FUNCTIONALLY IMPAIRED ANTIBODY RESPONSE TO BNT162B2 BOOSTER VACCINATION IN CVID IgG RESPONDERS.

BACKGROUND: While previous studies described the production of IgG-antibodies in a subgroup of CVID-patients following mRNA-vaccinations with bnt162b2 SARS-CoV2 (CVID responders), the functionality of these antibodies in terms of avidity as measured by the dissociation rate constant (kdis) and the antibody response to booster immunization has not been studied. OBJECTIVE: In CVID responders and healthy individuals the avidity of anti-SARS-CoV-2 serum-antibodies and their neutralization capacity as measured by surrogate virus neutralizing antibodies were analyzed in addition to IgG-, IgM- and IgA-antibody levels and the response of circulating follicular T-helper cells after a third vaccination with BNT162b2 SARS-CoV2 mRNA-vaccine. METHODS: Binding IgG, IgA and IgM serum levels were analyzed by ELISA in CVID-patients responding to the primary vaccination (CVID responders, n=10) and healthy controls (n=41). The binding-avidity of anti-spike antibodies was investigated using biolayer interferometry in combination with biotin-labelled receptor-binding-domain (RBD) of SARS-CoV2 spike-protein and streptavidin-labelled sensors. Antigen-specific recall T-cell responses were assessed by measuring activation-induced markers by flow cytometry. RESULTS: After the third vaccination with BNT162b2 IgG-, IgM and IgA-antibody levels, sVNT levels and antibody avidity were lower in CVID responders as compared to healthy. In contrast αSpike-avidity was comparable in CVID responders and healthy individuals following primary vaccination. Follicular T-helper cell response to booster vaccination in CVID-responders was significantly reduced when compared to healthy individuals. CONCLUSION: Impaired affinity-maturation during booster-response provides new insight into CVID pathophysiology.

Antigen-Specific CD4+ T-Cell Activation in Primary Antibody Deficiency After BNT162b2 mRNA COVID-19 Vaccination

Previous studies on immune responses following COVID-19 vaccination in patients with common variable immunodeficiency (CVID) were inconclusive with respect to the ability of the patients to produce vaccine-specific IgG antibodies, while patients with milder forms of primary antibody deficiency such as immunoglobulin isotype deficiency or selective antibody deficiency have not been studied at all. In this study we examined antigen-specific activation of CXCR5-positive and CXCR5-negative CD4+ memory cells and also isotype-specific and functional antibody responses in patients with CVID as compared to other milder forms of primary antibody deficiency and healthy controls six weeks after the second dose of BNT162b2 vaccine against SARS-CoV-2. Expression of the activation markers CD25 and CD134 was examined by multi-color flow cytometry on CD4+ T cell subsets stimulated with SARS-CoV-2 spike peptides, while in parallel IgG and IgA antibodies and surrogate virus neutralization antibodies against SARS-CoV-2 spike protein were measured by ELISA. The results show that in CVID and patients with other milder forms of antibody deficiency normal IgG responses (titers of spike protein-specific IgG three times the detection limit or more) were associated with intact vaccine-specific activation of CXCR5-negative CD4+ memory T cells, despite defective activation of circulating T follicular helper cells. In contrast, CVID IgG nonresponders showed defective vaccine-specific and superantigen-induced activation of both CD4+T cell subsets. In conclusion, impaired TCR-mediated activation of CXCR5-negative CD4+ memory T cells following stimulation with vaccine antigen or superantigen identifies patients with primary antibody deficiency and impaired IgG responses after BNT162b2 vaccination.

Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research.

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

Heparin-Functionalized Adsorbents Eliminate Central Effectors of Immunothrombosis, including Platelet Factor 4, High-Mobility Group Box 1 Protein and Histones.

Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents.