Robust humoral and cellular immune responses and low risk for reinfection at least 8 months following asymptomatic to mild COVID-19.

BACKGROUND: Emerging data support detectable immune responses for months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination, but it is not yet established to what degree and for how long protection against reinfection lasts. METHODS: We investigated SARS-CoV-2-specific humoral and cellular immune responses more than 8 months post-asymptomatic, mild and severe infection in a cohort of 1884 healthcare workers (HCW) and 51 hospitalized COVID-19 patients. Possible protection against SARS-CoV-2 reinfection was analyzed by a weekly 3-month polymerase chain reaction (PCR) screening of 252 HCW that had seroconverted 7 months prior to start of screening and 48 HCW that had remained seronegative at multiple time points. RESULTS: All COVID-19 patients and 96% (355/370) of HCW who were anti-spike IgG positive at inclusion remained anti-spike IgG positive at the 8-month follow-up. Circulating SARS-CoV-2-specific memory T cell responses were detected in 88% (45/51) of COVID-19 patients and in 63% (233/370) of seropositive HCW. The cumulative incidence of PCR-confirmed SARS-CoV-2 infection was 1% (3/252) among anti-spike IgG positive HCW (0.13 cases per 100 weeks at risk) compared to 23% (11/48) among anti-spike IgG negative HCW (2.78 cases per 100 weeks at risk), resulting in a protective effect of 95.2% (95% CI 81.9%-99.1%). CONCLUSIONS: The vast majority of anti-spike IgG positive individuals remain anti-spike IgG positive for at least 8 months regardless of initial COVID-19 disease severity. The presence of anti-spike IgG antibodies is associated with a substantially reduced risk of reinfection up to 9 months following asymptomatic to mild COVID-19.

Patients With COVID-19 Have Elevated Levels of Circulating Extracellular Vesicle Tissue Factor Activity That Is Associated With Severity and Mortality-Brief Report.

OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) have a high rate of thrombosis. We hypothesized that severe acute respiratory syndrome coronavirus 2 infection leads to induction of TF (tissue factor) expression and increased levels of circulating TF-positive extracellular vesicles (EV) that may drive thrombosis. Approach and Results: We measured levels of plasma EV TF activity in 100 patients with COVID-19 with moderate and severe disease and 28 healthy controls. Levels of EV TF activity were significantly higher in patients with COVID-19 compared with controls. In addition, levels of EV TF activity were associated with disease severity and mortality. Finally, levels of EV TF activity correlated with several plasma markers, including D-dimer, which has been shown to be associated with thrombosis in patients with COVID-19. CONCLUSIONS: Our results indicate that severe acute respiratory syndrome coronavirus 2 infection induces the release of TF-positive EVs into the circulation that are likely to contribute to thrombosis in patients with COVID-19. EV TF activity was also associated with severity and mortality.

Circulating Markers of Neutrophil Extracellular Traps Are of Prognostic Value in Patients With COVID-19.

OBJECTIVE: The full spectrum of coronavirus disease 2019 (COVID-19) infection ranges from asymptomatic to acute respiratory distress syndrome, characterized by hyperinflammation and thrombotic microangiopathy. The pathogenic mechanisms are poorly understood, but emerging evidence suggest that excessive neutrophil extracellular trap (NET) formation plays a key role in COVID-19 disease progression. Here, we evaluate if circulating markers of NETs are associated with COVID-19 disease severity and clinical outcome, as well as to markers of inflammation and in vivo coagulation and fibrinolysis. Approach and Results: One hundred six patients with COVID-19 with moderate to severe disease were enrolled shortly after hospital admission and followed for 4 months. Acute and convalescent plasma samples as well as plasma samples from 30 healthy individuals were assessed for markers of NET formation: citrullinated histone H3, cell-free DNA, NE (neutrophil elastase). We found that all plasma levels of NET markers were elevated in patients with COVID-19 relative to healthy controls, that they were associated with respiratory support requirement and short-term mortality, and declined to those found in healthy individuals 4 months post-infection. The levels of the NET markers also correlated with white blood cells, neutrophils, inflammatory cytokines, and C-reactive protein, as well as to markers of in vivo coagulation, fibrinolysis, and endothelial damage. CONCLUSIONS: Our findings suggest a role of NETs in COVID-19 disease progression, implicating their contribution to an immunothrombotic state. Further, we observed an association between circulating markers of NET formation and clinical outcome, demonstrating a potential role of NET markers in clinical decision-making, as well as for NETs as targets for novel therapeutic interventions in COVID-19.

