Études chimiques et immunologiques des capsules polysaccharidiques de Streptococcus suis

Streptococcus suis est l’un des plus importants pathogènes bactériens du porc, causant des pertes économiques substantielles à l’industrie porcine. De plus, c’est un agent zoonotique représentant de sérieux risques pour la santé humaine. Cette bactérie cause diverses pathologies, dont la méningite, la mort subite et le choc septique sont les plus fréquentes. De plus, il demeure qu’à ce jour, aucun vaccin efficace n’est disponible pour prévenir les infections à S. suis. Étant encapsulé, S. suis est classifié en 35 sérotypes définis par l’antigénicité de sa capsule polysaccharidique (CPS). Malgré cela, seule la structure de la CPS du sérotype 2 est connue à présent. Immunologiquement parlant, les CPS purifiées sont généralement des antigènes T-indépendant de par leur nature, faisant d’elles de très pauvres immunogènes. Ceci a déjà été démontré pour la CPS du sérotype 2. Paradoxalement, les anticorps anti-CPS sont fortement opsonisants et protecteurs, ce qui les rend désirables. Ainsi, les objectifs de cette thèse sont, d’abord, de poursuivre la caractérisation chimique des structures des CPS de différents sérotypes de S. suis, puis d’étudier les propriétés immunostimulatrices et immunogènes de ces nouvelles CPS. De plus, les travaux de cette thèse ont tenté d’améliorer l’immunogénicité de la CPS du sérotype 2 pour pouvoir l’utiliser comme antigène vaccinal. Dans un premier temps, un recensement de la littérature a permis de dresser un portrait à jour et mondial des infections chez le porc et chez l’homme, et d’identifier les sérotypes les plus importants. À l’aide de ces données épidémiologiques, les CPS des sérotypes choisis ont été purifiées, ce qui a permis de déterminer la structure pour huit CPS additionnelles (1, 1/2, 3, 7, 8, 9, 14 et 18). D’autre part, ces nouvelles connaissances sur la structure nous ont permis d’évaluer l’antigénicité et l’immunogénicité de ces nouvelles CPS. Ainsi, nous avons pu observer pour la première fois que la CPS du sérotype 3 de S. suis est hautement immunogène en produisant une réponse opsonisante d’IgG et d’IgM par un mécanisme T-indépendant. De plus, nous avons développé pour la première fois un vaccin glycoconjugué constitué de CPS du sérotype 2 couplé au toxoïde tétanique afin d’obtenir une réponse anticorps T-dépendante protectrice. Une preuve de concept chez le porc a permis de démontrer la protection conférée par notre vaccin glycoconjugué contre S. suis, et plus largement le potentiel des vaccins glycoconjugués pour combattre les infections bactériennes invasives en médecine vétérinaire. Enfin, l’étude de l’immunogénicité du vaccin glycoconjugué chez la souris a permis d’évaluer le rôle du choix du modèle animal et des adjuvants sur la réponse anti-CPS, en plus de produire trois nouveaux anticorps monoclonaux qui ont permis notamment d’étudier les mécanismes humoraux impliqués dans l’élimination de S. suis par opsonophagocytose. L’ensemble de ces études va permettre de souligner l’importance des anticorps anti-CPS dans la protection face aux infections bactériennes invasives.Streptococcus suis is one of the most important porcine bacterial pathogens and is responsible for substantial economic losses to the swine industry. Moreover, it is also a zoonotic agent representing serious risks to human health. This bacterium causes a variety of clinical signs, of which meningitis, sudden death, and septic shock are the most frequent. Furthermore, no efficient vaccine is available to protect against infections caused by S. suis. Being encapsulated, S. suis is classified into 35 serotypes based on the antigenicity of their capsular polysaccharide (CPS). However, only the structure of the serotype 2 CPS is known to date. Immunologically, purified CPSss generally behave as T-independent antigens, making them very poor immunogens. Indeed, this has been previously demonstrated to be the case for the serotype 2 CPS. Paradoxically, anti-CPS antibodies are strongly opsonizing and protective, making them attractive targets for vaccine development. Therefore, the objectives of this thesis are, firstly, to further characterize the chemical composition and structure of the S. suis CPSs from different serotypes, and secondly, to study the immunostimulatory and immunogenic properties of these additional CPSs. Moreover, this thesis will attempt to increase the serotype 2 CPS immunogenicity for potential use it as a vaccine antigen. In a first step, a review of the literature provided an updated and global view of S. suis infections in swine and humans and identified the most important serotypes. With these epidemiological data in hand, we then purified the CPSs of selected serotypes, which allowed us to determine the structures for eight additional CPSs (1, 1/2, 3, 7, 8, 9, 14, and 18). The knowledge obtained regarding the structure of theses CPSs allowed us to then evaluate their antigenicity and immunogenicity. We demonstrated for the first time that serotype 3 is highly immunogenic S. suis CPS, as it induces an opsonizing response composed of IgG and IgM by a T-independent mechanism. In addition, we have developped for the first time a glycoconjugate vaccine composed of serotype 2 CPS conjugated to tetanus toxoid in order to obtain a protective T-dependent antibody response. A proof of concept performed with pigs demonstrated protection conferred by our glycoconjugate vaccine against S. suis, and more importantly the potential of glycoconjugate vaccines in the fight against invasive bacterial infections in veterinary medicine. Finally, immunogenicity studies performed in mice with this glycoconjugate vaccine allowed us to evaluate the effect of the animal model and adjuvant choice on the anti-CPS response. It also allowed us to produce three new monoclonal antibodies that enabled us to study, among other aspects, the humoral mechanisms involved in S. suis clearance by opsonophagocytosis. Consequently, the work conducted during this thesis will serve to highlight the importance of anti-CPS antibodies in the protection against invasive bacterial diseases

