Regulatory B Cells-Immunopathological and Prognostic Potential in Humans.

The aim of the following review is to shed light on the putative role of regulatory B cells (Bregs) in various human diseases and highlight their potential prognostic and therapeutic relevance in humans. Regulatory B cells are a heterogeneous group of B lymphocytes capable of suppressing inflammatory immune reactions. In this way, Bregs contribute to the maintenance of tolerance and immune homeostasis by limiting ongoing immune reactions temporally and spatially. Bregs play an important role in attenuating pathological inflammatory reactions that can be associated with transplant rejection, graft-versus-host disease, autoimmune diseases and allergies but also with infectious, neoplastic and metabolic diseases. Early studies of Bregs identified IL-10 as an important functional molecule, so the IL-10-secreting murine B10 cell is still considered a prototype Breg, and IL-10 has long been central to the search for human Breg equivalents. However, over the past two decades, other molecules that may contribute to the immunosuppressive function of Bregs have been discovered, some of which are only present in human Bregs. This expanded arsenal includes several anti-inflammatory cytokines, such as IL-35 and TGF-ß, but also enzymes such as CD39/CD73, granzyme B and IDO as well as cell surface proteins including PD-L1, CD1d and CD25. In summary, the present review illustrates in a concise and comprehensive manner that although human Bregs share common functional immunosuppressive features leading to a prominent role in various human immunpathologies, they are composed of a pool of different B cell types with rather heterogeneous phenotypic and transcriptional properties.

Real-world data suggest effectiveness of the allogeneic mesenchymal stromal cells preparation MSC-FFM in ruxolitinib-refractory acute graft-versus-host disease.

BACKGROUND: Patients with steroid-refractory acute graft-versus-host disease (aGvHD) not tolerating/responding to ruxolitinib (RR-aGvHD) have a dismal prognosis. METHODS: We retrospectively assessed real-world outcomes of RR-aGvHD treated with the random-donor allogeneic MSC preparation MSC-FFM, available via Hospital Exemption in Germany. MSC-FFM is provided as frozen cell dispersion for administration as i.v. infusion immediately after thawing, at a recommended dose of 1-2 million MSCs/kg body weight in 4 once-weekly doses. 156 patients, 33 thereof children, received MSC-FFM; 5% had Grade II, 40% had Grade III, and 54% had Grade IV aGvHD. Median (range) number of prior therapies was 4 (1-10) in adults and 7 (2-11) in children. RESULTS: The safety profile of MSC-FFM was consistent with previous reports for MSC therapies in general and MSC-FFM specifically. The overall response rate at Day 28 was 46% (95% confidence interval [CI] 36-55%) in adults and 64% (45-80%) in children; most responses were durable. Probability of overall survival at 6, 12 and 24 months was 47% (38-56%), 35% (27-44%) and 30% (22-39%) for adults, and 59% (40-74%), 42% (24-58%) and 35% (19-53%) for children, respectively (whole cohort: median OS 5.8 months). CONCLUSION: A recent real-world analysis of outcomes for 64 adult RR-aGvHD patients not treated with MSCs reports survival of 20%, 16% and 10% beyond 6, 12 and 24 months, respectively (median 28 days). Our data thus suggest effectiveness of MSC-FFM in RR-aGvHD.

Phorbol-12-myristate-13-acetate is a potent enhancer of B cells with a granzyme B+ regulatory phenotype.

Introduction: The infusion of ex-vivo-generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including graft-versus-host disease. Methods: Previously, we developed a protocol for the generation of a novel population of regulatory B cells, which are characterized by secretion of enzymatically active granzyme B (GraB cells). This protocol uses recombinant interleukin 21 (IL-21) and goat-derived F(ab)'2 fragments against the human B cell receptor (anti-BCR). Generally, the use of xenogeneic material for the manufacturing of advanced therapy medicinal products should be avoided to prevent adverse immune reactions as well as potential transmission of so far unknown diseases. Results: In the present work we demonstrated that phorbol-12-myristate-13-acetate (PMA/TPA), a phorbol ester with a particular analogy to the second messenger diacylglycerol (DAG), is a potent enhancer of IL-21-induced differentiation of pre-activated B cells into GraB cells. The percentage of GraB cells after stimulation of pre-activated B cells with IL-21 and PMA/TPA was not significantly lower compared to stimulation with IL-21 and anti-BCR. Discussion: Given that PMA/TPA has already undergone encouraging clinical testing in patients with certain haematological diseases, our results suggest that PMA/TPA may be a safe and feasible alternative for ex-vivo manufacturing of GraB cells.

