Immunogenicity and safety of COVID-19 booster vaccination: A population-based clinical trial to identify the best vaccination strategy.

BACKGROUND: Various SARS-CoV-2 variants of concerns (VOCs) characterized by higher transmissibility and immune evasion have emerged. Despite reduced vaccine efficacy against VOCs, currently available vaccines provide protection. Population-based evidence on the humoral immune response after booster vaccination is crucial to guide future vaccination strategies and in preparation for imminent COVID-19 waves. METHODS: This multicenter, population-based cohort study included 4697 individuals ≥18 years of age who received a booster vaccination. Antibody levels against SARS-CoV-2 receptor binding domain (RBD) and neutralizing antibodies against wild-type (WT) virus and Omicron variants were assessed at baseline (day of booster vaccination) and after four weeks. Safety was evaluated daily within the first week using a participant-completed electronic diary. Antibody levels were compared across different vaccination strategies, taking into account individual host factors. RESULTS: Our main model including 3838 participants revealed that individuals who received a booster with mRNA-1273 compared to BNT162b2 vaccine had a significantly higher increase (95 %CI) in anti-RBD-antibody levels (37,707 BAU/mL [34,575-40,839] vs. 27,176 BAU/mL [26,265-28,087]), and of neutralization levels against WT (1,681 [1490-1872] vs. 1141 [1004-1278] and Omicron variant (422 [369-474] vs. 329 [284-374]). Neutralizing antibody titres highly correlated with anti-RBD antibodies, with neutralizing capacity 4.4 fold higher against WT compared to Omicron. No differences in safety were found between the two booster vaccines. CONCLUSION: Our study underlines the superiority of a booster vaccination with mRNA-1273, independent of the primary vaccination and therefore provides guidance on the vaccination strategy.

Association between reactogenicity and immunogenicity after BNT162b2 booster vaccination: a secondary analysis of a prospective cohort study.

OBJECTIVES: A weak correlation between symptom severity and antibody levels after primary immunization against COVID-19 has already been shown. This study aimed to describe the association between reactogenicity and immunogenicity after booster vaccination. METHODS: This secondary analysis of a prospective cohort study included 484 healthcare workers who received a booster vaccination with BNT162b2. Anti-receptor binding domain (RBD) antibodies were assessed at baseline and 28 days after booster vaccination. Side effects were graded (none, mild, moderate, or severe) and reported daily for 7 days after booster vaccination. Spearman correlation coefficient (rho) was used to determine the correlations between the severity of each symptom and anti-RBD levels before vaccination and 28 days after. The Bonferroni method was used to adjust p values for multiple comparisons. RESULTS: Most of the 484 participants reported at least one local (451 [93.2%]) or systemic (437 [90.3%]) post-booster symptom. No correlations between the severity of local symptoms and antibody levels were found. Except for nausea, systemic symptoms showed weak but statistically significant correlations with 28-day anti-RBD levels (fatigue [rho = 0.23, p < 0.01], fever [rho = 22, p < 0.01], headache [rho = 0.15, p 0.03], arthralgia [rho = 0.2, p < 0.01], myalgia [rho = 0.17, p < 0.01]). There was no association between post-booster symptoms and pre-booster antibody levels. DISCUSSION: This study showed only a weak correlation between the severity of systemic post-booster symptoms and anti-SARS-CoV-2 antibody levels at 28 days. Therefore, self-reported symptom severity cannot be used to predict immunogenicity after booster vaccination.

Antibodies against citrullinated proteins of IgA isotype are associated with progression to rheumatoid arthritis in individuals at-risk.

OBJECTIVE: Events triggering disease outbreak in individuals at-risk for rheumatoid arthritis (RA at-risk) remain unclear, and the role of the various anticitrullinated protein antibody (ACPA) isotypes in this process is still to be established. We aimed to investigate the prevalence of IgA ACPA in RA at-risk individuals, their role in the transition from the RA at-risk status to RA and their dynamics during this transition. METHODS: Cross-sectional measurement of serum IgA1 and IgA2 ACPA levels was conducted in healthy controls, RA at-risk individuals and patients with RA and compared with the frequency of RA development in at risk individuals during a follow-up of 14 months. In addition, longitudinal measurements of serum IgA1 and IgA2 ACPA levels prior to, at and after the onset of RA were performed. RESULTS: Approximately two-thirds of RA at-risk individuals were positive for serum IgA1 and IgA2 ACPA in levels comparable to IgG ACPA positive patients with RA. IgA1, but not IgA2 ACPA positivity was associated with the transition from the RA at-risk state to RA within the following 14 months. Interestingly, during this transition process, IgA1 ACPA levels declined at RA onset and also thereafter during the early phase of RA. This decline was confirmed in a second, independent cohort. CONCLUSION: Both IgA1 and IgA2 ACPA are present in RA at-risk individuals, but only IgA1 ACPA are associated with the progression to RA. The observed decline in serum IgA1 ACPA levels before the onset of RA might indicate starting barrier leakiness prior to disease outbreak.

