Background: Early-life microbial exposure to microbes may play a role in asthma development. This study tests the hypothesis that mothers born in low-income countries might have been exposed to diverse microbes-leading to greater diversity and higher levels of transplacental anti-microbial antibodies, thereby conferring protection against asthma in offspring. Methods: In the prospective Boston Birth Cohort, IgG antibody reactome against microbes (2740 species; 1311 genera) was profiled in cord blood, using Phage ImmunoPrecipitation Sequencing. Multinomial regression models were applied to examine the associations of cord blood IgG reactome with child risk of physician-diagnosed atopy and asthma. Mediation analysis was performed to examine the inter-relationships among maternal nativity, IgG reactome and risk of developing childhood asthma. Results: This report included 943 mother-child dyads enrolled at birth and followed prospectively. Compared to children of US-born mothers, children of foreign-born mothers had a lower prevalence of asthma (17.5% vs. 30.5%) and greater diversity of cord blood IgG antibodies against hundreds of microbes (FDR < 0.05). Cord blood seropositivity to peptides or proteins from 6 microbes was inversely associated with the risk of asthma in children, and children with more seropositivity to these microbes were at a lower risk of developing asthma (p < 0.001). IgG seropositivity to A. actinomycetemcomitans, H. pylori, S. flexneri, and T. parva each mediated 17%-62% of the association between maternal nativity and child risk of asthma. Conclusion: In this US prospective birth cohort, maternal transplacental IgG reactivity to four microbes (A. actinomycetemcomitans, H. pylori, S. flexneri and T. parva) was associated with a lower risk of childhood asthma, and partly explains the lower risk of asthma in children of mothers born outside the US.

Autoantibodies have been shown to be implied in COVID-19 but the emerging autoantibody repertoire remains largely unexplored. We investigated the new-onset autoantibody repertoire in 525 healthcare workers and hospitalized COVID-19 patients at five time points over a 16-month period in 2020 and 2021 using proteome-wide and targeted protein and peptide arrays. Our results show that prevalent new-onset autoantibodies against a wide range of antigens emerged following SARS-CoV-2 infection in relation to pre-infectious baseline samples and remained elevated for at least 12 months. We found an increased prevalence of new-onset autoantibodies after severe COVID-19 and demonstrated associations between distinct new-onset autoantibodies and neuropsychiatric symptoms post-COVID-19. Using epitope mapping, we determined the main epitopes of selected new-onset autoantibodies, validated them in independent cohorts of neuro-COVID and pre-pandemic healthy controls, and identified sequence similarities suggestive of molecular mimicry between main epitopes and the conserved fusion peptide of the SARS-CoV-2 Spike glycoprotein. Our work describes the complexity and dynamics of the autoantibody repertoire emerging with COVID-19 and supports the need for continued analysis of the new-onset autoantibody repertoire to elucidate the mechanisms of the post-COVID-19 condition.

The antibody repertoire is the ensemble of antibodies found in an individual at a given time. It displays high heterogeneity between individuals while being both largely temporally stable within an individual and rapidly responsive to immunological challenge. As distinct collections of antibodies within the repertoire contribute to the function and malfunction of the immune system, studying the many aspects of the antibody repertoire can give increased knowledge on antibody-mediated pathogen defense and autoimmune conditions.

There are several emergent techniques for assessing different properties of the antibody repertoire as well as determining distinct antibodies of interest in health and disease. The studies presented in this thesis use planar and bead-based arrays to investigate parts of the antibody repertoire consisting of antibodies against SARS-CoV-2 proteins in serological studies, as well as autoantibodies against the large collection of antigens in the Human Protein Atlas. Paper I explores the autoantibody repertoires of patients with psychosis using planar arrays of 42 000 antigens followed by targeted bead arrays and identifies associations to specific symptoms. Paper II defines the baseline serological characteristics of a longitudinal cohort using a then recently developed multiplex serological assay and gives an early description of COVID-19 symptomatology. Paper III investigates the four-month persistence and antigen diversity of antibodies against SARS-CoV-2 following infection. This work is continued in Paper IV which examines the persistence of the humoral and cellular response to infection and their protective effect against reinfection. Paper V connects these parts by exploring the autoantibody repertoire of this longitudinal cohort and identifying new-onset autoantibodies emerging at infection using arrays of human and viral antigens. It associates three new-onset autoantibodies to post-COVID-19 symptoms and demonstrates sequence similarity between human and viral epitopes, which may indicate molecular mimicry.

Antibody repertoires are heterogeneous and multifaceted, requiring several methods for full comprehension. The present investigation encompasses the analysis of one facet using antigen arrays and contributes to knowledge on disease-associated autoantibody repertoires as well as the prevalence and persistence of the serological and autoantibody response emerging after viral infection. This work represents a small step towards the goal of understanding the full repertoire complexity. Emergent large-scale techniques combined with the herein described analysis are together poised to identify clinically relevant antigens and advance knowledge on the diversity and heterogeneity of the antibody repertoire.