SARS-CoV-2 vaccination of convalescents boosts neutralization capacity against Omicron subvariants BA.1, BA.2 and BA.5 and can be predicted by anti-S antibody concentrations in serological assays.

Background: Recent data on immune evasion of new SARS-CoV-2 variants raise concerns about the efficacy of antibody-based COVID-19 therapies. Therefore, in this study the in-vitro neutralization capacity against SARS-CoV-2 variant B.1 and the Omicron subvariants BA.1, BA.2 and BA.5 of sera from convalescent individuals with and without boost by vaccination was assessed. Methods and findings: The study included 313 serum samples from 155 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=25) and with SARS-CoV-2 vaccination (n=130). We measured anti-SARS-CoV-2 antibody concentrations by serological assays (anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S) and neutralizing titers against B.1, BA.1, BA.2 and BA.5 in a pseudovirus neutralization assay. Sera of the majority of unvaccinated convalescents did not effectively neutralize Omicron sublineages BA.1, BA.2 and BA.5 (51.7%, 24.1% and 51.7%, resp.). In contrast, 99.3% of the sera of superimmunized individuals (vaccinated convalescents) neutralized the Omicron subvariants BA.1 and BA.5 and 99.6% neutralized BA.2. Neutralizing titers against B.1, BA.1, BA.2 and BA.5 were significantly higher in vaccinated compared to unvaccinated convalescents (p<0.0001) with 52.7-, 210.7-, 141.3- and 105.4-fold higher geometric mean of 50% neutralizing titers (NT50) in vaccinated compared to unvaccinated convalescents. 91.4% of the superimmunized individuals showed neutralization of BA.1, 97.2% of BA.2 and 91.5% of BA.5 with a titer ≥ 640. The increase in neutralizing titers was already achieved by one vaccination dose. Neutralizing titers were highest in the first 3 months after the last immunization event. Concentrations of anti-S antibodies in the anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S assays predicted neutralization capacity against B.1 and Omicron subvariants BA.1, BA.2 and BA.5. Conclusions: These findings confirm substantial immune evasion of the Omicron sublineages, which can be overcome by vaccination of convalescents. This informs strategies for choosing of plasma donors in COVID-19 convalescent plasma programs that shall select specifically vaccinated convalescents with very high titers of anti-S antibodies.

Immune Plasma for the Treatment of COVID-19: Lessons Learned so far.

COVID-19 convalescent plasma (CCP) has been explored as one of the treatment options for COVID-19. Results of many cohort studies and clinical trials have been recently published. At first glance, the results of the CCP studies appear to be inconsistent. However, it became clear that CCP is not beneficial if CCP with low anti-SARS-CoV-2 antibody concentrations is used, if it is administered late in advanced disease stages, and to patients who already mounted an antibody response against SARS-CoV-2 at the time of CCP transfusion. On the other hand, CCP may prevent progression to severe COVID-19 when very high-titer CCP is given early in vulnerable patients. Immune escape of new variants is a challenge for passive immunotherapy. While new variants of concern developed resistance to most clinically used monoclonal antibodies very rapidly, immune plasma from individuals immunized by both a natural SARS-CoV-2 infection and SARS-CoV-2 vaccination retained neutralizing activity against variants. This review briefly summarizes the evidence on CCP treatment to date and identifies further research needs. Ongoing research on passive immunotherapy is not only relevant for improving care for vulnerable patients in the ongoing SARS-CoV-2 pandemic, but even more as a model for passive immunotherapy in case of future pandemics with a newly evolving pathogen. Compared to other drugs, which must be newly developed in a pandemic (e.g., monoclonal antibodies, antiviral drugs), convalescent plasma is rapidly available, inexpensive to produce, and can be adaptive to viral evolution by selection of contemporary convalescent donors.