Accelerated waning of immune responses to a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases.

BACKGROUND: A 3rd COVID-19 vaccination is currently recommended for patients under immunosuppression. However, a fast decline of antibodies against the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein has been observed. Currently it remains unclear whether immunosuppressive therapy affects kinetics of humoral and cellular immune responses. METHODS: 50 patients under immunosuppression and 42 healthy controls (HCs) received a 3rd dose of an mRNA-based vaccine and were monitored over a 12-weeks period. Humoral immune response was assessed 4 and 12 weeks after 3rd dose. Antibodies were quantified using the Elecsys Anti-SARS-CoV-2 Spike immunoassay against the receptor-binding domain (RBD) of the spike protein. SARS-CoV-2-specific T cell responses were quantified by IFN-γ ELISpot assays. Adverse events, including SARS-CoV-2 infections, were monitored over a 12-week period. RESULTS: At week 12, reduced anti-RBD antibody levels were observed in IMID patients as compared to HCs (median antibody level 5345 BAU/ml [1781-10,208] versus 9650 BAU/ml [6633-16,050], p < 0.001). Reduction in relative antibody levels was significantly higher in IMID patients as compared to HCs at week 12 (p < 0.001). Lowest anti-RBD antibody levels were detected in IMID patients who received biological disease-modifying anti-rheumatic drugs (DMARDs) or a combination therapy with conventional synthetic and biological DMARDs. Number of SARS-CoV-2-specific T cells against wildtype and Omicron variants remained stable over 12 weeks in IMID patients. No serious adverse events were reported. CONCLUSION: Due to a fast decline in anti-RBD antibodies in IMID patients an early 4th vaccination should be considered in this vulnerable group of patients.

Reduced immunogenicity of BNT162b2 booster vaccination in combination with a tetravalent influenza vaccination: results of a prospective cohort study in 838 health workers.

OBJECTIVE: To investigate the immunogenicity and safety of BNT162b2 booster vaccination with and without a tetravalent influenza vaccine. METHODS: A prospective, open-label cohort study on immunogenicity and safety of COVID-19 booster vaccination with or without a tetravalent influenza vaccine was performed. Eight hundred thirty-eight health care workers were included in the following study arms: BNT162b2 booster-only, influenza-vaccine-only or combination of both. Levels of antibodies against SARS-CoV-2 spike receptor binding domain, and haemagglutinin inhibition tested for four different influenza strains (A(H1N1)pdm09, A(H3N2), B/Victoria, B/Yamagata) were measured at the time of vaccination and 4 weeks later. RESULTS: After 4 weeks, median (interquartile range) levels of antibodies against the receptor binding domain of the viral spike (S) protein and relative change from baseline were high in individuals who received BNTb162b2 booster vaccination only (absolute: 16 600 [10 980-24 360] vs. 12 630 [8198-18 750] BAU/mL [p < 0.0001]; relative increase: 49% [23.6-95.3] vs. 40% [21.9-80.6] [p 0.048]; booster-only n = 521 vs. combination-arm n = 229 respectively). Results were confirmed after matching for sex, age, body mass index, baseline antibody levels and vaccine compound received for primary immunization (absolute: 13 930 [10 610-22 760] vs. 12 520 [8710-17 940]; [p 0.031]; relative increase: 55.7% [27.8-98.5] vs. 42.2% [22.9-74.5]; p 0.045). Adverse events were almost identical in the booster-only and the combination-arm, but numerically low in the influenza arm (525/536 [97.9%] vs. 235/240 [97.9%] vs. 26/33 [78.8 %]). DISCUSSION: Although no safety concerns occurred, our study provides evidence on reduced immunogenicity of a BNT162b2 booster vaccination in combination with a tetravalent influenza vaccine. Further studies investigating new influenza variants as well as potential differences vaccine effectiveness are needed.

