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Objectives Methotrexate (MTX) is one of the most commonly used medications to treat rheumatoid arthritis (RA). However, the effect of MTX treatment on cellular immune responses remains incompletely understood. This raises concerns about the vulnerability of these patients to emerging infections and following vaccination.Methods In the current study, we investigated the impact of MTX treatment in patients with immune-mediated inflammatory disease on B and CD4 T cell SARS-CoV-2 vaccination responses. Eighteen patients with RA and two patients with psoriatic arthritis on MTX monotherapy were included, as well as 10 patients with RA without immunosuppressive treatment, and 29 healthy controls. CD4 T and B cell responses were analysed 7 days and 3–6 months after two SARS-CoV-2 messenger RNA vaccinations. High-dimensional flow cytometry analysis was used to analyse fresh whole blood, an activation-induced marker assay to measure antigen-specific CD4 T cells, and spike probes to study antigen-specific B cells.Results Seven days following two SARS-CoV-2 vaccinations, total B and T cell counts were similar between MTX-treated patients and controls. In addition, spike-specific B cell frequencies were unaffected. Remarkably, the frequency of antigen-specific CD4 T cells was reduced in patients using MTX and correlated strongly with anti-RBD IgG antibodies. These results suggest that decreased CD4 T cell activity may result in slower vaccination antibody responses in MTX-treated patients.Conclusion Taken together, MTX treatment reduces vaccine-induced CD4 T cell activation, which correlates with lower antibody responses.Trial registration number NL8900.

Objective Rheumatoid arthritis (RA) is characterised by the presence of autoantibodies, among which those targeting the constant region of immunoglobulin G (IgG), called rheumatoid factors (RF). Despite this link, RFs can also be found in other disorders and the healthy population, which hampers its use as a diagnostic tool. We recently showed that a subset of RA-derived RFs target a distinct epitope on the IgG-Fc, a feature that is currently not used in the clinic.Methods We determined immunoglobulin M (IgM)-RF levels specific against an RA-associated epitope (using our engineered next-generation RF antigen ‘T3-17’) in a prospective cohort of 475 patients with seropositive (for IgM-RF or aCCP) arthralgia that were followed for 5 years or until the development of arthritis.Results The presence of RFs targeting T3-17 was more strongly associated with progression to arthritis in comparison to traditional RF measurements. Within the group of patients positive for T3-17 RF the risk of arthritis development was increased as compared with wild-type RF, HR=3.2 (95% CI 2.4 to 4.3) vs HR=2.2 (95% CI 1.7 to 3.0). Predictive power of T3-17 RF was improved in combination with aCCP titres, HR=6.4 (4.7–8.7) vs HR=5.1 (3.9–6.8). This combination performed better than aCCP detection on its own.Conclusion The detection of disease-specific RF is feasible and seems to improve the diagnostic power of RF and should be considered to be implemented in the clinic.

Introduction Treatment with the immune checkpoint inhibitor anti-programmed cell death protein-1 (PD-1) often causes immune-related adverse events (irAEs). Since irAEs resemble autoimmune diseases, autoantibodies might play a role and could potentially be used to identify patients at risk. Therefore, we investigated the association between autoantibody-positivity and toxicity as well as clinical response in patients with melanoma treated with anti-PD-1.Materials and methods This two-center, retrospective study included 143 patients with melanoma treated with anti-PD-1. Toxicities grade ≥2 and recurrences/responses were captured until 6 months after treatment initiation. Autoantibody measurements were performed at baseline and 3 months after treatment initiation, including IgM-rheumatoid factor (RF), antinuclear antibodies (ANA), extractable nuclear antigen, anti-cyclic citrullinated peptide antibodies (anti-CCP2) and anti-thyroid antibodies.Results 169 irAEs were experienced by 86/143 patients (137 grades 1–2, 32 grades 3–4), the most common being thyroiditis (n=25), dermatitis (n=24), and sicca problems (n=19). Patients with autoantibodies at baseline experienced more irAEs (p=0.001), predominantly associated with anti-thyroid antibodies and thyroid dysfunction. No association was observed between any irAE and anti-CCP2, RF or ANA. In women, baseline and on-treatment anti-thyroid antibody-positivity as well as seroconversion during treatment was associated with thyroid dysfunction. In men, this association was only observed on-treatment. The presence of autoantibodies was not associated with melanoma recurrence (p=0.776) or response (p=0.597).Conclusion The presence of autoantibodies prior to anti-PD-1 therapy is associated with irAEs in patients with melanoma. Both baseline positivity and seroconversion of anti-thyroid antibodies were strongly associated with thyroid dysfunction. This association was stronger in women, with all women who were baseline positive developing thyroid dysfunction.

New therapies to treat or prevent viral infections are essential, as recently observed during the COVID-19 pandemic. Here, we propose a therapeutic strategy based on monoclonal antibodies that block the specific interaction between the host receptor Siglec-1/CD169 and gangliosides embedded in the viral envelope. Antibodies are an excellent option for treating infectious diseases based on their high specificity, strong targeting affinity, and relatively low toxicity. Through a process of humanization, we optimized monoclonal antibodies to eliminate sequence liabilities and performed biophysical characterization. We demonstrated that they maintain their ability to block viral entry into myeloid cells. These molecular improvements during the discovery stage are key if we are to maximize efforts to develop new therapeutic strategies. Humanized monoclonal antibodies targeting CD169 provide new opportunities in the treatment of infections caused by ganglioside-containing enveloped viruses, which pose a constant threat to human health. In contrast with current neutralizing antibodies that bind antigens on the infectious particle, our antibodies can prevent several types of enveloped viruses interacting with host cells because they target the host CD169 protein, thus becoming a potential pan-antiviral therapy.

Background: The burden of post COVID-19 condition (PCC) is not well studied in patients with advanced kidney disease. Methods: A large prospective cohort of SARS-CoV-2 vaccinated patients with chronic kidney disease stages G4–G5 (CKD G4/5), on dialysis, and kidney transplant recipients (KTR) were included. Antibody levels were determined after vaccination. Presence of long-lasting symptoms was assessed in patients with and without prior COVID-19 and compared using logistic regression. In patients with prior COVID-19, PCC was defined according to the WHO definition. Results: Two hundred sixteen CKD G4/5 patients, 375 dialysis patients, and 2005 KTR were included. Long-lasting symptoms were reported in 204/853 (24%) patients with prior COVID-19 and in 297/1743 (17%) patients without prior COVID-19 (aOR: 1.45 (1.17–1.78)], P < 0.001). PCC was prevalent in 29% of CKD G4/5 patients, 21% of dialysis patients, and 24% of KTR. In addition, 69% of patients with PCC reported (very) high symptom burden. Odds of PCC was lower per 10-fold increase in antibody level after vaccination (aOR 0.82 [0.70–0.96], P = 0.01) and higher in case of COVID-19 related hospital admission (aOR 4.64 [2.61–8.25], P = 0.003). Conclusions: CKD G4/5 patients, dialysis patients, and KTR are at risk for PCC with high symptom burden after SARS-CoV-2 vaccination, especially if antibody levels are low and in case of hospitalization due to COVID-19.

