CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment.

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

Successful lung transplantation using an allograft from a COVID-19-recovered donor: a potential role for subgenomic RNA to guide organ utilization.

Although the risk of SARS-CoV-2 transmission through lung transplantation from acutely infected donors is high, the risks of virus transmission and long-term lung allograft outcomes are not as well described when using pulmonary organs from COVID-19-recovered donors. We describe successful lung transplantation for a COVID-19-related lung injury using lungs from a COVID-19-recovered donor who was retrospectively found to have detectable genomic SARS-CoV-2 RNA in the lung tissue by multiple highly sensitive assays. However, SARS-CoV-2 subgenomic RNA (sgRNA), a marker of viral replication, was not detectable in the donor respiratory tissues. One year after lung transplantation, the recipient has a good functional status, walking 1 mile several times per week without the need for supplemental oxygen and without any evidence of donor-derived SARS-CoV-2 transmission. Our findings highlight the limitations of current clinical laboratory diagnostic assays in detecting the persistence of SARS-CoV-2 RNA in the lung tissue. The persistence of SARS-CoV-2 RNA in the donor tissue did not appear to represent active viral replication via sgRNA testing and, most importantly, did not negatively impact the allograft outcome in the first year after lung transplantation. sgRNA is easily performed and may be a useful assay for assessing viral infectivity in organs from donors with a recent infection.

Yearlong COVID-19 Infection Reveals Within-Host Evolution of SARS-CoV-2 in a Patient With B-Cell Depletion.

B-cell-depleting therapies may lead to prolonged disease and viral shedding in individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and this viral persistence raises concern for viral evolution. We report sequencing of early and late samples from a 335-day infection in an immunocompromised patient. The virus accumulated a unique deletion in the amino-terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts.

Year-long COVID-19 infection reveals within-host evolution of SARS-CoV-2 in a patient with B cell depletion.

BACKGROUND: B-cell depleting therapies may lead to protracted disease and prolonged viral shedding in individuals infected with SARS-CoV-2. Viral persistence in the setting of immunosuppression raises concern for viral evolution. METHODS: Amplification of sub-genomic transcripts for the E gene (sgE) was done on nasopharyngeal samples over the course of 355 days in a patient infected with SARS-CoV-2 who had previously undergone CAR T cell therapy and had persistently positive SARS-CoV-2 nasopharyngeal swabs. Whole genome sequencing was performed on samples from the patient's original presentation and 10 months later. RESULTS: Over the course of almost a year, the virus accumulated a unique in-frame deletion in the amino-terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Also, minority variants that were identified in the early samples-reflecting the heterogeneity of the initial infection-were found to be fixed late in the infection. Remdesivir and high-titer convalescent plasma treatment were given, and the infection was eventually cleared after 335 days of infection. CONCLUSIONS: The unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts, and the implication of these mutations in the emergence of viral variants. SUMMARY: We report an immunocompromised patient with persistent symptomatic SARS-CoV-2 infection for 335 days. During this time, the virus accumulated a unique in-frame deletion in the spike, and a complete deletion of ORF7b and ORF8 which is the first report of its kind in an immunocompromised patient.

Microbial signatures in the lower airways of mechanically ventilated COVID-19 patients associated with poor clinical outcome.

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.

Microbial signatures in the lower airways of mechanically ventilated COVID19 patients associated with poor clinical outcome.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

Microbial signatures in the lower airways of mechanically ventilated COVID19 patients associated with poor clinical outcome.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

Functional lower airways genomic profiling of the microbiome to capture active microbial metabolism.

BACKGROUND: Microbiome studies of the lower airways based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short-chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary. METHODS: Here we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole-genome shotgun (WGS) and RNA metatranscriptome sequencing. SCFAs were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed. RESULTS: Functional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome data were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFA levels were compared with WGS and metatranscriptome data. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing. CONCLUSIONS: Functional characterisation of the lower airway microbiota through metatranscriptome data identifies metabolically active organisms capable of producing metabolites with immunomodulatory capacity, such as SCFAs.