* *

RUSSIAN

ACADEMY OF MEDICALSCIE

NCES

Research Institute of Influenza, Russian Academy of Medical Science, Saint Petersburg

Molecular epidemiology of influenza viruses in Russia in 2005-2008.

Development of new influenza vaccines.

2008

Ñàõà (ßêóòèÿ) ðåñï.

Ìàãàäàíñêàÿ îáë.

Àðõàíãåëüñêàÿ îáë.

Ìóðìàíñêàÿ îáë.

Êîìè (ðåñï.)

Ñòàâðîïîëüñêèé êðàé

Âîðîíåæñêàÿ îáë. Êðàñíîÿðñêèé êðàéÈðêóòñêàÿ îáë.

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Ëåíèíãðàäñêàÿ îáë.

Àëòàéñêèé êðàé

Ñâåðäëîâñêàÿ îáë.

Íîâîñèáèðñêàÿ îáë.Ðîñòîâñêàÿ îáë.

Ìîñêîâñêàÿ îáë.

Îìñêàÿ îáë.

Ñàõàëèíñêàÿ îáë.

×åëÿáèíñêàÿ îáë.Áóðÿòèÿ (ðåñï.)

×èòèíñêàÿ îáë.

Ïðèìîðñêèé êðàé

Òàéìûðñêèé àâò. îêðóã

Ñòàðûå îï. áàçû ÔÖÃ (34)Íîâûå îï. áàçû ÔÖÃ (14)Îï. áàçû èíñò. Èâàíîâñêîãî (10)

Old basic labs 35New basic labs 14Labs of the Ivanovskiy Institute 10

Territories of Russian Federation under surveillance of Federal/National Influenza Center and Center for Ecology and Epidemiology

Virus isolation and characterization

(serological and IFA analysis, sequencing, etc.)

Federal Influenza CenterNational Influenza centers

Ministry of Public HealthAcademy of Med. Science

Department of Epidemiology

Institute of Influenza

Diagnostics and vaccine development

and production

WHO

Department of Vaccine clinical trials

Department of Biotechnology &

diagnostics

Department of Evolution of Influenza viruses

Department of Molecular Virology

Regional isolates

Department of Vaccine development

Department of Antivirals

Structure of seasonal influenza virus population in Russia in 2003-2008

Antigenic structure and HA phylogenetic tree of influenza A H1N1viruses isolated in 2007-2008

Influenza virus A strain HA (number of AA changes) NA

(number of AA changes)

A/New Caledonia/20/99 Asp 35 Ser, Tre 82 Lys, Tyr 94 His,

Lys 140 Glu, Val 165 Ala, Arg 188 Lys

(6 AA

)

Glu 214 Gly, Arg 222 Gln, Gly 249 lys,

Tre 287 Ile, Lys 329 Glu, Asp 344 Asn,Gly 354 Asp

(7 – 9 AA)A/Solomon Islands/3/06 Asp 35 Ser, Ser 73Lys,

Tre 128 Val, Lys 145 Arg, Arg 188 Lys

(5 AA)

2006-2007 Russian isolates Asp 35 Ser, Asn 36 Ser, Lys 140 Glu,

Lys 145 Arg, Lys 169 Glu, Tre 183 Asn,

Ser 188 Lys, Tre 189 Ala(8 AA)

No data

Influenza virus A strain HA (number of AA changes) NA

(number of AA changes)

A/New Caledonia/20/99 Asp 35 Ser, Tre 82 Lys, Tyr 94 His,

Lys 140 Glu, Val 165 Ala, Arg 188 Lys

(6 AA

)

Glu 214 Gly, Arg 222 Gln, Gly 249 lys,

Tre 287 Ile, Lys 329 Glu, Asp 344 Asn,Gly 354 Asp

(7 – 9 AA)A/Solomon Islands/3/06 Asp 35 Ser, Ser 73Lys,

Tre 128 Val, Lys 145 Arg, Arg 188 Lys

(5 AA)

2006-2007 Russian isolates Asp 35 Ser, Asn 36 Ser, Lys 140 Glu,

Lys 145 Arg, Lys 169 Glu, Tre 183 Asn,

Ser 188 Lys, Tre 189 Ala(8 AA)

No data

Differences in HA and NA sequences of influenza virus A strains (H1N1) isolated in 2007-2008 comparing to vaccine strains A/New Caledonia/20/99

and A/Solomon Islands/3/06 and 2006-2007 Russian isolates.

Strains of the end of 2008 epidemic season

(April) are marked in red

HA phylogenetic tree of influenza viruses A H3N2isolated in RF in 2006-2008

Influenza virus A strain HA (number of AA changes)

A/Brisbane/10/2007 Asn53Asp, Ser157Leu,Asn173Lys, Ile182Val

(4AA

)

A/Wisconsin/67/2005 Glu50Gly, Ile140Lys,Asn53Asp, Ser157Leu,Asn173Lys, Ile182Val

(6 AA)

2006-2007 Russian isolates Val112Ile (2006), Arg142Gly (2007)Glu50Gly, Ile140Lys,

Asn53Asp, Ser157Leu,Asn173Lys, Ile182Val

(8 AA)

Differences in HA sequences of influenza virus A strains (H3N2) isolated in 2007-2008 comparing to vaccine strains

