Marta Tontini

Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans

GNA2132 is a Neisseria meningitidis antigen of unknown function, discovered by reverse vaccinology, which has been shown to induce bactericidal antibodies in animal models. Here we show that this antigen induces protective immunity in humans and it is recognized by sera of patients after meningococcal disease. The protein binds heparin in vitro through an Arg-rich region and this property correlates with increased survival of the unencapsulated bacterium in human serum. Furthermore, two proteases, the meningococcal NalP and human lactoferrin, cleave the protein upstream and downstream from the Arg-rich region, respectively. We conclude that GNA2132 is an important protective antigen of N. meningitidis and we propose to rename it, Neisserial Heparin Binding Antigen (NHBA).

Rational Design of a Meningococcal Antigen Inducing Broad Protective Immunity

A single chimeric protein induces protective immunity against all meningococcal B strains with implications for producing broadly protective vaccines. All for One and One for All The three musketeers were a formidable team, but imagine combining all of their skills and valor into just one musketeer. That is precisely the approach that Rappuoli and his colleagues have taken with their design of a vaccine against meningococcus B, the bacterial pathogen that causes meningitis. Although mining of the genome sequence of this pathogen has yielded excellent targets that could be used in a vaccine, many of these antigens show a high degree of variation that has stymied attempts to use them as vaccine immunogens. For example, factor H binding protein is essential for the survival of meningococcus B in the human host because it protects the pathogen from the onslaught of the human immune system’s complement pathway. Because it is essential for survival, factor H binding protein should be a valuable immunogen, but because it has at least 300 sequence variants, it is impractical to make one vaccine that contains all of these variants. Rappuoli and his colleagues have tackled this problem by dividing the 300 sequence variants of factor H binding protein into three major groups. Using detailed structural information about these three major variants, they engineered variant 1 to carry patches of amino acids from the surfaces of variants 2 and 3. They then introduced groups of point mutations into the amino acids of these transplanted patches to mimic the natural variation of variant 2 and 3 strains of meningococcus B. They then tested which of the 54 engineered single chimeric immunogens could elicit bactericidal antibodies against many different strains of meningococcus B. To do this, they injected the immunogens into mice and assayed mouse sera in vitro for bactericidal activity against multiple bacterial strains. One chimeric immunogen, called G1, was capable of inducing bactericidal antibodies that could kill all strains of meningococcus B, suggesting that it could be used to produce a broadly protective vaccine. This structure-based approach to vaccine design may be useful not only for meningococcus B but also for other pathogens like HIV that show a high degree of antigenic variation. The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.

Beta-glucan-CRM197 conjugates as candidates antifungal vaccines.

A laminarin-diphtheria toxoid (CRM197) conjugate vaccine conferred protection against fungal infections in mice. We have now generated novel beta-glucan-CRM197 vaccines, with either natural (Curd-CRM197) or synthetic linear (15mer-CRM197), or beta-(1,6)-branched (17mer-CRM197) beta-(1,3)-oligosaccharides, formulated with the human-acceptable adjuvant MF59. Curd-CRM197 and 15mer-CRM197 conjugates, which induced high titers of anti-beta-(1,3)-glucan IgG, but no antibodies against beta-(1,6)-glucan, conferred protection to mice lethally challenged with C. albicans. In contrast, the 17mer-CRM197 conjugate, which induced anti-beta-(1,6)-glucan antibodies in addition to the anti-beta-(1,3)-glucan IgG, was non-protective. These data provide some insights on beta-glucan epitope(s) mediating antifungal protection and open the way to develop a synthetic oligosaccharide vaccine against fungal diseases.

