ABSTRACT
Genotypic definition of monogenic inborn errors of immunity (IEIs) continues to accelerate with broader access to next generation sequencing, underscoring this aggregated group of disorders as a major health burden impacting both civilian and military populations. At an estimated prevalence of 1 in 1200 individuals, IEIs affect ~8,000 patients within the Military Health System (MHS). Despite access to targeted gene/exome panels at military treatment facilities, most affected patients never receive a definitive genetic diagnosis that would significantly improve clinical care. To address this gap, we established the first registry of IEI patients within the MHS with the goal of identifying known and novel pathogenic genetic defects to increase diagnosis rates and enhance clinical care. Using the registry, a research protocol was opened in July 2022. Since July we have enrolled 75 IEI patients encompassing a breadth of phenotypes including severe and recurrent infections, bone marrow failure, autoimmunity/autoinflammation, atopic disease, and malignancy. Enrolled patients provide blood and bone marrow samples for whole genome, ultra-deep targeted panel and comprehensive transcriptome sequencing, plus cryopreservation of peripheral blood mononuclear cells for future functional studies. We are also implementing and developing analytical methods for identifying and interrogating non-coding and structural variants. Suspected pathogenic variants are adjudicated by a clinical molecular geneticist using state-of-the-art analysis pipelines. These analyses subsequently inform in vitro experiments to validate causative mutations using cell reporter systems and primary patient cells. Clinical variant validation and return of genetic results are planned with genetic counseling provided. As a proof of principle, this integrated genetic evaluation pipeline revealed a novel, candidate TLR7 nonsense variant in two adolescent brothers who both endured critical COVID-19 pneumonia, requiring mechanical ventilation and extracorporeal membrane oxygenation. Our protocol is therefore poised to greatly enrich clinical genetics resources available in the MHS for IEI patients, contributing to better diagnosis rates, informed family counseling, and targeted treatments that collectively improve the health and readiness of the military community. Moreover, our efforts should yield new mechanistic insights on immune pathogenesis for a broad variety of known and novel IEIs.Copyright © 2023 Elsevier Inc.
ABSTRACT
Background. T cells are central to the early identification and clearance of viral infections and support antibody generation by B cells, making them desirable for assessing the immune response to SARS-CoV-2 infection and vaccines. We combined 2 high-throughput immune profiling methods to create a quantitative picture of the SARS-CoV-2 T-cell response that is highly sensitive, durable, diagnostic, and discriminatory between natural infection and vaccination. Methods. We deeply characterized 116 convalescent COVID-19 subjects by experimentally mapping CD8 and CD4 T-cell responses via antigen stimulation to 545 Human Leukocyte Antigen (HLA) class I and 284 class II viral peptides. We also performed T-cell receptor (TCR) repertoire sequencing on 1815 samples from 1521 PCR-confirmed SARS-CoV-2 cases and 3500 controls to identify shared public TCRs from SARS-CoV-2-associated CD8 and CD4 T cells. Combining these approaches with additional samples from vaccinated individuals, we characterized the response to natural infection as well as vaccination by separating responses to spike protein from other viral targets. Results. We find that T-cell responses are often driven by a few immunodominant, HLA-restricted epitopes. As expected, the SARS-CoV-2 T-cell response peaks about 1-2 weeks after infection and is detectable at least several months after recovery. Applying these data, we trained a classifier to diagnose past SARS-CoV-2 infection based solely on TCR sequencing from blood samples and observed, at 99.8% specificity, high sensitivity soon after diagnosis (Day 3-7 = 85.1%;Day 8-14 = 94.8%) that persists after recovery (Day 29+/convalescent = 95.4%). Finally, by evaluating TCRs binding epitopes targeting all non-spike SARS-CoV-2 proteins, we were able to separate natural infection from vaccination with > 99% specificity. Conclusion. TCR repertoire sequencing from whole blood reliably measures the adaptive immune response to SARS-CoV-2 soon after viral antigenic exposure (before antibodies are typically detectable) as well as at later time points, and distinguishes post-infection vs. vaccine immune responses with high specificity. This approach to characterizing the cellular immune response has applications in clinical diagnostics as well as vaccine development and monitoring.
ABSTRACT
Background. The initial response of immune cells against respiratory viruses often determines the severity and duration of disease. The early trajectory of the immune response during infection with SARS-CoV-2 remains poorly understood. Dysregulation of innate immune factors that facilitate viral clearance and the adaptive response, such as type I interferons, have been implicated in severe COVID-19. However, collection of biological samples during the first seven days post-symptom onset has posed a logistical challenge, limiting our knowledge surrounding the immune responses that drive protection versus immunopathology. Methods. From March 2020, Military Health System beneficiaries presenting with a positive SARS-CoV-2 test, a COVID-19 like illness, or a high-risk SARS-CoV-2 exposure at nine military medical treatment facilities across the United States were eligible for enrollment in our longitudinal cohort study, which included collection of respiratory sample, sera, plasma, and peripheral blood mononuclear cells (PBMCs). Twenty-five SARS-CoV-2 infected study participants provided samples with in the first seven days of symptom onset, fifteen of whom were hospitalized with COVID-19. We employed multiparameter spectral flow cytometry to comprehensively analyze the early trajectory of the innate and adaptive immune responses. Results. We discovered that early activation of critical antigen presenting cell subsets was impaired upon comparing inpatients with outpatients, correlating with decreased antigen-experienced T cell responses. Specifically, we noted reduced expression of key costimulatory molecules, CD80 and CD86, on conventional dendritic cells that are required for viral antigen-specific T cell priming. Reduction in CD38, a marker of activation was also observed on inpatient dendritic cell subsets. Conclusion. Reduced antigen presenting cell activation and expression of ligands that facilitate T cell engagement may impede the efficient clearance of SARS-CoV-2, coinciding with more severe disease in our cohort. Further analysis of the functional activation of early innate immune responses triggered by SARS-CoV-2 may unveil new immune biomarkers and therapeutic targets to predict and prevent severe disease associated with inadequate T cell immunity.