Safety and immunogenicity of a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases compared with healthy controls.

OBJECTIVES: A third COVID-19 vaccination is recommended for immunosuppressed patients. However, data on immunogenicity and safety of a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMIDs) are sparse and therefore addressed within this clinical trial. METHODS: 60 immunosuppressed patients and 48 healthy controls (HCs) received a third vaccination with an mRNA vaccine. The primary endpoint was defined as the presence of antibody levels against the receptor-binding domain (RBD)>1500 BAU/mL in patients with IMIDs versus HCs. Further endpoints included differences in neutralising antibodies and cellular immune responses after the third vaccination. Reactogenicity was recorded for 7 days, and safety was evaluated until week 4. RESULTS: Rate of individuals with anti-RBD antibodies>1500 BAU/mL was not significantly different after the third vaccination between patients with IMIDs and HCs (91% vs 100% p=0.101). Anti-RBD and neutralising antibody levels were significantly lower in patients with IMIDs after the third vaccination than in HCs (p=0.002 and p=0.016, respectively). In contrast, fold increase in antibody levels between week 0 and 4 was higher in patients with IMIDs. Treatment with biological (b) disease-modifying anti-rheumatic drugs (DMARD) or combination of bDMARDs and conventional synthetic DMARDs was associated with reduced antibody levels. Enhanced cellular immune response to wild type and Omicron peptide stimulation was observed after the third vaccination. No serious adverse event was attributed to the third vaccination. CONCLUSION: Our clinical trial data support the immunogenicity and safety of a third COVID-19 vaccination in patients with IMIDs. However, effects of DMARD therapy on immunogenicity should be considered. TRIAL REGISTRATION NUMBER: EudraCT No: 2021-002693-10.

Increasing test specificity without impairing sensitivity: lessons learned from SARS-CoV-2 serology.

BACKGROUND: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the 'sensitivity improved two-test' or 'SIT²' algorithm. METHODS: SIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives). RESULTS: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. CONCLUSION: For SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

Multiparametric Prediction Models for Coronavirus Disease 2019 Vaccine Selection: Results of a Comparative Population-Based Cohort Study.

BACKGROUND: An understanding vaccine-dependent effects on protective and sustained humoral immune response is crucial to planning future vaccination strategies against coronavirus disease 2019 (COVID-19). METHODS: In this multicenter, population-based, cohort study including 4601 individuals after primary vaccination against COVID-19 ≥ 4 months earlier we compared factors associated with residual antibody levels against severe acute respiratory syndrome coronavirus-2 receptor-binding domain (RBD) across different vaccination strategies (BNT162b2, mRNA-1273, or ChAdOx1). RESULTS: Our main model including 3787 individuals (2 × BNT162b2, n = 2271; 2 × mRNA-1273, n = 251; 2 × ChAdOx1, n = 1265), predicted significantly lower levels of anti-RBD antibodies after 6 months in individuals vaccinated with ChAdOx1 (392.7 binding antibody units per milliliter [BAU/mL]) compared with those vaccinated with BNT162b2 (1179.5 BAU/mL) or mRNA-1273 (2098.2 BAU/mL). Vaccine-dependent association of antibody levels was found for age with a significant predicted difference in BAU/ml per year for BNT162b2 (-21.5; 95% confidence interval [CI], -24.7 to -18.3) and no significant association for mRNA-1273 (-4.0; 95% CI, -20.0 to 12.1) or ChAdOx1 (1.7; 95% CI, .2 to 3.1). The predicted decrease over time since full immunization was highest in mRNA-1273 (-23.4; 95% CI, -31.4 to -15.4) compared with BNT162b2 (-5.9; 95% CI, -7 to -4.8). CONCLUSIONS: Our study revealed population-based evidence of vaccine-dependent effects of age and time since full immunization on humoral immune response. Findings underline the importance of individualized vaccine selection, especially in elderly individuals.

Preanalytical stability of SARS-CoV-2 anti-nucleocapsid antibodies.

