The circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease, as shown in animal models. However, ...the limited efficacy of the PfCSP-based vaccine RTS,S calls for a better understanding of the mechanisms driving the development of the most potent human PfCSP antibodies and identification of their target epitopes. By characterizing 200 human monoclonal PfCSP antibodies induced by sporozoite immunization, we establish that the most potent antibodies bind around a conserved (N/D)PNANPN(V/A) core. High antibody affinity to the core correlates with protection from parasitemia in mice and evolves around the recognition of NANP motifs. The data suggest that the rational design of a next-generation PfCSP vaccine that elicits high-affinity antibody responses against the core epitope will promote the induction of protective humoral immune responses.
We have established a highly efficient 96-well format based strategy to characterize the expressed murine antibody repertoire by combining immunoglobulin (Ig) gene cloning with antibody expression ...and reactivity profiling at the single cell level. Individual mouse B lineage cells are isolated based on defined surface marker expression patterns by fluorescence-activated cell sorting (FACS) and corresponding full-length Ig heavy (H) and Ig light (L) chain variable (V) region gene transcripts are amplified by RT-PCR. Cloning of the amplified products into eukaryotic expression vectors enables the
in vitro production of monoclonal antibodies with antigen specificities identical to the initial B cell antigen receptors. IgH and IgL chain gene sequence information is obtained as part of the cloning procedure and can be directly linked to reactivity profiles of the recombinant antibodies. In summary, our RT-PCR based strategy to generate recombinant monoclonal antibodies from single mouse B cells allows the highly efficient and unbiased characterization of the expressed murine antibody repertoire by sequence analysis and parallel antibody reactivity testing.
Mucosal antibody responses play a major role in mediating homeostasis with the intestinal flora. It has been suggested that imbalance in the IgA+ and IgG+ intestinal B cell repertoire may be ...associated with the development of diseases such as inflammatory bowel disease. Despite this, little is known about the antibody specificity of human intestinal plasmablasts. Here, we have determined the reactivity profile of single isolated IgA+ and IgG+ plasmablasts from human terminal ileum using antibody cloning and in vitro expression. We found that approximately 25% of intestinal IgA and IgG plasmablast antibodies were polyreactive; the majority were antigen-specific. Antigen specificity was not only directed against enteropathogenic microbes but also against commensal microbes and self antigens. Regardless of their reactivity, all intestinal antibodies were somatically mutated and showed signs of antigen-mediated selection, suggesting that they developed from antigen-specific B cell responses. Together, our data indicate that antigen-specific immune responses to intestinal microbes are largely responsible for the maintenance of intestinal homeostasis and thus provide a basis for understanding the deregulated immune responses observed in patients with inflammatory bowel disease.
In humans, several grams of IgA are secreted every day in the intestinal lumen. While only one IgA isotype exists in mice, humans secrete IgA1 and IgA2, whose respective relations with the microbiota ...remain elusive. We compared the binding patterns of both polyclonal IgA subclasses to commensals and glycan arrays and determined the reactivity profile of native human monoclonal IgA antibodies. While most commensals are dually targeted by IgA1 and IgA2 in the small intestine, IgA1+IgA2+ and IgA1-IgA2+ bacteria coexist in the colon lumen, where Bacteroidetes is preferentially targeted by IgA2. We also observed that galactose-α terminated glycans are almost exclusively recognized by IgA2. Although bearing signs of affinity maturation, gut-derived IgA monoclonal antibodies are cross-reactive in the sense that they bind to multiple bacterial targets. Private anticarbohydrate-binding patterns, observed at clonal level as well, could explain these apparently opposing features of IgA, being at the same time cross-reactive and selective in its interactions with the microbiota.
We have developed an efficient strategy that combines immunoglobulin (Ig) gene repertoire analysis and Ig reactivity profiling at the single cell level. Based on surface marker expression individual ...cells at different stages of human B cell development are isolated by fluorescence-activated cell sorting. For each cell Ig heavy and corresponding Ig light chain gene transcripts are amplified by nested RT-PCR and cloned into eukaryotic expression vectors to produce monoclonal human antibodies of the same specificity in vitro. All reactions are performed in 96-well plates and allow cloning of large numbers of Ig genes. The recombinant antibodies are tested for reactivity with diverse self- and non-self antigens and the reactivity profile can be directly linked to the complete Ig heavy and Ig light chain gene sequence information that is obtained as part of the cloning strategy. In summary, our method to clone and express human monoclonal antibodies is unbiased, highly efficient, requires only small cell numbers and the recombinant antibodies allow direct conclusions on the frequency of specific human B cells in a diverse repertoire.
Single‐cell PCR and sequencing of full‐length Ig heavy (Igh) and Igk and Igl light chain genes is a powerful tool to measure the diversity of antibody repertoires and allows the functional assessment ...of B‐cell responses through direct Ig gene cloning and the generation of recombinant mAbs. However, the current methodology is not high‐throughput compatible. Here we developed a two‐dimensional bar‐coded primer matrix to combine Igh and Igk/Igl chain gene single‐cell PCR with next‐generation sequencing for the parallel analysis of the antibody repertoire of over 46 000 individual B cells. Our approach provides full‐length Igh and corresponding Igk/Igl chain gene‐sequence information and permits the accurate correction of sequencing errors by consensus building. The use of indexed cell sorting for the isolation of single B cells enables the integration of flow cytometry and Ig gene sequence information. The strategy is fully compatible with established protocols for direct antibody gene cloning and expression and therefore advances over previously described high‐throughput approaches to assess antibody repertoires at the single‐cell level.
Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and ...full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaired. We show at the single cell level that natural Pf infection induces the development of classical memory B cells (CM) and atypical memory B cells (AtM) that produce broadly neutralizing antibodies against blood stage Pf parasites. CM and AtM contribute to anti-Pf serum IgG production, but only AtM show signs of active antibody secretion. AtM and CM were also different in their IgG gene repertoire, suggesting that they develop from different precursors. The findings provide direct evidence that natural Pf infection leads to the development of protective memory B cell antibody responses and suggest that constant immune activation rather than impaired memory function leads to the accumulation of AtM in malaria. Understanding the memory B cell response to natural Pf infection may be key to the development of a malaria vaccine that induces long-lived protection.
Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens. We determined the molecular mechanism ...underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite
(PfCSP). We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.
Antibodies against the central repeat of the
(Pf) circumsporozoite protein (CSP) inhibit parasite activity and correlate with protection from malaria. However, the humoral response to the PfCSP C ...terminus (C-PfCSP) is less well characterized. Here, we describe B cell responses to C-PfCSP from European donors who underwent immunization with live Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), and were protected against controlled human malaria infection. Out of 215 PfCSP-reactive monoclonal antibodies, only two unique antibodies were specific for C-PfCSP, highlighting the rare occurrence of C-PfCSP-reactive B cells in PfSPZ-CVac-induced protective immunity. These two antibodies showed poor sporozoite binding and weak inhibition of parasite traversal and development, and did not protect mice from infection with PfCSP transgenic
sporozoites. Structural analyses demonstrated that one antibody interacts with a polymorphic region overlapping two T cell epitopes, suggesting that variability in C-PfCSP may benefit parasite escape from humoral and cellular immunity. Our data identify important features underlying C-PfCSP shortcomings as a vaccine target.
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