Characterization of reverse genetics-derived cold-adapted master donor virus A/Leningrad/134/17/57 (H2N2) and reassortants with H5N1 surface genes in a mouse model.

Isakova-Sivak I1, Chen LM, Bourgeois M, Matsuoka Y, Voeten JT, Heldens JG, van den Bosch H, Klimov A, Rudenko L, Cox NJ, Donis RO.

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Abstract

Live attenuated influenza vaccines offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing process and more cross-protective immune responses.Using an established reverse genetics (rg) system for wild type A/Leningrad/134/1957 and cold-adapted (ca) A/Leningrad/134/17/1957 (Len17) master donor virus (MDV) we produced and characterized three rg H5N1 reassortant viruses carrying modified HA and intact NA genes from either A/Vietnam/1203/2004 (H5N1, VN1203, clade 1) or A/Egypt/321/2007 (H5N1, EG321, clade 2) viruses. A mouse model of infection was used to determine the infectivity and tissue tropism of the parent wt viruses as compared to the ca master donor viruses as well as the H5N1 resassortants. All ca viruses showed reduced replication in lungs and enhanced replication in nasal epithelium. In addition, the H5N1 HA and NA enhanced replication in lungs unless it was restricted by the internal genes of the ca MDV. Mice inoculated twice four weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum HI and IgA antibody titers to the homologous and heterologous viruses consistent with protective immunity. These animals remained healthy after challenge inoculation with a lethal dose with homologous or heterologous wt H5N1 HPAI. The profiles of viral replication in respiratory tissues, immunogenicity and protective efficacy characteristics of the two ca H5N1 candidate LAIV warrant further development into a vaccine for human use.