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.

Live attenuated influenza vaccines (LAIV) offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing processes and more cross-protective immune responses. Using an established reverse genetics (rg) system for wild-type (wt) 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) virus. A mouse model of infection was used to determine the infectivity and tissue tropism of the parental wt viruses compared to the ca master donor viruses as well as the H5N1 reassortants. 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 4 weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum hemagglutination inhibition 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 highly pathogenic avian influenza (HPAI) viruses. The profiles of viral replication in respiratory tissues and the immunogenicity and protective efficacy characteristics of the two ca H5N1 candidate LAIV viruses warrant further development into a vaccine for human use.

Comparative immunogenicity and cross-clade protective efficacy of mammalian cell-grown inactivated and live attenuated H5N1 reassortant vaccines in ferrets.

Continued H5N1 virus infection in humans highlights the need for vaccine strategies that provide cross-clade protection against this rapidly evolving virus. We report a comparative evaluation in ferrets of the immunogenicity and cross-protective efficacy of isogenic mammalian cell-grown, live attenuated influenza vaccine (LAIV) and adjuvanted, whole-virus, inactivated influenza vaccine (IIV), produced from a clade 1 H5N1 6:2 reassortant vaccine candidate (caVN1203-Len17rg) based on the cold-adapted A/Leningrad/134/17/57 (H2N2) master donor virus. Two doses of LAIV or IIV provided complete protection against lethal homologous H5N1 virus challenge and a reduction in virus shedding and disease severity after heterologous clade 2.2.1 H5N1 virus challenge and increased virus-specific serum and nasal wash antibody levels. Although both vaccines demonstrated cross-protective efficacy, LAIV induced higher levels of nasal wash IgA and reduction of heterologous virus shedding, compared with IIV. Thus, enhanced respiratory tract antibody responses elicited by LAIV were associated with improved cross-clade protection.

Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2).

Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/134/17/57 (H2N2) (caLen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV caLen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV caLen17. In addition, we show that caLen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development.

Master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortants used in live attenuated influenza vaccine (LAIV) do not display neurovirulent properties in a mouse model.

Demonstration of the absence of neurovirulent properties of reassortant viruses contained in live attenuated influenza vaccine (LAIV) is a regulatory requirement. A mouse model was used to detect neurovirulent properties of the cold-adapted, temperature-sensitive and attenuated influenza master donor viruses (MDVs) A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortant influenza viruses. A/NWS/33 (H1N1), which is known to be neurovirulent in mice, was used as a positive control. Under conditions where the positive control virus induced symptoms of disease and showed viral replication in the upper respiratory tract as well as in the brain, replication of the influenza master donor viruses and reassortant influenza A and B viruses was limited to the upper respiratory tract where they were administered. None of the mice inoculated with MDVs or reassortant influenza viruses suffered from disease, and no virus or viral replication was observed in the brains of these mice. The results demonstrate the absence of neurovirulent properties of the MDVs and reassortant influenza viruses derived therefrom used in LAIV.

PB2 and PA genes control the expression of the temperature-sensitive phenotype of cold-adapted B/USSR/60/69 influenza master donor virus.

The cold-adapted (ca) and temperature-sensitive (ts) influenza master donor virus (MDV) B/USSR/60/69 was derived from its wild-type parental virus after successive passages in eggs at 32 degrees C and 25 degrees C. This strain is currently in use for preparing reassortant influenza B vaccine viruses which are used in the Russian trivalent live attenuated influenza vaccine. Vaccine viruses are obtained by classical reassortment of MDV and a currently circulating wild-type virus. The phenotypic properties cold adaptation and temperature sensitivity are inherited from the six genes encoding the internal proteins of the MDV. However, the role of the individual gene segments in temperature sensitivity and thus attenuation is not known. In this study, 35 reassortant viruses of B/USSR/60/69 MDV with current wild-type non-ts influenza B viruses were generated in eggs or MDCK cells and studied in order to identify the genes responsible for their ts phenotype. For each virus the exact genome composition was determined as well as its ts phenotype. The results demonstrated that the polymerase PB2 and PA gene segments of B/USSR/60/69 MDV independently controlled expression of the ts phenotype of B/USSR/60/69 MDV-based reassortant viruses. The other genes coding for internal proteins played no role in this respect. This suggests that mutations in the polymerase genes PB2 and PA play an essential role in attenuation of B/USSR/60/69 MDV-based reassortant influenza B vaccine viruses.