A Single Intranasal Vaccination with a Rationally Attenuated SARS-CoV-2 Elicits Strong Humoral Immune Response and Is Protective in Syrian Hamsters

Very few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are currently in pre-clinical or clinical development. We rationally attenuated SARS-CoV-2 (isolate WA1/2020) by removing the polybasic cleavage site within the spike protein and the open reading frames (ORFs) 6-8, and by introducing a pair of mutations into the non-structural protein 1 (Nsp1). The derived virus (WA1-DPRRA-ORF6-8-Nsp1K164A/H165A) became severely attenuated in both the K18-human ACE2 (hACE2) transgenic mice and in Syrian hamsters. Transcriptomic profiling of nasal turbinates and lung tissues of infected Syrian hamsters confirmed that WA1-DPRRA-ORF6-8-Nsp1K164A/H165A attenuated the upregulation of proinflammatory pathways. A single intranasal immunization of just 100 PFU of the WA1-DPRRA-ORF6-8-Nsp1K164A/H165A elicited binding and neutralizing antibody responses in Syrian hamsters and completely protected against SARS-CoV-2-induced weight loss and pneumonia. These data demonstrate the feasibility of rational attenuation of SARS-CoV-2. WA1-DPRRA-ORF6-8-Nsp1K164A/H165A represents a promising live attenuated vaccine candidate. Overall design: For challenge studies, adult (6-12 months old, male) Syrian hamsters were used. To test attenuation of recombinant SARS-CoV-2 viruses in vivo, adult K18-hACE2 transgenic mice were divided into five groupsand intranasally inoculated with 10^5 plaque forming unit (PFU) of WA1/2020 (n=3), DPRRA (n=4), Nsp1-K164A/H165A(n=3), and Nsp1-N128S/K129E (n=4) or left uninfected (n=2). To assess changes at the molecular level, we isolated RNA from both nasal turbinates and lung homogenates at 4 dpi (days post infection) and performed RNAseq analyses.