Differentially induced immunity in buccal and nasal mucosae after vaccination for SARS-CoV-2: Prospects for mass scale immunity-screening in large populations.

Introduction: Humoral immunity after SARS-CoV-2 vaccination has been extensively investigated in blood. Aim of this study was to develop an ELISA method in order to determine the prevalence of IgG and IgA SARS-CoV-2 domain 1 spike-protein (S) specific antibodies (Abs) in buccal and nasal mucosal surfaces of vaccinees. Methods: To this end, we analyzed 69 individuals who received their first vaccine dose between February and July 2021. Vaccines administered were BNT162b2, mRNA-1273 or ChAdOx1-nCoV-19. Detection of IgG and IgA Abs was performed using commercial ELISA kits for both blood and swab samples after protocol modification for the latter. Results: Anti-spike IgG and IgA Abs in the buccal and/or nasal swabs were detectable in >81% of the study subjects after the second dose. The IgG measurements in buccal swabs appeared to correlate in a more consistent way with the respective measurements in blood with a correlation coefficient of r=0.74. It is of note that IgA Abs appeared to be significantly more prevalent in the nasal compared to the buccal mucosa. Optimal selection of the assay cut-off for the IgG antibody detection in buccal swabs conferred a sensitivity of 91.8% and a specificity of 100%. Last, individuals vaccinated with mRNA-based vaccines exhibited higher antibody levels in both blood and mucosal surfaces compared to those receiving ChAdOx1-nCoV-19 confirming previously reported results. Conclusion: In conclusion, our findings show a differential prevalence of anti-S Abs on mucosal surfaces after vaccination for SARS-CoV-2, while they also set the basis for potential future use of IgG antibody detection in buccal swabs for extended immunity screening in large populations.

High antibody levels and reduced cellular response in children up to one year after SARS-CoV-2 infection.

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation.

One-year follow-up of the CAPSID randomized trial for high-dose convalescent plasma in severe COVID-19 patients.

BACKGROUNDResults of many randomized trials on COVID-19 convalescent plasma (CCP) have been reported, but information on long-term outcome after CCP treatment is limited. The objectives of this extended observation of the randomized CAPSID trial are to assess long-term outcome and disease burden in patients initially treated with or without CCP.METHODSOf 105 randomized patients, 50 participated in the extended observation. Quality of life (QoL) was assessed by questionnaires and a structured interview. CCP donors (n = 113) with asymptomatic to moderate COVID-19 were included as a reference group.RESULTSThe median follow-up of patients was 396 days, and the estimated 1-year survival was 78.7% in the CCP group and 60.2% in the control (P = 0.08). The subgroup treated with a higher cumulative amount of neutralizing antibodies showed a better 1-year survival compared with the control group (91.5% versus 60.2%, P = 0.01). Medical events and QoL assessments showed a consistent trend for better results in the CCP group without reaching statistical significance. There was no difference in the increase in neutralizing antibodies after vaccination between the CCP and control groups.CONCLUSIONThe trial demonstrated a trend toward better outcome in the CCP group without reaching statistical significance. A predefined subgroup analysis showed a significantly better outcome (long-term survival, time to discharge from ICU, and time to hospital discharge) among those who received a higher amount of neutralizing antibodies compared with the control group. A substantial long-term disease burden remains after severe COVID-19.Trial registrationEudraCT 2020-001310-38 and ClinicalTrials.gov NCT04433910.FundingBundesministerium für Gesundheit (German Federal Ministry of Health).

Cytokine levels associated with favorable clinical outcome in the CAPSID randomized trial of convalescent plasma in patients with severe COVID-19.