BackgroundRheumatoid factors (RFs) are autoantibodies that target the Fc region of IgG, and are found in patients with rheumatic diseases as well as in the healthy population. Many studies suggest that an immune trigger may (transiently) elicit RF responses. However, discrepancies between different studies make it difficult to determine if and to which degree RF reactivity can be triggered by vaccination or infection.ObjectiveWe quantitatively explored longitudinal RF responses after SARS-CoV-2 vaccination and infection in a well-defined, large cohort using a dual ELISA method that differentiates between true RF reactivity and background IgM reactivity. In addition, we reviewed existing literature on RF responses after vaccination and infection.Methods151 healthy participants and 30 RA patients were included to measure IgM-RF reactivity before and after SARS-CoV-2 vaccinations by ELISA. Additionally, IgM-RF responses after a SARS-CoV-2 breakthrough infection were studied in 51 healthy participants.ResultsPublished prevalence studies in subjects after infection report up to 85% IgM-RF seropositivity. However, seroconversion studies (both infection and vaccination) report much lower incidences of 2-33%, with a trend of lower percentages observed in larger studies. In the current study, SARS-CoV-2 vaccination triggered low-level IgM-RF responses in 5.5% (8/151) of cases, of which 1.5% (2/151) with a level above 10 AU/mL. Breakthrough infection was accompanied by development of an IgM-RF response in 2% (1/51) of cases.ConclusionOur study indicates that de novo RF induction following vaccination or infection is an uncommon event, which does not lead to RF epitope spreading.

Immunoglobulin G (IgG) antibodies are a critical component of the adaptive immune system, binding to and neutralizing pathogens and other foreign substances. Recent advances in molecular antibody biology and structural protein engineering enabled the modification of IgG antibodies to enhance their therapeutic potential. This review summarizes recent progress in both natural and engineered structural modifications of IgG antibodies, including allotypic variation, glycosylation, Fc engineering, and Fc gamma receptor binding optimization. We discuss the functional consequences of these modifications to highlight their potential for therapeutical applications.

Background Dupilumab is prescribed in one dosage across adult atopic dermatitis patients. Differences in drug exposure may explain variation in treatment response. Objective Investigating the clinical relevance of dupilumab serum concentration in atopic dermatitis in real-world practice. Methods In two centers (Netherlands, UK), adults treated with dupilumab for atopic dermatitis were evaluated for effectiveness and safety pretreatment and at 2, 12, 24, and 48 weeks; trough serum samples were analyzed for dupilumab concentration at corresponding time points. Results In 149 patients, median dupilumab levels during follow-up ranged from 57.4 to 72.4 μg/mL. Levels showed high inter-patient and low intra-patient variability. No correlation was found between levels and ΔEASI. At 2 weeks, levels of ≥64.1 μg/mL predict EASI ≤7 at 24 weeks (specificity:100%, sensitivity:60%; p = .022). At 12 weeks, ≤32.7 μg/mL predicts EASI >7 at 24 weeks (sensitivity:95%, specificity:26%; p = .011). Inverse correlations were found between baseline EASI and levels at 2, 12, and 24 weeks (r = −0.25 to 0.36; p ≤ .023). Low levels were particularly observed in patients with adverse events, treatment interval deviation, and discontinuation. Conclusion At the on-label dosage, the measured range of dupilumab levels does not seem to yield differences in treatment effectiveness. However, disease activity does seem to influence dupilumab levels - higher baseline disease activity results in lower levels at follow-up.

Abstract Background Patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressants (ISPs) may have impaired long-term humoral immune responses and increased disease activity after SARS-CoV-2 infection. We aimed to investigate long-term humoral immune responses against SARS-CoV-2 and increased disease activity after a primary SARS-CoV-2 infection in unvaccinated IMID patients on ISPs. Methods IMID patients on active treatment with ISPs and controls (i.e. IMID patients not on ISP and healthy controls) with a confirmed SARS-CoV-2 infection before first vaccination were included from an ongoing prospective cohort study (T2B! study). Clinical data on infections and increased disease activity were registered using electronic surveys and health records. A serum sample was collected before first vaccination to measure SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies. Results In total, 193 IMID patients on ISP and 113 controls were included. Serum samples from 185 participants were available, with a median time of 173 days between infection and sample collection. The rate of seropositive IMID patients on ISPs was 78% compared to 100% in controls (p

Summary: IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.

Abstract Although elevated levels of anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA), the in vivo functions of these antibodies remain unclear. Here, we have expressed monoclonal ACPAs derived from patients with RA, and analyzed their functions in mice, as well as their specificities. None of the ACPAs showed arthritogenicity nor induced pain-associated behavior in mice. However, one of the antibodies, clone E4, protected mice from antibody-induced arthritis. E4 showed a binding pattern restricted to skin, macrophages and dendritic cells in lymphoid tissue, and cartilage derived from mouse and human arthritic joints. Proteomic analysis confirmed that E4 strongly binds to macrophages and certain RA synovial fluid proteins such as α-enolase. The protective effect of E4 was epitope-specific and dependent on the interaction between E4-citrullinated α-enolase immune complexes with FCGR2B on macrophages, resulting in increased IL-10 secretion and reduced osteoclastogenesis. These findings suggest that a subset of ACPAs have therapeutic potential in RA.

Disrupting the association between the Immunoglobulin G constant fragment (Fc) and the neonatal Fc receptor (FcRn) by engineered antibodies is a promising strategy to reduce autoantibody levels in autoimmune diseases. Here authors show that the variable fragment (Fab) of immunoglobulins could disturb the Fc-FcRn interaction, therefore the therapeutic effect of Fc-only fragments might surpass that of Fc-engineered antibodies with enhanced binding to FcRn.

