Large libraries of single-chain trimer peptide-MHCs enable antigen-specific CD8+ T cell discovery and analysis.

The discovery and characterization of antigen-specific CD8+ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8+ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.

Reinfection with SARS-CoV-2 and Waning Humoral Immunity: A Case Report.

Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a different strain harboring the spike variant D614G. This case of reinfection was one of the first cases of reinfection reported in 2020. With antibody, B cell and T cell analytics, we show correlates of adaptive immunity at reinfection, including a differential response in neutralizing antibodies to a D614G pseudovirus. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.

Multiple early factors anticipate post-acute COVID-19 sequelae.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

Vitamin D Supplementation and Covid 19: Results from the U.S. N3C Data Enclave

Background It is estimated that 18% of adults in the U.S. take Vitamin D supplements. Some observational studies suggest that vitamin D supplementation activates the innate immune system and reduces the incidence and severity of viral infections. During the SARS-CoV-2 pandemic, vitamin D supplements were touted as a potential therapy to prevent the disease and/or complications. However, supportive evidence is lacking. Methods The National COVID Cohort Collaborative (N3C) enclave is the largest COVID-19 data base with nearly 1.4 million positive patients at 56 sites in the U.S. We performed a retrospective analysis of vitamin D supplementation, either prescribed before or during hospitalization for SARS-CoV-2. Results 137,399 people took vitamin D supplements out of 1.4 million. Females prescribed vitamin D outnumbered males by almost 2:1, whereas in non-users there were no sex differences. Most supplement users were older than 50. African Americans constituted 13% of the non-users, but 23% of those prescribed vitamin D. Infected individuals with any vitamin D supplementation, pre-Covid, post-Covid or both, had a 6.66% mortality rate vs 2% mortality in non-users. Similarly, nearly a third of the supplement users were hospitalized compared to 11% in the non-users. The Charlson Co-Morbidity Index was 3.0±3 (SD) in users vs 1.0±2 (SD) in non-users. Conclusion 10% of SARS-CoV-2 infected patients were taking vitamin D. They tended to be older, more likely to be African American and have significant co-morbidities. Hospitalization and mortality were higher among those taking Vitamin D in this cohort. Vitamin D is widely used to prevent and treat SARS-CoV-2 but without evidence of efficacy. Disclosures Sally L. Hodder, M.D., Gilead (Advisor or Review Panel member)Merck (Grant/Research Support, Advisor or Review Panel member)Viiv Healthcare (Grant/Research Support, Advisor or Review Panel member)

Angiotensin II receptor I auto-antibodies following SARS-CoV-2 infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with endothelial activation and coagulopathy, which may be related to pre-existing or infection-induced pro-thrombotic autoantibodies such as those targeting angiotensin II type I receptor (AT1R-Ab). METHODS: We compared prevalence and levels of AT1R-Ab in COVID-19 cases with mild or severe disease to age and sex matched negative controls utilizing multivariate logistic and quantile regression adjusted for comorbidities including hypertension, diabetes, and heart disease. RESULTS: There were trends toward increased prevalence (50% vs. 33%, p = 0.1) and level of AT1R-Ab (median 9.8 vs. 6.1 U/mL, p = 0.06) in all cases versus controls. When considered by COVID-19 disease severity, there was a trend toward increased prevalence of AT1R-Ab (55% vs. 31%, p = 0.07), as well as significantly higher AT1R-Ab levels (median 10.7 vs. 5.9 U/mL, p = 0.03) amongst individuals with mild COVID-19 versus matched controls. In contrast, the prevalence (42% vs. 37%, p = 0.9) and level (both medians 6.7 U/mL, p = 0.9) of AT1R-Ab amongst those with severe COVID-19 did not differ from matched controls. CONCLUSIONS: These findings support an association between COVID-19 and AT1R-Ab, emphasizing that vascular pathology may be present in individuals with mild COVID-19 as well as those with severe disease.

Integrated analysis of plasma and single immune cells uncovers metabolic changes in individuals with COVID-19.

A better understanding of the metabolic alterations in immune cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may elucidate the wide diversity of clinical symptoms experienced by individuals with coronavirus disease 2019 (COVID-19). Here, we report the metabolic changes associated with the peripheral immune response of 198 individuals with COVID-19 through an integrated analysis of plasma metabolite and protein levels as well as single-cell multiomics analyses from serial blood draws collected during the first week after clinical diagnosis. We document the emergence of rare but metabolically dominant T cell subpopulations and find that increasing disease severity correlates with a bifurcation of monocytes into two metabolically distinct subsets. This integrated analysis reveals a robust interplay between plasma metabolites and cell-type-specific metabolic reprogramming networks that is associated with disease severity and could predict survival.

Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

Reinfection with SARS-CoV-2 and Failure of Humoral Immunity: a case report.

Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a new strain harboring the spike variant D614G. With antibody and B cell analytics, we show correlates of adaptive immunity, including a differential response to D614G. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.