OBJECTIVES: Anti-nucleocapsid (NC) antibodies are produced in response to SARS-CoV-2 infection. Therefore, they are well suited for the detection of a previous infection. Especially in the case of seroprevalence studies or during the evaluation of a novel in-vitro diagnostic test, samples have been stored at <-70 °C (short- and long-term) or 2-10 °C (short-term) before analysis. This study aimed to assess the impact of different storage conditions relevant to routine biobanking on anti-NC antibodies. METHODS: The preanalytical impact of short-term storage (84 [58-98] days) on <-70 °C and for 14 days at 2-10 °C was evaluated using samples from 111 donors of the MedUni Vienna Biobank. Long-term effects (443 [409-468] days) were assessed using 208 samples from Biobank Graz and 49 samples from Biobank Vienna. Anti-Nucleocapsid antibodies were measured employing electrochemiluminescence assays (Roche Anti-SARS-CoV-2). RESULTS: After short-term storage, the observed changes did not exceed the extent that could be explained by analytical variability. In contrast, results after long-term storage were approximately 20% higher and seemed to increase with storage duration. This effect was independent of the biobank from which the samples were obtained. Accordingly, the sensitivity increased from 92.6 to 95.3% (p=0.008). However, comparisons with data from Anti-Spike protein assays, where these deviations were not apparent, suggest that this deviation could also be explained by the analytical variability of the qualitative Anti-NC assay. CONCLUSIONS: Results from anti-NC antibodies are stable during short-term storage at <-70 °C and 2-10 °C. After long-term storage, a slight increase in sensitivity could not be ruled out.

Heterologous vector versus homologous mRNA COVID-19 booster vaccination in non-seroconverted immunosuppressed patients: a randomized controlled trial.

Impaired response to COVID-19 vaccination is of particular concern in immunosuppressed patients. To determine the best vaccination strategy for this vulnerable group we performed a single center, 1:1 randomized blinded clinical trial. Patients who failed to seroconvert upon two mRNA vaccinations (BNT162b2 or mRNA-1273) are randomized to receive either a third dose of the same mRNA or the vector vaccine ChAdOx1 nCoV-19. Primary endpoint is the difference in SARS-CoV-2 spike antibody seroconversion rate between vector and mRNA vaccinated patients four weeks after the third dose. Secondary outcomes include cellular immune responses. Seroconversion rates at week four are significantly higher in the mRNA (homologous vaccination, 15/24, 63%) as compared to the vector vaccine group (heterologous vaccination, 4/22, 18%). SARS-CoV-2-specific T-cell responses are reduced but could be increased after a third dose of either vector or mRNA vaccine. In a multivariable logistic regression analysis, patient age and vaccine type are associated with seroconversion. No serious adverse event is attributed to COVID-19 booster vaccination. Efficacy and safety data underline the importance of a booster vaccination and support the use of a homologous mRNA booster vaccination in immunosuppressed patients.Trial registration: EudraCT No.: 2021-002693-10.

Immunogenicity and safety of a fourth COVID-19 vaccination in rituximab-treated patients: an open-label extension study.

OBJECTIVES: Patients under rituximab therapy are at high risk for a severe COVID-19 disease course. Humoral immune responses to SARS-CoV-2 vaccination are vastly diminished in B-cell-depleted patients, even after a third vaccine dose. However, it remains unclear whether these patients benefit from a fourth vaccination and whether continued rituximab therapy affects antibody development. METHODS: In this open-label extension trial, 37 rituximab-treated patients who received a third dose with either a vector or mRNA-based vaccine were vaccinated a fourth time with an mRNA-based vaccine (mRNA-1273 or BNT162b2). Key endpoints included the humoral and cellular immune response as well as safety after a fourth vaccination. RESULTS: The number of patients who seroconverted increased from 12/36 (33%) to 21/36 (58%) following the fourth COVID-19 vaccination. In patients with detectable antibodies to the spike protein's receptor-binding domain (median: 8.0 binding antibody units (BAU)/mL (quartiles: 0.4; 13.8)), elevated levels were observed after the fourth vaccination (134.0 BAU/mL (quartiles: 25.5; 1026.0)). Seroconversion and antibody increase were strongly diminished in patients who received rituximab treatment between the third and the fourth vaccination. The cellular immune response declined 12 weeks after the third vaccination, but could only be slightly enhanced by a fourth vaccination. No unexpected safety signals were detected, one serious adverse event not related to vaccination occurred. CONCLUSIONS: A fourth vaccine dose is immunogenic in a fraction of rituximab-treated patients. Continuation of rituximab treatment reduced humoral immune response, suggesting that rituximab affects a second booster vaccination. It might therefore be considered to postpone rituximab treatment in clinically stable patients. TRIAL REGISTRATION NUMBER: 2021-002348-57.

