Recombinant influenza A/H1N1pdm09 vaccine expressing streptococcal surface epitope for dual protection

Aim: Develop A/H1N1pdm09-based live attenuated influenza vaccines (LAIVs) presenting chimeric hemagglutinin (HA) fused to fragments of Streptococcus pneumoniae (PspA, Spr1875) or S. agalactiae (ScaAB) to elicit combined anti-influenza and anti-bacterial immunity. Methods: Recombinant LAIVs were generated by reverse genetics. Replicative fitness was measured in embryonated chicken eggs (CE; log10 EID50/0.1 mL, n = 5 per delution) and MDCK cells (log10 TCID50/mL, n = 5). BALB/c mice (n = 20 per group; serology n = 6 per group; lung titers n = 5 per group) received intranasal 106 EID50. Systemic IgG and mucosal IgA to influenza and to the recombinant pneumococcal peptide were quantified by ELISA (GMT ± SD). Early cytokine responses were profiled in THP-1 cells. Results: All recombinant strains replicated in CE at 33°C but were temperature-sensitive at 39°C. H1-ScaAB retained relatively high replication and exhibited a cold-adapted phenotype despite a large N-terminal insert. In MDCK cells, H1-PspA showed significantly reduced replication compared with the parental LAIV. In mouse lungs, replication on day 3 post-immunization was significantly lower for H1-ScaAB and H1-PspA compared with the parental LAIV strain (p < 0.05). The parental LAIV induced robust systemic anti-influenza IgG and, uniquely for H1-ScaAB, significant mucosal anti-influenza IgA (p < 0.05). H1-Spr generated stronger antibody responses to the inserted pneumococcal peptide (p < 0.05). THP-1 assays revealed construct-specific cytokine patterns (unmodified H1N1: highest IFN-α; H1-Spr: elevated IL-6; H1-ScaAB: greatest MCP-1). Conclusions: Multiple A/H1N1pdm09-based recombinant LAIVs with chimeric HA can replicate in eggs and murine respiratory tract and induce dual influenza/pneumococcal antigen responses. Expanded biophysical validation, functional antibody assays and challenge studies are needed to optimize insert design without compromising viral fitness.