Objectives: To determine the profile of cytokines in patients with severe COVID-19 who were enrolled in a trial of COVID-19 convalescent plasma (CCP). Methods: Patients were randomized to receive standard treatment and 3 CCP units or standard treatment alone (CAPSID trial, ClinicalTrials.gov NCT04433910). The primary outcome was a dichotomous composite outcome (survival and no longer severe COVID-19 on day 21). Time to clinical improvement was a key secondary endpoint. The concentrations of 27 cytokines were measured (baseline, day 7). We analyzed the change and the correlation between serum cytokine levels over time in different subgroups and the prediction of outcome in receiver operating characteristics (ROC) analyses and in multivariate models. Results: The majority of cytokines showed significant changes from baseline to day 7. Some were strongly correlated amongst each other (at baseline the cluster IL-1ß, IL-2, IL-6, IL-8, G-CSF, MIP-1α, the cluster PDGF-BB, RANTES or the cluster IL-4, IL-17, Eotaxin, bFGF, TNF-α). The correlation matrix substantially changed from baseline to day 7. The heatmaps of the absolute values of the correlation matrix indicated an association of CCP treatment and clinical outcome with the cytokine pattern. Low levels of IP-10, IFN-γ, MCP-1 and IL-1ß on day 0 were predictive of treatment success in a ROC analysis. In multivariate models, low levels of IL-1ß, IFN-γ and MCP-1 on day 0 were significantly associated with both treatment success and shorter time to clinical improvement. Low levels of IP-10, IL-1RA, IL-6, MCP-1 and IFN-γ on day 7 and high levels of IL-9, PDGF and RANTES on day 7 were predictive of treatment success in ROC analyses. Low levels of IP-10, MCP-1 and high levels of RANTES, on day 7 were associated with both treatment success and shorter time to clinical improvement in multivariate models. Conclusion: This analysis demonstrates a considerable dynamic of cytokines over time, which is influenced by both treatment and clinical course of COVID-19. Levels of IL-1ß and MCP-1 at baseline and MCP-1, IP-10 and RANTES on day 7 were associated with a favorable outcome across several endpoints. These cytokines should be included in future trials for further evaluation as predictive factors.

Persistent but atypical germinal center reaction among 3rd SARS-CoV-2 vaccination after rituximab exposure.

Background: Durable vaccine-mediated immunity relies on the generation of long-lived plasma cells and memory B cells (MBCs), differentiating upon germinal center (GC) reactions. SARS-CoV-2 mRNA vaccination induces a strong GC response in healthy volunteers (HC), but limited data is available about response longevity upon rituximab treatment. Methods: We evaluated humoral and cellular responses upon 3rd vaccination in seven patients with rheumatoid arthritis (RA) who initially mounted anti-spike SARS-CoV-2 IgG antibodies after primary 2x vaccination and got re-exposed to rituximab (RTX) 1-2 months after the second vaccination. Ten patients with RA on other therapies and ten HC represented the control groups. As control for known long-lived induced immunity, we analyzed humoral and cellular tetanus toxoid (TT) immune responses in steady-state. Results: After 3rd vaccination, 5/7 seroconverted RTX patients revealed lower anti-SARS-CoV-2 IgG levels but similar neutralizing capacity compared with HC. Antibody levels after 3rd vaccination correlated with values after 2nd vaccination. Despite significant reduction of circulating total and antigen-specific B cells in RTX re-exposed patients, we observed the induction of IgG+ MBCs upon 3rd vaccination. Notably, only RTX treated patients revealed a high amount of IgA+ MBCs before and IgA+ plasmablasts after 3rd vaccination. IgA+ B cells were not part of the steady state TT+ B cell pool. TNF-secretion and generation of effector memory CD4 spike-specific T cells were significantly boosted upon 3rd vaccination. Summary: On the basis of pre-existing affinity matured MBCs within primary immunisation, RTX re-exposed patients revealed a persistent but atypical GC immune response accompanied by boosted spike-specific memory CD4 T cells upon SARS-CoV-2 recall vaccination.

SARS-CoV2 mRNA Vaccine-Specific B-, T- and Humoral Responses in Adolescents After Kidney Transplantation.

