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A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent formulations, showing excellent safety profiles and immunogenicity. However, responses were suboptimal in unprimed individuals after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for a single-dose vaccine.
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Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen-GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity.
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The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.
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Nanopartículas , Neisseria meningitidis , Ratones , Animales , Ferritinas , Antígenos Bacterianos , Antígenos Virales , Anticuerpos Bloqueadores , Vacunas Combinadas , Nanopartículas/químicaRESUMEN
The ability of Neisseria meningitidis Outer Membrane Vesicles (OMV) to induce protective responses in humans is well established and mainly attributed to Porin A (PorA). However, the contribution of additional protein antigens to protection remains to be elucidated. In this study we dissected the immunogenicity of antigens originating from the OMV component of the 4CMenB vaccine in mice and humans. We collected functional data on a panel of strains for which bactericidal responses to 4CMenB in infants was attributable to the OMV component and evaluated the role of 30 OMV-specific protein antigens in cross-coverage. By using tailor-made protein microarrays, the immunosignature of OMV antigens was determined. Three of these proteins, OpcA, NspA, and PorB, triggered mouse antibodies that were bactericidal against several N. meningitidis strains. Finally, by genetic deletion and/or serum depletion studies, we demonstrated the ability of OpcA and PorB to induce functional immune responses in infant sera after vaccination. In conclusion, while confirming the role of PorA in eliciting protective immunity, we identified two OMV antigens playing a key role in protection of infants vaccinated with the 4CMenB vaccine against different N. meningitidis serogroup B strains.
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A 59-year-old woman was referred to the emergency room with acute abdominal pain. A CT scan revealed multiple dissections and microaneurysms of the superior mesenteric, the hepatic and the renal arteries. Stenting of the superior mesenteric artery was required. A non-invasive diagnostic procedure was instrumental to establish the diagnosis and guide appropriate treatment, which resulted in a rapid and sustained recovery.
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Arteria Mesentérica Superior , Enfermedades Raras , Disección , Femenino , Humanos , Arteria Mesentérica Superior/diagnóstico por imagen , Persona de Mediana Edad , Arteria Renal/diagnóstico por imagen , StentsRESUMEN
Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines.
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Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein of Neisseria meningitidis and a component of the Bexsero vaccine. NHBA is characterized by the presence of a highly conserved Arg-rich region involved in binding to heparin and heparan sulphate proteoglycans present on the surface of host epithelial cells, suggesting a possible role of NHBA during N. meningitidis colonization. NHBA has been shown to be cleaved by the meningococcal protease NalP and by human lactoferrin (hLF), a host protease presents in different body fluids (saliva, breast milk and serum). Cleavage occurs upstream or downstream the Arg-rich region. Since the human nasopharynx is the only known reservoir of infection, we further investigated the susceptibility of NHBA to human proteases present in the saliva to assess whether proteolytic cleavage could happen during the initial steps of colonization. Here we show that human saliva proteolytically cleaves NHBA, and identified human kallikrein 1 (hK1), a serine protease, as responsible for this cleavage. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and in the central nervous system. They are present in plasma, lymph, urine, saliva, pancreatic juices, and other body fluids where they catalyze the proteolysis of several human proteins. Here we report the characterization of NHBA cleavage by the tissue kallikrein, expressed in saliva and the identification of the cleavage site on NHBA both, as recombinant protein or as native protein, when expressed on live bacteria. Overall, these findings provide new insights on NHBA as target of host proteases, highlights thepotential role of NHBA in the Neisseria meningitidis nasopharyngeal colonization, and of kallikrein as a defensive agent against meningococcal infection.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Infecciones Meningocócicas/microbiología , Proteolisis , Saliva/química , Calicreínas de Tejido/metabolismo , Secuencia de Aminoácidos , Humanos , Neisseria meningitidis/fisiología , ProteómicaRESUMEN
Background: Although the relevance of humoral immunity for protection against S. aureus skin and soft tissue infections (SSTIs) has been suggested by several animal and human studies, the question of which human antibodies may be protective has so far impeded the development of a safe and effective vaccine. Because most adults have developed certain anti-S. aureus antibodies due to S. aureus colonization or infection, we hypothesized that the titers of antibodies to S. aureus in uninfected controls would differ from those in infected patients and would also differ in infected patients from the time of acute infection to a 40-day convalescent serum. Methods: To test these hypotheses, we measured human antibody levels against a panel of 134 unique antigens comprising the S. aureus surfome and secretome in subjects with active culture-confirmed S. aureus SSTIs (cases) and in controls with no infection, using a novel S. aureus protein microarray. Results: Most S. aureus SSTI patients (n = 60) and controls (n = 142) had antibodies to many of the tested S. aureus antigens. Univariate analysis showed statistically weak differences in the IgG levels to some antigens in the SSTI patient (case) sera compared with controls. Antibody levels to most tested antigens did not increase comparing acute with 40-day serum. Multiple logistic regression identified a rich subset of antigens that, by their antibody levels, together correctly differentiated all cases from all controls. Conclusions: Antibodies directed against S. aureus antigens were present both in patients with S. aureus SSTIs and in uninfected control patients. We found that SSTI patients and controls could be distinguished only based on differences in antibody levels to many staphylococcal surface and secreted antigens. Our results demonstrate that in the studied population, the levels of anti-S. aureus antibodies appear largely fixed, suggesting that there may be some level of unresponsiveness to natural infection.
