No abstract

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.

Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin–Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44+CD62L−) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP366); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.