S-Layer From Lactobacillus brevis Modulates Antigen-Presenting Cell Functions via the Mincle-Syk-Card9 Axis

C-type lectin receptors (CLRs) are pattern recognition receptors that are crucial in the innate immune response. The gastrointestinal tract contributes significantly to the maintenance of immune homeostasis; it is the shelter for billions of microorganisms including many genera of Lactobacillus sp. Previously, it was shown that host-CLR interactions with gut microbiota play a crucial role in this context. The Macrophage-inducible C-type lectin (Mincle) is a Syk-coupled CLR that contributes to sensing of mucosa-associated commensals. In this study, we identified Mincle as a receptor for the Surface (S)-layer of the probiotic bacteria Lactobacillus brevis modulating GM-CSF bone marrow-derived cells (BMDCs) functions. We found that the S-layer/Mincle interaction led to a balanced cytokine response in BMDCs by triggering the release of both pro- and anti-inflammatory cytokines. In contrast, BMDCs derived from Mincle−/−, CARD9−/− or conditional Syk−/− mice failed to maintain this balance, thus leading to an increased production of the pro-inflammatory cytokines TNF and IL-6, whereas the levels of the anti-inflammatory cytokines IL-10 and TGF-β were markedly decreased. Importantly, this was accompanied by an altered CD4+ T cell priming capacity of Mincle−/− BMDCs resulting in an increased CD4+ T cell IFN-γ production upon stimulation with L. brevis S-layer. Our results contribute to the understanding of how commensal bacteria regulate antigen-presenting cell (APC) functions and highlight the importance of the Mincle/Syk/Card9 axis in APCs as a key factor in host-microbiota interactions.Fil: Prado Acosta, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Goyette Desjardins, Guillaume. University of Veterinary Medicine; AustriaFil: Scheffel, Jörg. Humboldt-Universität zu Berlin; AlemaniaFil: Dudeck, Anne. Otto-von-Guericke-Universität Magdeburg; AlemaniaFil: Ruland, Jürgen. Universitat Technical Zu Munich; AlemaniaFil: Lepenies, Bernd. University of Veterinary Medicine; Austri

Glycoengineered outer membrane vesicles: A novel platform for bacterial vaccines

The World Health Organization has indicated that we are entering into a post-antibiotic era in which infections that were routinely and successfully treated with antibiotics can now be lethal due to the global dissemination of multidrug resistant strains. Conjugate vaccines are an effective way to create a long-lasting immune response against bacteria. However, these vaccines present many drawbacks such as slow development, high price, and batch-to-batch inconsistencies. Alternate approaches for vaccine development are urgently needed. Here we present a new vaccine consisting of glycoengineered outer membrane vesicles (geOMVs). This platform exploits the fact that the initial steps in the biosynthesis of most bacterial glycans are similar. Therefore, it is possible to easily engineer non-pathogenic Escherichia coli lab strains to produce geOMVs displaying the glycan of the pathogen of interest. In this work we demonstrate the versatility of this platform by showing the efficacy of geOMVs as vaccines against Streptococcus pneumoniae in mice, and against Campylobacter jejuni in chicken. This cost-effective platform could be employed to generate vaccines to prevent infections caused by a wide variety of microbial agents in human and animals

Characterization and Protective Activity of Monoclonal Antibodies Directed against Streptococcus suis Serotype 2 Capsular Polysaccharide Obtained Using a Glycoconjugate