Protection of adult kidney transplant recipients against SARS-CoV2 was shown to be strongly impaired owing to low reactogenicity of available vaccines. So far, data on vaccination outcomes in adolescents are scarce due to later vaccination approval for this age group. We therefore comprehensively analyzed vaccination-specific humoral-, T- and B-cell responses in kidney transplanted adolescents aged 12-18 years in comparison to healthy controls 6 weeks after standard two-dose BNT162b2 ("Comirnaty"; Pfizer/BioNTech) vaccination. Importantly, 90% (18/20) of transplanted adolescents showed IgG seroconversion with 75% (15/20) developing neutralizing titers. Still, both features were significantly diminished in magnitude compared to controls. Correspondingly, spike-specific B cells were quantitatively reduced and enriched for non-isotype-class-switched IgD+27+ memory cells in patients. Whereas spike specific CD4+ T cell frequencies were similar in both groups, cytokine production and memory differentiation were significantly impaired in transplant recipients. Although our data identify limitations in all arms of vaccine-specific immunity, the majority of our adolescent patients showed robust humoral responses despite antimetabolite-based treatment being associated with poor vaccination outcomes in adults.

BNT162b2 Booster Vaccination Elicits Cross-Reactive Immunity Against SARS-CoV-2 Variants B.1.1.529 and B.1.617.2 in Convalescents of All Ages.

In this prospective observational cohort study we analyzed cellular and serological immune response parameters against SARS-CoV-2 and current variants of concern (VOC) in 147 COVID-19-convalescent and 39 COVID-19-naïve individuals before and after BNT162b2 booster vaccination. No significant differences regarding immunological response parameters were observed between younger and older individuals. Booster vaccination induced full recovery of both cellular and serological response parameters including IFN-γ secretion and anti-spike antibody titers with strong neutralization capacities against wild type SARS-COV-2 and Delta. Surprisingly, even serological neutralization capacity against Omicron was detectable one month after second vaccination and four months before it had been first observed in South Africa. As a result, more than 90% of convalescent individuals exhibited detectable and 75% strong Omicron neutralization capacity after booster vaccination, compared with 72% and 46% of COVID-19-naïve individuals. Our results support the notion that broad and cross-reactive immune memory against SARS-CoV-2 including currently known VOCs can be established by booster vaccination with spike-based mRNA vaccines like BNT162b2, particularly in COVID-19-convalescent individuals of all ages. Nevertheless, especially in COVID-19-naïve individuals future variants escaping the memory immune response may require vaccine approaches such as inactivated whole virus vaccines, which include all antigenic components of the virus.

B Cell Characteristics at Baseline Predict Vaccination Response in RTX Treated Patients.

Background: Vaccination is considered as most efficient strategy in controlling SARS-CoV-2 pandemic spread. Nevertheless, patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) are at increased risk to fail humoral and cellular responses upon vaccination. The ability to predict vaccination responses is essential to guide adequate safety and optimal protection in these patients. Methods: B- and T- cell data before vaccination were evaluated for characteristics predicting vaccine responses in altogether 15 patients with autoimmune inflammatory rheumatic diseases receiving RTX. Eleven patients with rheumatoid arthritis (RA) on other therapies, 11 kidney transplant recipients (KTR) on regular immunosuppression and 15 healthy controls (HC) served as controls. A multidimensional analysis of B cell subsets via UMAP algorithm and a correlation matrix were performed in order to identify predictive markers of response in patients under RTX therapy. Results: Significant differences regarding absolute B cell counts and specific subset distribution pattern between the groups were identified at baseline. In this context, the majority of B cells from vaccination responders of the RTX group (RTX IgG+) were naïve and transitional B cells, whereas vaccination non-responders (RTX IgG-) carried preferentially plasmablasts and double negative (CD27-IgD-) B cells. Moreover, there was a positive correlation between neutralizing antibodies and B cells expressing HLA-DR and CXCR5 as well as an inverse correlation with CD95 expression and CD21low expression by B cells among vaccination responders. Summary: Substantial repopulation of the naïve B cell compartment after RTX therapy appeared to be essential for an adequate vaccination response, which seem to require the additional capability of antigen presentation and germinal center formation. Moreover, expression of exhaustion markers represent negative predictors of vaccination responses.

