Plates were further incubated for 20?min while rocking

Plates were further incubated for 20?min while rocking. levels of mucosal and serum IgG and IgA to SARS-CoV-2?S or its receptor-binding domain. Serum antibodies from MPV/S-2P-immunized animals efficiently inhibited ACE2 receptor binding to S proteins of variants of concern. Based on its attenuation and immunogenicity in macaques, MPV/S-2P will be further evaluated as a live-attenuated vaccine for intranasal immunization against SARS-CoV-2. Subject areas: Virology, Immunology, Immune response Graphical abstract Open in a separate window Highlights ? Murine pneumonia virus (MPV) was used as a vector to express SARS-CoV-2 spike protein ? Macaques were immunized by the respiratory mucosal route with MPV/S or MPV/S-2P ? MPV/S-2P induced strong mucosal and serum anti-spike antibody responses in macaques ? MPV/S-2P will be evaluated as a live-attenuated intranasal COVID-19 vaccine Virology; Immunology; Immune response Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Deoxycholic acid sodium salt emerged in late 2019 and has been a worldwide public health burden since early 2020.1,2 SARS-CoV-2 is the causative agent of coronavirus infectious disease (COVID-19), which predominantly causes disease of the respiratory tract, though other symptoms can also occur.3,4 mRNA-based COVID-19 vaccines became available under emergency use authorization (EUA) in late 2020, and a non-replicating adenovirus-vector-based vaccine as well as a protein subunit vaccine were authorized under EUA shortly thereafter.5,6,7,8,9 These Deoxycholic acid sodium salt COVID-19 vaccines were effective in reducing the burden of severe Serpinf2 COVID-19 and COVID-19 mortality.10 However, these injectable vaccines do not directly stimulate immunity in the respiratory mucosa, and booster immunizations are required to maintain protection. Furthermore, current Deoxycholic acid sodium salt COVID-19 vaccines are not highly effective in preventing infection and replication of SARS-CoV-2 at the respiratory mucosal entry sites; transmission rates remain high, allowing for the continued emergence of new variants. SARS-CoV-2 vaccines are needed that induce a robust mucosal immune response.11,12 Here, we used murine pneumonia virus (MPV) as a viral vector to express the SARS-CoV-2 spike (S)?protein. MPV is the murine homolog of respiratory syncytial virus (RSV) and there is no or minimal pre-existing immunity to MPV in humans.13 Our group previously evaluated MPV as a vector to express the RSV fusion (F)?protein. We found that MPV was suitable for stable expression of a foreign gene.14 Furthermore, MPV replication in non-human primates (NHPs) was highly restricted, presumably due to a strong host-range restriction,13,14 but MPV vectors expressing RSV F still induced strong serum RSV-neutralizing antibody responses that were comparable to those induced by wild-type RSV.14 These features along with its tropism for epithelial cells in the respiratory tract identify MPV as an attractive candidate for mucosal vaccination via the respiratory route. The live-attenuated MPV vector vaccine candidates of the present study were designed for intranasal immunization to directly induce mucosal immunity to SARS-CoV-2 in the respiratory tract, in addition to stimulating systemic immunity. Most of the current injectable COVID-19 vaccines are based on prefusion-stabilized versions of the SARS-CoV-2?S protein, the major neutralization and protective antigen of SARS-CoV-2.15,16 Prefusion-stabilization by introduction of proline substitutions prevents this antigen from forming a post-fusion conformation and keeps the protective epitopes of the receptor binding domain (RBD) exposed. Indeed, in previous studies, the prefusion-stabilized versions of S were shown to be more physically stable and immunogenic than wild-type S.17,18 In the present report, we constructed MPV vectors expressing the native or prefusion-stabilized version of the SARS-CoV-2?S protein, performed comparisons, and evaluated the two vectors for safety and immunogenicity in rhesus macaques. Results Generation of MPV/S and MPV/S-2P and characterization in cell culture We previously generated an MPV vaccine vector based on a recombinant version of Deoxycholic acid sodium salt MPV strain 15.14 Because of concerns of possible over-attenuation due to the addition of a supernumerary gene into the MPV vector, we partially codon-pair optimized the L open reading frame (ORF) encoding the MPV polymerase14. To generate a version of MPV expressing the SARS-CoV-2?S protein, the ORF encoding the S protein (aa 1-1,273) of the ancestral SARS-CoV-2?S protein (GenBank MN908947) was codon-optimized for expression in humans. A second version of this optimized S ORF incorporated two proline substitutions ([K986P] and [V987P]), which stabilized S in the prefusion conformation, and.