SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection.

Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10-23 and 2*10-13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys.

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.

Multiomics Profiling of Alzheimer's Disease Serum for the Identification of Autoantibody Biomarkers.

New biomarkers of Alzheimer's disease (AD) with a diagnostic value in preclinical and prodromal stages are urgently needed. AD-related serum autoantibodies are potential candidate biomarkers. Here, we aimed at identifying AD-related serum autoantibodies using protein microarrays and mass spectrometry-based methods. To this end, an untargeted complementary screening using high-density (42,100 antigens) and low-density (384 antigens) planar protein-epitope signature tag (PrEST) arrays and an immunoprecipitation protocol coupled to mass spectrometry analysis were used for serum autoantibody profiling. From the untargeted screening phase, 377 antigens corresponding to 338 proteins were selected for validation. Out of them, IVD, CYFIP1, and ADD2 seroreactivity was validated using 128 sera from AD patients and controls by PrEST-suspension bead arrays, and ELISA or luminescence Halotag-based bead immunoassay using full-length recombinant proteins. Importantly, IVD, CYFIP1, and ADD2 showed in combination a noticeable AD diagnostic ability. Moreover, IVD protein abundance in the prefrontal cortex was significantly two-fold higher in AD patients than in controls by western blot and immunohistochemistry, whereas CYFIP1 and ADD2 were significantly down-regulated in AD patients. The panel of AD-related autoantigens identified by a comprehensive multiomics approach may provide new insights of the disease and should help in the blood-based diagnosis of Alzheimer's disease. Mass spectrometry raw data are available in the ProteomeXchange database with the access number PXD028392.

Autoantibody profiles associated with clinical features in psychotic disorders.

Autoimmune processes are suspected to play a role in the pathophysiology of psychotic disorders. Better understanding of the associations between auto-immunoglobulin G (IgG) repertoires and clinical features of mental illness could yield novel models of the pathophysiology of psychosis, and markers for biological patient stratification. We undertook cross-sectional detection and quantification of auto-IgGs in peripheral blood plasma of 461 people (39% females) with established psychotic disorder diagnoses. Broad screening of 24 individuals was carried out on group level in eight clinically defined groups using planar protein microarrays containing 42,100 human antigens representing 18,914 proteins. Autoantibodies indicated by broad screening and in the previous literature were measured using a 380-plex bead-based array for autoantibody profiling of all 461 individuals. Associations between autoantibody profiles and dichotomized clinical characteristics were assessed using a stepwise selection procedure. Broad screening and follow-up targeted analyses revealed highly individual autoantibody profiles. Females, and people with family histories of obesity or of psychiatric disorders other than schizophrenia had the highest overall autoantibody counts. People who had experienced subjective thought disorder and/or were treated with clozapine (trend) had the lowest overall counts. Furthermore, six autoantibodies were associated with specific psychopathology symptoms: anti-AP3B2 (persecutory delusions), anti-TDO2 (hallucinations), anti-CRYGN (initial insomnia); anti-APMAP (poor appetite), anti-OLFM1 (above-median cognitive function), and anti-WHAMMP3 (anhedonia and dysphoria). Future studies should clarify whether there are causal biological relationships, and whether autoantibodies could be used as clinical markers to inform diagnostic patient stratification and choice of treatment.

An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses.

Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.

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.

Antibodies to SARS-CoV-2 and risk of past or future sick leave.

The extent that antibodies to SARS-CoV-2 may protect against future virus-associated disease is unknown. We invited all employees (n = 15,300) at work at the Karolinska University Hospital, Stockholm, Sweden to participate in a study examining SARS-Cov-2 antibodies in relation to registered sick leave. For consenting 12,928 healthy hospital employees antibodies to SARS-CoV-2 could be determined and compared to participant sick leave records. Subjects with viral serum antibodies were not at excess risk for future sick leave (adjusted odds ratio (OR) controlling for age and sex: 0.85 [95% confidence interval (CI) (0.85 (0.43-1.68)]. By contrast, subjects with antibodies had an excess risk for sick leave in the weeks prior to testing [adjusted OR in multivariate analysis: 3.34 (2.98-3.74)]. Thus, presence of viral antibodies marks past disease and protection against excess risk of future disease. Knowledge of whether exposed subjects have had disease in the past or are at risk for future disease is essential for planning of control measures.Trial registration: First registered on 02/06/20, ClinicalTrials.gov NCT04411576.

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.

Array-Based Profiling of Proteins and Autoantibody Repertoires in CSF.

Protein profiling enabled through affinity proteomics represents a powerful strategy for analysis of complex samples such as human body fluids. Cerebrospinal fluid (CSF) is the proximal fluid of the central nervous system and is commonly analyzed in the context of neurological diseases. Through the presence of brain-derived proteins, this fluid can offer insight into the physiological state of the brain. Here, we describe multiplex and flexible protein and autoantibody profiling approaches using suspension bead arrays. Through minimal sample processing, these methods enable high-throughput analysis of hundreds of samples and proteins in one single assay and thereby provide powerful approaches for discovery of disease-associated proteins and autoantigens.