Erytra blood group analyser and kode technology testing of SARS-CoV-2 antibodies among convalescent patients and vaccinated individuals.

Surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires tests to monitor antibody formation and prevalence. We detected SARS-CoV-2 antibodies using red cells coated by Kode technology with short peptides derived from the SARS-CoV-2 spike protein (SP). Such modified red cells, called C19-kodecytes, can be used as reagent cells in any manual or automated column agglutination assay. We investigated the presence of SARS-CoV-2 antibodies in 130 samples from COVID-19 convalescent plasma donors using standard manual technique, two FDA-authorized enzyme-linked immunosorbent assay (ELISA) assays and a virus neutralisation assay. The sensitivity of the C19-kodecyte assay was 88%, comparable to the anti-SP and anti-nucleocapsid protein (NCP) ELISAs (86% and 83%) and the virus neutralisation assay (88%). The specificity of the C19-kodecyte assay was 90% (anti-SP 100% and anti-NCP 97%). Likewise, 231 samples from 73 vaccinated individuals were tested with an automated analyser, and we monitored the appearance and persistence of SARS-CoV-2 antibodies. The C19-kodecyte assay is a robust tool for SARS-CoV-2 antibody detection. Automated blood group analyser use enables large-scale SARS-CoV-2 antibody testing for vaccination monitoring in population surveys.

Temporary antimetabolite treatment hold boosts SARS-CoV-2 vaccination-specific humoral and cellular immunity in kidney transplant recipients.

Transplant recipients exhibit an impaired protective immunity after SARS-CoV-2 vaccination, potentially caused by mycophenolate (MPA) immunosuppression. Recent data from patients with autoimmune disorders suggest that temporary MPA hold might greatly improve booster vaccination outcomes. We applied a fourth dose of SARS-CoV-2 vaccine to 29 kidney transplant recipients during a temporary (5 weeks) MPA/azathioprine hold, who had not mounted a humoral immune response to previous vaccinations. Seroconversion until day 32 after vaccination was observed in 76% of patients, associated with acquisition of virus-neutralizing capacity. Interestingly, 21/25 (84%) calcineurin inhibitor-treated patients responded, but only 1/4 belatacept-treated patients responded. In line with humoral responses, counts and relative frequencies of spike receptor binding domain-specific (RBD-specific) B cells were markedly increased on day 7 after vaccination, with an increase in RBD-specific CD27++CD38+ plasmablasts. Whereas overall proportions of spike-reactive CD4+ T cells remained unaltered after the fourth dose, frequencies were positively correlated with specific IgG levels. Importantly, antigen-specific proliferating Ki67+ and in vivo-activated programmed cell death 1-positive T cells significantly increased after revaccination during MPA hold, whereas cytokine production and memory differentiation remained unaffected. In summary, antimetabolite hold augmented all arms of immunity during booster vaccination. These data suggest further studies of antimetabolite hold in kidney transplant recipients.

Robust and durable serological response following pediatric SARS-CoV-2 infection.

The quality and persistence of children's humoral immune response following SARS-CoV-2 infection remains largely unknown but will be crucial to guide pediatric SARS-CoV-2 vaccination programs. Here, we examine 548 children and 717 adults within 328 households with at least one member with a previous laboratory-confirmed SARS-CoV-2 infection. We assess serological response at 3-4 months and 11-12 months after infection using a bead-based multiplex immunoassay for 23 human coronavirus antigens including SARS-CoV-2 and its Variants of Concern (VOC) and endemic human coronaviruses (HCoVs), and additionally by three commercial SARS-CoV-2 antibody assays. Neutralization against wild type SARS-CoV-2 and the Delta VOC are analysed in a pseudotyped virus assay. Children, compared to adults, are five times more likely to be asymptomatic, and have higher specific antibody levels which persist longer (96.2% versus 82.9% still seropositive 11-12 months post infection). Of note, symptomatic and asymptomatic infections induce similar humoral responses in all age groups. SARS-CoV-2 infection occurs independent of HCoV serostatus. Neutralization responses of children and adults are similar, although neutralization is reduced for both against the Delta VOC. Overall, the long-term humoral immune response to SARS-CoV-2 infection in children is of longer duration than in adults even after asymptomatic infection.

