Development of safe and immunogenic reassortant viruses with 5:3 genotype for live attenuated influenza vaccine
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1 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 January 2013, Hong Kong Baptist University, Hong Kong SAR, China 1 of 16
2 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
3 Reassortant LAIV PB2 PB1 PA HA NP NA M NS Wild-type virus PB2 PB1 PA HA NP NA M NS Master Donor Virus LAIV 6:2 reassortant 5:3? 3 of 16
4 Which wt internal gene would be advantageous over ca and can be included into LAIV? PB2 PB1 PA NP M????? NS Retain attenuated and high-growth phenotype of LAIV;? Improve immunogenicity and cross-protection. 4 of 16
5 Mutations in internal genes of MDV A/Leningrad/134/17/57 (H2N2) PB2 PB1 PA NP M NS Master Donor Virus A/Leningrad/134/17/57 (H2N2) Val-478-Leu 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) Unique mutations for MDV 5 of 16
6 PB2 gene Ts phenotype correlates with LAIV attenuation Can NOT be replaced by wt 6 of 16
7 PB1 gene Can NOT be replaced by wt 7 of 16
8 PB2+PB1 genes PB2 and PB1 genes are crucial for LAIV ts phenotype (i.e. attenuation) 8 of 16
9 log pfu/ml PA gene Growth characteristics of mutant viruses Plaque Assay Titers at 33oC, 37oC and 38oC (SGR) oC 37oC 38oC calen-rg Len-ca wtlen-rg Len-wt capb2 capb1 capa canp cam cans Viruses (Parents and Mutants) PA gene plays role in LAIV high-growth phenotype 9 of 16
10 PA gene Virus reproduction at low temperature (25-26oC) 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 10 of 16
11 NS gene Minor effect on LAIV ts phenotype Better not to replaced by wt 11 of 16
12 log pfu/ml M gene Growth characteristics of mutant viruses Plaque Assay Titers at 33oC, 37oC and 38oC (SGR) oC 37oC 38oC calen-rg Len-ca wtlen-rg Len-wt capb2 capb1 capa canp cam cans Viruses (Parents and Mutants) M gene plays role in LAIV high-growth phenotype May be replaced by wt if it doesn t alter high-growth phenotype 12 of 16
13 NP gene Can be replaced by wt 13 of 16
14 NP gene major target for CTL response Len/17 Len/17 Len/17 Len/17 Len/17 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 14 of 16
15 Retrospective analysis of 5:3 LAIVs Reassortants Wild-type virus Genes from wt virus Age No Reactogenicity Temperature (%) Immunogenicicty (seronegaive) % 4-fold rise of Ab titer st dose 2 nd dose A/9/30/37/88 (H3N2) A/Leningrad/37/88 (H3N2) HA, NA, NP Adults ( 18) (clinic) Adults ( 18) Children (4.3) ND A/Leningrad/92/ 89 (H1N1), R-1 A/Leningrad/92/89 A/Leningrad/92/ (H1N1) 89 (H1N1), R-5 HA, NA, NP HA, NA, NP Adults ( 18) Adults ( 18) 20 3 (15) /25/1 A(H1N1) A/Brazil/11/79 (H1N1) HA, NA, PA 1 (0.6) (3.4) Children (1.54) Children /Ph A(H3N2) A/Philippine/2/87 (H3N2) HA, NA, PB2 Children (3.9) 0 ND 60.0 Children (22.6) 0 ND 48.5 All 5:3 LAIVs were safe and immunogenic 15 of 16
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. 16 of 16
17 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
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