Targeted plasma proteomics reveals signatures discriminating COVID-19 from sepsis with pneumonia.

BACKGROUND: COVID-19 remains a major public health challenge, requiring the development of tools to improve diagnosis and inform therapeutic decisions. As dysregulated inflammation and coagulation responses have been implicated in the pathophysiology of COVID-19 and sepsis, we studied their plasma proteome profiles to delineate similarities from specific features. METHODS: We measured 276 plasma proteins involved in Inflammation, organ damage, immune response and coagulation in healthy controls, COVID-19 patients during acute and convalescence phase, and sepsis patients; the latter included (i) community-acquired pneumonia (CAP) caused by Influenza, (ii) bacterial CAP, (iii) non-pneumonia sepsis, and (iv) septic shock patients. RESULTS: We identified a core response to infection consisting of 42 proteins altered in both COVID-19 and sepsis, although higher levels of cytokine storm-associated proteins were evident in sepsis. Furthermore, microbiologic etiology and clinical endotypes were linked to unique signatures. Finally, through machine learning, we identified biomarkers, such as TRIM21, PTN and CASP8, that accurately differentiated COVID-19 from CAP-sepsis with higher accuracy than standard clinical markers. CONCLUSIONS: This study extends the understanding of host responses underlying sepsis and COVID-19, indicating varying disease mechanisms with unique signatures. These diagnostic and severity signatures are candidates for the development of personalized management of COVID-19 and sepsis.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells

Natural killer (NK) cells eliminate virus-infected cells. Hammer et al. show that SARS-CoV-2 encodes for a peptide that does not bind to an inhibitory receptor of NK cells, thereby facilitating NK cell activation. This missing self recognition could enable NK cells to detect and kill SARS-CoV-2-infected cells.

High-dimensional profiling reveals phenotypic heterogeneity and disease-specific alterations of granulocytes in COVID-19.

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion, and migration of granulocytes (e.g., CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers, supporting pathophysiologic relevance. Furthermore, clinical features, including multiorgan dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.

Systematic evaluation of SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

OBJECTIVE: The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. METHODS: More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. RESULTS: Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. CONCLUSION: These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

SARS-CoV-2-specific humoral and cellular immunity persists through 9 months irrespective of COVID-19 severity at hospitalisation.

OBJECTIVES: Humoral and cellular immunity to SARS-CoV-2 following COVID-19 will likely contribute to protection from reinfection or severe disease. It is therefore important to characterise the initiation and persistence of adaptive immunity to SARS-CoV-2 amidst the ongoing pandemic. METHODS: Here, we conducted a longitudinal study on hospitalised moderate and severe COVID-19 patients from the acute phase of disease into convalescence at 5 and 9 months post-symptom onset. Utilising flow cytometry, serological assays as well as B cell and T cell FluoroSpot assays, we assessed the magnitude and specificity of humoral and cellular immune responses during and after human SARS-CoV-2 infection. RESULTS: During acute COVID-19, we observed an increase in germinal centre activity, a substantial expansion of antibody-secreting cells and the generation of SARS-CoV-2-neutralising antibodies. Despite gradually decreasing antibody levels, we show persistent, neutralising antibody titres as well as robust specific memory B cell responses and polyfunctional T cell responses at 5 and 9 months after symptom onset in both moderate and severe COVID-19 patients. CONCLUSION: Our findings describe the initiation and, importantly, persistence of cellular and humoral SARS-CoV-2-specific immunological memory in hospitalised COVID-19 patients long after recovery, likely contributing towards protection against reinfection.

Shedding of infectious SARS-CoV-2 by hospitalized COVID-19 patients in relation to serum antibody responses.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic. The understanding of the transmission and the duration of viral shedding in SARS-CoV-2 infection is still limited. OBJECTIVES: To assess the timeframe and potential risk of SARS-CoV-2 transmission from hospitalized COVID-19 patients in relation to antibody response. METHOD: We performed a cross-sectional study of 36 COVID-19 patients hospitalized at Karolinska University Hospital. Patients with more than 8 days of symptom duration were sampled from airways, for PCR analysis of SARS-CoV-2 RNA and in vitro culture of replicating virus. Serum SARS-CoV-2-specific immunoglobulin G (IgG) and neutralizing antibodies titers were assessed by immunofluorescence assay (IFA) and microneutralization assay. RESULTS: SARS-CoV-2 RNA was detected in airway samples in 23 patients (symptom duration median 15 days, range 9-53 days), whereas 13 patients were SARS-CoV-2 RNA negative (symptom duration median 21 days, range 10-37 days). Replicating virus was detected in samples from 4 patients at 9-16 days. All but two patients had detectable levels of SARS-CoV-2-specific IgG in serum, and SARS-CoV-2 neutralizing antibodies were detected in 33 out of 36 patients. Total SARS-CoV-2-specific IgG titers and neutralizing antibody titers were positively correlated. High levels of both total IgG and neutralizing antibody titers were observed in patients sampled later after symptom onset and in patients where replicating virus could not be detected. CONCLUSIONS: Our data suggest that the presence of SARS-Cov-2 specific antibodies in serum may indicate a lower risk of shedding infectious SARS-CoV-2 by hospitalized COVID-19 patients.

