Deep profiling of antigen-specific B cells from different pathogens identifies novel compartments in the IgG memory B cell and antibody-secreting cell lineages (preprint)

A better understanding of the bifurcation of human B cell differentiation into memory B cells (MBC) and antibody-secreting cells (ASC) and identification of MBC and ASC precursors is crucial to optimize vaccination strategies or block undesired antibody responses. To unravel the dynamics of antigen-induced B cell responses, we compared circulating B cells reactive to SARS-CoV-2 (Spike, RBD and Nucleocapsid) in COVID-19 convalescent individuals to B cells specific to Influenza-HA, RSV-F and TT, induced much longer ago. High-dimensional spectral flow cytometry indicated that the decision point between ASC- and MBC-formation lies in the CD43+CD71+IgG+ Activated B cell compartment, showing properties indicative of recent germinal center activity and recent antigen encounter. Within this Activated B cells compartment, CD86+ B cells exhibited close phenotypical similarity with ASC, while CD86- B cells were closely related to IgG+ MBCs. Additionally, different activation stages of the IgG+ MBC compartment could be further elucidated. The expression of CD73 and CD24, regulators of survival and cellular metabolic quiescence, discerned activated MBCs from resting MBCs. Activated MBCs (CD73-CD24lo) exhibited phenotypical similarities with CD86- IgG+ Activated B cells and were restricted to SARS-CoV-2 specificities, contrasting with the resting MBC compartment (CD73-/CD24hi) that exclusively encompassed antigen-specific B cells established long ago. Overall, these findings identify novel stages for IgG+ MBC and ASC formation and bring us closer in defining the decision point for MBC or ASC differentiation. ImportanceIn this study, researchers aimed to better understand human B cell differentiation and their role in establishing long-lived humoral immunity. Using high-dimensional flow cytometry, they studied B cells reactive to three SARS-CoV-2 antigens in individuals convalescent for COVID-19, and compared their phenotypes to B cells reactive to three distinct protein antigens derived from vaccines or viruses encountered months to decades before. Their findings showed that Activated B cells reflect recent germinal center graduates that may have diverse fates; with some feeding the pool of antibody-secreting cells and others fueling the resting memory B cell compartment. Activated B cells gradually differentiate into resting memory B cells through an activated MBC phase. Increased expression of the cellular metabolic regulators CD73 and CD24 in resting memory B cells distinguishes them from the activated memory B cells phase, and is likely involved in sustaining a durable memory of humoral immunity. These findings are crucial for the development of vaccines that provide lifelong protection and may show potential to define reactive B cells in diseases where the cognate-antigen is still unknown such as in autoimmunity, cancers, or novel viral outbreaks.

Suppressed IgG4 class switching in dupilumab- and TNF inhibitor-treated patients after repeated SARS-CoV-2 mRNA vaccination (preprint)

Background Repeated mRNA vaccination against SARS-CoV-2 has been shown to induce class switching to IgG4, a non-inflammatory human antibody subclass linked to tolerance. Although poorly understood, prolonged antigenic stimulation and IL-4 signalling may be instrumental in IgG4 switching. We and others have previously shown that widely used immunosuppressive drugs such as methotrexate (MTX) and TNF inhibitors (TNFi) have a minor inhibitory impact on humoral SARS-CoV-2 mRNA vaccination responses. However, the impact of such immunosuppressive drugs on IgG4 switching is unknown. Aim To study the impact of widely used immunosuppressive drugs (TNFi, MTX, or the IL-4 receptor-blocking antibody dupilumab on IgG4 skewing upon repeated SARS-CoV-2 mRNA vaccination. Methods Antibody responses to the receptor-binding domain (RBD) of the spike protein upon repeated SARS-CoV-2 mRNA vaccination were measured in 604 individuals including patients with immune-mediated inflammatory diseases treated with TNFi and/or MTX, or dupilumab, as well as healthy controls and untreated patients. Results We observed a substantial increase in the proportion of RBD-specific IgG4 antibodies (median 21%) in healthy/untreated controls after a third mRNA vaccination. This IgG4 skewing was absent when primary vaccination was adenoviral vector-based and was profoundly reduced in both dupilumab- and TNFi-treated patients (<1%), but only moderately in patients treated with MTX (7%). Conclusion Our results imply a major role for both IL-4/IL-13 as well as TNF in IgG4 class switching. These novel findings advance our understanding of IgG4 class switch dynamics, and may benefit future mRNA vaccine strategies, humoral tolerance induction, as well as treatment of IgG4 pathologies.

Primary SARS-CoV-2 infection in patients with immune-mediated inflammatory diseases: long-term humoral immune responses and effects on disease activity (preprint)

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<0.001). Seropositivity rates were lowest in patients on anti-CD20 (40.0%) and anti-tumor necrosis factor (TNF) agents (60.5%), as compared to other ISPs (p<0.001 and p<0.001, respectively). Increased disease activity after infection was reported by 68 of 260 patients (26.2%; 95% CI 21.2–31.8%), leading to ISP intensification in 6 out of these 68 patients (8.8%). Conclusion: IMID patients using ISPs showed reduced long-term humoral immune responses after primary SARS-CoV-2 infection, which was mainly attributed to treatment with anti-CD20 and anti-TNF agents. Increased disease activity after SARS-CoV-2 infection was reported commonly, but was mostly mild. Trial registration NL74974.018.20, Trial ID: NL8900. Registered on 9 September 2020.