Kidney transplant recipients (KTRs) elicit an impaired immune response after COVID-19 vaccination; however, the exact clinical impact remains unclear. We therefore analyse the relationship between antibody levels after vaccination and the risk of COVID-19 in a large cohort of KTRs. All KTRs living in the Netherlands were invited to send a blood sample 28 days after their second COVID-19 vaccination for measurement of their IgG antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein (anti-RBD IgG). Information on COVID-19 was collected from the moment the blood sample was obtained until 6 months thereafter. Multivariable Cox and logistic regression analyses were performed to analyse which factors affected the occurrence and severity (i.e., hospitalization and/or death) of COVID-19. In total, 12,159 KTRs were approached, of whom 2885 were included in the analyses. Among those, 1578 (54.7%) became seropositive (i.e., anti-RBD IgG level >50 BAU/mL). Seropositivity was associated with a lower risk for COVID-19, also after adjusting for multiple confounders, including socio-economic status and adherence to COVID-19 restrictions (HR 0.37 (0.19–0.47), p = 0.005). When studied on a continuous scale, we observed a log-linear relationship between antibody level and the risk for COVID-19 (HR 0.52 (0.31–0.89), p = 0.02). Similar results were found for COVID-19 severity. In conclusion, antibody level after COVID-19 vaccination is associated in a log-linear manner with the occurrence and severity of COVID-19 in KTRs. This implies that if future vaccinations are indicated, the aim should be to reach for as high an antibody level as possible and not only seropositivity to protect this vulnerable patient group from disease.

Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions.

Abstract Background Studies have suggested incremental short-term adverse events (AE) after repeated vaccination. In this report, we assessed occurrence and risk factors for short-term AEs following repeated SARS-CoV-2 vaccination in patients with various immune-mediated inflammatory diseases (IMIDs). Methods Self-reported daily questionnaires on AEs during the first 7 days after vaccination were obtained of 2259 individuals (2081 patients and 178 controls) participating in an ongoing prospective multicenter cohort study on SARS-CoV-2 vaccination in patients with various IMIDs in the Netherlands (T2B-COVID). Relative risks were calculated for potential risk factors associated with clinically relevant AE (rAE), defined as AE lasting longer than 2 days or impacting daily life. Results In total, 5454 vaccinations were recorded (1737 first, 1992 second and 1478 third vaccinations). Multiple sclerosis, Crohn’s disease and rheumatoid arthritis were the largest disease groups. rAEs were reported by 57.3% (95% CI 54.8–59.8) of patients after the first vaccination, 61.5% (95% CI 59.2–63.7) after the second vaccination and 58% (95% CI 55.3–60.6) after the third vaccination. At day 7 after the first, second and third vaccination, respectively, 7.6% (95% CI 6.3–9.1), 7.4% (95% CI 6.2–8.7) and 6.8% (95% CI 5.4–8.3) of patients still reported AEs impacting daily life. Hospital admissions and allergic reactions were uncommon (

Abstract The neonatal Fc receptor (FcRn) is known to mediate placental transfer of IgG from mother to unborn. IgE is widely known for triggering immune responses to environmental antigens. Recent evidence suggests FcRn-mediated transplacental passage of IgE during pregnancy. However, direct interaction of FcRn and IgE was not investigated. Here, we compared binding of human IgE and IgG variants to recombinant soluble human FcRn with β2-microglobulin (sFcRn) in surface plasmon resonance (SPR) at pH 7.4 and pH 6.0. No interaction was found between human IgE and human sFcRn. These results imply that FcRn can only transport IgE indirectly, and thereby possibly transfer allergenic sensitivity from mother to fetus.

Targeted biologic therapies can elicit an undesirable host immune response characterized by the development of antidrug antibodies (ADA), an important cause of treatment failure. The most widely used biologic across immune-mediated diseases is adalimumab, a tumor necrosis factor inhibitor. This study aimed to identify genetic variants that contribute to the development of ADA against adalimumab, thereby influencing treatment failure. In patients with psoriasis on their first course of adalimumab, in whom serum ADA had been evaluated 6–36 months after starting treatment, we observed a genome-wide association with ADA against adalimumab within the major histocompatibility complex (MHC). The association signal mapped to the presence of tryptophan at position 9 and lysine at position 71 of the HLA-DR peptide-binding groove, with both residues conferring protection against ADA. Underscoring their clinical relevance, these residues were also protective against treatment failure. Our findings highlight antigenic peptide presentation via MHC class II as a critical mechanism in the development of ADA against biologic therapies and downstream treatment response.

Abstract Objectives The complement system is an important component of innate immunity. The alternative pathway (AP) amplification loop is considered an essential feed forward mechanism for complement activation. However, the role of the AP in classical pathway (CP) activation has only been studied in ELISA settings. Here, we investigated its contribution on physiologically relevant surfaces of human cells and bacterial pathogens and in antibody‐mediated complement activation, including in autoimmune haemolytic anaemia (AIHA) setting with autoantibodies against red blood cells (RBCs). Methods We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP‐depleted sera or antibodies against factor B and factor D. Results We show that the amplification loop is redundant when efficient CP activation takes place. This is independent of the presence of membrane‐bound complement regulators. The role of the AP may become significant when insufficient CP complement activation occurs, but this depends on antibody levels and (sub)class. Our data indicate that therapeutic intervention in the amplification loop will most likely not be effective to treat antibody‐mediated diseases. Conclusion The AP can be bypassed through efficient CP activation. The AP amplification loop has a role in complement activation during conditions of modest activation via the CP, when it can allow for efficient complement‐mediated killing.

Of the four human immunoglobulin G (IgG) subclasses, IgG4 is considered the least inflammatory, in part because it poorly activates the complement system. Regardless, in IgG4 related disease (IgG4-RD) and in autoimmune disorders with high levels of IgG4 autoantibodies, the presence of these antibodies has been linked to consumption and deposition of complement components. This apparent paradox suggests that conditions may exist, potentially reminiscent of in vivo deposits, that allow for complement activation by IgG4. Furthermore, it is currently unclear how variable glycosylation and Fab arm exchange may influence the ability of IgG4 to activate complement. Here, we used well-defined, glyco-engineered monoclonal preparations of IgG4 and determined their ability to activate complement in a controlled system. We show that IgG4 can activate complement only at high antigen and antibody concentrations, via the classical pathway. Moreover, elevated or reduced Fc galactosylation enhanced or diminished complement activation, respectively, with no apparent contribution from the lectin pathway. Fab glycans slightly reduced complement activation. Lastly, we show that bispecific, monovalent IgG4 resulting from Fab arm exchange is a less potent activator of complement than monospecific IgG4. Taken together, these results imply that involvement of IgG4-mediated complement activation in pathology is possible but unlikely.

