Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19) 1 – 4 . However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

CLIA-certified laboratories were enrolled through the IMPACT biorepository study 15. In the IMPACT study, biospecimens including blood, nasopharyngeal swabs, saliva, urine and stool samples were collected at study enrolment (baseline denotes the first time point) and longitudinally on average every 3 to 7 days (serial time points). The detailed demographics and clinical characteristics of these 98 participants are shown in Extended Data Table 1. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood, and plasma was used for titre measurements of SARS-CoV-2 spike S1 protein-specific IgG and IgM antibodies (anti-S1-IgG and-IgM) and cytokine or chemokine measurements. Freshly isolated PBMCs were stained and analysed by flow cytometry 15. We obtained longitudinal serial time-point samples from a subset of these 98 study participants (n = 48; information in Extended Data Table 1). To compare the immune phenotypes between sexes, two sets of data analyses were performed in parallel-baseline and longitudinal, as described below. As a control group, healthcare workers (HCWs) from Yale-New Haven Hospital were enrolled who were uninfected with COVID-19. Demographics and background information for the HCW group and the demographics of HCWs for cytokine assays and flow cytometry assays for the primary analyses are in Extended Data Table 1. Demographic data, time-point information of the samples defined by the days from the symptom onset (DFSO) in each patient, treatment information, and raw data used to generate figures and tables is in Supplementary Table 1. Baseline analysis The baseline analysis was performed on samples from the first time point of patients who met the following criteria: not in intensive care unit (ICU), had not received tocilizumab, and had not received high doses of corticosteroids (prednisone equivalent of more than 40 mg) before the first sample collection date. This patient group, cohort A, consisted of 39 patients (17 male and 22 female) (Extended Data Tables 1, 2). Intersex and transgender individuals were not represented in this study. Figures 1-4 represent analyses of baseline raw values obtained from patients in cohort A. In cohort A patients, male and female patients were matched in terms of age, body mass index (BMI), and DFSO at the first time point sample collection (Extended Data Fig. 1a). However, there were significant differences in age and BMI between HCW controls and patients (patients had higher age and BMI values) (Extended Data Table 1), and therefore an age-and BMI-adjusted difference-indifferences analysis was also performed in parallel (Extended Data Table 3). Longitudinal analysis As parallel secondary analyses, we performed longitudinal analysis on a total patient cohort (cohort B) to evaluate the difference in immune response over the course of the disease between male and female patients. Cohort B included all patient samples from cohort A (including several time-point samples from the cohort A patients) as well as an additional 59 patients who d...

Rapid and accurate SARS-CoV-2 diagnostic testing is essential for controlling the ongoing COVID-19 pandemic. The current gold standard for COVID-19 diagnosis is real-time RT-PCR detection of SARS-CoV-2 from nasopharyngeal swabs. Low sensitivity, exposure risks to healthcare workers, and global shortages of swabs and personal protective equipment, however, necessitate the validation of new diagnostic approaches. Saliva is a promising candidate for SARS-CoV-2 diagnostics because (1) collection is minimally invasive and can reliably be self-administered and (2) saliva has exhibited comparable sensitivity to nasopharyngeal swabs in detection of other respiratory pathogens, including endemic human coronaviruses, in previous studies. To validate the use of saliva for SARS-CoV-2 detection, we tested nasopharyngeal and saliva samples from confirmed COVID-19 patients and self-collected samples from healthcare workers on COVID-19 wards. When we compared SARS-CoV-2 detection from patient-matched nasopharyngeal and saliva samples, we found that saliva yielded greater detection sensitivity and NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.consistency throughout the course of infection. Furthermore, we report less variability in self-sample collection of saliva. Taken together, our findings demonstrate that saliva is a viable and more sensitive alternative to nasopharyngeal swabs and could enable at-home self-administered sample collection for accurate large-scale SARS-CoV-2 testing.

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COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1–8. While pathological innate immune activation is well documented in severe disease1, the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the “exoproteome”). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with a high prevalence of autoantibodies against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.

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Nuclear factor erythroid-related factor 2 (NRF2) encodes a transcription factor that induces expression of cytoprotective proteins upon oxidative stress and oncogenic NRF2 mutations have been found in lung and head/neck cancers that inactivate KEAP1-mediated degradation of NRF2. The aim of this study was to catalogue NRF2 mutations in other human cancers. For this, we analysed 1145 cancer tissues from carcinomas from oesophagus, skin, uterine cervix, lung, larynx, breast, colon, stomach, liver, prostate, urinary bladder, ovary, uterine cervix, and kidney, and meningiomas, multiple myelomas, and acute leukaemias by single-strand conformation polymorphism (SSCP) assay. We detected NRF2 mutations in oesophagus (8/70; 11.4%), skin (1/17; 6.3%), lung (10/125; 8.0%), and larynx (3/23; 13.0%) cancers. Of note, all of the 22 mutations except one were found in squamous cell carcinomas (SCCs) (95.5%). The mutations were observed within or near DLG and ETGE motifs that are important in NRF2 and KEAP1 interaction. All of the oesophageal SCCs and skin SCCs with the NRF2 mutations showed increased NRF2 expression in the nuclei. However, none of the SCCs from oesophagus and skin harboured KEAP1 mutation. Our study demonstrated here that NRF2 mutation occurs not only in lung and head/neck cancers, but also in oesophageal and skin cancers. Our data suggest that the NRF2 mutation plays a role in the development of SCC and is a feature of SCC.

OBJECTIVE-Progranulin is an important molecule in inflammatory response. Chronic inflammation is frequently associated with central obesity and associated disturbances; however, the role of circulating progranulin in human obesity, type 2 diabetes, and dyslipidemia is unknown.RESEARCH DESIGN AND METHODS-For the measurement of progranulin serum concentrations, we developed an enzymelinked immunosorbent assay (ELISA). Using this ELISA, we assessed circulating progranulin in a cross-sectional study of 209 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance and in 60 individuals with normal (NGT) or impaired (IGT) glucose tolerance or type 2 diabetes before and after a 4-week physical training program. Progranulin mRNA and protein expression was measured in paired samples of omental and subcutaneous adipose tissue (adipocytes and cells of the stromal vascular fraction) from 55 lean or obese individuals. Measurement of Erk activation and chemotactic activity induced by progranulin in vitro was performed using THP-1-based cell migration assays.RESULTS-Progranulin serum concentrations were significantly higher in individuals with type 2 diabetes compared with NGT and in obese subjects with predominant visceral fat accumulation. Circulating progranulin significantly correlates with BMI, macrophage infiltration in omental adipose tissue, C-reactive protein (CRP) serum concentrations, A1C values, and total cholesterol. Multivariable linear regression analyses revealed CRP levels as the strongest independent predictor of circulating progranulin. The extent of in vitro progranulin-mediated chemotaxis is similar to that of monocyte chemoattractant protein-1 but independent of G␣. Moreover, in type 2 diabetes, but not in IGT and NGT individuals, physical training for 4 weeks resulted in significantly decreased circulating progranulin levels.CONCLUSIONS-Elevated progranulin serum concentrations are associated with visceral obesity, elevated plasma glucose, and dyslipidemia. We identified progranulin as a novel marker of chronic inflammation in obesity and type 2 diabetes that closely reflects omental adipose tissue macrophage infiltration. Physical training significantly reduces elevated circulating progranulin in patients with type 2 diabetes. Diabetes 58:627-636, 2009