Objective: Identification of those at high and low risk of disease relapse is a major unmet need in the management of patients with ANCA-associated vasculitis (AAV). Precise stratification would allow tailoring of immunosuppressive medication. We profiled the autoantibody repertoire of AAV patients in remission to identify novel autoantibodies associated with relapse risk. Methods: Plasma samples collected from 246 AAV patients in remission were screened for novel autoantibodies using in-house generated protein arrays including 42 000 protein fragments representing 18 000 unique human proteins. Patients were categorized based on the occurrence and frequency of relapses. We modelled the association between these antibodies and relapse occurrence using descriptive and high dimensional regression approaches. Results: We observed nine autoantibodies at higher frequency in samples from AAV patients experiencing multiple relapses compared with patients in long-term remission off therapy. LASSO analysis identified six autoantibodies that exhibited an association with relapse occurrence after sample collection. Antibodies targeting homeostatic iron regulator (HFE) and synaptotagmin 5 (SYT5) were identified as associated with relapse in both analyses. Conclusion: Through a broad protein array-based autoantibody screening, we identified two novel autoantibodies directed against HFE and SYT5 as candidate biomarkers of relapse in AAV.

Systemic sclerosis (SSc) is a rare autoimmune systemic disease that leads to decreased survival and quality of life due to fibrosis, inflammation, and vascular damage in the skin and/or vital organs. Early diagnosis is crucial for clinical benefit in SSc patients. Our study aimed to identify autoantibodies in the plasma of SSc patients that are associated with fibrosis in SSc. Initially, we performed a proteome-wide screening on sample pools from SSc patients by untargeted autoantibody screening on a planar antigen array (including 42,000 antigens representing 18,000 unique proteins). The selection was complemented with proteins reported in the literature in the context of SSc. A targeted antigen bead array was then generated with protein fragments representing the selected proteins and used to screen 55 SSc plasma samples and 52 matched controls. We found eleven autoantibodies with a higher prevalence in SSc patients than in controls, eight of which bound to proteins associated with fibrosis. Combining these autoantibodies in a panel could lead to the subgrouping of SSc patients with fibrosis. Anti-Phosphatidylinositol-5-phosphate 4-kinase type 2 beta (PIP4K2B)- and anti-AKT Serine/Threonine Kinase 3 (AKT3)-antibodies should be further explored to confirm their association with skin and lung fibrosis in SSc patients.

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.

Autoimmune disease diagnosis and definition of prognosis can be challenging. Patients with the same autoimmune disease could present with very heterogeneous symptoms. Therefore, there is a need to understand the disease better to improve patients’ diagnosis, and classification, and tailor the treatment. Autoantibodies are a hallmark of many autoimmune diseases. They are antibodies produced by B cells and targeting self-antigens. Autoantibodies could be useful as diagnostic and prognostic markers of autoimmune disease.

Protein arrays enable multiplex and high-throughput profiling of autoantibodies, therefore representing a good tool for autoantibody markers discovery and validation. This thesis presents the work performed to study autoantibodies in ANCA Associated Vasculitis (AAV) and Systemic Sclerosis (SSc) by employing planar and bead-based antigen arrays. Moreover, this thesis also includes the work done to optimize the application of a multiplex serology assay for the detection of anti-SARS-CoV-2 antibodies in saliva.

In paper 1, we aimed to perform a broad autoantibody profiling in serum samples of AAV patients to search for new autoantibodies associated with the disease or disease subgroups and activity. The main result is related to the identification of anti-KIF5C antibodies at high prevalence in anti-MPO-positive patients and patients with microscopic polyangiitis (MPA). Anti-KIF4A also showed a higher prevalence in anti-MPO positive and MPA patients.

In Paper 2 an in-depth autoantibody profiling was performed to identify autoantibodies as candidates for future relapse prediction in the plasma of AAV patients. We tested samples from patients classified as long-term remission-off-therapy (LTROT) and patients suffering future flares. Nine autoantibodies were found with higher reactivity in the relapse group. Among these, anti-ATF3 antibodies had increased reactivity in patients with kidney and ENT symptoms.

In paper 3, plasma samples from SSc patients and controls were tested to detect autoantibodies associated with fibrosis. We identified eleven autoantibodies with increased prevalence in patients with systemic sclerosis compared to the control group. Eight of these autoantibodies are new in the context of systemic sclerosis and all bind to proteins that are involved in fibrosis. Among these, the anti-AKT3 was shown to be more reactive in patients with skin and lung fibrosis and anti-PIP4K2B in patients that were negative for the available diagnostic marker.

Finally, in paper 4, a multiplex bead-based array serological assay was optimized to detect anti-SARS-CoV-2–specific IgG and IgA in saliva samples. This method was developed to measure antibodies, using SARS-CoV-2 spike and nucleocapsid proteins.

