It would have also been informative to incorporate analysis on antigen-specific T cell- and B cell-mediated cellular immunity and how this may relate to the observed serological response. to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at comparative timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one contamination and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 contamination (n = 30). Results Rituximab-treated patients experienced lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7C38.8) LY2140023 (LY404039) nM vs. 4.6 (2.3C44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximab-treated patients [median (range) KD: 1.05 (0.45C1.84) nM vs. 20.25 (13.2C38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients experienced comparable anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination. Conversation Our results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response. Keywords: antibody affinity, rituximab, SARS-CoV-2 vaccination, neutralisation capacity, Omicron (B.1.1.529), antibody LY2140023 (LY404039) concentration, immunocompromised, SARS-CoV-2 contamination Introduction SARS-CoV-2 contamination (COVID-19) is of ongoing clinical concern for patients with primary systemic vasculitis, particularly those receiving repeated dosing with B cell-depleting therapies, such as the anti-CD20 agent, rituximab. Patients with autoimmune/inflammatory conditions on immunosuppressive therapy, particularly those on anti-CD20 therapies (1, 2), are vulnerable to poorer clinical outcomes following SARS-CoV-2 contamination, including hospitalisation and death (3, 4). Given this and the suboptimal humoral immune responses after SARS-CoV-2 vaccination observed in those receiving anti-CD20 therapies (5C7), subsequent SARS-CoV-2 vaccine doses, i.e., boosters, have been recommended for these patient LY2140023 (LY404039) groups in several jurisdictions (8C10), including those with main systemic vasculitis (11). Humoral immune responses after main vaccination using vaccines targeted against the original (ancestral, wild-type) strain of SARS-CoV-2, e.g., the mRNA vaccines Pfizer BioNTech BNT162b2 (Pfizer) and mRNA-1273 (Moderna) or the adenovirus-vector based vaccines Oxford-AstraZeneca ChAdOx1 nCoV-19 (AZ) or Ad26.COV2-S (Johnson & Johnson), are often suboptimal among patients receiving anti-CD20 therapies. Specifically, titres of antibodies directed against the spike protein subunit (anti-spike IgG) and/or receptor binding domain name (anti-RBD IgG) of SARS-CoV-2 and the proportion of patients on anti-CD20 therapy who seroconvert following main vaccination are lower compared to those not on such therapy and healthy controls (5, 6, 12, 13). Furthermore, of patients on anti-CD20 therapies who seroconvert after main vaccination, many have lower neutralisation titres compared to those on other immunosuppressants and healthy controls (14C16). Rabbit polyclonal to AADACL3 Although neutralising antibody titres and, to a lesser extent, anti-spike IgG and anti-RBD IgG titres, derived from main vaccination with vaccines targeting the original strain of SARS-CoV-2, correlate with protection against symptomatic contamination from your ancestral computer virus (17), significant reductions in the neutralisation capacity of these antibodies have been observed against subsequent SARS-CoV-2 variants of concern, such as the B.1.617.2 variant (Delta) (17) and the B.1.1.529 variant and its sublineages (Omicron) (18C20), which harbour mutations in the spike protein that LY2140023 (LY404039) modify the critical domain for virus-neutralising antibodies (21, 22). SARS-CoV-2-specific T-cell responses following vaccination, which may be protective against severe contamination (23, 24), appear largely preserved among anti-CD20-treated patients (5), even though clinical significance of these responses in such patients remains unclear. Consequently, main systemic vasculitis patients receiving anti-CD20 therapies may still be vulnerable to severe SARS-CoV-2 infection even if they develop antibodies following main vaccination, particularly given the emergence of variants with humoral immune escape properties. We have recently developed a novel immunoassay, microfluidic antibody affinity profiling (MAAP), for in-solution quantification of the fundamental parameters of the antibody response, namely, affinity and concentration, directly in serum (25), and used it to characterise.