Some patients who survive severe COVID-19 develop persistent respiratory symptoms. Although increased activation of T-cells has been reported in severe acute disease, little is known about the long-term evolution of T-cells after SARS-CoV-2 infection in patients with long-haul symptoms. Circulating T-cells were tracked in a sample of a COVID-19 cohort (n=88) consisting of patients with persistent respiratory symptoms. Cells were obtained during severe acute COVID-19 illness and at 6 weeks and 6-11 months after hospital discharge. Cells were analyzed by high-dimensional immunophenotyping using spectral flow cytometry. Longitudinal changes in complex cell signatures were identified using the T-REX algorithm. Antibodies to SARS-CoV-2 proteins were assessed by ImmunoCAP assay. Patients with long-haul symptoms who were sampled at 6 weeks after hospital discharge had higher frequencies of activated (HLA-DR+CD38+) and tissue-homing (CCR5+) CD4+ and CD8+ T-cells compared to healthy subjects and patients with mild acute COVID-19. Higher numbers of terminally differentiated (CCR7-CD27-) CD8+ T-cells were also evident. T-REX identified multiple CD4+ and CD8+ T cell signatures that expanded or contracted by ≥95% up to 6 months after acute infection, including highly activated subtypes (CD3+6CD4+7CD45RA+7CD45RO+1CD38+1HLA-DR+4CD95+1T-BET+2TCF1+1KI-67+2 and CD3+6CD8+9CD45RA+6CD38+1HLA-DR+5CD95+2CCR5+2TCF1+1T-BET+6KI-67+2). Fluxes in T-cell signatures were detectable several months after acute infection, even in the presence of declining antibodies to SARS-CoV-2 proteins. Patients with long-haul respiratory symptoms after severe COVID-19 illness display activated T-cell signatures and marked immune perturbations, consistent with trafficking of T-cells with pathogenic potential and dysregulated homeostasis. Activated T-cells may contribute to airway inflammation long after acute illness resolves.

This review summarizes published findings of the beneficial and harmful effects on the heart, lungs, immune system, kidney, liver, and central nervous system of 47 drugs that have been proposed to treat COVID-19. Many of the repurposed drugs were chosen for their benefits to the pulmonary system, as well as immunosuppressive and anti-inflammatory effects. However, these drugs have mixed effects on the heart, liver, kidney, and central nervous system. Drug treatments are critical in the fight against COVID-19, along with vaccines and public health protocols. Drug treatments are particularly needed as variants of the SARS-Cov-2 virus emerge with some mutations that could diminish the efficacy of the vaccines. Patients with comorbidities are more likely to require hospitalization and greater interventions. The combination of treating severe COVID-19 symptoms in the presence of comorbidities underscores the importance of understanding the effects of potential COVID-19 treatments on other organs.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

Human rhinovirus (RV) is a major cause of the common cold and is known to induce acute asthma episodes in allergic subjects. Despite established roles for CD4+ T cells in both anti-viral immunity and allergic disease, the T cell mechanisms of RV-induced asthma are poorly understood. Current theory suggests a role for deficient anti-viral responses, as well as pro-allergic Th2 responses. In this study, we leveraged new pMHCII tetramer reagents to specifically probe both RV- and allergen-specific circulating CD4+ T cell responses to experimental RV infection in allergic asthmatic and control subjects. T cells were monitored for 3 weeks to capture acute and convalescent phases. RV-specific cells were readily detected prior to infection in both asthmatics and controls, whereas allergen-specific cells were only detected at appreciable frequencies in asthmatic subjects. While allergen-specific cells displayed predominantly Th2 phenotypes (CCR4+, CRTH2+) in allergic asthmatics, RV-specific cells were Th1-like (CXCR3+, CCR5+). The RV-specific Th1 signature was enhanced during acute infection and persisted into convalescence. A minority of cells of both specificities expressed hybrid Th1/Th2 molecular signatures. As predicted, RV-specific cells expanded and became activated during infection with RV-A16. However, this response was augmented in allergic asthmatics, and was accompanied by increased numbers of allergen-specific cells. Additionally, plasmacytoid dendritic cells (pDCs) increased during acute infection, but only in asthmatics. Taken together, these data suggest that RV-induced asthma is characterized by an augmented anti-viral Th1 response that is mediated by pDCs, and which co-opts allergen-specific Th2 cells.

Modified allergens that display reduced IgE reactivity along with T-cell activating properties, are strong candidates for immunotherapy, because of the potential to decrease side-effects due to IgE cross-linking, but still retain immunogenicity. Single and multiple Bla g 2 mutants were designed according to prior knowledge of the antigenic structure of the allergen and expressed in Pichia pastoris. Folding of the mutants was assessed by CD spectrometry or X-ray crystallography. IgE reactivity was measured by antibody binding and mast cell release assays. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and CD4+ T-cell phenotype in PBMC cultures. Single and multiple mutations of residues implicated in binding to monoclonal antibodies (K132A, K251A and/or F162Y) reduced IgE reactivity but did not influence the native molecular fold, as proven by comparing the triple mutant with wild type Bla g 2 by X-ray crystallography. As compared with wild type allergen, mutants KK and KKF showed from at least 100-fold to a total reduction in IgE antibody binding. Whereas similar T-cell activating capacity was retained based on CD25 expression, both mutants were weaker inducers of the Th2 cytokine, IL-13. Furthermore, both mutants induced high levels of IL-10 from a non-T-cell source, and levels induced by the triple mutant exceeded those induced by Bla g 2 (p=0.004). A rational design of site-directed mutagenesis was effective in producing candidate molecules for immunotherapy that maintain the same fold as wild type Bla g 2, but display reduced IgE reactivity with T-cell modulatory potential.