Heterologous ChAdOx1 nCoV-19 and BNT162b2 prime-boost vaccination elicits potent neutralizing antibody responses and T cell reactivity against prevalent SARS-CoV-2 variants.

BACKGROUND: Heterologous COVID-19 vaccination regimens combining vector- and mRNA-based vaccines are already administered, but data on solicited adverse reactions, immunological responses and elicited protection are limited. METHODS: To evaluate the reactogenicity and humoral as well as cellular immune responses towards most prevalent SARS-CoV-2 variants after a heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, we analysed a cohort of 26 clinic employees aged 25-46 (median 30.5) years who received a ChAdOx1 nCoV-19 prime followed by a BNT162b2 boost after an 8-week interval. Serological data were compared to a cohort which received homologous BNT162b2 vaccination with a 3-week interval (14 individuals aged 25-65, median 42). FINDINGS: Self-reported solicited symptoms after ChAdOx1 nCoV-19 prime were in line with previous reports and more severe than after the BNT162b2 boost. Antibody titres increased significantly over time resulting in strong neutralization titres two weeks after the BNT162b2 boost and subsequently slightly decreased over the course of 17 weeks. At the latest time point measured, all analysed sera retained neutralizing activity against the currently dominant Delta (B.1.617.2) variant. Two weeks post boost, neutralizing activity against the Alpha (B.1.1.7) and immune-evading Beta (B.1.351) variant was ∼4-fold higher than in individuals receiving homologous BNT162b2 vaccination. No difference was observed in neutralization of Kappa (B.1.617.1). In addition, heterologous vaccination induced CD4+ and CD8+ T cells reactive to SARS-CoV-2 spike peptides of all analysed variants; Wuhan-Hu-1, Alpha, Beta, Gamma (P.1), and Delta. INTERPRETATION: In conclusion, heterologous ChAdOx1 nCoV-19 / BNT162b2 prime-boost vaccination is not associated with serious adverse events and induces potent humoral and cellular immune responses. The Alpha, Beta, Delta, and Kappa variants of spike are potently neutralized by sera from all participants and reactive T cells recognize spike peptides of all tested variants. These results suggest that this heterologous vaccination regimen is at least as immunogenic and protective as homologous vaccinations and also offers protection against current variants of concern. FUNDING: This project has received funding from the European Union's Horizon 2020 research and innovation programme, the German Research Foundation, the BMBF, the Robert Koch Institute (RKI), the Baden-Württemberg Stiftung, the county of Lower Saxony, the Ministry for Science, Research and the Arts of Baden-Württemberg, Germany, and the National Institutes of Health.

B Cell Numbers Predict Humoral and Cellular Response Upon SARS-CoV-2 Vaccination Among Patients Treated With Rituximab.

OBJECTIVE: Patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) therapy are at higher risk of poor COVID-19 outcomes and show substantially impaired humoral immune response to anti-SARS-CoV-2 vaccine. However, the complex relationship between antigen-specific B cells and T cells and the level of B cell repopulation necessary to achieve anti-vaccine responses remain largely unknown. METHODS: Antibody responses to SARS-CoV-2 vaccines and induction of antigen-specific B and CD4/CD8 T cell subsets were studied in 19 patients with rheumatoid arthritis (RA) or antineutrophil cytoplasmic antibody-associated vasculitis receiving RTX, 12 patients with RA receiving other therapies, and 30 healthy controls after SARS-CoV-2 vaccination with either messenger RNA or vector-based vaccines. RESULTS: A minimum of 10 B cells per microliter (0.4% of lymphocytes) in the peripheral circulation appeared to be required for RTX-treated patients to mount seroconversion to anti-S1 IgG upon SARS-CoV-2 vaccination. RTX-treated patients who lacked IgG seroconversion showed reduced receptor-binding domain-positive B cells (P = 0.0005), a lower frequency of Tfh-like cells (P = 0.0481), as well as fewer activated CD4 (P = 0.0036) and CD8 T cells (P = 0.0308) compared to RTX-treated patients who achieved IgG seroconversion. Functionally relevant B cell depletion resulted in impaired interferon-γ secretion by spike-specific CD4 T cells (P = 0.0112, r = 0.5342). In contrast, antigen-specific CD8 T cells were reduced in both RA patients and RTX-treated patients, independently of IgG formation. CONCLUSION: In RTX-treated patients, a minimum of 10 B cells per microliter in the peripheral circulation is a candidate biomarker for a high likelihood of an appropriate cellular and humoral response after SARS-CoV-2 vaccination. Mechanistically, the data emphasize the crucial role of costimulatory B cell functions for the proper induction of CD4 responses propagating vaccine-specific B cell and plasma cell differentiation.