BackgroundKidney transplant recipients (KTRs) have an impaired immune response after vaccination against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Iron deficiency (ID) may adversely affect immunity and vaccine efficacy. We aimed to investigate whether ferric carboxymaltose (FCM) treatment improves humoral and cellular responses after SARS-CoV-2 vaccination in iron-deficient KTRs.MethodsWe randomly assigned 48 iron-deficient KTRs to intravenous FCM (1-4 doses of 500mg with six-week intervals) or placebo. Co-primary endpoints were SARS-CoV-2-specific anti-Receptor Binding Domain (RBD) Immunoglobulin G (IgG) titers and T-lymphocyte reactivity against SARS-CoV-2 at four weeks after the second vaccination with mRNA-1273 or mRNA-BNT162b2.ResultsAt four weeks after the second vaccination, patients receiving FCM had higher plasma ferritin and transferrin saturation (P

Summary: Background: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced FcγRIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. Methods: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). Findings: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen-experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen-experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. Interpretation: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. Funding: LSBR 1721, 1908; ZonMW 10430012010021, 09150161910033, 10430012010008; DFG 398859914, 400912066, 390884018; PMI; DOI4-Nr. 3; H2020-MSCA-ITN 721815.

The generation of high-affinity antibodies requires an efficient germinal center (GC) response. As differentiating B cells cycle between GC dark and light zones they encounter different oxygen pressures (pO2). However, it is essentially unknown if and how variations in pO2 affect B cell differentiation, in particular for humans. Using optimized in vitro cultures together with in-depth assessment of B cell phenotype and signaling pathways, we show that oxygen is a critical regulator of human naive B cell differentiation and class switch recombination. Normoxia promotes differentiation into functional antibody secreting cells, while a population of CD27++ B cells was uniquely generated under hypoxia. Moreover, time-dependent transitions between hypoxic and normoxic pO2 during culture - reminiscent of in vivo GC cyclic re-entry - steer different human B cell differentiation trajectories and IgG class switch recombination. Taken together, we identified multiple mechanisms trough which oxygen pressure governs human B cell differentiation.