Comparison of SARS-CoV-2 spike-specific IgA and IgG in nasal secretions, saliva and serum.

Introduction: Several novel vaccine platforms aim at mucosal immunity in the respiratory tract to block SARS-CoV-2 transmission. Standardized methods for mucosal sample collection and quantification of mucosal antibodies are therefore urgently needed for harmonized comparisons and interpretations across mucosal vaccine trials and real-world data. Methods: Using commercial electrochemiluminescence antibody panels, we compared SARS-CoV-2 spike-specific IgA and IgG in paired saliva, nasal secretions, and serum from 1048 healthcare workers with and without prior infection. Results: Spike-specific IgA correlated well in nasal secretions and saliva (r>0.65, p<0.0001), but the levels were more than three-fold higher in nasal secretions as compared to in saliva (p<0.01). Correlations between the total population of spike-specific IgA and spike-specific secretory IgA (SIgA) were significantly stronger (p<0.0001) in nasal secretions (r=0.96, p<0.0001) as opposed to in saliva (r=0.77, p<0.0001), and spike-specific IgA correlated stronger (p<0.0001) between serum and saliva (r=0.73, p<0.001) as opposed to between serum and nasal secretions (r=0.54, p<0.001), suggesting transudation of monomeric spike specific IgA from the circulation to saliva. Notably, spike-specific SIgA had a markedly higher SARS-CoV-2 variant cross-binding capacity as compared to the total population of spike specific IgA and IgG in both nasal secretions, saliva and serum, (all p<0.0001), which emphasizes the importance of taking potential serum derived monomeric IgA into consideration when investigating mucosal immune responses. Discussion: Taken together, although spike-specific IgA can be reliably measured in both nasal secretions and saliva, our findings imply an advantage of higher levels and likely also a larger proportion of SIgA in nasal secretions as compared to in saliva. We further corroborate the superior variant cross-binding capacity of SIgA in mucosal secretions, highlighting the potential protective benefits of a vaccine targeting the upper respiratory tract.

Prognostic value of circulating markers of neutrophil activation, neutrophil extracellular traps, coagulation and fibrinolysis in patients with terminal cancer.

Predicting survival accurately in patients with advanced cancer is important in guiding interventions and planning future care. Objective tools are therefore needed. Blood biomarkers are appealing due to their rapid measurement and objective nature. Thrombosis is a common complication in cancer. Recent data indicate that tumor-induced neutrophil extracellular traps (NETs) are pro-thrombotic. We therefore performed a comprehensive investigation of circulating markers of neutrophil activation, NET formation, coagulation and fibrinolysis in 106 patients with terminal cancer. We found that neutrophil activation and NET markers were prognostic in terminal cancer patients. Interestingly, markers of coagulation and fibrinolysis did not have a prognostic value in this patient group, and there were weak or no correlations between these markers and markers of neutrophil activation and NETs. This suggest that NETs are linked to a poor prognosis through pathways independent of coagulation. Additional studies are needed to determine the utility of circulating neutrophil activation and NET markers, alone or in concert with established clinical parameters, as objective and reliable prognostic tools in advanced cancer.

SARS-CoV-2 exposure, symptoms and seroprevalence in healthcare workers in Sweden.

SARS-CoV-2 may pose an occupational health risk to healthcare workers. Here, we report the seroprevalence of SARS-CoV-2 antibodies, self-reported symptoms and occupational exposure to SARS-CoV-2 among healthcare workers at a large acute care hospital in Sweden. The seroprevalence of IgG antibodies against SARS-CoV-2 was 19.1% among the 2149 healthcare workers recruited between April 14th and May 8th 2020, which was higher than the reported regional seroprevalence during the same time period. Symptoms associated with seroprevalence were anosmia (odds ratio (OR) 28.4, 95% CI 20.6-39.5) and ageusia (OR 19.2, 95% CI 14.3-26.1). Seroprevalence was also associated with patient contact (OR 2.9, 95% CI 1.9-4.5) and covid-19 patient contact (OR 3.3, 95% CI 2.2-5.3). These findings imply an occupational risk for SARS-CoV-2 infection among healthcare workers. Continued measures are warranted to assure healthcare workers safety and reduce transmission from healthcare workers to patients and to the community.

Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.

BACKGROUND: Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. OBJECTIVES: We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. METHODS: We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. RESULTS: We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. CONCLUSIONS: Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.