Changes in Phenotypic and Molecular Features of Naïve and Central Memory T Helper Cell Subsets following SARS-CoV-2 Vaccination.

Molecular changes in lymphocytes following SARS-CoV-2 vaccination are incompletely understood. We hypothesized that studying the molecular (transcriptomic, epigenetic, and T cell receptor (TCR) repertoire) changes in CD4+ T cells following SARS-CoV-2 vaccination could inform protective mechanisms and refinement of future vaccines. We tested this hypothesis by reporting alterations in CD4+ T cell subsets and molecular features of CD4+ naïve and CD4+ central memory (CM) subsets between the unvaccinated and vaccinated groups. Compared with the unvaccinated, the vaccinated had higher HLA-DR expression in CD4+ T subsets, a greater number of differentially expressed genes (DEGs) that overlapped with key differentially accessible regions (DARs) along the chromatin linked to inflammasome activation, translation, regulation (of apoptosis, inflammation), and significant changes in clonal architecture beyond SARS-CoV-2 specificity. Several of these differences were more pronounced in the CD4+CM subset. Taken together, our observations imply that the COVID-19 vaccine exerts its protective effects via modulation of acute inflammation to SARS-CoV-2 challenge.

Shared neutrophil and T cell dysfunction is accompanied by a distinct interferon signature during severe febrile illnesses in children.

Severe febrile illnesses in children encompass life-threatening organ dysfunction caused by diverse pathogens and other severe inflammatory syndromes. A comparative approach to these illnesses may identify shared and distinct features of host immune dysfunction amenable to immunomodulation. Here, using immunophenotyping with mass cytometry and cell stimulation experiments, we illustrate trajectories of immune dysfunction in 74 children with multi-system inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, 30 with bacterial infection, 16 with viral infection, 8 with Kawasaki disease, and 42 controls. We explore these findings in a secondary cohort of 500 children with these illnesses and 134 controls. We show that neutrophil activation and apoptosis are prominent in multi-system inflammatory syndrome, and that this is partially shared with bacterial infection. We show that memory T cells from patients with multi-system inflammatory syndrome and bacterial infection are exhausted. In contrast, we show viral infection to be characterized by a distinct signature of decreased interferon signaling and lower interferon receptor gene expression. Improved understanding of immune dysfunction may improve approaches to immunomodulator therapy in severe febrile illnesses in children.

INVESTIGATION INTO P2Y RECEPTOR FUNCTION IN PLATELETS FROM PATIENTS WITH SEPSIS.

ABSTRACT: Key underlying pathological mechanisms contributing to sepsis are hemostatic dysfunction and overwhelming inflammation. Platelet aggregation is required for hemostasis, and platelets are also separately involved in inflammatory responses that require different functional attributes. Nevertheless, P2Y receptor activation of platelets is required for this dichotomy of function. The aim of this study was to elucidate whether P2YR-dependent hemostatic and inflammatory functions were altered in platelets isolated from sepsis patients, compared with patients with mild sterile inflammation. Platelets from patients undergoing elective cardiac surgery (20 patients, 3 female) or experiencing sepsis after community-acquired pneumonia (10 patients, 4 female) were obtained through the IMMunE dysfunction and Recovery from SEpsis-related critical illness in adults (IMMERSE) Observational Clinical Trial. In vitro aggregation and chemotaxis assays were performed with platelets after stimulation with ADP and compared with platelets isolated from healthy control subjects (7 donors, 5 female). Cardiac surgery and sepsis both induced a robust inflammatory response with increases in circulating neutrophil counts with a trend toward decreased circulating platelet counts being observed. The ability of platelets to aggregate in response to ex vivo ADP stimulation was preserved in all groups. However, platelets isolated from patients with sepsis lost the ability to undergo chemotaxis toward N -formylmethionyl-leucyl-phenylalanine, and this suppression was evident at admission through to and including discharge from hospital. Our results suggest that P2Y 1 -dependent inflammatory function in platelets is lost in patients with sepsis resulting from community-acquired pneumonia. Further studies will need to be undertaken to determine whether this is due to localized recruitment to the lungs of a platelet responsive population or loss of function as a result of dysregulation of the immune response.

Comparison of systemic inflammatory profiles in COVID-19 and community-acquired pneumonia patients: a prospective cohort study.

BACKGROUND: Inflammatory responses contribute to tissue damage in COVID-19 and community-acquired pneumonia (CAP). Although predictive values of different inflammatory biomarkers have been reported in both, similarities and differences of inflammatory profiles between these conditions remain uncertain. Therefore, we aimed to determine the similarities and differences of the inflammatory profiles between COVID-19 and CAP, and their association with clinical outcomes. METHODS: We report a prospective observational cohort study; conducted in a reference hospital in Latin America. Patients with confirmed COVID-19 pneumonia and CAP were included. Multiplex (Luminex) cytokine assays were used to measure the plasma concentration of 14 cytokines at hospital admission. After comparing similarities and differences in the inflammatory profile between COVID-19 and CAP patients, an unsupervised classification method (i.e., hierarchical clustering) was used to identify subpopulations within COVID-19 and CAP patients. RESULTS: A total of 160 patients were included, 62.5% were diagnosed with COVID-19 (100/160), and 37.5% with CAP (60/160). Using the hierarchical clustering, COVID-19 and CAP patients were divided based on its inflammatory profile: pauci, moderate, and hyper-inflammatory immune response. COVID-19 hyper-inflammatory subpopulation had the highest mortality. COVID-19 hyper-inflammatory subpopulation, compared to pauci-inflammatory, had higher levels of IL-10 (median [IQR] 61.4 [42.0-109.4] vs 13.0 [5.0-24.9], P: < 0.001), IL-6 (48.1 [22.3-82.6] vs 9.1 [0.1-30.4], P: < 0.001), among others. Hyper-inflammatory vs pauci-inflammatory CAP patients were characterized by elevation of IFN2 (48.8 [29.7-110.5] vs 3.0 [1.7-10.3], P: < 0.001), TNFα (36.3 [24.8-53.4] vs 13.1 [11.3-16.9], P: < 0.001), among others. Hyper-inflammatory subpopulations in COVID-19 and CAP compared to the corresponding pauci-inflammatory subpopulations had higher MCP-1. CONCLUSIONS: Our data highlights three distinct subpopulations in COVID-19 and CAP, with differences in inflammatory marker profiles and risks of adverse clinical outcomes. TRIAL REGISTRATION: This is a prospective study, therefore no health care intervention were applied to participants and trial registration is not applicable.

