Next-generation single-cycle respiratory syncytial virus vaccines with increased type I interferon induction yield robust systemic and mucosal responses in mice

We previously demonstrated that vaccination with RSV-Mnull, a prototype single-cycle live vaccine lacking the matrix (M) gene, generated anti-viral serum IgG and memory T cell responses, and reduced challenge virus shedding and pulmonary dysfunction in mice. Here we further characterized the response to RSV-Mnull, and designed and tested second generation Mnull vaccines. In mice, prime-boost vaccination with RSV-Mnull generated pre-fusion (preF) and attachment protein (G) -specific serum IgG and lung IgA, and protected from lung pathology, showing that a single-cycle live vaccine was effective in this model. In an effort to enhance efficacy for future human application, second generation Mnull vaccines were designed, in which nonstructural protein 1 (NS1), a known interferon (IFN) antagonist, was relocated to reduce expression. In addition, the G or F genes were moved to the first genome position (RSV-Mnull/G1 and RSV-Mnull/F1 respectively). In vitro , RSV-Mnull/G1 and RSV-Mnull/F1 showed reduced NS1 levels and increased IFN-β induction, whereas IFN-λ levels were not affected. Viruses with relocated NS1 also displayed enhanced anti-viral state in uninfected cells. RSV-Mnull/G1 induced higher levels of anti-G serum antibodies (Abs), whereas RSV-Mnull/F1 increased the ratio of anti-preF:anti-G IgG Abs. RSV-Mnull induced lower levels of lung IgA than wildtype RSV; However, relocation of NS1 increased early IgA induction and restored IgA levels to those seen with wildtype RSV. These findings suggest differences in G and F presentation or processing and Ab induction, and indicate that the genomic location of NS1, G, and F can impact/improve IgG and IgA levels and timing. All vaccines induced Abs that neutralized RSV in vitro and protected animals against a high challenge dose of wildtype RSV. In terms of protection against lung pathology, we did not see major improvements of RSV-Mnull/G1 and RSV-Mnull/F1 over the RSV-Mnull prototype, potentially due to limitations of the model. Nevertheless, RSV-Mnull/G1 and RSV-Mnull/F1 elicited high Ab levels against both major antigens, enhanced serum anti-G IgG or lung anti-F IgA levels, and protected mice from lung pathology in spite of single-cycle replication. Thus, the single-cycle approach has potential to be a platform for development of safe and efficacious live RSV vaccines.