- Variation of receptor specificity of influenza viruses in - Variation of receptor specificity of influenza viruses in

different avian speciesdifferent avian species

- Receptor specificity and cell tropism of H1N1pdm and of - Receptor specificity and cell tropism of H1N1pdm and of

its mutants associated with severe cases of infectionits mutants associated with severe cases of infection

Topics Topics

3- and 6-linked Sia receptors in different species3- and 6-linked Sia receptors in different species

2-3

2-32-3

2-6

2-62-32-3

2-6

2-3

2-32-3 2-6

Characterization of receptor-binding specificity of avian Characterization of receptor-binding specificity of avian influenza viruses, 2002 influenza viruses, 2002 present present

Alexandra Gambaryan, Nikolai Bovin, Mikhail Matrosovich, Svetlana Alexandra Gambaryan, Nikolai Bovin, Mikhail Matrosovich, Svetlana Yamnikova, Dmitri Lvov, Alexander Klimov, Robert Webster, Ilaria Yamnikova, Dmitri Lvov, Alexander Klimov, Robert Webster, Ilaria

Capua, Jiovanni Cattoli, Ron Fouchier, Vincent Munster, Jill Banks et al., Capua, Jiovanni Cattoli, Ron Fouchier, Vincent Munster, Jill Banks et al.,

Alexandra GambaryanAlexandra Gambaryan

M.P.Chumakov Institute of M.P.Chumakov Institute of Poliomyelitis, MoscowPoliomyelitis, Moscow

Nikolai BovinNikolai Bovin

Institute of Bio-organic Institute of Bio-organic Chemistry, MoscowChemistry, Moscow

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

Receptor specificity of avian viruses Receptor specificity of avian viruses

(H9 and H7)

Fuc

Fuc

Molecular features that determine binding of avian viruses to sulfated Molecular features that determine binding of avian viruses to sulfated and fucosylated receptorsand fucosylated receptors

No sterical conflicts between fucose and No sterical conflicts between fucose and amino acid in position 222 amino acid in position 222 (gull viruses, some poultry viruses)(gull viruses, some poultry viruses)

Ionic bond between sulfate (type II core) and Ionic bond between sulfate (type II core) and conserved lysine/arginine in position 193 conserved lysine/arginine in position 193 (H5, H7, some H13)(H5, H7, some H13)

193193

222222

SuSu

193193

222222

FucFuc

FucFuc

SiaSia

SiaSia

Crystal structure of H7 HA: Russell, Gamblin, Skehel et al. Crystal structure of H7 HA: Russell, Gamblin, Skehel et al.

- Receptor specificity of influenza viruses is not limited - Receptor specificity of influenza viruses is not limited

to to recognition of the type of Sia-Gal linkage. recognition of the type of Sia-Gal linkage.

- Receptor specificity of avian viruses is not uniform. Receptor specificity of avian viruses is not uniform.

Viruses of gulls and poultry are closer to each other than Viruses of gulls and poultry are closer to each other than

to duck viruses. This may facilitate virus exchange to duck viruses. This may facilitate virus exchange

between gulls and poultrybetween gulls and poultry. .

- Expression of distinct 3-linked sequences varies Expression of distinct 3-linked sequences varies

depending on the animal species, tissue and cell type. depending on the animal species, tissue and cell type.

Avian viruses may differ by their tissue and cell tropism Avian viruses may differ by their tissue and cell tropism

in humans.in humans.

- 6-linked Sia are present on epithelial cells in 6-linked Sia are present on epithelial cells in

galliformes; evolution of viruses in these species may galliformes; evolution of viruses in these species may

increase binding to receptors in humans. increase binding to receptors in humans.

Further studies are needed to Further studies are needed to

characterize role of receptor specificity characterize role of receptor specificity

of avian viruses in their host-range, of avian viruses in their host-range,

tissue tropism and pathogenicitytissue tropism and pathogenicity

Ten FeiziTen Feizi

Alan HayAlan Hay

The neoglycolipid-based carbohydrate microarrays The neoglycolipid-based carbohydrate microarrays (Ten Feizi lab, Imperial College London, UK)(Ten Feizi lab, Imperial College London, UK)

- Sequence-defined natural and synthetic Sequence-defined natural and synthetic

oligosaccharides are coupled to synthetic lipid oligosaccharides are coupled to synthetic lipid

to allow efficient immobilization on solid to allow efficient immobilization on solid

supports. supports.