IgA rheumatoid factor in rheumatoid arthritis.

OBJECTIVES: Rheumatoid factor (RF) is a well-established marker for the diagnosis and classification of rheumatoid arthritis (RA). Most studies evaluated IgM RF or isotype-nonspecific total RF assays. We evaluated the added value of IgA RF in this context. METHODS: An international sample cohort consisting of samples from 398 RA patients and 1073 controls was tested for IgA RF with 3 commercial assays. For all RA patients and 100 controls essential clinical and serological data for ACR/EULAR classification were available. RESULTS: The sensitivity of IgA RF for diagnosing RA was lower than the sensitivity of IgM RF. Differences in numerical values between IgA RF assays were observed. With all assays, the highest IgA RF values were found in patients with primary Sjögren's syndrome. Double positivity for IgM RF and IgA RF had a higher specificity for RA than either IgM RF or IgA RF. The sensitivity of double positivity was lower than the sensitivity of either IgA RF or IgM RF. Single positivity for IgA RF was at least as prevalent in controls than in RA patients. Adding IgA RF to IgM RF and anti-citrullinated protein antibodies (ACPA) did not affect RA classification. However, combined positivity for IgA RF, IgM RF and IgG ACPA had a higher specificity and lower sensitivity for RA classification than positivity for either of the antibodies. CONCLUSIONS: IgA RF showed a lower sensitivity than IgM RF. Combining IgA RF with IgM RF and ACPA did not improve sensitivity of RA classification. Combined positivity (IgA-RF/IgM-RF/ACPA) increased specificity.

Standardisation of ACPA tests: evaluation of a new candidate reference preparation.

INTRODUCTION: Commercial assays measuring antibodies to citrullinated protein/peptide (ACPA) show poor quantitative agreement. The diagnostic industry has never adopted the International Union of Immunological Societies-Centers for Disease Control and Prevention (IUIS-CDC) ACPA reference standard. Recently, the National Institute for Biological Standards and Control (NIBSC) prepared a new candidate ACPA standard (18/204). We evaluated both reference materials using different commercially available ACPA assays. MATERIALS AND METHODS: This is an international study in which the NIBSC candidate ACPA standard and the IUIS-CDC ACPA reference material were analysed together with 398 diagnostic samples from individuals with rheumatoid arthritis (RA) and in 1073 individuals who did not have RA using nine commercial ACPA assays. RESULTS: For both reference materials and samples from individuals with RA and individuals who did not have RA, there were large differences in quantitative ACPA results between assays. For most assays, values for the IUIS-CDC standard were lower than values for NIBSC 18/204 and the IUIS-CDC/NIBSC ratio was comparable for several, but not all assays. When NIBSC 18/204 was used as a calibrator, an improvement in alignment of ACPA results across several of the evaluated assays was obtained. Moreover, NIBSC 18/204 could align clinical interpretation for some but not all assays. CONCLUSION: Adoption of an international standard for ACPA determination is highly desirable. The candidate NIBSC 18/204 standard improved the standardisation and alignment of most ACPA assays and might therefore be recommended to be used as reference in commercial assays.

Prospective Studies on the Risk of Rheumatoid Arthritis: The European Risk RA Registry.

Background: The accumulation of risk for the development of rheumatoid arthritis (RA) is regarded as a continuum that may start with interacting environmental and genetic factors, proceed with the initiation of autoimmunity, and result in the formation of autoantibodies such as anti-citrullinated peptide antibodies (ACPA). In parallel, at-risk individuals may be asymptomatic or experience joint pain (arthralgia) that is itself non-specific or clinically suspicious for evolving RA, even in the absence of overt arthritis. Optimal strategies for the management of people at-risk of RA, both for symptom control and to delay or prevent progression to classifiable disease, remain poorly understood. Methods: To help address this, groups of stakeholders from academia, clinical rheumatology, industry and patient research partners have collaborated to advance understanding, define and study different phases of the at-risk state. In this current report we describe different European initiatives in the field and the successful effort to build a European Registry of at-risk people to facilitate observational and interventional research. Results: We outline similarities and differences between cohorts of at-risk individuals at institutions spanning several countries, and how to best combine them within the new database. Over the past 2 years, besides building the technical infrastructure, we have agreed on a core set of variables that all partners should strive to collect for harmonization purposes. Conclusion: We emphasize to address this process from different angles and touch on the biologic, epidemiologic, analytic, and regulatory aspects of collaborative studies within a meta-database of people at-risk of RA.

