Search

Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct ex vivo staining of PBMC with tetramer reagents was used to define immunodominant and subdominant T cell epitopes and estimate the frequencies of these T cells in SARS-CoV-2 exposed and naïve individuals. Majority of SARS-CoV-2 epitopes identified have, Author summary Previous studies with activation induced marker assays in monitoring antigen-specific CD4+ T cells have shown that common cold coronavirus T cells can cross-react with SARS-CoV-2 antigens and these cross-reactive T cells are present in up to 60% of the unexposed population. In this current study, sets of overlapping peptides for Spike, Membrane, and Nucleocapsid proteins were used to identify epitopes across 11 HLA-DR and 1 HLA-DP alleles in SARS-CoV-2 convalescent samples using peptide-loaded MHC-II tetramers. Using these tetramers ex vivo, frequencies of these epitope-specific T cells were estimated in convalescent and pre-pandemic samples. Based on these frequencies, epitopes were stratified into immunodominant and subdominant epitopes. Amino acid sequences of epitopes identified were compared with 4 common cold coronaviruses. Potential cross-reactive epitopes were defined as having ≥67% sequence identity between common cold viruses and SARS-CoV-2. Four potential Spike specific cross-reactive epitopes were identified and functional cross-reactivity was demonstrated. Of the four cross-reactive epitopes identified, three were subdominant epitopes eliciting relatively low frequencies in both unexposed and convalescent subjects. In contrast to the results from the activation induced marker assays, the current data suggests that only a limited number of high avidity SARS-CoV-2 T cells as detected by tetramers are cross-reactive.

Adaptation of Staphylococcus aureus to host microenvironments during chronic infection involves spontaneous mutations, yet changes underlying adaptive phenotypes remain incompletely explored. Here, we employed artificial selection and whole-genome sequencing to better characterize spontaneous chromosomal mutations that alter two pathogenicity phenotypes relevant to chronic infection in S. aureus: intracellular invasiveness and intracellular cytotoxicity. We identified 23 genes whose alteration coincided with enhanced virulence, 11 that were previously known and 12 (52%) that had no previously described role in S. aureus pathogenicity. Using precision genome editing, transposon mutants, and gene complementation, we empirically assessed the contributions of individual genes to the two virulence phenotypes. We functionally validated 14 of 21 genes tested as measurably influencing invasion and/or cytotoxicity, including 8 newly implicated by this study. We identified inactivating mutations (murA, ndhC, and a hypothetical membrane protein) and gain-of-function mutations (aroE Thr182Ile, yhcF Thr74Ile, and Asp486Glu in a hypothetical peptidase) in previously unrecognized S. aureus virulence genes that enhance pathogenesis when introduced into a clean genetic background, as well as a novel activating mutation in the known virulence regulator gene saeS (Ala106Thr). Investigation of potentially epistatic interactions identified a tufA mutation (Ala271Val) that enhances virulence only in the context of purine operon repressor gene (purR) inactivation. This project reveals a functionally diverse range of genes affected by gain- or loss-of-function mutations that contribute to S. aureus adaptive virulence phenotypes. More generally, the work establishes artificial selection as a means to determine the genetic mechanisms underlying complex bacterial phenotypes relevant to adaptation during infection.

Vaccination can prevent infection and disease due to SARS-CoV-2. Early reports indicate that immune suppressed or immune compromised populations have reduced immune responses to US emergency use authorized (EUA) vaccines. Patients with autoimmune disorders are at risk for severe COVID-19, and are frequently immune suppressed related to therapy, the underlying disease, or both. Myasthenia gravis (MG) is an autoimmune disorder characterized by antibodies that interrupt neuromuscular transmission. Chronic immune suppressive therapy is typically required. We report the case of a 74 year old woman with MG receiving mycophenolate, prednisone, and eculizumab in whom mRNA vaccination failed to elicit detectable circulating vaccine-specific IgG or IFN-γ T cell responses. Eculizumab was discontinued, and repeat vaccination with two doses of an alternative EUA mRNA vaccine led to circulating IgG specific for the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and to detectable S-specific T cell responses. While it is not known if these responses will protect against SARS-CoV-2 infection or disease, a repeat course of mRNA vaccination appears to be safe and was broadly immunogenic in this individual., Highlights • T and B cell immune responses to SARS-CoV-2 mRNA vaccine reduced in an immune suppressed patient. • Repeat vaccination course with an alternative mRNA vaccine led to detectable, specific T and B cell responses. • Persons with autoimmune disease receiving immune suppression may benefit from additional doses of vaccine.