- Neoglycolipids are spotted on nitrocellulose-Neoglycolipids are spotted on nitrocellulose-

coated glass slides.coated glass slides.

- The lipid-linked probes have some lateral The lipid-linked probes have some lateral

mobility. Clustering of glycolipids allows their mobility. Clustering of glycolipids allows their

optimal multivalent presentation that mimics optimal multivalent presentation that mimics

presentation at the cell surface in vivo.presentation at the cell surface in vivo.Liu et al., Biol.Chem. 2009Liu et al., Biol.Chem. 2009

Examples of binding profilesExamples of binding profiles

80 sialic acid-containing oligosaccharide probes with differing backbone types, chain 80 sialic acid-containing oligosaccharide probes with differing backbone types, chain

lengths and branching patternslengths and branching patterns

2-3, 2-6, 2-8-linked sialyl epitopes and their combinations (depicted by different colors)2-3, 2-6, 2-8-linked sialyl epitopes and their combinations (depicted by different colors)

6 neutral probes (negative control; gray) 6 neutral probes (negative control; gray)

Examples of glycan probesExamples of glycan probes

2-3 2-6 2-8 2-3 2-6 2-8

Virus 1 Virus 2

Binding profilesBinding profiles

H1N1pdmH1N1pdm (Hamburg/5/09) (Hamburg/5/09) H1N1 seasonalH1N1 seasonal (Memphis/14/96) (Memphis/14/96) H3N2 pandemicH3N2 pandemic (Aichi/2/68) (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, H1N1pdm, similar to 1918, 1957 and 1968 pandemic viruses, preferentially binds to 6-linked Siapreferentially binds to 6-linked Sia

- H1N1pdm differs from seasonal H1N1 virus by a broader binding - H1N1pdm differs from seasonal H1N1 virus by a broader binding specificity and by low-avidity binding to 3-linked Siaspecificity and by low-avidity binding to 3-linked Sia

No binding of H1N1pdm to 2-3-probes in several assaysNo binding of H1N1pdm to 2-3-probes in several assaysBlue – 2-3 probes, red: 2-6 probes

Maines et alMaines et al.,., Science 2009 Science 2009 Yang, Carney & Stevens, PLoS Curr Influenza 2010Yang, Carney & Stevens, PLoS Curr Influenza 2010

1.1. 2.2.

0

10

20

30

40

50

Ham/09(H1N1v)

Mem/96 (H1N1) HK/68 (H3N2) duck/98 (H1N1)

Kas

s, 1

/uM

Neu

5Ac

2-3-fetuin 2-6-fetuin

Binding of Binding of soluble probes, 3- and 6-fetuin,soluble probes, 3- and 6-fetuin, to the solid-phase immobilized virus to the solid-phase immobilized virus

3.3.

Binding to the glycoarray of Binding to the glycoarray of recombinant HA proteinrecombinant HA protein

Neoglycolipid-based arrays:Neoglycolipid-based arrays:Utilization of whole virus particles and clustered glycolipid probes Utilization of whole virus particles and clustered glycolipid probes

could facilitate detection of low-affinity polyvalent interactionscould facilitate detection of low-affinity polyvalent interactions

H1N1pdm H1N1 seasonalH1N1pdm H1N1 seasonal

Biological significance of binding to 2-3 Sia ?Biological significance of binding to 2-3 Sia ?

6- and 3-linked Sia in human respiratory tract6- and 3-linked Sia in human respiratory tract

Shinya et al. 2006 van Riel et al., 2007Shinya et al. 2006 van Riel et al., 2007

Binding:Binding:

Lectins Lectins 2-6 (green), 2-3 (red) 2-6 (green), 2-3 (red) Viruses Viruses

Nasal and tracheo-bronchial epithelial Nasal and tracheo-bronchial epithelial cells mainly express 6-linked Sia. cells mainly express 6-linked Sia. Binding Binding to 6-linked receptors seems to be to 6-linked receptors seems to be essential for virus replication in the upper essential for virus replication in the upper respiratory tract and efficient human-to-respiratory tract and efficient human-to-human transmission.human transmission.