Antibody Development and Disease Severity of Covid-19 in Non-Immunized Patients with Rheumatic Immune-Mediated Inflammatory Diseases: Data from a Prospective Cohort Study (preprint)

Background: Research on the disease severity of COVID-19 in patients with rheumatic immune-mediated inflammatory diseases (IMIDs) has been inconclusive, and long-term prospective data on the development of SARS-CoV-2 antibodies in these patients are lacking. We conducted an investigator-driven prospective cohort study to compare the disease severity of COVID-19, and the development of SARS-CoV-2 antibodies over time between patients with rheumatic IMIDs and healthy controls.Methods: Adult patients with rheumatic IMIDs from the Amsterdam Rheumatology & immunology Center, Amsterdam were invited to participate. All patients were asked to recruit their own sex- and age-matched control subject. We developed a new platform for collecting clinical data in large patient groups with online questionnaires (at baseline, and after 1-4 and 5-9 months of follow-up). Serum samples were collected two times during follow-up; after completing the follow-up questionnaires and prior to COVID-19 vaccination. All serum samples were analyzed for the presence of SARS-CoV-2 specific antibodies with a total-antibody bridging ELISA. IgG titers were quantified in samples with a positive test result in the bridging assay. Logistic regression analyses, and linear and logistic mixed model analyses were used to compare COVID-19 related hospitalization rates, proportions of SARS-CoV-2 seropositivity and IgG antibody titers between patients and controls, and between patients stratified for major immunosuppressive drug categories (i.e. biological [b] or conventional synthetic [cs] disease modifying anti-rheumatic drugs [DMARDs]).Findings: In total, 3080 consecutive patients and 1102 healthy controls with comparable age and sex distribution were included for analyses. The incidence of COVID-19 was slightly lower in patients compared to controls (14.7% vs. 16.0% had detectable SARS-CoV-2 antibodies), but patients were more frequently hospitalized compared to controls; 23 of 347 (7%) patients vs. 1 of 134 (0.7%) controls (adjusted OR: 7.33, 95% CI: 0.96 – 55.77, P 0.055). Three (13%) of 23 patients were admitted to the intensive care unit (ICU), and one of these died. Only treatment with B-cell targeting therapy was independently associated with an increased risk of COVID-19 related hospitalization (adjusted OR: 14.62, 95% CI: 2.31 – 92.39, P 0.004). Proportions of SARS-CoV-2 seropositivity in participants with a PCR confirmed COVID-19 diagnosis were similar for patients and controls ( P 0.73), and did not significantly decrease during the first twelve months after infection ( P 0.10). IgG antibody titers were higher in hospitalized patients compared to non-hospitalized patients, and slowly declined with time (rate per month: 0.86, 95% CI: 0.81 – 0.91, P < 0.0001) in similar patterns for patients in all treatment subgroups and controls.Interpretation: We observed that patients with rheumatic IMIDs, especially those treated with B-cell targeting therapy, were more likely to be hospitalized when infected with SARS-CoV-2, although subsequent ICU admissions and/or death were infrequent. In addition, treatment with cs- or bDMARDs other than B-cell targeting agents is unlikely to have negative effects on the development of long-lasting humoral immunity against SARS-CoV-2.Funding Information: ZonMw and Reade Foundation.Declaration of Interests: None to declare. Ethics Approval Statement: The research protocol was approved by the medical ethical committee of the VU University medical center (registration number 2020.169). All participants gave written informed consent.

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors (preprint)

The novel SARS-CoV-2 virus emerged in late 2019 and has caused a global health and economic crisis. The characterization of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes as well for treatment options such as transfusion with convalescent plasma or immunoglobin products derived from convalescent plasma. In this study, we measured antibody responses in 844 longitudinal samples from 151 RT-PCR positive SARS-CoV-2 convalescent adults during the first 34 weeks after onset of symptoms. 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 slowly declined with median half-lifes of 62 and 59 days during 2-5 months after symptom onset, respectively. The rate of decline of antibody levels diminished during extended follow-up. In addition, the magnitude of the IgG response correlated with neutralization capacity measured in a classic plaque reduction assay as well in our in-house developed competition assay. The result of this study gives valuable insight into the longitudinal response of antibodies to SARS-CoV-2.

Complex Systems Analysis Informs on the Spread of COVID-19 (preprint)

The non-linear progression of new infection numbers in a pandemic poses challenges to the evaluation of its management. The tools of complex systems research may aid in attaining information that would be difficult to extract with other means. To study the COVID-19 pandemic, we utilize the reported new cases per day for the globe, nine countries and six US states through October 2020. Fourier and univariate wavelet analyses inform on periodicity and extent of change. Evaluating time-lagged data sets of various lag lengths, we find that the autocorrelation function, average mutual information and box counting dimension represent good quantitative readouts for the progression of new infections. Bivariate wavelet analysis and return plots give indications of containment versus exacerbation. Homogeneity or heterogeneity in the population response, uptick versus suppression, and worsening or improving trends are discernible, in part by plotting various time lags in three dimensions. The analysis of epidemic or pandemic progression with the techniques available for observed (noisy) complex data can aid decision making in the public health response.