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Anticuerpos Antibacterianos/sangre , Inmunoglobulina G/sangre , Enfermedades Cutáneas Bacterianas/inmunología , Infecciones de los Tejidos Blandos/inmunología , Infecciones Estafilocócicas/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Enfermedades Cutáneas Bacterianas/sangre , Infecciones de los Tejidos Blandos/sangre , Infecciones Estafilocócicas/sangre , Staphylococcus aureus/inmunología , Adulto JovenRESUMEN
Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and constitutes one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, cleave NHBA protein upstream or downstream of a conserved Arg-rich region, respectively. The cleavage results in the release of the C-terminal portion of the protein. The C-terminal fragment originating from the processing of meningococcal proteases, referred to as C2 fragment, exerts a toxic effect on endothelial cells altering the endothelial permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistent with previous findings showing that Neisseria meningitidis traverses the epithelial barrier without disrupting the junctional structures. We showed that epithelial cells constantly secrete proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich region, a putative docking domain reported to be essential for C2-mediated toxic effect. Moreover, we found that the C3-convertase of the alternative complement pathway is one of the proteases responsible for this processing. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. NHBA cleavage may occur at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.
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Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , C3 Convertasa de la Vía Alternativa del Complemento/metabolismo , Neisseria/metabolismo , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Portadoras/química , Línea Celular , Ácido Edético/farmacología , Células Epiteliales/citología , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , Magnesio/química , Magnesio/metabolismo , Péptido Hidrolasas/metabolismo , Proteolisis/efectos de los fármacos , Proteómica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Zinc/química , Zinc/metabolismoRESUMEN
High incidence, severity and increasing antibiotic resistance characterize Pseudomonas aeruginosa infections, highlighting the need for new therapeutic options. Vaccination strategies to prevent or limit P. aeruginosa infections represent a rational approach to positively impact the clinical outcome of risk patients; nevertheless this bacterium remains a challenging vaccine target. To identify novel vaccine candidates, we started from the genome sequence analysis of the P. aeruginosa reference strain PAO1 exploring the reverse vaccinology approach integrated with additional bioinformatic tools. The bioinformatic approaches resulted in the selection of 52 potential antigens. These vaccine candidates were conserved in P. aeruginosa genomes from different origin and among strains isolated longitudinally from cystic fibrosis patients. To assess the immune-protection of single or antigens combination against P. aeruginosa infection, a vaccination protocol was established in murine model of acute respiratory infection. Combinations of selected candidates, rather than single antigens, effectively controlled P. aeruginosa infection in the in vivo model of murine pneumonia. Five combinations were capable of significantly increase survival rate among challenged mice and all included PA5340, a hypothetical protein exclusively present in P. aeruginosa. PA5340 combined with PA3526-MotY gave the maximum protection. Both proteins were surface exposed by immunofluorescence and triggered a specific immune response. Combination of these two protein antigens could represent a potential vaccine to prevent P. aeruginosa infection.
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Genoma Bacteriano , Genómica , Infecciones por Pseudomonas/inmunología , Vacunas contra la Infección por Pseudomonas/genética , Vacunas contra la Infección por Pseudomonas/inmunología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Fibrosis Quística/complicaciones , Modelos Animales de Enfermedad , Estudio de Asociación del Genoma Completo , Genómica/métodos , Humanos , Ratones , Sistemas de Lectura Abierta , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/mortalidad , Infecciones por Pseudomonas/prevención & control , Vacunas contra la Infección por Pseudomonas/administración & dosificación , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization.