Rhinovirus (RV) is a major cause of common cold. Over 100 RV serotypes have been identified to date, and infections with different strains occur repeatedly. Despite this, pre-existing T cells that might confer protection have not been characterized. We sought to develop a panel of MHC II tetramers displaying RV peptides with a view to phenotyping circulating RV-specific CD4+ T cells. T-cell epitopes of RV-16 and RV-39 capsid proteins were identified by tetramer-guided epitope mapping and their conservation assessed by Jalview. Circulating RV-specific CD4+ T cells were identified in uninfected subjects by enriching PBMCs for tetramer+ cells, counterstaining for other surface markers, and analyzing by flow cytometry. T-cell epitopes of the RV capsid proteins, VP1 and VP2, mapped to regions that were highly conserved across all RV strains. Additionally, identical and overlapping RV capsid peptides were recognized in the context of multiple HLA alleles, indicating HLA promiscuity among RV epitopes. In healthy subjects, circulating RV-specific CD4+ T cells were predominantly inactivated (CD127+, CD25neg) memory cells (CD45RO+), including both central (CCR7+) and effector (CCR7neg) memory populations. Circulating RV-specific memory CD4+ T cells were heterogeneous as judged by the presence of CXCR5-PD-1- and CXCR5+PD-1+and subsets. Circulating RV-specific memory CD4+ T cells directed against conserved epitopes are readily identified in uninfected subjects that comprise T helper (CXCR5-PD-1-) and T follicular helper (CXCR5+PD-1+) phenotypes. Our findings suggest that further studies are warranted to explore the potential for pre-existing antigen-experienced RV-specific T cells to confer protection across multiple RV strains.

Little is known about T cell mechanisms of human rhinovirus (HRV) infection, despite the link to asthma. Our objective was to provide proof-of-concept for elucidating properties of circulating virus-specific CD4+ T cells in HRV-infected individuals using novel MHC II/peptide tetramers. HRV-39 seronegative, non-allergic subjects who were HLA-DR*0401-positive were infected intranasally with HRV-39. PBMCs isolated 28 days before inoculation, at day 0, and days 5 and 21 post-infection, were stained with PE-labeled HLA-DR*0401 tetramers containing HRV-39 epitopes. Cells were enriched using an anti-PE column, then stained for surface markers and analyzed by flow cytometry. A discrete population of tetramer-positive CD4+ T cells was detected at each time point using tetramers containing two novel epitopes of HRV-39 capsid proteins. Before infection, circulating HRV-specific T cells constituted 0.2-1.2% of CD4+ T cells following enrichment, corresponding to a precursor frequency of 4/10,000 to 3/1,000 CD4+ T cells. Five days post-infection, numbers increased to 1.2-10% of enriched cells (precursor frequency = 3/10,000 to 6/1,000 CD4+ T cells). Additionally, virus-specific T cells with a central memory phenotype (CCR7+ CD45RO+) decreased, whereas effector memory (CCR7- CD45RO+) and lung-homing (CCR5+) cells increased. At day 21, HRV-specific T cell numbers contracted, or else were further expanded among a subset of subjects. In subjects infected with HRV-39, circulating virus-specific CD4+ T cells expand and convert to an effector memory phenotype. Our findings demonstrate the feasibility of using tetramers containing epitopes of viral capsid proteins to accurately enumerate and phenotype HRV-specific CD4+ T cells in infected subjects.

One hundred twenty-one Liberian children were admitted in coma to the ELWA Hospital, Monrovia, Liberia. Admitting diagnoses, before lumbar puncture, were compared with discharge diagnoses. Ninety-four children were discharged with a final diagnosis of cerebral malaria and 27 with a diagnosis of meningitis. The admitting diagnosis was correct in 76.6% (72 of 94) of patients with cerebral malaria and 59.3% (16 of 27) of patients with meningitis. The cerebrospinal fluid leukocyte count was the single most significant factor in determining the correct diagnosis. Without the cerebrospinal fluid analysis, the discriminant accuracy (77%), i.e. definitive separation of the two illnesses, was comparable to the physician's admission diagnosis (73%). Other data contributing to the differential diagnosis of cerebral malaria and meningitis included the number of days of fever before admission, the presence or absence of nuchal rigidity, fontanelle fullness and peripheral blood malaria smear. Mortality rates for cerebral malaria and meningitis were 14.9 and 29.6%, respectively. These data suggest that physicians cannot reliably discriminate between cerebral malaria and meningitis without cerebrospinal fluid analysis.