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Some HIV-infected individuals develop broadly neutralizing antibodies (bNAbs), whereas most develop antibodies that neutralize only a narrow range of viruses (nNAbs). bNAbs, but not nNAbs, protect animals from experimental infection and are likely a key component of an effective vaccine. nNAbs and bNAbs target the same regions of the viral envelope glycoprotein (Env), but for reasons that remain unclear only nNAbs are elicited by Env immunization. We show that in contrast to germline-reverted (gl) bNAbs, glnNAbs recognized diverse recombinant Envs. Moreover, owing to binding affinity differences, nNAb B cell progenitors had an advantage in becoming activated and internalizing Env compared with bNAb B cell progenitors. We then identified an Env modification strategy that minimized the activation of nNAb B cells targeting epitopes that overlap those of bNAbs.

An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum open reading frames for proteins that elicit parasite-inhibitory Abs. This has led to the identification of the cysteine-rich protective Ag (CyRPA). CyRPA is a cysteine-rich protein harboring a predicted signal sequence. The stage-specific expression of CyRPA in late schizonts resembles that of proteins known to be involved in merozoite invasion. Immunofluorescence staining localized CyRPA at the apex of merozoites. The entire protein is conserved as shown by sequencing of the CyRPA encoding gene from a diverse range of P. falciparum isolates. CyRPA-specific mAbs substantially inhibited parasite growth in vitro as well as in a P. falciparum animal model based on NOD-scid IL2Rγnull mice engrafted with human erythrocytes. In contrast to other P. falciparum mouse models, this system generated very consistent results and evinced a dose-response relationship and therefore represents an unprecedented in vivo model for quantitative comparison of the functional potencies of malaria-specific Abs. Our data suggest a role for CyRPA in erythrocyte invasion by the merozoite. Inhibition of merozoite invasion by CyRPA-specific mAbs in vitro and in vivo renders this protein a promising malaria asexual blood-stage vaccine candidate Ag.

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Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.DOI: http://dx.doi.org/10.7554/eLife.20383.001

Background Diarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality. Methods Flexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa . The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here. Findings Flexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection. Interpretation Collectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field. Funding Funding for this study was provided through a Wellcome Trust grant.

Background Shigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O -polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A . We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis. Methods In this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed. Findings Sixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% ( p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% ( p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score ( p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease ( p = 0.0016) and with a decreased shigellosis disease score ( p = 0.002). Interpretation The Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti- S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.) Funding Funding for this study was through a grant from the Wellcome Trust.

BackgroundThe generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and purity is a tedious undertaking and can be demanding especially in the case of membrane proteins. Furthermore, antibodies generated against a purified recombinant protein are frequently incapable of binding to the endogenous protein in its native context.ResultsWe describe a strategy to generate monoclonal antibodies against membrane or membrane-associated proteins that completely bypasses any need for purified recombinant antigen. This approach utilises stably transfected mammalian cells expressing recombinant antigens on their cell surface for immunisation of mice. The transfected cells are also used for measuring seroconversion, hybridoma selection and antibody characterisation. By presenting the antigen in its native conformation for immunisation and hybridoma selection, this procedure promotes the generation of antibodies capable of binding to the endogenous protein. In the present study, we applied this approach successfully for three predicted GPI-anchored proteins of the malaria parasite Plasmodium falciparum.ConclusionsThe described entirely cell-based technology is a fast and efficient approach for obtaining antibodies reactive with endogenous cell-surface proteins in their native conformation.