Plasmablasts in previously immunologically naïve COVID-19 patients express markers indicating mucosal homing and secrete antibodies cross-reacting with SARS-CoV-2 variants and other beta-coronaviruses.

Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin ß1, only some integrin ß7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.

Brain injury in COVID-19 is associated with dysregulated innate and adaptive immune responses.

COVID-19 is associated with neurological complications including stroke, delirium and encephalitis. Furthermore, a post-viral syndrome dominated by neuropsychiatric symptoms is common, and is seemingly unrelated to COVID-19 severity. The true frequency and underlying mechanisms of neurological injury are unknown, but exaggerated host inflammatory responses appear to be a key driver of COVID-19 severity. We investigated the dynamics of, and relationship between, serum markers of brain injury [neurofilament light (NfL), glial fibrillary acidic protein (GFAP) and total tau] and markers of dysregulated host response (autoantibody production and cytokine profiles) in 175 patients admitted with COVID-19 and 45 patients with influenza. During hospitalization, sera from patients with COVID-19 demonstrated elevations of NfL and GFAP in a severity-dependent manner, with evidence of ongoing active brain injury at follow-up 4 months later. These biomarkers were associated with elevations of pro-inflammatory cytokines and the presence of autoantibodies to a large number of different antigens. Autoantibodies were commonly seen against lung surfactant proteins but also brain proteins such as myelin associated glycoprotein. Commensurate findings were seen in the influenza cohort. A distinct process characterized by elevation of serum total tau was seen in patients at follow-up, which appeared to be independent of initial disease severity and was not associated with dysregulated immune responses unlike NfL and GFAP. These results demonstrate that brain injury is a common consequence of both COVID-19 and influenza, and is therefore likely to be a feature of severe viral infection more broadly. The brain injury occurs in the context of dysregulation of both innate and adaptive immune responses, with no single pathogenic mechanism clearly responsible.

Upper Respiratory Tract Levels of Severe Acute Respiratory Syndrome Coronavirus 2 RNA and Duration of Viral RNA Shedding Do Not Differ Between Patients With Mild and Severe/Critical Coronavirus Disease 2019.

This study reports longitudinal viral RNA loads from the nasopharynx/throat in patients with mild and severe/critical coronavirus disease 2019 (COVID-19). We also investigated whether the duration of symptoms correlated with the duration of viral RNA shedding. A total of 56 patients were included. The highest viral loads occurred early after onset of symptoms. Neither the viral RNA loads in the upper respiratory tract nor the time to viral RNA clearance differed between patients with mild or severe/critical disease. There was a moderate correlation between number of days with symptoms and number of days with viral RNA shedding in patients with mild COVID-19.

Longitudinal single-cell analysis of SARS-CoV-2-reactive B cells uncovers persistence of early-formed, antigen-specific clones.

Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from patients with severe COVID-19 every third to seventh day during hospitalization and every third month after recovery. We profiled their antigen-specific immune cell dynamics by combining single-cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and B cell receptor-Seq (BCR-Seq) with oligo-tagged antigen baits. While the proportion of Spike receptor binding domain-specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen-specific cells, and affinity maturation was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to 1 year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen-specific B cell responses in longitudinally sampled patients infected with COVID-19.

No effect of remdesivir or betamethasone on upper respiratory tract SARS-CoV-2 RNA kinetics in hospitalised COVID-19 patients: a retrospective observational study.

BACKGROUND: The viral kinetics of SARS-CoV-2 has been considered clinically important. While remdesivir and corticosteroids are recommended for COVID-19 patients requiring oxygen support, there is a limited number of published reports on viral kinetics in hospitalised patients with COVID-19 treated with remdesivir or corticosteroids. METHODS: We conducted a retrospective study by collecting longitudinal samples from the nasopharynx/throat of 123 hospitalised patients (median age 55 years, 74% male) with COVID-19, to evaluate the effects of remdesivir and corticosteroid treatment on viral RNA levels. The subjects were divided into four groups: those receiving remdesivir (n = 25), betamethasone (n = 41), both (n = 15), or neither (n = 42). Time to viral RNA clearance was analysed using Kaplan-Meier plots, categorical data were analysed using Fisher's exact test, and Kruskal-Wallis for continuous data. Viral RNA decline rate was analysed using a mixed effect model. RESULTS: We found no significant difference in SARS-CoV-2 RNA decline rate or time to SARS-CoV-2 RNA clearance between the groups. Moreover, clinical status at baseline was not correlated with time to viral clearance. CONCLUSIONS: Since SARS-CoV-2 RNA kinetics was not affected by treatment, repeated sampling from the upper respiratory tract cannot be used to evaluate treatment response.