3-linked Sia are mainly present in 3-linked Sia are mainly present in bronchioles and alveoli. bronchioles and alveoli. Binding to 3-Binding to 3-linked receptors could facilitate virus linked receptors could facilitate virus replication in the lower respiratory tract replication in the lower respiratory tract and increase pathogenicity.and increase pathogenicity.

Can low-avidity binding of H1N1pdm Can low-avidity binding of H1N1pdm

to 3-linked Sia contribute to viral to 3-linked Sia contribute to viral

capacity to causecapacity to cause

severe disease in humans ?severe disease in humans ?

HA polymorphism in position 222 HA polymorphism in position 222 (225, H3 numb.)(225, H3 numb.)

- Major variant, Major variant, 222D222D. Substitutions . Substitutions G,E,N. G,E,N.

- 222G222G found in 7-10% of sequences in fatal and severe cases, but not in clinically found in 7-10% of sequences in fatal and severe cases, but not in clinically mild cases. mild cases. The mutants seem to occur sporadically with no evidence of The mutants seem to occur sporadically with no evidence of sustained transmission.sustained transmission.

- 222E222E – No apparent correlation with disease severity; – No apparent correlation with disease severity; transmissible virus.transmissible virus.

- 222N – Not enough data. 222N – Not enough data.

Two of five HA sequences Two of five HA sequences

from the victims of the from the victims of the

1918 pandemic had 1918 pandemic had

mutation D222Gmutation D222G

Amino acid 222 Amino acid 222

- Correlates with the virus host species. Avian viruses have 222G, human and swine viruses 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 have 222D/E. Propagation of human viruses in hen’s eggs often leads to mutations D222G/N.D222G/N.

- Mutation D222G increases binding to 3-linked receptors of human virusesMutation D222G increases binding to 3-linked receptors of human viruses (Gambaryan et al., (Gambaryan et al.,

1997,1999; Glaser et al., 2005; Stevens et al., 2006),1997,1999; Glaser et al., 2005; Stevens et al., 2006), including H1N1pdmincluding H1N1pdm ((Yang, Carney & Stevens, Yang, Carney & Stevens,

PLoS Curr Influenza 2010)PLoS Curr Influenza 2010)..

- Mutation D222G decreased airborne transmission of the 1918 virus in ferretsMutation D222G decreased airborne transmission of the 1918 virus in ferrets (Tumpey et al., (Tumpey et al.,

2007)2007)..

Can mutations D222G/E in H1N1pdm Can mutations D222G/E in H1N1pdm

change viral cell tropism and replication change viral cell tropism and replication

efficiency in human respiratory tract ?efficiency in human respiratory tract ?

Cultures of differentiated human tracheo-bronchial epithelial Cultures of differentiated human tracheo-bronchial epithelial

cells (HTBE) - an cells (HTBE) - an in vitroin vitro model of human airway epithelium model of human airway epithelium

Immuno-staining of cilia in fixed cultureImmuno-staining of cilia in fixed culture

Beating of cilia in live cultureBeating of cilia in live culture

Cross-section of 6-wk-old culture

Primary airway epithelial cells grow on membrane Primary airway epithelial cells grow on membrane supports at air-liquid interfacesupports at air-liquid interface

6- and 3-linked sialic acids in HTBE cultures6- and 3-linked sialic acids in HTBE cultures

Sambucus nigraSambucus nigra agglutinin agglutinin Maackia amurensisMaackia amurensis lectin lectin SNASNA MAL-1 MAL-1

2-62-6 2-32-3

Expression pattern agrees with that Expression pattern agrees with that in vivoin vivo ((Baum and Paulson, 1990; Baum and Paulson, 1990;

Gagneux et al., 2003; Shinya et al., 2006; Nicholls et al., 2007; Yao et al., 2007Gagneux et al., 2003; Shinya et al., 2006; Nicholls et al., 2007; Yao et al., 2007) )

Human and avian viruses target different types of cellsHuman and avian viruses target different types of cellsRedRed, viral antigen; , viral antigen; graygray, cilia of ciliated cells, cilia of ciliated cells

Seasonal human virus Avian virusSeasonal human virus Avian virus

<5 <5 % of infected ciliated cells % of infected ciliated cells >70 >70

Cell tropism of H1N1pdm in HTBE culturesCell tropism of H1N1pdm in HTBE cultures

222D: 222D: 222G: 222G:

Moldova/G186/09, Cyprus/S2487/09Moldova/G186/09, Cyprus/S2487/09 Lviv/N6/09 (fatal), Lviv/N6/09 (fatal),

Hamburg/5/09Hamburg/5/09 Norway/3206-3/09 (fatal)Norway/3206-3/09 (fatal)

222E: 222E: Hamburg/5/09Hamburg/5/09-e-e (egg-derived)(egg-derived)

Dakar/37/09Dakar/37/09

3-5 3-5 % of infected ciliated cells % of infected ciliated cells 20-3020-30

Receptor specificity, glycoarraysReceptor specificity, glycoarrays D222G mutation increases binding to 3-linked SiaD222G mutation increases binding to 3-linked Sia

2-6

2-3

2-6

2-3

Binding to resialylated soluble 3- and 6-fetuinBinding to resialylated soluble 3- and 6-fetuinD222G mutation decreases binding to 6-linked SiaD222G mutation decreases binding to 6-linked Sia

Tropism, Tropism, % ciliated cells% ciliated cells 3-5 20-30 3-5 20-30Circulation in humans Yes NoCirculation in humans Yes No

Replication kinetics in HTBE culturesReplication kinetics in HTBE culturesHamburg/5/09 (H1N1pdm) vs its D222G mutantHamburg/5/09 (H1N1pdm) vs its D222G mutant

wt (222D) D222G

*

*p=0.0001

Sharon Brookes Ian BrownSharon Brookes Ian Brown

Selection of receptor-binding variants during Selection of receptor-binding variants during replication and transmission in pigsreplication and transmission in pigs

Inoculum:Inoculum: Egg-grown A/California/7/09 Egg-grown A/California/7/09

Direct infectionDirect infection

TransmissionTransmission

Middle lung Middle lung

lobe tissueslobe tissues

1 and 2 dpi1 and 2 dpi

wt + 222Gwt + 222G

wt (wt (222D222D) ) wt + 222G wt + 222G

4 and 7 dpi4 and 7 dpi

222G222G

wt + D222G + Q223Rwt + D222G + Q223R

3 - 6 dpi3 - 6 dpi

Figure modified from Figure modified from Brookes et al., 2010Brookes et al., 2010

Expression of 3- and 6-Sia in pigs is similar to that in humans 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)(van Riel et al., 2007; Van Poucke et al., 2010; Nelli et al., 2010)

Humans Pigs Humans Pigs

Shinya et al. 2006 Shinya et al. 2006 N Nelli et al. BMC Veterinary Research, 2010elli et al. BMC Veterinary Research, 2010

2,6 (green); 2,3 (red)2,6 (green); 2,3 (red)

I.I. Mutation D222EMutation D222E has marginal effect on receptor has marginal effect on receptor

specificity and does not affect viral cell tropism in HTBE specificity and does not affect viral cell tropism in HTBE

cultures.cultures. This is consistent with apparent unrestricted This is consistent with apparent unrestricted

circulation of the variant in humans and with lack of circulation of the variant in humans and with lack of

correlation of 222E with severe disease. correlation of 222E with severe disease.

II. Mutation D222GII. Mutation D222G reduces viral replication efficiency in reduces viral replication efficiency in

HTBE cultures and prevents transmissibility in HTBE cultures and prevents transmissibility in

experimentally infected pigs.experimentally infected pigs. These findings could These findings could

explain why the mutants do not transmit in humans. explain why the mutants do not transmit in humans.

III.III. Mutation D222G alters viral receptor specificity and Mutation D222G alters viral receptor specificity and cell tropism in human epithelium, provides the mutant cell tropism in human epithelium, provides the mutant with replicative advantage in the LRT of pigs.with replicative advantage in the LRT of pigs.

