A Molecular Analysis of Memory B Cell and Antibody Responses Against Plasmodium falciparum Merozoite Surface Protein 1 in Children and Adults From Uganda.

Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum (Pf) merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against Pf develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, full-length Pf merozoite surface protein 1 (PfMSP1FL), in individuals from a region in Uganda with high Pf transmission. Our results showed that PfMSP1FL-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ PfMSP1FL-specific classical MBCs. In contrast, anti-PfMSP1FL plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against PfMSP1FL, with broadening of the response against non-3D7 strains in adults. The B cell receptors encoded by PfMSP1FL-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable PfMSP1 protein. Proteomics analysis of PfMSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to B cell receptors of PfMSP1FL-specific MBCs, anti-PfMSP119 IgGs had high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the PfMSP1-specific humoral immune response with cumulative Pf exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of PfMSP1 variants by the plasma IgG repertoire.

COVID19 disease severity influences the expression of markers of durability in memory B cells

OBJECTIVES/GOALS: Studies have shown that SARS-CoV-2 specific memory B cells can be maintained at least a year after exposure. However, reports show an altered B cell response during infection in severe COVID-19 cases. This study aims to describe the B cell response during COVID-19 convalescence with a focus on signatures that contribute to durable and robust immunity. METHODS/STUDY POPULATION: Our study cohort consisted of individuals who had recovered from non-severe (hospitalized) or severe (hospitalized and requiring invasive mechanical ventilation) COVID-19. In our comparative analysis, samples from both groups were carefully matched to fall within 4-5 weeks post-symptom onset. We also performed a longitudinal analysis of non-severe patients with sampling ending 5 months post-symptom onset. Using high parameter flow cytometry, we characterized the phenotype of memory B cells using 19 distinct cell markers and fluorescently labeled probes to identify B cells reactive with SARS-CoV-2 spike and receptor-binding domain protein. Additionally, serum collected from individuals was used to quantify antibody titers. RESULTS/ANTICIPATED RESULTS: The frequency of spike-specific B cells and serum antibody titers were similar between severe and non-severe groups. However, we observed that individuals recovered from severe COVID-19 have a significantly reduced frequency of spike specific IgG+ memory B cells expressing Tbet and FcRL5 (markers associated with long lived immunity). In the non-severe patients, we observed IgG+Tbet+ B cells targeting the spike protein peak at 2-3 weeks post-symptom onset, decrease by almost fifty percent 4-5 weeks post-symptom onset, and return to baseline 5 months post-symptom onset. Our study also validated previous findings of a short-lived primary response of IgM+ B cells targeting the spike protein. DISCUSSION/SIGNIFICANCE: Our findings highlight potential implications for long-term immunity against re-infection or severity of the resulting disease in patients with severe COVID-19. Further investigation will be necessary to determine whether the maintenance of immunological protection is hindered in patients who overcame severe COVID-19.

SARS-CoV-2 spike-specific memory B cells express higher levels of T-bet and FcRL5 after non-severe COVID-19 as compared to severe disease.

SARS-CoV-2 infection elicits a robust B cell response, resulting in the generation of long-lived plasma cells and memory B cells. Here, we aimed to determine the effect of COVID-19 severity on the memory B cell response and characterize changes in the memory B cell compartment between recovery and five months post-symptom onset. Using high-parameter spectral flow cytometry, we analyzed the phenotype of memory B cells with reactivity against the SARS-CoV-2 spike protein or the spike receptor binding domain (RBD) in recovered individuals who had been hospitalized with non-severe (n = 8) or severe (n = 5) COVID-19. One month after symptom onset, a substantial proportion of spike-specific IgG+ B cells showed an activated phenotype. In individuals who experienced non-severe disease, spike-specific IgG+ B cells showed increased expression of markers associated with durable B cell memory, including T-bet and FcRL5, as compared to individuals who experienced severe disease. While the frequency of T-bet+ spike-specific IgG+ B cells differed between the two groups, these cells predominantly showed an activated switched memory B cell phenotype in both groups. Five months post-symptom onset, the majority of spike-specific memory B cells had a resting phenotype and the percentage of spike-specific T-bet+ IgG+ memory B cells decreased to baseline levels. Collectively, our results highlight subtle differences in the B cells response after non-severe and severe COVID-19 and suggest that the memory B cell response elicited during non-severe COVID-19 may be of higher quality than the response after severe disease.