Streptococcus suis serotype 2 is an encapsulated bacterium and an important swine pathogen. Opsonizing antibody responses targeting capsular polysaccharides (CPSs) are protective against extracellular pathogens. To elucidate the protective activity of monoclonal antibodies (mAbs) directed against S. suis serotype 2 CPS, mice were immunized with a serotype 2 CPS-glycoconjugate and three hybridomas were isolated; of which, two were murine IgMs and the other a murine IgG1. Whereas the IgMs (mAbs 9E7 and 13C8) showed different reactivity levels with S. suis serotypes 1, 1/2, 2 and 14, the IgG1 (mAb 16H11) was shown to be serotype 2-specific. All mAbs targeted the sialylated chain of the CPSs. Using an opsonophagocytosis assay, the IgMs were opsonizing towards the S. suis serotypes to which they cross-react, while the IgG1 failed to induce bacterial elimination. In a model of mouse passive immunization followed by a lethal challenge with S. suis serotype 2, the IgG1 and IgM cross-reacting only with serotype 14 (mAb 13C8) failed to protect, while the IgM cross-reacting with serotypes 1, 1/2, and 14 (mAb 9E7) was shown to be protective by limiting bacteremia. These new mAbs show promise as new S. suis diagnostic tools, as well as potential for therapeutic applications

Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota

Porcine Dendritic Cells as an In Vitro Model to Assess the Immunological Behaviour of Streptococcus suis Subunit Vaccine Formulations and the Polarizing Effect of Adjuvants

An in vitro porcine bone marrow-derived dendritic cell (DC) culture was developed as a model for evaluating immune polarization induced by adjuvants when administered with immunogens that may become vaccine candidates if appropriately formulated. The swine pathogen Streptococcus suis was chosen as a prototype to evaluate proposed S. suis vaccine candidates in combination with the adjuvants Poly I:C, Quil A ®, Alhydrogel ®, TiterMax Gold ® and Stimune ®. The toll-like receptor ligand Poly I:C and the saponin Quil A ® polarized swine DC cytokines towards a type 1 phenotype, with preferential production of IL-12 and TNF-α. The water-in-oil adjuvants TiterMax Gold ® and Stimune ® favoured a type 2 profile as suggested by a marked IL-6 release. In contrast, Alhydrogel ® induced a type 1/type 2 mixed cytokine profile. The antigen type differently modified the magnitude of the adjuvant effect, but overall polarization was preserved. This is the first comparative report on swine DC immune activation by different adjuvants. Although further swine immunization studies would be required to better characterize the induced responses, the herein proposed in vitro model is a promising approach that helps assessing behaviour of the vaccine formulation rapidly at the pre-screening stage and will certainly reduce numbers of animals used while advancing vaccinology science

The endosomal proteome of macrophage and dendritic cells.

International audienceThe essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types

Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

The capsular polysaccharide (CPS) represents a key virulence factor for most encapsulated streptococci. Streptococcus suis and Group B Streptococcus (GBS) are both well-encapsulated pathogens of clinical importance in veterinary and/or human medicine and responsible for invasive systemic diseases. S. suis and GBS are the only Gram-positive bacteria which express a sialylated CPS at their surface. An important difference between these two sialylated CPSs is the linkage between the side-chain terminal galactose and sialic acid, being α-2,6 for S. suis but α-2,3 for GBS. It is still unclear how sialic acid may affect CPS production and, consequently, the pathogenesis of the disease caused by these two bacterial pathogens. Here, we investigated the role of sialic acid and the putative effect of sialic acid linkage modification in CPS synthesis using inter-species allelic exchange mutagenesis. To this aim, a new molecular biogenetic approach to express CPS with modified sialic acid linkage was developed. We showed that sialic acid (and its α-2,6 linkage) is crucial for S. suis CPS synthesis, whereas for GBS, CPS synthesis may occur in presence of an α-2,6 sialyltransferase or in absence of sialic acid moiety. To evaluate the effect of the CPS composition/structure on sialyltransferase activity, two distinct capsular serotypes within each bacterial species were compared (S. suis serotypes 2 and 14 and GBS serotypes III and V). It was demonstrated that the observed differences in sialyltransferase activity and specificity between S. suis and GBS were serotype unrestricted. This is the first time that a study investigates the interspecies exchange of capsular sialyltransferase genes in Gram-positive bacteria. The obtained mutants represent novel tools that could be used to further investigate the immunomodulatory properties of sialylated CPSs. Finally, in spite of common CPS structural characteristics and similarities in the cps loci, sialic acid exerts differential control of CPS expression by S. suis and GBS