Phosphorylation of the Synthetic Hexasaccharide Repeating Unit Is Essential for the Induction of Antibodies to Clostridium difficile PSII Cell Wall Polysaccharide

Clostridium difficile is emerging worldwide as a major cause of nosocomial infections. The negatively charged PSII polysaccharide has been found in different strains of C. difficile and, thereby, represents an important target molecule for a possible carbohydrate-based vaccine. In order to identify a synthetic fragment that after conjugation to a protein carrier could be able to induce anti-PSII antibodies, we exploited a combination of chemical synthesis with immunochemistry, confocal immunofluorescence microscopy, and solid state NMR. We demonstrate that the phosphate group is crucial in synthetic glycans to mimic the native PSII polysaccharide; both native PSII and a phosphorylated synthetic hexasaccharide repeating unit conjugated to CRM(197) elicit comparable immunogenic responses in mice. This finding can aid design and selection of carbohydrate antigens to be explored as vaccine candidates.

Synthesis of a well-defined glycoconjugate vaccine by a tyrosine-selective conjugation strategy

An anti-candidiasis glycoconjugate vaccine was prepared via a tyrosine-selective alkynylation and a click chemistry mediated glycoconjugation sequence. It features a well-defined glycan, protein carrier, and connectivity. The construct, although with significantly lower carbohydrate loading and a shorter β-(1,3) glucan chain than the well-established anti-candidiasis vaccine derived from the random conjugation of laminarin at lysines, elicited a comparable level of specific IgG antibodies.

Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X

Significance Meningococcal serogroup X has recently emerged as a cause of meningitis outbreaks with epidemic potential in sub-Saharan Africa. Novel conjugation technologies, compatible with a reproducible production process, have been successfully used to develop immunogenic polysaccharide conjugate vaccine candidates that are likely to protect against meningococcal X disease. The timely development of an anti-meningococcal X conjugate vaccine appears a logical next step in the broadest control of meningococcal disease and requires commitment now. Neisseria meningitidis is a major cause of bacterial meningitis worldwide, especially in the African meningitis belt, and has a high associated mortality. The meningococcal serogroups A, W, and X have been responsible for epidemics and almost all cases of meningococcal meningitis in the meningitis belt over the past 12 y. Currently no vaccine is available against meningococcal X (MenX). Because the development of a new vaccine through to licensure takes many years, this leaves Africa vulnerable to new epidemics of MenX meningitis at a time when the epidemiology of meningococcal meningitis on the continent is changing rapidly, following the recent introduction of a glycoconjugate vaccine against serogroup A. Here, we report the development of candidate glycoconjugate vaccines against MenX and preclinical data from their use in animal studies. Following optimization of growth conditions of our seed MenX strain for polysaccharide (PS) production, a scalable purification process was developed yielding high amounts of pure MenX PS. Different glycoconjugates were synthesized by coupling MenX oligosaccharides of varying chain length to CRM197 as carrier protein. Analytical methods were developed for in-process control and determination of purity and consistency of the vaccines. All conjugates induced high anti-MenX PS IgG titers in mice. Antibodies were strongly bactericidal against African MenX isolates. These findings support the further development of glycoconjugate vaccines against MenX and their assessment in clinical trials to produce a vaccine against the one cause of epidemic meningococcal meningitis that currently cannot be prevented by available vaccines.

Synthesis of Laminarin Fragments and Evaluation of a β-(1,3) Glucan Hexasaccaride-CRM197 Conjugate as Vaccine Candidate against Candida albicans

Laminarin-CRM197 glycoconjugates were previously demonstrated to be immunogenic and confer protection against Candida albicans in mice. Laminarin consists of β-(1,3) glucan repeating units, with sporadic β-(1,6) branches. A set of short glucans was used to study the effect of the β-(1,6) branch on the antigenicity of linear β-(1,3) glucans. A linear β-(1,3) glucan hexasaccharide was selected as the best fragment able to inhibit the binding between laminarin and antilaminarin antibodies. The hexamer was then conjugated to CRM197 and induced, in mice, significant titers of specific antilaminarin antibodies comparable with those raised by the Lam-CRM197 conjugate.