Abstract Objectives It is currently thought that most—but not all—individuals infected with SARS-CoV-2 develop symptoms, but the infectious period starts on average 2 days before the first overt symptoms appear. It is estimated that pre- and asymptomatic individuals are responsible for more than half of all transmissions. By detecting infected individuals before they have overt symptoms, wearable devices could potentially and significantly reduce the proportion of transmissions by pre-symptomatic individuals. Using laboratory-confirmed SARS-CoV-2 infections (detected via serology tests [to determine if there are antibodies against the SARS-CoV-2 in the blood] or SARS-CoV-2 infection tests such as polymerase chain reaction [PCR] or antigen tests) as the gold standard, we will determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the following two algorithms to detect first time SARS-CoV-2 infection including early or asymptomatic infection: • The algorithm using Ava bracelet data when coupled with self-reported Daily Symptom Diary data (Wearable + Symptom Data Algo; experimental condition) • The algorithm using self-reported Daily Symptom Diary data alone (Symptom Only Algo; control condition) In addition, we will determine which of the two algorithms has superior performance characteristics for detecting SARS-CoV-2 infection including early or asymptomatic infection as confirmed by SARS-CoV-2 virus testing. Trial design The trial is a randomized, single-blinded, two-period, two-sequence crossover trial. The study will start with an initial learning phase (maximum of 3 months), followed by period 1 (3 months) and period 2 (3 months). Subjects entering the study at the end of the recruitment period may directly start with period 1 and will not be part of the learning phase. Each subject will undergo the experimental condition (the Wearable + Symptom Data Algo) in either period 1 or period 2 and the control condition (Symptom Only Algo) in the other period. The order will be randomly assigned, resulting in subjects being allocated 1:1 to either sequence 1 (experimental condition first) or sequence 2 (control condition first). Based on demographics, medical history and/or profession, each subject will be stratified at baseline into a high-risk and normal-risk group within each sequence. Participants The trial will be conducted in the Netherlands. A target of 20,000 subjects will be enrolled. Based on demographics, medical history and/or profession, each subject will be stratified at baseline into a high-risk and normal-risk group within each sequence. This results in approximately 6500 normal-risk individuals and 3500 high-risk individuals per sequence. Subjects will be recruited from previously studied cohorts as well as via public campaigns and social media. All data for this study will be collected remotely through the Ava COVID-RED app, the Ava bracelet, surveys in the COVID-RED web portal and self-sampling serology and PCR kits. More information on the study can be found in www.covid-red.eu . During recruitment, subjects will be invited to visit the COVID-RED web portal. After successfully completing the enrolment questionnaire, meeting eligibility criteria and indicating interest in joining the study, subjects will receive the subject information sheet and informed consent form. Subjects can enrol in COVID-RED if they comply with the following inclusion and exclusion criteria: Inclusion criteria: • Resident of the Netherlands • At least 18 years old • Informed consent provided (electronic) • Willing to adhere to the study procedures described in the protocol • Must have a smartphone that runs at least Android 8.0 or iOS 13.0 operating systems and is active for the duration of the study (in the case of a change of mobile number, the study team should be notified) • Be able to read, understand and write Dutch Exclusion criteria: • Previous positive SARS-CoV-2 test result (confirmed either through PCR/antigen or antibody tests; self-reported) • Current suspected (e.g. waiting for test result) COVID-19 infection or symptoms of a COVID-19 infection (self-reported) • Participating in any other COVID-19 clinical drug, vaccine or medical device trial (self-reported) • Electronic implanted device (such as a pacemaker; self-reported) • Pregnant at the time of informed consent (self-reported) • Suffering from cholinergic urticaria (per the Ava bracelet’s user manual; self-reported) • Staff involved in the management or conduct of this study Intervention and comparator All subjects will be instructed to complete the Daily Symptom Diary in the Ava COVID-RED app daily, wear their Ava bracelet each night and synchronize it with the app each day for the entire period of study participation. Provided with wearable sensor and/or self-reported symptom data within the last 24 h, the Ava COVID-RED app’s underlying algorithms will provide subjects with a real-time indicator of their overall health and well-being. Subjects will see one of three messages, notifying them that no seeming deviations in symptoms and/or physiological parameters have been detected; some changes in symptoms and/or physiological parameters have been detected and they should self-isolate; or alerting them that deviations in their symptoms and/or physiological parameters could be suggestive of a potential COVID-19 infection and to seek additional testing. We will assess the intraperson performance of the algorithms in the experimental condition (Wearable + Symptom Data Algo) and control conditions (Symptom Only Algo). Note that both algorithms will also instruct to seek testing when any SARS-CoV-2 symptoms are reported in line with those defined by the Dutch national institute for public health and the environment ‘Rijksinstituut voor Volksgezondheid en Milieu’ (RIVM) guidelines. Main outcomes The trial will evaluate the use and performance of the Ava COVID-RED app and Ava bracelet, which uses sensors to measure breathing rate, pulse rate, skin temperature and heart rate variability for the purpose of early and asymptomatic detection and monitoring of SARS-CoV-2 in general and high-risk populations. Using laboratory-confirmed SARS-CoV-2 infections (detected via serology tests, PCR tests and/or antigen tests) as the gold standard, we will determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for each of the following two algorithms to detect first-time SARS-CoV-2 infection including early or asymptomatic infection: the algorithm using Ava bracelet data when coupled with the self-reported Daily Symptom Diary data and the algorithm using self-reported Daily Symptom Diary data alone. In addition, we will determine which of the two algorithms has superior performance characteristics for detecting SARS-CoV-2 infection including early or asymptomatic infection as confirmed by SARS-CoV-2 virus testing. The protocol contains an additional twenty secondary and exploratory objectives which address, among others, infection incidence rates, health resource utilization, symptoms reported by SARS-CoV-2-infected participants and the rate of breakthrough and asymptomatic SARS-CoV-2 infections among individuals vaccinated against COVID-19. PCR or antigen testing will occur when the subject receives a notification from the algorithm to seek additional testing. Subjects will be advised to get tested via the national testing programme and report the testing result in the Ava COVID-RED app and a survey. If they cannot obtain a test via the national testing programme, they will receive a nasal swab self-sampling kit at home, and the sample will be tested by PCR in a trial-affiliated laboratory. In addition, all subjects will be asked to take a capillary blood sample at home at baseline (between month 0 and 3.5 months after the start of subject recruitment), at the end of the learning phase (month 3; note that this sampling moment is skipped if a subject entered the study at the end of the recruitment period), period 1 (month 6) and period 2 (month 9). These samples will be used for SARS-CoV-2-specific antibody testing in a trial-affiliated laboratory, differentiating between antibodies resulting from a natural infection and antibodies resulting from COVID-19 vaccination (as vaccination will gradually be rolled out during the trial period). Baseline samples will only be analysed if the sample collected at the end of the learning phase is positive, or if the subject entered the study at the end of the recruitment period, and samples collected at the end of period 1 will only be analysed if the sample collected at the end of period 2 is positive. When subjects obtain a positive PCR/antigen or serology test result during the study, they will continue to be in the study but will be moved into a so-called COVID-positive mode in the Ava COVID-RED app. This means that they will no longer receive recommendations from the algorithms but can still contribute and track symptom and bracelet data. The primary analysis of the main objective will be executed using the data collected in period 2 (months 6 through 9). Within this period, serology tests (before and after period 2) and PCR/antigen tests (taken based on recommendations by the algorithms) will be used to determine if a subject was infected with SARS-CoV-2 or not. Within this same time period, it will be determined if the algorithms gave any recommendations for testing. The agreement between these quantities will be used to evaluate the performance of the algorithms and how these compare between the study conditions. Randomization All eligible subjects will be randomized using a stratified block randomization approach with an allocation ratio of 1:1 to one of two sequences (experimental condition followed by control condition or control condition followed by experimental condition). Based on demographics, medical history and/or profession, each subject will be stratified at baseline into a high-risk and normal-risk group within each sequence, resulting in approximately equal numbers of high-risk and normal-risk individuals between the sequences. Blinding (masking) In this study, subjects will be blinded to the study condition and randomization sequence. Relevant study staff and the device manufacturer will be aware of the assigned sequence. The subject will wear the Ava bracelet and complete the Daily Symptom Diary in the Ava COVID-RED app for the full duration of the study, and they will not know if the feedback they receive about their potential infection status will only be based on the data they entered in the Daily Symptom Diary within the Ava COVID-RED app or based on both the data from the Daily Symptom Diary and the Ava bracelet. Numbers to be randomized (sample size) A total of 20,000 subjects will be recruited and randomized 1:1 to either sequence 1 (experimental condition followed by control condition) or sequence 2 (control condition followed by experimental condition), taking into account their risk level. This results in approximately 6500 normal-risk and 3500 high-risk individuals per sequence. Trial status Protocol version: 3.0, dated May 3, 2021. Start of recruitment: February 19, 2021. End of recruitment: June 3, 2021. End of follow-up (estimated): November 2021 Trial registration The Netherlands Trial Register on the 18th of February, 2021 with number NL9320 ( https://www.trialregister.nl/trial/9320 ) Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol.

Abstract Background Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. Methods Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. Results Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. Conclusions We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

IntroductionDuring the COVID-19 pandemic, certain disease modifying therapies (DMTs) used in multiple sclerosis (MS), such as anti-CD20 therapies, have been associated with decreased humoral responses after SARS-CoV-2 vaccination. Hybrid immunity, referring to immunity after both vaccination and SARS-CoV-2 infection might increase humoral responses.MethodsThis was a substudy of two prospective cohort studies on SARS-CoV-2 antibodies after SARS-CoV-2 infection and vaccination. RBD-specific IgG titers of patients with MS and healthy controls who had experienced SARS-CoV-2 infection prior to the first vaccination were compared with those patients and healthy controls without prior infection. Humoral responses were measured at various time points after SARS-CoV-2 infection in convalescent patients and all patients prior to the first vaccination, 28 days after the first vaccination, and 28 days after the second vaccination.ResultsOne hundred and two individuals [of which 34 patients with MS and DMTs (natalizumab or ocrelizumab), 30 patients without DMTs, and 38 healthy controls] were included. Fifty one of these individuals were convalescent. Median SARS-CoV-2 antibody titers were higher after the first vaccination in convalescent individuals compared with individuals without infection prior to vaccination. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers were comparable after the second vaccination in patients with MS with and without prior infection. However, in the convalescent ocrelizumab-treated patients, SARS-CoV-2 antibody titers did not increase after vaccinations.ConclusionIn patients with MS without anti-CD20 therapies, SARS-CoV-2 infection before vaccination increases humoral responses after the first vaccination, similar to the healthy controls. In patients with MS treated with ocrelizumab (convalescent and non-convalescent), humoral responses remained low.