In conclusion, the described work shows the application of the protein array technology to identify (auto)antibodies in different body fluids and within autoimmune diseases and SARS-CoV-2 infection. Moreover, we have identified autoantibody targets that are worth further investigation for their usefulness in better understanding some autoimmune conditions.

Autoimmuna sjukdomar diagnos och definition av prognos kan vara utmanande. Patienter med samma autoimmuna sjukdom kan uppvisa mycket heterogena symtom. Därför finns det ett behov av att förstå sjukdomen bättre för att förbättra patienternas diagnostik, bättre klassificering av patienter och skräddarsydd behandling. Autoantikroppar är ett kännetecken för många autoimmuna sjukdomar. De är antikroppar som produceras av B-celler och riktar sig mot självantigener. Autoantikroppar kan vara användbara som diagnostiska och prognostiska markörer för autoimmun sjukdom.

Proteinmatriser möjliggör multiplex och högkapacitetsprofilering av autoantikroppar, och representerar därför ett bra verktyg för upptäckt och validering av autoantikroppsmarkörer. I den presenterade avhandlingen utfördes arbetet med att studera autoantikroppar i ANCA Associated Vasculitis (AAV) och Systemic Sclerosis (SSc) genom att använda plana och kulbaserade antigenarrayer. Dessutom inkluderar denna avhandling också det arbete som gjorts för att optimera tillämpningen av en multiplex serologisk analys för detektion av anti-SARS-CoV-2 antikroppar i saliv.

I artikel 1 syftade vi till att utföra en bred autoantikroppsprofilering i serumprover av AAV-patienter för att söka efter nya autoantikroppar associerade med sjukdomen eller sjukdomens undergrupper och aktivitet. Huvudresultatet är relaterat till identifiering av anti-KIF5C-antikroppar med hög prevalens hos anti-MPO-positiva patienter och hos patienter med mikroskopisk polyangit (MPA). Anti-KIF4A visade också en högre prevalens hos anti-MPO-positiva och MPA-patienter.

I artikel 2 utfördes en djupgående profilering av autoantikroppar för att identifiera autoantikroppar som kandidater för framtida återfallsförutsägelse i plasma från AAV-patienter. Vi testade prover från patienter som klassificerats som långvarig remission-off-terapi (LTROT) och patienter som lider av framtida flare. Nio autoantikroppar hittades med högre reaktivitet i återfallsgruppen. Bland dessa hade anti-ATF3-antikroppar ökad reaktivitet hos patienter med njure och ÖNH-symtom.

I artikel 3 testades plasmaprover från SSc-patienter och kontroller för att detektera autoantikroppar associerade med fibros. Vi identifierade elva autoantikroppar med ökad prevalens hos patienter med systemisk skleros jämfört med kontrollgruppen. Åtta av dessa autoantikroppar är nya i samband med systemisk skleros och alla binder till proteiner som är involverade i fibros. Bland dessa visade sig anti-AKT3 vara mer reaktiv hos patienter med hud- och lungfibros och anti-PIP4K2B hos patienter som var negativa för den tillgängliga diagnostiska markören.

Slutligen i papper 4 optimerades multiplex pärlor-baserad array-teknologi för att detektera anti-SARS-CoV-2-specifika IgG och IgA i salivprover. Denna metod utvecklades för att mäta antikroppar, med hjälp av SARS-CoV-2-spik och nukleokapsidproteiner.

Sammanfattningsvis visar det beskrivna arbetet tillämpningen av protein array-teknologin för att identifiera (auto)antikroppar i olika kroppsvätskor och i synnerhet inom autoimmunitet, men även SARS-CoV-2-infektion. Dessutom har vi identifierat autoantikroppsmål som är värda ytterligare undersökning för deras användbarhet för att bättre förstå vissa autoimmuna tillstånd.

Background. Declining humoral immunity in coronavirus disease 2019 (COVID-19) patients and possible reinfection have raised concern. Mucosal immunity, particularly salivary antibodies, may be short lived although long-term studies are lacking. Methods. Using a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (n = 74, cohort 1), undiagnosed individuals with self-reported questionnaires (n = 147, cohort 2), and individuals sampled prepandemic (n = 35, cohort 3). Results. Salivary IgG antibody responses in cohort 1 (mainly mild COVID-19) were detectable up to 9 months postrecovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in blood and saliva in most patients. Salivary IgA was rarely detected at this time point. In cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19-like symptoms. Salivary IgG tolerated temperature and detergent pretreatments. Conclusions. Unlike SARS-CoV-2 salivary IgA that appeared short lived, specific saliva IgG appeared stable even after mild COVID-19, as for blood serology. This noninvasive saliva-based SARS-CoV-2 antibody test with home self-collection may be a complementary alternative to conventional blood serology.

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.