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Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/metabolismo , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Células Epiteliales/microbiología , Infecciones Meningocócicas/tratamiento farmacológico , Neisseria meningitidis/fisiología , Anticuerpos Antibacterianos/química , Sitios de Unión , Línea Celular , Técnicas de Inactivación de Genes , Proteoglicanos de Heparán Sulfato/metabolismo , Heparina/metabolismo , Humanos , Lactoferrina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Mutación , Neisseria meningitidis/inmunología , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
BACKGROUND: Non-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways. A broadly protective vaccine against NTHi has been a long-unmet medical need, as the high genetic variability of this bacterium has posed great challenges. RESULTS: In this study, we developed a robust serum bactericidal assay (SBA) to optimize the selection of protective antigens against NTHi. SBA takes advantage of the complement-mediated lysis of bacterial cells and is a key in vitro method for measuring the functional activity of antibodies. As a proof of concept, we assessed the bactericidal activity of antibodies directed against antigens known to elicit a protective response, including protein D used as carrier protein in the Synflorix pneumococcal polysaccharide conjugate vaccine. Prior to SBA screening, the accessibility of antigens to antibodies and the capacity of the latter to induce C3 complement deposition was verified by flow cytometry. Using baby rabbit serum as a source of complement, the proposed assay not only confirmed the bactericidal activity of the antibodies against the selected vaccine candidates, but also showed a significant reproducibility. CONCLUSIONS: Considering the rapidity and cost-effectiveness of this novel SBA protocol, we conclude that it is likely to become an important tool to prove the capability of antibodies directed against recombinant antigens to induce NTHi in vitro killing and to both select new protective vaccine candidates, and predict vaccine efficacy.
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Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Actividad Bactericida de la Sangre , Haemophilus influenzae/inmunología , Haemophilus influenzae/fisiología , Viabilidad Microbiana/efectos de los fármacos , Animales , Cobayas , Haemophilus influenzae/efectos de los fármacos , Inmunoensayo/métodos , Ratones , ConejosRESUMEN
Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.
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Adyuvantes Inmunológicos/farmacología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/química , Receptor Toll-Like 7/química , Absceso/patología , Inmunidad Adaptativa , Animales , Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Antígenos/inmunología , Humanos , Ratones , Modelos Animales , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus , Células TH1/inmunologíaRESUMEN
Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.
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Adhesinas Bacterianas/inmunología , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Mapeo Epitopo/métodos , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Neisseria meningitidis Serogrupo B/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Multimerización de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , TemperaturaRESUMEN
Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.
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Adhesinas Bacterianas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Staphylococcus aureus/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Carga Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Femenino , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/inmunología , Alineación de Secuencia , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/genéticaRESUMEN
In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.
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Antígenos Bacterianos/química , Proteínas Bacterianas/química , Proteoma/química , Staphylococcus aureus/metabolismo , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Minería de Datos , Ratones , Ratones Endogámicos , Proteoma/genética , Proteoma/metabolismo , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/inmunologíaRESUMEN
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.
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Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Infecciones por Clostridium/inmunología , Enterotoxinas/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/inmunología , Clostridioides difficile/inmunología , Infecciones por Clostridium/prevención & control , Cricetinae , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Ratones , Proteínas Recombinantes/inmunologíaRESUMEN
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (Collagen binding protein A), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG-like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent Kd in the order of 10(-9 ) M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild-type C. difficile 630 strain was indistinguishable to the cbpA knock-out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM-producing cells. The binding activity of L. lactis-CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface-exposed adhesin contributing to the C. difficile interaction with the host.
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Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Clostridioides difficile/fisiología , Colágeno/metabolismo , Interacciones Huésped-Patógeno , Animales , Fibroblastos/metabolismo , Fibroblastos/microbiología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Cinética , Lactococcus lactis/genética , Lactococcus lactis/fisiología , Ratones , Microscopía Confocal , Unión ProteicaRESUMEN
Most of the vaccines available today, albeit very effective, have been developed using traditional "old-style" methodologies. Technologies developed in recent years have opened up new perspectives in the field of vaccinology and novel strategies are now being used to design improved or new vaccines against infections for which preventive measures do not exist. The Reverse Vaccinology (RV) approach is one of the most powerful examples of biotechnology applied to the field of vaccinology for identifying new protein-based vaccines. RV combines the availability of genomic data, the analyzing capabilities of new bioinformatic tools, and the application of high throughput expression and purification systems combined with serological screening assays for a coordinated screening process of the entire genomic repertoire of bacterial, viral, or parasitic pathogens. The application of RV to Neisseria meningitidis serogroup B represents the first success of this novel approach. In this chapter, we describe how this revolutionary approach can be easily applied to any pathogen.