Multicentre study to improve clinical interpretation of rheumatoid factor and anti-citrullinated protein/peptide antibodies test results.

BACKGROUND: Rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA) are important biomarkers for diagnosis of rheumatoid arthritis (RA). However, there is poor harmonisation of RF and ACPA assays. The aim of this study was to refine RF and ACPA interpretation across commercial assays. MATERIALS AND METHODS: Six total RF isotype-non-specific assays, 3 RF IgM isotype-specific assays and 9 ACPA immunoglobulin G assays of 13 different companies were evaluated using 398 diagnostic samples from patients with RA and 1073 disease controls. RESULTS: Using cut-offs proposed by the manufacturer, there was a large variability in diagnostic sensitivity and specificity between assays. Thresholds of antibody levels were determined based on predefined specificities and used to define test result intervals. Test result interval-specific likelihood ratios (LRs) were concordant across the different RF and ACPA assays. For all assays, the LR for RA increased with increasing antibody level. Higher LRs were found for ACPA than for RF. ACPA levels associated with LRs >80 were found in a substantial fraction (>22%) of patients with RA. CONCLUSION: Defining thresholds for antibody levels and assigning test result interval-specific LRs allows alignment of clinical interpretation for all RF and ACPA assays.

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis.

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT's 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.

Additional heterologous versus homologous booster vaccination in immunosuppressed patients without SARS-CoV-2 antibody seroconversion after primary mRNA vaccination: a randomised controlled trial.

OBJECTIVES: SARS-CoV-2-induced COVID-19 has led to exponentially rising mortality, particularly in immunosuppressed patients, who inadequately respond to conventional COVID-19 vaccination. METHODS: In this blinded randomised clinical trial, we compare the efficacy and safety of an additional booster vaccination with a vector versus mRNA vaccine in non-seroconverted patients. We assigned 60 patients under rituximab treatment, who did not seroconvert after their primary mRNA vaccination with either BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna), to receive a third dose, either using the same mRNA or the vector vaccine ChAdOx1 nCoV-19 (Oxford-AstraZeneca). Patients were stratified according to the presence of peripheral B cells. The primary efficacy endpoint was the difference in the SARS-CoV-2 antibody seroconversion rate between vector (heterologous) and mRNA (homologous) vaccinated patients by week 4. Key secondary endpoints included the overall seroconversion and cellular immune response; safety was assessed at week 1 and week 4. RESULTS: Seroconversion rates at week 4 were comparable between vector (6/27 patients, 22%) and mRNA (9/28, 32%) vaccines (p=0.6). Overall, 27% of patients seroconverted; specific T cell responses were observed in 20/20 (100%) vector versus 13/16 (81%) mRNA vaccinated patients. Newly induced humoral and/or cellular responses occurred in 9/11 (82%) patients. 3/37 (8%) of patients without and 12/18 (67%) of the patients with detectable peripheral B cells seroconverted. No serious adverse events, related to immunisation, were observed. CONCLUSIONS: This enhanced humoral and/or cellular immune response supports an additional booster vaccination in non-seroconverted patients irrespective of a heterologous or homologous vaccination regimen.

Impact of autoimmune serology test results on RA classification and diagnosis.

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease and also the most severe arthritic disorder. The measurement of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) in serum supports the diagnosis of RA, which gained increasing significance over the last 65 years. However, a high variability between RF and ACPA methods has been described, impacting the diagnostic performance of the current ACR/EULAR RA classification criteria. The great number of commercially available assays, often lacking traceability to an international standard, is a major factor attributing to this in-between assay variability. The adoption of an international standard for ACPA, as is since long available for rheumatoid factor, is therefore highly desirable. Further harmonization in clinical interpretation of RF/ACPA assays could be obtained by harmonization of the cut-offs, for both the low and high antibody levels, based on predefined specificity in disease controls. Reporting test result specific likelihood ratios (LR) adds value in the interpretation of autoantibody tests. However, a good understanding of the control population used to define antibody test result interval-associated LRs is crucial in defining the diagnostic performance characteristics of antibody serology. Finally, specificity in RA classification can be improved by refining serological weight scoring taking into account the nature of the antibody, the antibody level and double RF + ACPA positivity.

