Cross-reactive and mono-reactive SARS-CoV-2 CD4+ T cells in prepandemic and COVID-19 convalescent individuals.

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 <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies.

Artificial Selection for Pathogenicity Mutations in Staphylococcus aureus Identifies Novel Factors Relevant to Chronic Infection.

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.

Selective retention of virus-specific tissue-resident T cells in healed skin after recovery from herpes zoster.

Herpes zoster is a localized skin infection caused by reactivation of latent varicella-zoster virus. Tissue-resident T cells likely control skin infections. Zoster provides a unique opportunity to determine if focal reinfection of human skin boosts local or disseminated antigen-specific tissue-resident T cells. Here, we show virus-specific T cells are retained over one year in serial samples of rash site and contralateral unaffected skin of individuals recovered from zoster. Consistent with zoster resolution, viral DNA is largely undetectable on skin from day 90 and virus-specific B and T cells decline in blood. In skin, there is selective infiltration and long-term persistence of varicella-zoster virus-specific T cells in the rash site relative to the contralateral site. The skin T cell infiltrates express the canonical tissue-resident T cell markers CD69 and CD103. These findings show that zoster promotes spatially-restricted long-term retention of antigen-specific tissue-resident T cells in previously infected skin.

T cell response to intact SARS-CoV-2 includes coronavirus cross-reactive and variant-specific components.

SARS-CoV-2 provokes a brisk T cell response. Peptide-based studies exclude antigen processing and presentation biology and may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DC to activate CD8 and CD4 T cells from convalescent persons. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the alpha, beta, gamma, and delta variant lineages.

T cell response to intact SARS-CoV-2 includes coronavirus cross-reactive and variant-specific components.

SARS-CoV-2 provokes a robust T cell response. Peptide-based studies exclude antigen processing and presentation biology, which may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DCs to activate CD8 and CD4 T cells from convalescent people. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory tract cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the Alpha, Beta, Gamma, and Delta variant lineages.

Immunogenicity of repeat COVID-19 mRNA vaccinations in a patient with myasthenia gravis receiving mycophenolate, prednisone, and eculizumab.

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.