Antikroppsrepertoaren utgörs av den samling av antikroppar som återfinns i en individ vid ett givet tillfälle. Den uppvisar stor heterogenitet mellan individer samtidigt som den inom en individ både är övervägande stabil över tid och snabbt föränderlig vid immunologiska händelser. Eftersom avgränsade samlingar av antikroppar inom repertoaren bidrar till immunförsvarets funktion och dysfunktion är det av stor vikt att studera de många olika aspekterna av antikroppsrepertoaren för att öka förståelsen av både det försvar mot patogen och de autoimmuna tillstånd som tillkommer genom antikroppars verkan.

Det finns flera banbrytande tekniker som utvecklats för att undersöka olika aspekter av antikroppsrepertoaren samt identifiera särskilda antikroppar som kan bidra till kunskap om friska och sjuka tillstånd. De forskningsarbeten som presenteras i den här avhandlingen använde sig av analysmetoder som grundar sig på plana ytor och mikrosfärer för att undersöka olika delar av antikroppsrepertoaren. Dessa delar bestod av antikroppar mot proteiner hos SARS-CoV-2 som undersöktes i serologiska arbeten, samt autoantikroppar mot den stora samling av antigen som finns i HPA – atlasen över människans proteiner. Artikel I utforskar autoantikroppsrepertoarerna hos patienter med psykos med hjälp av 42 000 antigen från HPA arrangerade på plana ytor, följt av riktad analys med antigen fästa till mikrosfärer, och resulterar i identifierade kopplingar mellan antikroppar och specifika symptom. Artikel II definierar den grundläggande serologiska profilen hos en longitudinell kohort med hjälp av en då nyligen utvecklad serologisk metod för att mäta många virusproteiner, samt ger en tidig beskrivning av symtomatologin vid COVID-19. Artikel III undersöker varaktigheten av antikroppar fyra månader efter SARS-CoV-2-infektion och mångfalden av deras antigen. Detta arbete följs upp i Artikel IV som undersöker varaktigheten av det humorala och cellulära immunsvaret mot infektion och dess skyddande effekt mot återinfektion. Artikel V sammanbinder dessa delar genom att utforska antikroppsrepertoaren hos den beskrivna longitudinella kohorten och identifiera autoantikroppar som uppkommer vid infektion med hjälp av analysmetoder med både mänskliga och virala antigen. Artikeln kopplar tre nyuppkomna autoantikroppar till symtom efter COVID-19 och påvisar sekvenslikhet mellan mänskliga och virala epitop, vilket kan antyda molekylär mimikry.

Antikroppsrepertoarer är heterogena och mångfasetterade och kräver därför flera metoder för full förståelse. De forskningsarbeten som förs fram i den här avhandlingen omfattar analys av en fasett med hjälp av antigenbaserade analysmetoder. Dessa arbeten bidrar till kunskap om sjukdomskopplade autoantikroppsrepertoarer samt förekomsten och varaktigheten hos det serologiska svaret och autoantikroppar efter virusinfektion. Arbetet representerar ett litet steg mot det slutgiltiga målet att förstå helheten av repertoarens komplexitet. Banbrytande storskaliga tekniker i kombination med den analys som beskrivs i den här avhandlingen har stor potential att identifiera kliniskt relevanta antigen och ge ökad kunskap om antikroppsrepertoarens mångfald och heterogenitet.

The diagnostic criteria for schizophrenia (SCZ) and bipolar disorder (BD) are based on clinical assessments of symptoms. In this pilot study, we applied high-throughput antibody-based protein profiling to serum samples of healthy controls and individuals with SCZ and BD with the aim of identifying differentially expressed proteins in these disorders. Moreover, we explored the influence of polygenic burden for SCZ and BD on the serum levels of these proteins. Serum samples from 113 individuals with SCZ and 125 with BD from the PsyCourse Study and from 44 healthy controls were analyzed by using a set of 155 antibodies in an antibody-based assay targeting a selected panel of 95 proteins. For the cases, genotyping and imputation were conducted for DNA samples and SCZ and BD polygenic risk scores (PRS) were calculated. Univariate linear and logistic models were used for association analyses. The comparison between SCZ and BD revealed two serum proteins that were significantly elevated in BD after multiple testing adjustment: "complement C9" and "Interleukin 1 Receptor Accessory Protein". Moreover, the first principal component of variance in the proteomics dataset differed significantly between SCZ and BD. After multiple testing correction, SCZ-PRS, BD-PRS, and SCZ-vs-BD-PRS were not significantly associated with the levels of the individual proteins or the values of the proteome principal components indicating no detectable genetic effects. Overall, our findings contribute to the evidence suggesting that the analysis of circulating proteins could lead to the identification of distinctive biomarkers for SCZ and BD. Our investigation warrants replication in large-scale studies to confirm these findings.

Background: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. Methods: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFN gamma) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. Results: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFN gamma and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFN gamma in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. Conclusion: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.

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

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-gamma 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.

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.