of cilia in live culture Cultures of differentiated human tracheo-bronchial epithelial cells (HTBE) - an in vitro model of human airway epithelium

23. 6- and 3-linked sialic acids in HTBE cultures 2-62-3 Sambucus nigra agglutinin Maackia amurensis lectin SNAMAL-1 Expression pattern agrees with that in vivo(Baum and Paulson, 1990; Gagneux et al., 2003; Shinya et al., 2006; Nicholls et al., 2007; Yao et al., 2007)

24. Human and avian viruses target different types of cells Red, viral antigen; gray, cilia of ciliated cells Seasonal human virus Avian virus <5 % of infected ciliated cells >70

25. Cell tropism of H1N1pdm in HTBE cultures 222D: 222G: Moldova/G186/09, Cyprus/S2487/09 Lviv/N6/09 (fatal), Hamburg/5/09 Norway/3206-3/09 (fatal) 222E: Hamburg/5/09-e(egg-derived) Dakar/37/09 3-5 % of infected ciliated cells 20-30

26. Receptor specificity, glycoarraysD222G mutation increases binding to 3-linked Sia 2-6 2-3 2-6 2-3

27. Binding to resialylated soluble 3- and 6-fetuinD222G mutation decreases binding to 6-linked Sia Tropism, % ciliated cells 3-5 20-30 Circulation in humans Yes No

28. Replication kinetics in HTBE culturesHamburg/5/09 (H1N1pdm) vs its D222G mutant wt (222D) D222G * *p=0.0001

29. Sharon Brookes Ian Brown

30. Selection of receptor-binding variants during replication and transmission in pigs Inoculum: Egg-grown A/California/7/09 wt + D222G + Q223R wt + 222G wt (222D) Direct infection Transmission 3 - 6 dpi wt + 222G 222G Middle lung lobe tissues 1 and 2 dpi 4 and 7 dpi Figure modified from Brookes et al., 2010

31. Expression of 3- and 6-Sia in pigs is similar to that in humans (van Riel et al., 2007; Van Poucke et al., 2010; Nelli et al., 2010) 2,6 (green); 2,3 (red) Humans Pigs Shinya et al. 2006 Nelli et al. BMC Veterinary Research, 2010

32. I.Mutation D222Ehas marginal effect on receptor specificity and does not affect viral cell tropism in HTBE cultures. This is consistent with apparent unrestricted circulation of the variant in humans and with lack of correlation of 222E with severe disease. II. Mutation D222Greduces viral replication efficiency in HTBE cultures and prevents transmissibility in experimentally infected pigs. These findings could explain why the mutants do not transmit in humans.

33. III.Mutation D222G alters viral receptor specificity and cell tropism in human epithelium, provides the mutant with replicative advantage in the LRT of pigs. Potential explanations of association of D222G with severe disease:- Mutation causes severe disease owing to propensity of the mutant to reach and infect the LRTor/and- Mutation emerges as a consequence of severe disease owing to the virus replication in the LRT

34. It is important to closely monitor position 222 mutants and other potential mutants with altered receptor specificity and cell tropism

35. Tatyana Matrosovich Jennifer Uhlendorff Markus Eickmann Hans-Dieter Klenk Institute of Virology, Philipps University, Marburg, Germany

36. Collaborators M.P.Chumakov Institute of Poliomyelitis, Moscow, Russia Alexandra Gambaryan Carbohydrate laboratory, Institute of Bio-organic Chemistry, Moscow, Russia Nikolai Bovin Alexander Tuzikov Galina Pazynina D.I.Ivanovsky Institute of Virology, Moscow, Russia Svetlana Yamnikova Dmitri Lvov Natalia Lomakina Influenza Division, CDC, Atlanta, GA, USA Alexander Klimov Amanda Balish St.Jude Children’s Research Hospital, Memphis, TN, USA Robert Webster Scott Krauss Erasmus Medical Center, Rotterdam, The Netherlands Ron Fouchier Vincent Munster Members of EU Concortium FLUPATH Ilaria Capua Giovanni Cattoli Jill Banks Glycosciences laboratory, Faculty of Medicine, Imperial College, London, UK Ten Feizi Yan Liu Robert Childs Angelina Palma Wengang Chai Maria Campanero-Rhodes Yibing Zhang Division of Virology, MRC NIMR, Mill Hill, London, UK Alan Hay Steve Warton Rod Daniels Vicky Gregory VLA,Weybridge, Addlestone, Surrey, UK Ian Brown Sharon Brookes Alejandro Nunez Bhudipa Choudhury Stephen Essen Derek Clifford Marek Slomka Fanny Garson Bethany Nash Amanda Hanna Rebecca Gardner Richard Irvine Members of EU Concortium ESNIP-2 Emanuela Foni Gaelle Kuntz-Simon Michel Bublot Jaime Maldonado Garcia Willie Loefflen Kristien Van Reeth

37. Thanks to: Grant support: FLUPATH, EU Wellcome Trust, UK SFB 593, Germany LOEWE UGLMC, Hesse, Germany von Behring-Röntgen-Stiftung, Germany WHO Global Influenza Network

38. Marburg

40. Virus binding to selected 2-3 Sia sequences V V % infected ciliated cells3-5 20-30