BackgroundAs SARS-CoV-2 will likely continue to circulate, low-impact methods become more relevant to monitor antibody-mediated immunity. Saliva sampling could provide a non-invasive method with reduced impact on children. Studies reporting on the differences between systemic and mucosal humoral immunity to SARS-CoV-2 are inconsistent in adults and scarce in children. These differences may be further unraveled by exploring associations to demographic and clinical variables.MethodsTo evaluate the use of saliva antibody assays, we performed a cross-sectional cohort study by collecting serum and saliva of 223 children attending medical services in the Netherlands (irrespective of SARS-CoV-2 exposure, symptoms or vaccination) from May to October 2021. With a Luminex and a Wantai assay, we measured prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD) and nucleocapsid-specific IgG and IgA in serum and saliva and explored associations with demographic variables.FindingsThe S-specific IgG prevalence was higher in serum 39% (95% CI 32 – 45%) than in saliva 30% (95% CI 24 – 36%) (P ≤ 0.003). Twenty-seven percent (55/205) of children were S-specific IgG positive in serum and saliva, 12% (25/205) were only positive in serum and 3% (6/205) only in saliva. Vaccinated children showed a higher concordance between serum and saliva than infected children. Odds for saliva S-specific IgG positivity were higher in girls compared to boys (aOR 2.63, P = 0.012). Moreover, immunocompromised children showed lower odds for S- and RBD-specific IgG in both serum and saliva compared to healthy children (aOR 0.23 – 0.25, P ≤ 0.050).ConclusionsWe showed that saliva-based antibody assays can be useful for identifying SARS-CoV-2 humoral immunity in a non-invasive manner, and that IgG prevalence may be affected by sex and immunocompromisation. Differences between infection and vaccination, between sexes and between immunocompromised and healthy children should be further investigated and considered when choosing systemic or mucosal antibody measurement.

Abstract Background A substantial proportion of rheumatoid arthritis (RA) patients discontinues treatment with tumour necrosis factor inhibitors (TNFi) due to inefficacy or intolerance. After the failure of treatment with a TNFi, treatment can be switched to another TNFi or a bDMARD with a different mode of action (non-TNFi). Measurement of serum drug concentrations and/or anti-drug antibodies (therapeutic drug monitoring (TDM)) may help to inform the choice for the next step. However, the clinical utility of TDM to guide switching has not been investigated in a randomised test-treatment study. Methods ADDORA-switch is a 24-week, multi-centre, triple-blinded, superiority test-treatment randomised controlled trial. A total of 84 RA patients failing adalimumab treatment (treatment failure defined as DAS28-CRP > 2.9) will be randomised in a 1:1 ratio to a switching strategy to either TNFi or non-TNFi based on adalimumab serum trough level (intervention group) or random allocation (control group). The primary outcome is the between-group difference in mean time-weighted DAS28 over 24 weeks. Discussion The trial design differs in many aspects from previously published and ongoing TDM studies and is considered the first blinded test-treatment trial using TDM in RA. Several choices in the design of this trial are described, and overarching principles regarding test-treatment trials and clinical utility of TDM are discussed in further detail. Trial registration Dutch Trial Register NL8210 . Registered on 3 December 2019 (CMO NL69841.091.19).

Summary: Background: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. Methods: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). Findings: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p

Background: Patients affected by different types of autoimmune diseases, including common conditions such as multiple sclerosis (MS) and rheumatoid arthritis (RA), are often treated with immunosuppressants to suppress disease activity. It is not fully understood how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and cellular immunity induced by infection and/or upon vaccination is affected by immunosuppressants. Methods: The dynamics of cellular immune reactivation upon vaccination of SARS-CoV-2 experienced MS patients treated with the humanized anti-CD20 monoclonal antibody ocrelizumab (OCR) and RA patients treated with methotrexate (MTX) monotherapy were analyzed at great depth via high-dimensional flow cytometry of whole blood samples upon vaccination with the SARS-CoV-2 mRNA-1273 (Moderna) vaccine. Longitudinal B and T cell immune responses were compared to SARS-CoV-2 experienced healthy controls (HCs) before and 7 days after the first and second vaccination. Results: OCR-treated MS patients exhibit a preserved recall response of CD8+ T central memory cells following first vaccination compared to HCs and a similar CD4+ circulating T follicular helper 1 and T helper 1 dynamics, whereas humoral and B cell responses were strongly impaired resulting in absence of SARS-CoV-2-specific humoral immunity. MTX treatment significantly delayed antibody levels and B reactivation following the first vaccination, including sustained inhibition of overall reactivation marker dynamics of the responding CD4+ and CD8+ T cells. Conclusions: Together, these findings indicate that SARS-CoV-2 experienced MS-OCR patients may still benefit from vaccination by inducing a broad CD8+ T cell response which has been associated with milder disease outcome. The delayed vaccine-induced IgG kinetics in RA-MTX patients indicate an increased risk after the first vaccination, which might require additional shielding or alternative strategies such as treatment interruptions in vulnerable patients. Funding: This research project was supported by ZonMw (The Netherlands Organization for Health Research and Development, #10430072010007), the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#792532 and #860003), the European Commission (SUPPORT-E, #101015756) and by PPOC (#20_21 L2506), the NHMRC Leadership Investigator Grant (#1173871).

Background/MethodsFor mechanistic studies, in-vitro human B-cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T-cell-dependent (TD) and T-cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols make the interpretation of results challenging. The aim of the present study was to achieve the most optimal B-cell differentiation conditions using isolated CD19+ B cells and peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were analyzed after 6 and 9 days by measuring B-cell proliferation and expansion, plasmablast and plasma cell formation, and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared.ResultsThis study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2,500 and 25,000 B–cells per culture well for the TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B-cell cultures, which allows dismissal of additional B-cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B-cell differentiation and Ig secretion in B-cell but not in PBMC cultures. With this approach, efficient B-cell differentiation and Ig secretion were accomplished when starting from fresh or cryopreserved samples.ConclusionOur methodology demonstrates optimized TD and TI stimulation protocols for more in-depth analysis of B-cell differentiation in primary human B-cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B-cell differentiation of patient samples from different cohorts of B-cell-mediated diseases.