Potential explanations of association of D222G Potential explanations of association of D222G with severe disease:with severe disease:

- Mutation - Mutation causes severe diseasecauses severe disease owing to propensity owing to propensity of the mutant to reach and infect the LRTof the mutant to reach and infect the LRT

or/andor/and- Mutation - Mutation emerges as a consequence of severe emerges as a consequence of severe diseasedisease owing to the virus replication in the LRT owing to the virus replication in the LRT

It is important to closely monitor position 222 It is important to closely monitor position 222

mutants and other potential mutants with mutants and other potential mutants with

altered receptor specificity and cell tropismaltered receptor specificity and cell tropism

Hans-Dieter KlenkHans-Dieter Klenk

Markus EickmannMarkus Eickmann Jennifer UhlendorffJennifer UhlendorffTatyana MatrosovichTatyana Matrosovich

Institute of Virology, Philipps University, Marburg, GermanyInstitute of Virology, Philipps University, Marburg, Germany

M.P.Chumakov Institute of M.P.Chumakov Institute of Poliomyelitis, Moscow, RussiaPoliomyelitis, Moscow, RussiaAlexandra GambaryanAlexandra Gambaryan

Carbohydrate laboratory, Carbohydrate laboratory, Institute of Bio-organic Institute of Bio-organic Chemistry, Moscow, RussiaChemistry, Moscow, RussiaNikolai BovinNikolai BovinAlexander TuzikovAlexander TuzikovGalina PazyninaGalina Pazynina

D.I.Ivanovsky Institute of D.I.Ivanovsky Institute of Virology, Moscow, RussiaVirology, Moscow, RussiaSvetlana YamnikovaSvetlana YamnikovaDmitri LvovDmitri LvovNatalia LomakinaNatalia Lomakina

Influenza Division, CDC, Influenza Division, CDC, Atlanta, GA, USA Atlanta, GA, USA Alexander KlimovAlexander KlimovAmanda BalishAmanda Balish

St.Jude Children’s Research St.Jude Children’s Research Hospital, Memphis, TN, USAHospital, Memphis, TN, USARobert WebsterRobert WebsterScott KraussScott Krauss

Erasmus Medical Center,Erasmus Medical Center,Rotterdam, The NetherlandsRotterdam, The NetherlandsRon FouchierRon FouchierVincent MunsterVincent Munster

Members of EU Concortium Members of EU Concortium FLUPATHFLUPATHIlaria CapuaIlaria CapuaGiovanni Cattoli Giovanni Cattoli Jill BanksJill Banks

VLA,Weybridge, Addlestone, VLA,Weybridge, Addlestone, Surrey, UKSurrey, UKIan BrownIan BrownSharon BrookesSharon BrookesAlejandro NunezAlejandro NunezBhudipa ChoudhuryBhudipa ChoudhuryStephen EssenStephen EssenDerek CliffordDerek CliffordMarek SlomkaMarek SlomkaFanny GarsonFanny GarsonBethany NashBethany NashAmanda HannaAmanda HannaRebecca GardnerRebecca GardnerRichard IrvineRichard Irvine

Members of EU Concortium Members of EU Concortium ESNIP-2ESNIP-2Emanuela FoniEmanuela FoniGaelle Kuntz-SimonGaelle Kuntz-SimonMichel BublotMichel BublotJaime Maldonado GarciaJaime Maldonado GarciaWillie LoefflenWillie LoefflenKristien Van ReethKristien Van Reeth

Glycosciences laboratory, Glycosciences laboratory, Faculty of Medicine, Imperial Faculty of Medicine, Imperial College, London, UKCollege, London, UK Ten FeiziTen FeiziYan LiuYan LiuRobert ChildsRobert ChildsAngelina PalmaAngelina PalmaWengang ChaiWengang ChaiMaria Campanero-RhodesMaria Campanero-RhodesYibing ZhangYibing Zhang

Division of Virology, Division of Virology, MRC NIMR, MRC NIMR, Mill Hill, London, UKMill Hill, London, UKAlan HayAlan HaySteve WartonSteve WartonRod DanielsRod DanielsVicky GregoryVicky Gregory

CollaboratorsCollaborators

Thanks to:Thanks to:

WHO Global Influenza NetworkWHO Global Influenza Network

Grant support:Grant support:

FLUPATH, EUFLUPATH, EU

Wellcome Trust, UK Wellcome Trust, UK

SFB 593, GermanySFB 593, Germany

LOEWE UGLMC, Hesse, GermanyLOEWE UGLMC, Hesse, Germany

von Behring-Röntgen-Stiftung, Germany von Behring-Röntgen-Stiftung, Germany

Marburg

Virus binding to selected 2-3 Sia sequencesVirus binding to selected 2-3 Sia sequences

V

V

% infected ciliated cells% infected ciliated cells 3-5 20-303-5 20-30