No abstract

Influenza A viruses (IAVs) enter into cells by receptor-dependent endocytosis. Subsequently, conformational changes of haemagglutinin are triggered by low environmental pH and the N terminus of HA2 glycoprotein (gp) is inserted into the endosomal membrane, resulting in fusion pore formation and genomic vRNA release into the cytoplasm. However, the pH optimum of membrane fusion is host-and virus-specific and can have an impact on virus pathogenicity. We prepared mutants of neurotropic IAV A/WSN/33 (H1N1) with aa substitutions in HA2 gp at the site of HA1/HA2 interaction, namely T64 2 H (HA2 numbering position 64, H1 numbering position HA407; referred to as mutant '64'), V66 2 H ('66') (HA409); and a double mutant ('D') with two aa substitutions (T64 2 H, V66 2 H). These substitutions were hypothesized to influence the pH optimum of fusion. The pH optimum of fusion activity was measured by a luciferase assay and biological properties of viruses were monitored. The in vitro and in vivo replication ability and pathogenicity of mutants were comparable (64) or lower (66, D) than those of the wild-type virus. However, the HA2 mutation V66 2 H and double mutation T64 2 H, V66 2 H shifted the fusion pH maximum to lower values (ranging from 5.1 to 5.3) compared to pH from 5.4 to 5.6 for the wild-type and 64 mutant. The decreased replication ability and pathogenicity of 66 and D mutants was accompanied by higher titres in late intervals post-infection in lungs, and viral RNA in brains compared to wild-type virus-infected mice. These results have implications for understanding the pathogenicity of influenza viruses.

A promising candidate for developing the universal influenza vaccine is the ectodomain of the M2 protein (M2e). We designed and prepared an experimental DNA vaccine with an improved potential to induce anti-M2e immune response. The sequence for truncated NS1 protein followed by 4xM2e was inserted into the expression vector pTriEx-4 (pEx). M2e repeats were fused to the transmembrane domain and cytoplasmic tail of lysosome-associated membrane glycoprotein 2 isoform A (LAMP-2a) to target the M2e to the endo-lysosome pathway, facilitating increased antigen presentation by MHC II. Using confocal microscope immunofluorescence analysis, we confirmed a strong colocalization of pEx 4M2e-LAMP-2a with early endosomes and a weaker colocalization with late endosomes. BALB/c mice immunized with three doses of pEx 4M2e-LAMP-2a DNA vaccine and challenged with 2LD 50 mouse-adapted influenza virus developed significantly (up to 16 times) higher anti-M2e antibody response in comparison to mice immunized with pEx 4M2e vaccine using the same immunization protocol. This was in correlation with the increased survival rate (near to 67% vs 50%) observed in animals immunized with pEx 4M2e-LAMP-2a DNA in comparison to mice immunized with pEx 4M2e.