Monoclonal antibodies (mAbs), such as infliximab, are important treatment options for different diseases. Immunogenicity is a major risk, resulting in anti-drug antibodies (ADAs), being associated with adverse events and loss of response, influencing long-term outcomes. The development of ADAs against infliximab is primarily measured by immunoassays like radioimmunoassay (RIA). Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized across different fields, this technique is currently not used for ADAs against infliximab measurements. Therefore, we developed the first LC-MS/MS method. Stable isotopically labeled infliximab antigen-binding fragments (SIL IFX F(ab’)2) were used to bind and measure ADAs indirectly. Protein A magnetic beads were used to capture IgG, including ADAs, whereafter SIL IFX F(ab’)2 was added for labeling. After washing, internal standard addition, elution, denaturation and digestion samples were measured by LC-MS/MS. Internal validation showed good linearity between 0.1 and 16 mg/L (R2 > 0.998). Sixty samples were used for cross-validation with RIA, and no significant difference between ADA concentrations was found. The methods had high correlation (R = 0.94, p < 0.001) and excellent agreement, intraclass correlation coefficient = 0.912 (95% confidence interval 0.858–0.947, p < 0.001). We present the first ADA against the infliximab LC-MS/MS method. The method is amendable for quantifying other ADAs, making it applicable as a template for future ADA methods.

There are limited data on therapeutic drug monitoring (TDM) in inflammatory bowel disease (IBD) patients treated with vedolizumab (VDZ). Although an exposure–response relation has been demonstrated in the post-induction phase, this relationship is more uncertain in the maintenance phase of treatment. The aim of our study was to determine whether there is an association between VDZ trough concentration and clinical and biochemical remission in the maintenance phase. A prospective, observational multicenter study has been performed on patients with IBD on VDZ in the maintenance treatment (≥14 weeks). Patient demographics, biomarkers, and VDZ serum trough concentrations were collected. Clinical disease activity was scored by the Harvey Bradshaw Index (HBI) for Crohn’s disease (CD) and the Simple Clinical Colitis Activity Index (SCCAI) for ulcerative colitis (UC). Clinical remission was determined as HBI < 5 and SCCAI < 3. Biochemical remission was defined as fecal calprotectin p = 0.019). In this population, higher trough VDZ concentrations were associated with biochemical remission but not with clinical remission.

Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct.

Variation in response to biologic therapy for inflammatory diseases, such as psoriasis, is partly driven by variation in drug exposure. Real‐world psoriasis data were used to develop a pharmacokinetic/pharmacodynamic (PK/PD) model for the first‐line therapeutic antibody ustekinumab. The impact of differing dosing strategies on response was explored. Data were collected from a UK prospective multicenter observational cohort (491 patients on ustekinumab monotherapy, drug levels, and anti‐drug antibody measurements on 797 serum samples, 1,590 measurements of Psoriasis Area Severity Index (PASI)). Ustekinumab PKs were described with a linear one‐compartment model. A maximum effect (Emax) model inhibited progression of psoriatic skin lesions in the turnover PD mechanism describing PASI evolution while on treatment. A mixture model on half‐maximal effective concentration identified a potential nonresponder group, with simulations suggesting that, in future, the model could be incorporated into a Bayesian therapeutic drug monitoring “dashboard” to individualize dosing and improve treatment outcomes.

Many studies already reported on the association between patient characteristics on the severity of COVID-19 disease outcome, but the relation with SARS-CoV-2 antibody levels is less clear. To investigate this in more detail, we performed a retrospective observational study in which we used the IgG antibody response from 11,118 longitudinal antibody measurements of 2,082 unique COVID convalescent plasma donors. COVID-19 symptoms and donor characteristics were obtained by a questionnaire. Antibody responses were modelled using a linear mixed-effects model. Our study confirms that the SARS-CoV-2 antibody response is associated with patient characteristics like body mass index and age. Antibody decay was faster in male than in female donors (average half-life of 62 versus 72 days). Most interestingly, we also found that three symptoms (headache, anosmia, nasal cold) were associated with lower peak IgG, while six other symptoms (dry cough, fatigue, diarrhoea, fever, dyspnoea, muscle weakness) were associated with higher IgG concentrations.

Abstract Objectives High‐magnitude CD8+ T cell responses are associated with mild COVID‐19 disease; however, the underlying characteristics that define CD8+ T cell‐mediated protection are not well understood. The antigenic breadth and the immunodominance hierarchies of epitope‐specific CD8+ T cells remain largely unexplored and are essential for the development of next‐generation broad‐protective vaccines. This study identified a broad spectrum of conserved SARS‐CoV‐2 CD8+ T cell epitopes and defined their respective immunodominance and phenotypic profiles following SARS‐CoV‐2 infection. Methods CD8+ T cells from 51 convalescent COVID‐19 donors were analysed for their ability to recognise 133 predicted and previously described SARS‐CoV‐2‐derived peptides restricted by 11 common HLA class I allotypes using heterotetramer combinatorial coding, which combined with phenotypic markers allowed in‐depth ex vivo profiling of CD8+ T cell responses at quantitative and phenotypic levels. Results A comprehensive panel of 49 mostly conserved SARS‐CoV‐2‐specific CD8+ T cell epitopes, including five newly identified low‐magnitude epitopes, was established. We confirmed the immunodominance of HLA‐A*01:01/ORF1ab1637–1646 and B*07:02/N105–113 and identified B*35:01/N325–333 as a third epitope with immunodominant features. The magnitude of subdominant epitope responses, including A*03:01/N361–369 and A*02:01/S269–277, depended on the donors' HLA‐I context. All epitopes expressed prevalent memory phenotypes, with the highest memory frequencies in severe COVID‐19 donors. Conclusion SARS‐CoV‐2 infection induces a predominant CD8+ T memory response directed against a broad spectrum of conserved SARS‐CoV‐2 epitopes, which likely contributes to long‐term protection against severe disease. The observed immunodominance hierarchy emphasises the importance of T cell epitopes derived from nonspike proteins to the overall protective and cross‐reactive immune response, which could aid future vaccine strategies.

Background: Plasma concentration of natalizumab falls above the therapeutic threshold in many patients who, therefore, receive more natalizumab than necessary and have higher risk of progressive multifocal leukoencephalopathy.Objective: To assess in a single study the individual and treatment characteristics that influence the pharmacokinetics and pharmacodynamics of natalizumab in multiple sclerosis (MS) patients in the real-world practice.Methods: Prospective observational study to analyse the impact of body weight, height, body surface area, body mass index, gender, age, treatment duration, and dosage scheme on natalizumab concentrations and the occupancy of α4-integrin receptor (RO) by natalizumab.Results: Natalizumab concentrations ranged from 0.72 to 67 μg/ml, and RO from 26 to 100%. Body mass index inversely associated with natalizumab concentration (beta = −1.78; p ≤ 0.001), as it did body weight (beta = −0.34; p = 0.001), but not height, body surface area, age or gender Extended vs. standard dose scheme, but not treatment duration, was inversely associated with natalizumab concentration (beta = −7.92; p = 0.016). Similar to natalizumab concentration, body mass index (beta = −1.39; p = 0.001) and weight (beta = −0.31; p = 0.001) inversely impacted RO. Finally, there was a strong direct linear correlation between serum concentrations and RO until 9 μg/ml (rho = 0.71; p = 0.003). Nevertheless, most patients had higher concentrations of natalizumab resulting in the saturation of the integrin.Conclusions: Body mass index and dosing interval are the main variables found to influence the pharmacology of natalizumab. Plasma concentration of natalizumab and/or RO are wide variable among patients and should be routinely measured to personalize treatment and, therefore, avoid either over and underdosing.