Longitudinal Follow Up of Immune Responses to SARS-CoV-2 in Health Care Workers in Sweden With Several Different Commercial IgG-Assays, Measurement of Neutralizing Antibodies and CD4+ T-Cell Responses.

Background: The risk of SARS-CoV-2 infection among health care workers (HCWs) is a concern, but studies that conclusively determine whether HCWs are over-represented remain limited. Furthermore, methods used to confirm past infection vary and the immunological response after mild COVID-19 is still not well defined. Method: 314 HCWs were recruited from a Swedish Infectious Diseases clinic caring for COVID-19 patients. IgG antibodies were measured using two commercial assays (Abbot Architect nucleocapsid (N)-assay and YHLO iFlash-1800 N and spike (S)-assays) at five time-points, from March 2020 to January 2021, covering two pandemic waves. Seroprevalence was assessed in matched blood donors at three time-points. More extensive analyses were performed in 190 HCWs in September/October 2020, including two additional IgG-assays (DiaSorin LiaisonXL S1/S2 and Abbot Architect receptor-binding domain (RBD)-assays), neutralizing antibodies (NAbs), and CD4+ T-cell reactivity using an in-house developed in vitro whole-blood assay based on flow cytometric detection of activated cells after stimulation with Spike S1-subunit or Spike, Membrane and Nucleocapsid (SMN) overlapping peptide pools. Findings: Seroprevalence was higher among HCWs compared to sex and age-matched blood donors at all time-points. Seropositivity increased from 6.4% to 16.3% among HCWs between May 2020 and January 2021, compared to 3.6% to 11.9% among blood donors. We found significant correlations and high levels of agreement between NAbs and all four commercial IgG-assays. At 200-300 days post PCR-verified infection, there was a wide variation in sensitivity between the commercial IgG-assays, ranging from <30% in the N-assay to >90% in the RBD-assay. There was only moderate agreement between NAbs and CD4+ T-cell reactivity to S1 or SMN. Pre-existing CD4+ T-cell reactivity was present in similar proportions among HCW who subsequently became infected and those that did not. Conclusions: HCWs in COVID-19 patient care in Sweden have been infected with SARS-CoV-2 at a higher rate compared to blood donors. We demonstrate substantial variation between different IgG-assays and propose that multiple serological targets should be used to verify past infection. Our data suggest that CD4+ T-cell reactivity is not a suitable measure of past infection and does not reliably indicate protection from infection in naive individuals.

Correction: Serum-IgG responses to SARS-CoV-2 after mild and severe COVID-19 infection and analysis of IgG non-responders.

[This corrects the article DOI: 10.1371/journal.pone.0241104.].

Serum-IgG responses to SARS-CoV-2 after mild and severe COVID-19 infection and analysis of IgG non-responders.

BACKGROUND: To accurately interpret COVID-19 seroprevalence surveys, knowledge of serum-IgG responses to SARS-CoV-2 with a better understanding of patients who do not seroconvert, is imperative. This study aimed to describe serum-IgG responses to SARS-CoV-2 in a cohort of patients with both severe and mild COVID-19, including extended studies of patients who remained seronegative more than 90 days post symptom onset. METHODS: SARS-CoV-2-specific IgG antibody levels were quantified using two clinically validated and widely used commercial serological assays (Architect, Abbott Laboratories and iFlash 1800, YHLO), detecting antibodies against the spike and nucleocapsid proteins. RESULTS: Forty-seven patients (mean age 49 years, 38% female) were included. All (15/15) patients with severe symptoms and 29/32 (90.6%) patients with mild symptoms of COVID-19 developed SARS-CoV-2-specific IgG antibodies in serum. Time to seroconversion was significantly shorter (median 11 vs. 22 days, P = 0.04) in patients with severe compared to mild symptoms. Of the three patients without detectable IgG-responses after >90 days, all had detectable virus-neutralizing antibodies and in two, spike-protein receptor binding domain-specific IgG was detected with an in-house assay. Antibody titers were preserved during follow-up and all patients who seroconverted, irrespective of the severity of symptoms, still had detectable IgG levels >75 days post symptom onset. CONCLUSIONS: Patients with severe COVID-19 both seroconvert earlier and develop higher concentrations of SARS-CoV-2-specific IgG than patients with mild symptoms. Of those patients who not develop detectable IgG antibodies, all have detectable virus-neutralizing antibodies, suggesting immunity. Our results showing that not all COVID-19 patients develop detectable IgG using two validated commercial clinical methods, even over time, are vital for the interpretation of COVID-19 seroprevalence surveys.