Mild SARS-CoV-2 infection results in long-lasting microbiota instability

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of SARS-CoV-2 infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2 positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls, as long as 154 days after their positive test. These results were confirmed and extended in the K18-hACE2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the United States), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology. IMPORTANCETaken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2 it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.

Limited cross-variant immunity after infection with the SARS-CoV-2 Omicron variant without vaccination

SARS-CoV-2 Delta and Omicron strains are the most globally relevant variants of concern (VOCs). While individuals infected with Delta are at risk to develop severe lung disease1, Omicron infection causes less severe disease, mostly upper respiratory symptoms2,3. The question arises whether rampant spread of Omicron could lead to mass immunization, accelerating the end of the pandemic. Here we show that infection with Delta, but not Omicron, induces broad immunity in mice. While sera from Omicron-infected mice only neutralize Omicron, sera from Delta-infected mice are broadly effective against Delta and other VOCs, including Omicron. This is not observed with the WA1 ancestral strain, although both WA1 and Delta elicited a highly pro-inflammatory cytokine response and replicated to similar titers in the respiratory tracts and lungs of infected mice as well as in human airway organoids. Pulmonary viral replication, pro-inflammatory cytokine expression, and overall disease progression are markedly reduced with Omicron infection. Analysis of human sera from Omicron and Delta breakthrough cases reveals effective cross-variant neutralization induced by both viruses in vaccinated individuals. Together, our results indicate that Omicron infection enhances preexisting immunity elicited by vaccines, but on its own may not induce broad, cross-neutralizing humoral immunity in unvaccinated individuals.

SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles

The Omicron SARS-CoV-2 virus contains extensive sequence changes relative to the earlier arising B.1, B.1.1 and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (SC2-VLPs), we examined mutations in all four structural proteins and found that Omicron showed 3-fold higher capsid assembly and cell entry relative to Delta, a property conferred by S and N protein mutations. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in 8 out of 8 subjects compared to 1 out of 8 pre-boost. Furthermore, the monoclonal antibody therapeutics Casirivimab and Imdevimab had robust neutralization activity against B.1, B.1.1 or Delta VLPs but no detectable neutralization of Omicron VLPs. Our results suggest that Omicron is more efficient at assembly and cell entry compared to Delta, and the antibody response triggered by existing vaccines or previous infection, at least prior to boost, will have limited ability to neutralize Omicron. In addition, some currently available monoclonal antibodies will not be useful in treating Omicron-infected patients. One-Sentence SummaryOmicron SARS-CoV-2 virus-like particles have enhanced infectivity that is only weakly neutralized by vaccination without boost or prior infection, or antibody therapeutics.

Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles

Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M). One-Sentence SummaryR203M substitution within SARS-CoV-2 N, found in delta variant, improves RNA packaging into virus-like particles by 10-fold.