Optimized properties of live vaccine influenza reassortant strains obtained by reverse genetics

Abstract

Classical reassortment in developing chicken eggs is a well-established technique for obtaining LAIV strains. Naturally generated reassortant vaccine strains are characterized by high reproductive capacity, genetically stable characteristics of temperature sensitivity and cold resistance, which correspond to the characteristics of the MDV involved in crossing with the epidemic virus. Along with antigenic relevance, natural reassortment ensures attenuation of vaccine strains, good reproduction capacity in upper respiratory tract cells and inability to reproduction in the lower respiratory tract. With classical reassortment, the speed and efficiency of obtaining vaccine reassortants largely depend on the properties of epidemic virus, and therefore cannot be stable. The potential of reverse genetics is attractive because it allows to obtain vaccine reassortants quickly and efficiently, reduce the likelihood of spontaneous mutations; however, the vaccine strain is deprived of the advantages of natural selection, in which the most viable clones are selected. This study presents the results of comparatively assessed A(H3N2) LAIVs obtained in parallel by classical reassortment and reverse genetics according to criteria confirming that vaccine strains inherit the necessary properties that guarantee their harmlessness and high reproduction in chicken embryos. Strains for LAIV obtained by both methods retained all attenuating mutations inherited from the MDV, were highly reproductive at the optimal temperature, with temperature sensitivity corresponded to the MDV. However, strains obtained by reverse genetics, was observed to have partial loss of cold resistance in comparison with that of the MDV and classical reassortants. Reduced cold adaptation may negatively affect vaccine effectiveness. It is important that after several additional passages in chicken embryos at low temperature, the cold resistance of the vaccine strain, assembled by reverse genetics, was increased. Credibly that cold resistance is a phenotypic trait, the degree of manifestation of which depends on the temperature conditions of virus multiplication. The selective factor of reduced incubation temperature is missing in reverse genetics. In order for the cold-adapted phenotype to be fully realized, additional passages at low temperature of RG-reassortants are necessary. Thus, the reverse genetics method using plasmid technology allows to effectively prepare reassortant strains for LAIV. An important stage in obtaining vaccine strains using genetic engineering techniques should be the control of their cold-adapted phenotype and its optimization by additional passages at low temperature.