Novel Spike-stabilized trimers with improved production protect K18-hACE2 mice and golden Syrian hamsters from the highly pathogenic SARS-CoV-2 Beta variant.

Most COVID-19 vaccines are based on the SARS-CoV-2 Spike glycoprotein (S) or their subunits. However, the S shows some structural instability that limits its immunogenicity and production, hampering the development of recombinant S-based vaccines. The introduction of the K986P and V987P (S-2P) mutations increases the production of the recombinant S trimer and, more importantly, its immunogenicity, suggesting that these two parameters are related. However, S-2P still shows some molecular instability and it is produced with low yield. Thus, S-2P production can be further optimized. Here we described a novel set of mutations identified by molecular modelling and located in the S2 region of the Spike that increase S-2P production up to five-fold. Besides their immunogenicity, the efficacy of two representative S-2P-based mutants, S-29 and S-21, protecting from a heterologous SARS-CoV-2 Beta variant challenge was assayed in K18-hACE2 mice (an animal model of severe SARS-CoV-2 disease) and golden Syrian hamsters (GSH) (a moderate disease model). S-21 induced higher level of WH1 and Delta variants neutralizing antibodies than S-2P in K18-hACE2 mice three days after challenge. Viral load in nasal turbinate and oropharyngeal samples were reduced in S-21 and S-29 vaccinated mice. Despite that, only the S-29 protein protected 100% of K18-hACE2 mice from severe disease. When GSH were analyzed, all immunized animals were protected from disease development irrespectively of the immunogen they received. Therefore, the higher yield of S-29, as well as its improved immunogenicity and efficacy protecting from the highly pathogenic SARS-CoV-2 Beta variant, pinpoint the S-29 spike mutant as an alternative to the S-2P protein for future SARS-CoV-2 vaccine development. Authors summaryThe rapid development of SARS-CoV-2 vaccines have been pivotal in the control of the COVID-19 pandemic worldwide. Most of these vaccines include the S glycoprotein as the main immunogen since this protein, and particularly its receptor binding domain (RBD), is the major target of neutralizing antibodies. SARS-CoV-2 have been evolving from the beginning of the pandemic and several variants with increased transmissibility, pathogenicity or resistance to infection- or vaccine-induced immunity have emerged. Different strategies have been adopted to improve vaccine protection including additional booster doses or the adaptation of the S immunogens to the novel SARS-CoV-2 variants. As a complementary strategy we have identified a combination of non-proline mutations that increase S production by 5-fold (S-29 protein). Despite the sequence of this novel S-29 immunogen is based on the ancestral SARS-CoV-2 WH1 variant, it effectively protects animal model from the highly pathogenic and neutralization resistant SARS-CoV-2 Beta variant. Thus, we describe a novel set of mutations that can increase the production and efficacy of S-based COVID-19 vaccines.