Development of safe and immunogenic reassortant viruses with 5:3 genotype for live attenuated influenza vaccine

Irina Isakova-Sivak, PhD

Institute of Experimental Medicine,

Saint Petersburg, Russia

The First WHO Integrated Meeting on Development and Clinical Trials of Influenza Vaccines that Induce Broadly Protective and Long-Lasting Immune Responses 24-26

January 2013, Hong Kong Baptist University, Hong Kong SAR, China 1 of 16

Reassortant LAIV in Russia

> 30 years on the Market;

> 100 million doses produced;

Effectiveness of LAIV for adults (summary from 126 trials : 500 to 45,000

adults per trial; total > 500,000 adults):

Effectiveness was confirmed for each trial.

Mean effectiveness for 126 trials was 1.80 (= 45%).

Index of effectiveness was 1.5 (= 33%), even when epidemics were

caused by new antigenic variants not presented in LAIV.

Nevertheless, new approaches to improve LAIV immunogenicity and

effectiveness are of great interest 2 of 16

PB1

PA

HA

NP

NA

M

PB2

NS

PB1

PA

HA

NP

NA

M

PB2

NS

Wild-type virus Master Donor Virus

LAIV 6:2 reassortant

Reassortant LAIV

5:3? 3 of 16

Which wt internal gene would be advantageous over ca and can be included into LAIV?

PB1

PA

NP

M

PB2

NS

Retain attenuated and high-growth phenotype of LAIV;

Improve immunogenicity and cross-protection.

?

?

?

?

?

?

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Mutations in internal genes of MDV A/Leningrad/134/17/57 (H2N2)

PB1

PA

NP

M

PB2

NS

Master Donor Virus A/Leningrad/134/17/57 (H2N2)

Val-478-Leu

Unique mutations for MDV

Lys-265-Asn; Val-591-Ile

Leu-28-Pro; Val-341-Leu

none (egg-grown); Asn-492-Ser (MDCK-grown)

Ile-15-Val (M1); Phe-144-Leu (M1)

Met-100-Ile (NS2)

5 of 16

PB2 gene

Ts phenotype correlates with LAIV attenuation

Can NOT be replaced by wt 6 of 16

PB1 gene

Can NOT be replaced by wt 7 of 16

PB2+PB1 genes

PB2 and PB1 genes are crucial for LAIV ts phenotype (i.e. attenuation) 8 of 16

0

1

2

3

4

5

6

7

8

9

10

caLen-rg Len-ca wtLen-rg Len-wt caPB2 caPB1 caPA caNP caM caNS

log

pfu

/ml

Viruses (Parents and Mutants)

Plaque Assay Titers at 33oC, 37oC and 38oC (SGR)

33oC

37oC

38oC

PA gene

PA gene plays role in LAIV high-growth phenotype

Growth characteristics of mutant viruses

9 of 16

Klimov et al. (2001) In: Options for the Control of Influenza IV.

PA gene is responsible for LAIV ca phenotype (important for replication in URT, i.e. for local immune response)

Can NOT be replaced by wt

PA gene Virus reproduction at low temperature (25-26oC)

10 of 16

NS gene

Better not to replaced by wt

Minor effect on LAIV ts phenotype

11 of 16

0

1

2

3

4

5

6

7

8

9

10

caLen-rg Len-ca wtLen-rg Len-wt caPB2 caPB1 caPA caNP caM caNS

log

pfu

/ml

Viruses (Parents and Mutants)

Plaque Assay Titers at 33oC, 37oC and 38oC (SGR)

33oC

37oC

38oC

M gene

M gene plays role in LAIV high-growth phenotype

Growth characteristics of mutant viruses

May be replaced by wt if it doesn’t alter high-growth phenotype 12 of 16

NP gene

Can be replaced by wt 13 of 16

NP gene – major target for CTL response

CTLs targeted to some NP epitopes of Len/17 MDV do not

recognize NP of currently circulated influenza A viruses

Inclusion of NP from currently circulated influenza A viruses into LAIV formulation would

bring advantages in terms of CTL response

wt NP is the best candidate for inclusion into LAIV 5:3

reassortants

Len/17

Len/17

Len/17

Len/17

Len/17

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Reassortants Wild-type virus

Genes from

wt virus

Age No

Reactogenicity Immunogenicicty

(seronegaive)

Temperature (%) %≥4-fold rise of Ab titer

37.1-37.5 ≥37.6-38.5 1st dose 2nd dose

A/9/30/37/88 (H3N2)

A/Leningrad/37/88 (H3N2)

HA, NA, NP

Adults (≥18) (clinic)

20 0 0 50.0 55.0

Adults (≥18) 80 0 0 50.0 60.0

Children 116 5 (4.3) 0 60.0 ND

A/Leningrad/92/89 (H1N1), R-1 A/Leningrad/92/89

(H1N1)

HA, NA, NP

Adults (≥18) 20 0 0 55.5 61.1

A/Leningrad/92/89 (H1N1), R-5

HA, NA, NP

Adults (≥18) 20 3 (15) 0 61.1 66.6

47/25/1 A(H1N1)

A/Brazil/11/79 (H1N1)

HA, NA, PA

Children 7-14 334 20 (3.4)

1 (0.6) 48.1 60.0

Children 3-6 262 2 (1.54) 48.1 59.4

47/Ph A(H3N2) A/Philippine/2/87

(H3N2) HA, NA,

PB2

Children 7-14 51 2 (3.9) 0 ND 60.0

Children 3-6 53 12 (22.6) 0 ND 48.5

Retrospective analysis of 5:3 LAIVs

All 5:3 LAIVs were safe and immunogenic 15 of 16

Summary There is a possibility of modifying LAIV genome composition by

including one additional gene from wild-type parental virus;

Polymerase genes (PB2, PB1 and PA) and NS genes of MDV A/Leningrad/134/17/57 (H2N2) can not be replaced due to their impact on LAIV ts/ca and attenuated phenotype;

In some cases M gene of MDV can be replaced by one of wild-type virus (if it doesn’t alter high-growth properties of LAIV strain);

Wild-type NP gene is the best candidate for inclusion into LAIV 5:3 reassortants due to its advantages in terms of cross-reactive CD8+ CTL response;

A pair of related LAIV reassortants with 6:2 and 5:3 genome compositions (with wt NP gene) will be evaluated for the induction of cross-reactive CD8+ CTL response.

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Acknowledgements

• Professor L. G. Rudenko (Head of Department of Virology, IEM, St.Petersburg, Russia);

• R. O. Donis, L.-M. Chen, A.I. Klimov (CDC, Atlanta, USA);

• Staff of Department of Virology, IEM;

• WHO