A/Wisconsin/67/2005 and A/Brisbane/10/2007 and 2006-2007 Russian isolates

B/Khabarovsk/10/07

B/Saint-Petersburg/18/07

B/Irkutsk/97/07

B/Saint-Petersburg/15/07

B/Saint-Petersburg/22/07

B/Saint-Petersburg/520/06

B/Saint-Petersburg/341/06

B/Malaysia/2506/2004

B/Murmansk/57/06

B/Hong Kong/330/2001

B/Victoria/02/1987

B/Sichuan/379/99

B/Novosibirsk/4/07

0.01

B/Yamagata/16/1988

HA phylogenetic tree of influenza viruses B isolated in RF in 2006-2008

Shift from Victorian lineage to Yamagata

Resistance to rimantadine (amantadine)

Resistance to oseltamivir (Tamiflu)

2005 – 2006 2006 – 2007 2007 – 2008

Russian H1N1 resistant strains have Tyr at 275 pos. of NA-proteinsensitive His

Russian resistant strains have Ala at 30 and Asn at 31 pos. of M2-proteinsensitive Ala/Pro Gly

Per cent of resistant strains

78 43 22

0 0 41

Mutation resulting in rimantadine resistance was observed mainly in isolates of H3N2 subtype and never in strains that were resistant to oseltamivir.

Drug resistance analysis

Origin Number of strainsDetection method

Number of resistant strainsRFLP* Sequencing

Saint-Petersburg 35 30 15 32

Moscow 4 3 4 0

Kaliningrad 18 13 6 1

Astrakhan 17 12 5 0Voronezh 10 6 4 2

Tula 3 0 3 0

Rostov-na-Donu 1 0 1 0

Kursk 1 1 0 1

Samara 1 0 1 1TOTAL 90 65 39 37

Resistance to oseltamivir (Tamiflu) among influenza virus A H1N1 strains isolated in Russia in 2008

*  ‐

protocol and chemistry were kindly provided by Dr. A.Klimov and Dr. L.Gubareva, CDC, Atlanta

1. Heterogenicity of influenza viruses A and B was observed on both the type and subtype levels.

2. Essential activation of influenza A H1N1 viruses during the last epidemic seasons in Russia could be associated with the appearance of new antigenic variants.

3. Influenza A H3N2 viruses are still one of the etiological factors causing flu epidemics. Last years were characterized by simultaneous circulation of different antigenic variants of H3N2 viruses on the territory of RF.

4. In the last epidemic season a large group of H1N1 viruses resistant to oseltamivir by its neuraminidase gene structure was revealed, whereas number of rimantadine resistant cases decreased and there were practically no such cases in H1N1 strains.

5. Influenza B viruses of two lineages continue to circulate on the territory of RF. During two last epidemic seasons the shift of prevalence of Victorian to Yamagata line was observed.

Conclusions

A.Egorov et al., 1998

Development of new influenza vaccines

Inactivated vaccines

•Do not replicate

•Limited cross-protection

(adjuvants, high dosing)

•No CTLs, no sIgA

•Injection

•Production in eggs,

cell culture

FLUVACC

LAIV vaccines

•Replicating virus

(shedding,

transmission)

•Cross-protection

•CTL, sIgA response

•Intranasal

administration

•Production in eggs

A.Egorov

New vaccine strategies

“New approaches to the development of influenza pandemic vaccines”

PB2

PB1

PA

HA

NA

NP

M

NS2

Deletion of NS1 gene

Modified HA cleavage site

Replication deficiency phenotype

AttenuationPR8

PR8

Avian

PQRERRRKKRGLFGAI PQTETRGLFGAIModification of HA cleavage site

Recombinant vaccine strain A/Vietnam/1203/04 Genes

modificationResult

The strain was obtained by reverse genetics method

Randomized placebo-controlled phase I dose-escalation study of the vaccine safety and immunogenicity in healthy adults

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RUSSIAN

ACADEMY OF MEDICALSCIE

NCES

◊

Intranasal vaccine application

◊

Combine properties of LAIV and inactivatedvaccines: safe and immunogenic

◊

Possible to produce the vaccine on Vero cells

“New approaches to the development of influenza pandemic vaccines”

Advantages:

Tuberculum

Non-vaccinated Vaccinated

CFU

/org

an(l

og10)

Control FLU/ESAT-6 INH FLU/ESAT6+INH

FLU/NS1-125

The prominent synergistic effect was reached when vaccination with Flu/ESAT-6 vectors

was applied together with isoniazid treatment,

reducing bacterial load in the lungs to

undetectable levels (P<0.05).

Effect of treatment with Flu/ESAT-6 viral vectors on the lung bacillary load of mice

Lungs morphology of mice 40 days after i.v. challenge with M. bovis

Influenza vectors expressing M.Tuberculosis ESAT-6 protein

FLU/ESAT-6 vaccine vectors are at least as efficient as BCG vaccine in animal models of TB infection.

M.Stukova et al., 2006)

O.I.Kiselev

Department of vaccine development, “reverse genetics” group

Department of Evolution of Influenza viruses

Department of Molecular Virology

M.Y.Eropkin

N.E.Konovalova

T.M.Gudkova

V.I.Grigorieva

M.P.Grudinin

M.A.Stukova

J.V.Busitskaya

A.B.Komissarov

A.V.Zadonskaya

M.M.Pisareva

A.Y.Egorov

J.R.Romanova

E.A.Romanovskaya-

Romanko

S.S.IlisavskiyDepartment of Antivirals

V.V.Zarubaev

P.N.Anfimov