B and T Cell Responses after a Third Dose of SARS-CoV-2 Vaccine in Kidney Transplant Recipients.

BACKGROUND: Accumulating evidence sugges ts solid organ transplant recipients, as opposed to the general population, show strongly impaired responsiveness toward standard SARS-CoV-2 mRNA-based vaccination, demanding alternative strategies for protectio n o f this vulnerable group. METHODS: In line with recent recommendations, a third dose of either heterologous ChAdOx1 (AstraZeneca) or homologous BNT162b2 (BioNTech) was administered to 25 kidney transplant recipients (KTR) without humoral response after two doses of BNT162b2, followed by analysis of serological responses and vaccine-specific B- and T-cell immunity. RESULTS: Nine out of 25 (36%) KTR under standard immunosuppressive treatment seroconverted until day 27 after the third vaccination, whereas one patient developed severe COVID-19 infection immediately after vaccination. Cellular analysis 7 days after the third dose showed significantly elevated frequencies of viral spike-protein receptor-binding domain-specific B cells in humor al responders as compared with nonresponders. Likewise, portions of spike-reactive CD4 + T helper cells were significantly elevated in patients who were seroconverting. Furthermore, overall frequencies of IL-2 + , IL-4 + , and polyfunctional CD4 + T cells significantly increased after the third dose, whereas memory/effector differentiation remained unaffected. CONCLUSIONS: Our data suggest a fraction of transplant recipients benefit from triple vaccination, where seroconversion is associated with quantitative and qualitative changes of cellular immunity. At the same time, the study highlights that modified vaccination approaches for immunosuppressed patients remain an urgent medical need. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/JASN/2021_11_23_briggsgriffin112321.mp3.

BNT162b2 Vaccination Elicits Strong Serological Immune Responses Against SARS-CoV-2 Including Variants of Concern in Elderly Convalescents.

Elderly residents of long-term care facilities (LTCFs) have long been underrepresented in studies on vaccine efficacy, particularly in light of currently emerging variants of concern (VOCs). In this prospective observational cohort study, we analyzed serological immune responses in 190 individuals before, 3 weeks after 1st and 3 weeks after 2nd vaccination with BNT162b2. Unvaccinated COVID-19-convalescent subjects served as reference. End points comprised serum anti-spike IgG and IgA titers as well as neutralization capacities against unmutated and mutated SARS-CoV-2 receptor binding domains including B.1.1.7, B.1.351 and P.1. We found that antibody titers and neutralization capacities up to 3 weeks after 2nd vaccination with BNT162b2 were significantly higher in COVID-19-convalescent as compared to COVID-19-naive vaccinees. Moreover, pre-vaccination anti-NCP IgG titers, but not age or gender, had a high impact on the strength and kinetics of post-vaccination neutralization capacity development. Most importantly, BNT162b2-induced neutralization capacity was cross-reactive with VOCs. In contrast to unvaccinated convalescents, vaccinated convalescent individuals of all ages acquired strong neutralizing capacities against current VOCs. The present study suggests that COVID-19-convalescent individuals with a broad age range between 18 and 98 years benefit from BNT162b2 vaccination by developing strong and broad neutralizing immune responses against SARS-CoV-2 including current VOCs.