ABSTRACT COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.

Abstract Background The aim of our study was to investigate the influence of conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and body mass index (BMI) on circulating drug levels and clinical response to tumour necrosis factor inhibitor (TNFi) therapy in axial spondyloarthritis (axSpA) patients. Methods Prospective observational study during 1 year with 2 cohorts (Madrid and Amsterdam) including 180 axSpA patients treated with standard doses of infliximab or adalimumab. Patients were stratified by BMI, being 78 (43%) normal weight (18.5–24.9 kg/m2) and 102 (57%) overweight/obese (≥ 25.0 kg/m2). After the first year of treatment, TNFi trough levels were measured by capture ELISA. Clinical response to TNFi was defined as ∆BASDAI ≥ 2 and clinical remission as BASDAI

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.

Abstract Objectives Characterisation of the human antibody response to SARS‐CoV‐2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT‐PCR‐positive SARS‐CoV‐2 convalescent adults during the first 250 days after onset of symptoms. Methods We measured antibody responses to the receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT‐PCR‐positive SARS‐CoV‐2 convalescent adults. With a median of 5 (range 2–18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed‐effects modelling. Results All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow‐up analysis. Anti‐RBD IgG and anti‐nucleocapsid IgG levels declined with median half‐lives of 62 and 59 days, respectively, 2–5 months after symptom onset, and several‐fold variation in half‐lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow‐up, which points towards long‐term immunological memory. The magnitude of the anti‐RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in‐house developed competitive assay. Conclusion The result of this study gives valuable insight into the long‐term longitudinal response of antibodies to SARS‐CoV‐2.

Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the N-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead CH2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants.

Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.

Objectives Anti-drug antibodies (ADA) are responsible for decreased adalimumab efficacy in axial spondyloarthritis (SpA). We aimed to evaluate the ability of methotrexate (MTX) to decrease adalimumab immunisation.Methods A total of 110 patients eligible to receive adalimumab 40 mg subcutaneously (s.c.) every other week were randomised (1:1 ratio) to receive, 2 weeks before adalimumab (W-2) and weekly, MTX 10 mg s.c. (MTX+) or not (MTX−). ADA detection and adalimumab serum concentration were assessed at weeks 4 (W4), 8 (W8), 12 (W12) and 26 (W26) after starting adalimumab (W0). The primary outcome was the proportion of patients with ADA at W26. Four years after the study completion, we retrospectively analysed adalimumab maintenance in relation with MTX co-treatment duration.Results We analysed data for 107 patients (MTX+; n=52; MTX-; n=55). ADA were detected at W26 in 39/107 (36.4%) patients: 13/52 (25%) in the MTX+ group and 26/55 (47.3%) in the MTX− group (p=0.03). Adalimumab concentration was significantly higher in the MTX+ than MTX− group at W4, W8, W12 and W26. The two groups did not differ in adverse events or efficacy. In the follow-up study, MTX co-treatment >W26 versus no MTX or ≤W26 was significantly associated with adalimumab long-term maintenance (p=0.04).Conclusion MTX reduces the immunogenicity and ameliorate the pharmacokinetics of adalimumab in axial SpA. A prolonged co-treatment of MTX>W26 seems to increase adalimumab long-term maintenance.

Tumor necrosis factor inhibitors (TNFi) have significantly improved treatment outcome of rheumatic diseases since their incorporation into treatment protocols two decades ago. Nevertheless, a substantial fraction of patients experiences either primary or secondary failure to TNFi due to ineffectiveness of the drug or adverse reactions. Secondary failure and adverse events can be related to the development of anti-drug antibodies (ADA). The earliest studies that reported ADA toward TNFi mainly used drug-sensitive assays. Retrospectively, we recognize this has led to an underestimation of the amount of ADA produced due to drug interference. Drug-tolerant ADA assays also detect ADA in the presence of drug, which has contributed to the currently reported higher incidence of ADA development. Comprehension and awareness of the assay format used for ADA detection is thus essential to interpret ADA measurements correctly. In addition, a concurrent drug level measurement is informative as it may provide insight in the extent of underestimation of ADA levels and improves understanding the clinical consequences of ADA formation. The clinical effects are dependent on the ratio between the amount of drug that is neutralized by ADA and the amount of unbound drug. Pharmacokinetic modeling might be useful in this context. The ADA response generally gives rise to high affinity IgG antibodies, but this response will differ between patients. Some patients will not reach the phase of affinity maturation while others generate an enduring high titer high affinity IgG response. This response can be transient in some patients, indicating a mechanism of tolerance induction or B-cell anergy. In this review several different aspects of the ADA response toward TNFi will be discussed. It will highlight the ADA assays, characteristics and regulation of the ADA response, impact of immunogenicity on the pharmacokinetics of TNFi, clinical implications of ADA formation, and possible mitigation strategies.

Antibody formation to human(ized) therapeutic antibodies in humans is highly skewed toward anti-idiotype responses, probably because the idiotype is the only ‘foreign’ part of the antibody molecule. Here, we analyzed antibody responses to F(ab’)2 fragments of a panel of 17 human(ized) therapeutic antibodies in rabbits. Homology between the rabbit germline and the human(ized) antibodies is moderate not only for the variable domains (both the complementarity-determining regions and the framework regions), but also for the constant domains (66% or less). Nevertheless, we observed a highly skewed anti-idiotype response in all cases, with up to >90% of the antibodies directed toward the idiotype. These results indicate that the idiotype may be inherently immunodominant. We used these biased responses to raise monoclonal rabbit anti-idiotype antibodies against secukinumab, ustekinumab, reslizumab, mepolizumab, palivizumab, and dupilumab and demonstrate the potential to develop sensitive pharmacokinetic assays with these antibodies.