The diagnostic and prognostic value of IgG and IgA anti-citrullinated protein antibodies in patients with early rheumatoid arthritis.

Objectives: Anti-citrullinated peptide antibodies (ACPA) are specific markers for rheumatoid arthritis (RA) and typically measured by assays employing a cyclic citrullinated peptide (CCP) as antigen. This study was aimed at investigating the diagnostic performance of anti-CCP2 and anti-CCP3 IgG and IgA assays in patients with early RA with a particular focus on the potential prognostic value of IgA ACPA. Methods: The anti-CCP3.1 assay (Inova Diagnostics) measuring IgG and IgA antibodies simultaneously was compared to anti-CCP2 IgG and IgA assays (Thermo Fisher Scientific) employing sera of 184 early RA patients, 360 disease controls and 98 healthy subjects. Results: Anti-CCP2 IgG and IgA assays showed high specificity versus disease controls (98.9%; 99.4%). Sensitivity was 52.2% (IgG) and 28.8% (IgA), resulting in positive likelihood ratios (LR+) of 47.5 (IgG) and 48.0 (IgA). The anti-CCP3.1 assay proved slightly more sensitive than the anti-CCP2 IgG assay (56%) but specificity was markedly lower (90.8% versus disease controls). However, when using a threefold higher cut-off specificity of the anti-CCP3.1 assay increased (97.5%) while sensitivity (52.7%) became comparable to the anti-CCP2 IgG assay resulting in a LR+ of 21.5. Anti-CCP2 IgA antibodies did not increase the diagnostic sensitivity of ACPA testing, but IgA positive patients showed diminished responses to treatment with anti-TNF biologicals compared to patients who had only IgG antibodies. Conclusion: Specificity of ACPA assays should be adjusted to reduce the risk of misclassification and a false positive diagnosis. Determination of ACPA IgA might provide important prognostic information concerning therapeutic responses.

Presence of anti-acetylated peptide antibodies (AAPA) in inflammatory arthritis and other rheumatic diseases suggests discriminative diagnostic capacity towards early rheumatoid arthritis.

AIMS: To determine the diagnostic value of anti-acetylated peptide antibodies (AAPA) in patients with rheumatoid arthritis (RA). METHODS: Three acetylated peptides (ac-lysine, ac-lysine.inv and ac-ornithine) derived from vimentin were employed to measure AAPA by enzyme-linked immunosorbent assay (ELISA) in sera of 120 patients with early RA (eRA), 195 patients with established RA (est RA), 99 healthy controls (HC), and 216 patients with other inflammatory rheumatic diseases. A carbamylated and a citrullinated version of the vimentin peptide were used additionally. Receiver operating characteristics and logistic regression analyses were used to assess the discriminative capacity of AAPA. RESULTS: AAPA were detected in 60% of eRA and 68.7% of estRA patients, 22.2% of HC, and 7.1- 30.6% of patients with other rheumatic diseases. Importantly, AAPA were also present in 40% of seronegative RA patients, while antibodies to the carbamylated peptide were detected less frequently. Diagnostic sensitivity of individual peptides for eRA was 28.3%, 35.8%, and 34% for ac-lysine, ac-ornithine, and ac-lysine.inv, respectively. Positive likelihood ratios (LR+) for eRA versus HC were 14.0, 7.1, and 2.1. While the presence of a single AAPA showed varying specificity (range: 84-98%), the presence of two AAPA increased specificity considerably since 26.7% of eRA, as compared with 6% of disease controls, were double positive. Thus, double positivity discriminated eRA from axial spondyloarthritis with a LR+ of 18.3. Remarkably, triple positivity was 100% specific for RA, being observed in 10% of eRA and 21.5% of estRA patients, even in the absence of RF and ACPA. CONCLUSION: AAPA are highly prevalent in early RA and occur also independently of RF and ACPA, thereby reducing the gap of seronegativity. Furthermore, multiple AAPA reactivity increased the specificity for RA, suggesting high diagnostic value of AAPA testing.