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 severe acute respiratory syndrome coronavirus 2 (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. These results were confirmed and extended in the K18-humanized angiotensin-converting enzyme 2 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 USA), 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.

mRNA vaccine-induced T cells respond identically to SARS-CoV-2 variants of concern but differ in longevity and homing properties depending on prior infection status.

While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-naïve and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-naïve individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-naïve vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-naïve counterparts.

Vaccination is one of the best ways to prevent severe COVID-19. Two doses of mRNA vaccine protect against serious illness caused by the coronavirus SARS-CoV-2. They do this, in part, by encouraging the immune system to make specialised proteins known as antibodies that recognise the virus. Most of the vaccine research so far has focussed on these antibodies, but they are only one part of the immune response. Vaccines also activate immune cells called T cells. These cells have two main roles, coordinating the immune response and killing cells infected with viruses. It is likely that they play a key role in preventing severe COVID-19. There are many kinds of T cells, each with a different role. Currently, the identity and characteristics of the T cells that protect against COVID-19 is unclear. Different types of T cells have unique proteins on their surface. Examining these proteins can reveal details about how the T cells work, which part of the virus they recognise, and which part of the body they protect. A tool called cytometry by time of flight allows researchers to measure these proteins, one cell at a time. Using this technique, Neidleman, Luo et al. investigated T cells from 11 people before vaccination and after their first and second doses. Five people had never had COVID-19 before, and six had already recovered from COVID-19. Neidleman, Luo et al. found that the T cells recognizing SARS-CoV-2 in the two groups differed. In people who had never had COVID-19 before, the second dose of vaccine improved the quality and quantity of the T cells. The same was not true for people who had already recovered from COVID-19. However, although their T cells did not improve further after a second vaccine dose, they did show signs that they might offer more protection overall. The proteins on the cells suggest that they might last longer, and that they might specifically protect the nose, throat and lungs. Neidleman, Luo et al. also found that, for both groups, T cells activated by vaccination responded in the same way to different variants of the virus. This work highlights the importance of getting both vaccine doses for people who have never had COVID-19. It also suggests that vaccination in people who have had COVID-19 may generate better T cells. Larger studies could show whether these patterns remain true across the wider population. If so, it is possible that delivering vaccines to the nose or throat could boost immunity by mimicking natural infection. This might encourage T cells to make the surface proteins that allow them to home to these areas.

Protracted yet Coordinated Differentiation of Long-Lived SARS-CoV-2-Specific CD8+ T Cells during Convalescence.

CD8+ T cells can potentiate long-lived immunity against COVID-19. We screened longitudinally-sampled convalescent human donors against SARS-CoV-2 tetramers and identified a participant with an immunodominant response against residues 322 to 311 of nucleocapsid (Nuc322-331), a peptide conserved in all variants of concern reported to date. We conducted 38-parameter cytometry by time of flight on tetramer-identified Nuc322-331-specific CD8+ T cells and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins, and took 32 serological measurements. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and Ab pillars of adaptive immunity. Over the approximately six month period of convalescence monitored, we observed a slow and progressive decrease in the activation state and polyfunctionality of Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph node-homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence into a state characteristic of long-lived, self-renewing memory.

Distinctive features of SARS-CoV-2-specific T cells predict recovery from severe COVID-19.

Although T cells are likely players in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe coronavirus disease 2019 (COVID-19). We analyze T cells from 34 individuals with COVID-19 with severity ranging from mild (outpatient) to critical, culminating in death. Relative to individuals who succumbed, individuals who recovered from severe COVID-19 harbor elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 cases display elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4+ T cells, as assessed by longitudinal sampling. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of individuals with severe COVID-19, these results support a model where lung-homing T cells activated through bystander effects contribute to immunopathology, whereas a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19.

Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts.

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.

SARS-CoV-2-Specific T Cells Exhibit Phenotypic Features of Helper Function, Lack of Terminal Differentiation, and High Proliferation Potential.

Convalescing coronavirus disease 2019 (COVID-19) patients mount robust T cell responses against SARS-CoV-2, suggesting an important role of T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells and predominantly Tcm cells with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can proliferate homeostatically, and can persist for over 2 months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection and support an important role of SARS-CoV-2-specific T cells in host control of COVID-19.