Preparation, characterization and immunogenicity of HIV-1 related high-mannose oligosaccharides-CRM197 glycoconjugates

The dense glycan shield on the surface of human immunodeficiency virus type 1 (HIV-1) gp120 masks conserved protein epitopes and facilitates virus entry via interaction to glycan binding proteins on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope is currently being considered for the design of a synthetic vaccine. The cluster nature of the 2G12 epitope suggests that a multivalent antigen presentation is important to develop a carbohydrate-based vaccine candidate. In this work we describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates. We exploited flexible polyamidoamine (PAMAM) scaffold to generate four- and eight-valent sugar clusters of HIV-1-related oligomannose antigens Man4, Man6 and Man9. The multivalent presentation of oligomannoses increased the avidity of Man4 and Man9 to 2G12. The synthetic glycodendrons were then covalently coupled to the protein carrier CRM197, formulated with the adjuvant MF59, and used to immunize two animal species. Oligomannose-specific IgG antibodies were generated; however, the antisera failed to recognize recombinant HIV-1 gp120 proteins. We conclude that further structural vaccinology work is needed to identify an antigen presentation that closely matches in vivo the structure of the epitope mapped by 2G12.

Deciphering the structure–immunogenicity relationship of anti-Candida glycoconjugate vaccines

The elucidation of the molecular details underlying the immune properties of glycoconjugate vaccines has largely focused on the carbohydrate moiety, while very little is known on the effect of the corresponding conjugation sites. Herein we constructed a set of β-(1 → 3) glucan oligosaccharide conjugates with a well-defined glycan structure, connected to patterns of predetermined tyrosine or lysine residues onto the CRM197 carrier protein. To evaluate the effect of multivalent architecture in the glycan presentation, a novel linker enabling tyrosine-directed ligation of couples of oligosaccharides was prepared. The potential of these constructs as anti-Candida vaccines was evaluated in vivo, using as controls glycoconjugates prepared by a conventional random coupling strategy, and the structure–immune properties relationship was established. We found that: (i) the tyrosine-directed ligation resulted in higher anti-glycan IgG levels in comparison to the conjugation at predetermined lysine residues; (ii) the presentation of the carbohydrate antigen with a biantennary cluster of glycans onto specific tyrosine residues did not further increase the anti-glycan antibody level; (iii) the sera deriving from immunization with defined conjugates at tyrosine and, particularly at lysine residues, were proven stronger inhibitors of fungal adhesion to human epithelial cells in comparison to those from conjugates prepared by classic random chemistry; (iv) the presence of antibodies directed to the linkers did not affect the anti-glycan immune response. These findings suggest that a careful choice of the defined sites of conjugation and the loading density of antigens are important factors to raise high-quality anti-carbohydrate antibodies.

Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines.

Glycoconjugate vaccines are among the most effective and safest vaccines ever developed. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM197 have been mostly used as protein carriers in licensed vaccines. We evaluated the immunogenicity of serogroup A, C, W-135 and Y meningococcal oligosaccharides conjugated to CRM197, DT and TT in naïve mice. The three carriers were equally efficient in inducing an immune response against the carbohydrate moiety in immunologically naïve mice. The effect of previous exposure to different dosages of the carrier protein on the anti-carbohydrate response was studied using serogroup A meningococcal (MenA) saccharide conjugates as a model. CRM197 showed a strong propensity to positively prime the anti-carbohydrate response elicited by its conjugates or those with the antigenically related carrier DT. Conversely in any of the tested conditions TT priming did not result in enhancement of the anti-carbohydrate response elicited by the corresponding conjugates. Repeated exposure of mice to TT or to CRM197 before immunization with the respective MenA conjugates resulted in a drastic suppression of the anti-carbohydrate response in the case of TT conjugate and only in a slight reduction in the case of CRM197. The effect of carrier priming on the anti-MenA response of DT-based conjugates varied depending on their carbohydrate to protein ratio. These data may have implications for human vaccination since conjugate vaccines are widely used in individuals previously immunized with DT and TT carrier proteins.