Cellular and molecular mechanisms of IMMunE dysfunction and Recovery from SEpsis-related critical illness in adults: An observational cohort study (IMMERSE) protocol paper.

Sepsis is a common illness. Immune responses are considered major drivers of sepsis illness and outcomes. However, there are no proven immunomodulator therapies in sepsis. We hypothesised that in-depth characterisation of sepsis-specific immune trajectory may inform immunomodulation in sepsis-related critical illness. We describe the protocol of the IMMERSE study to address this hypothesis. We include critically ill sepsis patients without documented immune comorbidity and age-sex matched cardiac surgical patients as controls. We plan to perform an in-depth biological characterisation of innate and adaptive immune systems, platelet function, humoral components and transcriptional determinants of the immune system responses in sepsis. This will be done at pre-specified time points during their critical illness to generate an illness trajectory. The sample size for each biological assessment is different and is described in detail. In summary, the overall aim of the IMMERSE study is to increase the granularity of longitudinal immunology model of sepsis to inform future immunomodulation trials.

Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR.

Tools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold (CT) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARS-CoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.

Virological Characterization of Critically Ill Patients With COVID-19 in the United Kingdom: Interactions of Viral Load, Antibody Status, and B.1.1.7 Infection.

BACKGROUND: Convalescent plasma containing neutralizing antibody to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is under investigation for coronavirus disease 2019 (COVID-19) treatment. We report diverse virological characteristics of UK intensive care patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomized controlled trial that potentially influence treatment outcomes. METHODS: SARS-CoV-2 RNA in nasopharyngeal swabs collected pretreatment was quantified by PCR. Antibody status was determined by spike-protein ELISA. B.1.1.7 was differentiated from other SARS-CoV-2 strains using allele-specific probes or restriction site polymorphism (SfcI) targeting D1118H. RESULTS: Of 1274 subjects, 90% were PCR positive with viral loads 118-1.7 × 1011IU/mL. Median viral loads were 40-fold higher in those IgG seronegative (n = 354; 28%) compared to seropositives (n = 939; 72%). Frequencies of B.1.1.7 increased from <1% in November 2020 to 82% of subjects in January 2021. Seronegative individuals with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians 5.8 × 106 and 2.0 × 105 IU/mL, respectively; P = 2 × 10-15). CONCLUSIONS: High viral loads in seropositive B.1.1.7-infected subjects and resistance to seroconversion indicate less effective clearance by innate and adaptive immune responses. SARS-CoV-2 strain, viral loads, and antibody status define subgroups for analysis of treatment efficacy.

Acute Immune Signatures and Their Legacies in Severe Acute Respiratory Syndrome Coronavirus-2 Infected Cancer Patients.

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19+ non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care.

Author Correction: A dynamic COVID-19 immune signature includes associations with poor prognosis.

A Correction to this paper has been published: https://doi.org/10.1038/s41591-020-01186-5.

Author Correction: A dynamic COVID-19 immune signature includes associations with poor prognosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A dynamic COVID-19 immune signature includes associations with poor prognosis.

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.

Peripheral immunophenotypes in children with multisystem inflammatory syndrome associated with SARS-CoV-2 infection.

Recent reports highlight a new clinical syndrome in children related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1-multisystem inflammatory syndrome in children (MIS-C)-which comprises multiorgan dysfunction and systemic inflammation2-13. We performed peripheral leukocyte phenotyping in 25 children with MIS-C, in the acute (n = 23; worst illness within 72 h of admission), resolution (n = 14; clinical improvement) and convalescent (n = 10; first outpatient visit) phases of the illness and used samples from seven age-matched healthy controls for comparisons. Among the MIS-C cohort, 17 (68%) children were SARS-CoV-2 seropositive, suggesting previous SARS-CoV-2 infections14,15, and these children had more severe disease. In the acute phase of MIS-C, we observed high levels of interleukin-1ß (IL-1ß), IL-6, IL-8, IL-10, IL-17, interferon-γ and differential T and B cell subset lymphopenia. High CD64 expression on neutrophils and monocytes, and high HLA-DR expression on γδ and CD4+CCR7+ T cells in the acute phase, suggested that these immune cell populations were activated. Antigen-presenting cells had low HLA-DR and CD86 expression, potentially indicative of impaired antigen presentation. These features normalized over the resolution and convalescence phases. Overall, MIS-C presents as an immunopathogenic illness1 and appears distinct from Kawasaki disease.