Evaluation of 11 SARS-CoV-2 antibody tests by using samples from patients with defined IgG antibody titers.

We evaluated the performance of 11 SARS-CoV-2 antibody tests using a reference set of heat-inactivated samples from 278 unexposed persons and 258 COVID-19 patients, some of whom contributed serial samples. The reference set included samples with a variation in SARS-CoV-2 IgG antibody titers, as determined by an in-house immunofluorescence assay (IFA). The five evaluated rapid diagnostic tests had a specificity of 99.0% and a sensitivity that ranged from 56.3 to 81.6% and decreased with low IFA IgG titers. The specificity was > 99% for five out of six platform-based tests, and when assessed using samples collected ≥ 22 days after symptom onset, two assays had a sensitivity of > 96%. These two assays also detected samples with low IFA titers more frequently than the other assays. In conclusion, the evaluated antibody tests showed a heterogeneity in their performances and only a few tests performed well with samples having low IFA IgG titers, an important aspect for diagnostics and epidemiological investigations.

Innate lymphoid cell composition associates with COVID-19 disease severity.

OBJECTIVES: The role of innate lymphoid cells (ILCs) in coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is unknown. Understanding the immune response in COVID-19 could contribute to unravel the pathogenesis and identification of treatment targets. Here, we describe the phenotypic landscape of circulating ILCs in COVID-19 patients and identified ILC phenotypes correlated to serum biomarkers, clinical markers and laboratory parameters relevant in COVID-19. METHODS: Blood samples collected from moderately (n = 11) and severely ill (n = 12) COVID-19 patients, as well as healthy control donors (n = 16), were analysed with 18-parameter flow cytometry. Using supervised and unsupervised approaches, we examined the ILC activation status and homing profile. Clinical and laboratory parameters were obtained from all COVID-19 patients, and serum biomarkers were analysed with multiplex immunoassays. RESULTS: Innate lymphoid cells were largely depleted from the circulation of COVID-19 patients compared with healthy controls. Remaining circulating ILCs revealed decreased frequencies of ILC2 in severe COVID-19, with a concomitant decrease of ILC precursors (ILCp) in all patients, compared with controls. ILC2 and ILCp showed an activated phenotype with increased CD69 expression, whereas expression levels of the chemokine receptors CXCR3 and CCR4 were significantly altered in ILC2 and ILCp, and ILC1, respectively. The activated ILC profile of COVID-19 patients was associated with soluble inflammatory markers, while frequencies of ILC subsets were correlated with laboratory parameters that reflect the disease severity. CONCLUSION: This study provides insights into the potential role of ILCs in immune responses against SARS-CoV-2, particularly linked to the severity of COVID-19.

MAIT cell activation and dynamics associated with COVID-19 disease severity.

Severe COVID-19 is characterized by excessive inflammation of the lower airways. The balance of protective versus pathological immune responses in COVID-19 is incompletely understood. Mucosa-associated invariant T (MAIT) cells are antimicrobial T cells that recognize bacterial metabolites, and can also function as innate-like sensors and mediators of antiviral responses. Here, we investigated the MAIT cell compartment in COVID-19 patients with moderate and severe disease, as well as in convalescence. We show profound and preferential decline in MAIT cells in the circulation of patients with active disease paired with strong activation. Furthermore, transcriptomic analyses indicated significant MAIT cell enrichment and pro-inflammatory IL-17A bias in the airways. Unsupervised analysis identified MAIT cell CD69high and CXCR3low immunotypes associated with poor clinical outcome. MAIT cell levels normalized in the convalescent phase, consistent with dynamic recruitment to the tissues and later release back into the circulation when disease is resolved. These findings indicate that MAIT cells are engaged in the immune response against SARS-CoV-2 and suggest their possible involvement in COVID-19 immunopathogenesis.

Expansion of SARS-CoV-2-Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific Abs are typically a major predictor of protective immunity, yet human B cell and Ab responses during COVID-19 are not fully understood. In this study, we analyzed Ab-secreting cell and Ab responses in 20 hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19 and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific Ab-secreting cells in all 20 COVID-19 patients using a multicolor FluoroSpot Assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing Abs by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG, and IgM Ab levels positively correlated with SARS-CoV-2-neutralizing Ab titers, suggesting that SARS-CoV-2-specific Ab levels may reflect the titers of neutralizing Abs in COVID-19 patients during the acute phase of infection. Last, we showed that IL-6 and C-reactive protein serum concentrations were higher in patients who were hospitalized for longer, supporting the recent observations that IL-6 and C-reactive protein could be used as markers for COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in 20 COVID-19 patients, with a focus on B cell and Ab responses, and describes tools to study immune responses to SARS-CoV-2 infection and vaccination.