RESUMEN
The identification and recombinant production of functional antigens and/or epitopes of pathogens represent a crucial step for the development of an effective protein-based vaccine. Many vaccine targets are outer membrane proteins anchored into the lipidic bilayer through an extended hydrophobic portion making their recombinant production challenging. Moreover, only the extracellular loops, and not the hydrophobic regions, are naturally exposed to the immune system. In this work, the Domain 3 (D3) from Group B Streptococcus (GBS) pilus 2a backbone protein has been identified and engineered to be used as a scaffold for the display of extracellular loops of two Neisseria gonorrhoeae membrane proteins (PorB.1b and OpaB). A computational structure-based approach has been applied to the design of both the scaffold and the model antigens. Once identified the best D3 engineerable site, several different chimeric D3 displaying PorB.1b and OpaB extracellular loops were produced as soluble proteins. Each molecule has been characterized in terms of solubility, stability, and ability to correctly display the foreign epitope. This antigen dissection strategy allowed the identification of most immunogenic extracellular loops of both PorB.1b and OpaB gonococcal antigens. The crystal structure of chimeric D3 displaying PorB.1b immunodominant loop has been obtained confirming that the engineerization did not alter the predicted native structure of this epitope. Taken together, the reported data suggest that D3 is a novel protein scaffold for epitope insertion and display, and a valid alternative to the production of whole membrane protein antigens. Finally, this work describes a generalized computational structure-based approach for the identification, design, and dissection of epitopes in target antigens through chimeric proteins.
Asunto(s)
Proteínas de la Membrana , Vacunas , Epítopos/genética , Antígenos Bacterianos/genética , Membrana Dobles de LípidosRESUMEN
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.
RESUMEN
OBJECTIVES: The main aim of this exploratory study was to evaluate functional activity of antibodies elicited by a maternal Group B Streptococcus (GBS) investigational vaccine composed of capsular polysaccharides Ia, Ib, and III conjugated to genetically detoxified Diphtheria toxin CRM197. The second objective was to investigate the relationship between serotype-specific IgG concentrations and functional activity in maternal and cord sera. METHODS: Maternal and cord sera collected at baseline and at delivery from vaccine and placebo recipients during a double-blind placebo-controlled Phase II study (www.clinicaltrials.gov, NCT01446289) were tested in an opsono-phagocytic bacterial killing assay. Cord sera from vaccine recipients were also passively transferred to newborn mice to investigate conferred protection against bacterial challenge. RESULTS: Antibody-mediated GBS phagocytic killing was significantly increased in maternal serum at delivery and in cord sera from the investigational vaccine group as compared to the placebo group. Anti-capsular IgG concentrations above 1 µg/mL mediated in vitro killing against GBS strains belonging to all three serotypes and IgG levels correlated with functional titers. Passively administered cord sera elicited a dose-dependent protective response against all GBS serotypes in the in vivo model. CONCLUSIONS: The maternal vaccine elicited functional antibodies that were placentally transferred. Anti-capsular IgG concentrations in maternal and cord sera were predictive of functional activity and in vivo protection in the mouse model.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Sangre Fetal/inmunología , Glicoconjugados/inmunología , Inmunización Pasiva , Vacunas Estreptocócicas/inmunología , Adolescente , Adulto , Animales , Animales Recién Nacidos , Método Doble Ciego , Femenino , Humanos , Inmunidad Materno-Adquirida , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Ratones , Embarazo , Serogrupo , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/administración & dosificación , Streptococcus agalactiae , Vacunas Conjugadas/administración & dosificación , Adulto JovenRESUMEN
Immunization of nonpregnant adults could help prevent invasive group B Streptococcus (GBS) infections, but adult immune responses have not been investigated. We defined capsular polysaccharide (CPS) and pilus island (PI) surface antigen distribution and expression and immune responses to GBS infection in nonpregnant adults. Prospective surveillance from 7 hospitals in Houston, Texas, USA, identified 102 adults with GBS bacteremia; 43% had skin/soft tissue infection, 16% bacteremia without focus, and 12% osteomyelitis. CPS-specific IgG was determined by ELISA and pilus-specific IgG by multiplex immunoassay. CPS types were Ia (24.5%), Ib (12.7%), II (9.8%), III (16.7%), IV (13.7%), and V (12.7%); 9.8% were nontypeable by serologic methods. Pili, expressed by 89%, were most often PI-2a. CPS and pilus-specific IgG increased during convalescence among patients with strains expressing CPS or PI. All GBS expressed CPS or PI; 79% expressed both. Increased antibodies to CPS and PI during recovery suggests that GBS bacteremia in adults is potentially vaccine preventable.
Asunto(s)
Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos/inmunología , Bacteriemia , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Tipificación Molecular , Polisacáridos Bacterianos/inmunología , Serotipificación , Infecciones Estreptocócicas/diagnóstico , Streptococcus agalactiae/clasificación , Streptococcus agalactiae/genética , Streptococcus agalactiae/aislamiento & purificaciónRESUMEN
A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Interleucina-17/metabolismo , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Receptor Toll-Like 7/metabolismo , Adyuvantes Inmunológicos , Animales , Anticuerpos Neutralizantes/inmunología , Linfocitos T CD4-Positivos/citología , Citocinas/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Bazo/metabolismo , Bazo/patología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/mortalidad , Staphylococcus aureus/genética , Tasa de Supervivencia , Células TH1/inmunología , Células Th17/inmunología , Receptor Toll-Like 7/inmunologíaRESUMEN
SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.
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Anticuerpos Antibacterianos/farmacología , Formación de Anticuerpos , Infecciones por Escherichia coli , Proteínas de Escherichia coli/inmunología , Polisacárido Liasas/antagonistas & inhibidores , Factores de Virulencia/inmunología , Animales , Animales no Consanguíneos , Anticuerpos Antibacterianos/metabolismo , Células Cultivadas , Escherichia coli Enteropatógena/crecimiento & desarrollo , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/metabolismo , Activación Enzimática/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Femenino , Intestinos/microbiología , Ratones , Ratones Endogámicos CBA , Polisacárido Liasas/inmunología , Polisacárido Liasas/metabolismo , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/metabolismoRESUMEN
Group A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interact in vitro with the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cells in vitro and that Lactococcus lactis expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro and to mice nasal mucosa in vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (Streptococcus pyogenes Adhesion and Division protein).
Asunto(s)
Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/inmunología , Streptococcus pyogenes/metabolismo , Antígenos Bacterianos , Proteínas Bacterianas/genética , Línea Celular , Clonación Molecular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/microbiología , Eliminación de Gen , Humanos , Lactococcus lactis/metabolismo , Unión Proteica , Streptococcus pyogenes/citología , Streptococcus pyogenes/genéticaRESUMEN
UNLABELLED: Streptolysin O is a potent pore-forming toxin produced by group A Streptococcus. The aims of the present study were to dissect the relative contributions of different structural domains of the protein to hemolytic activity, to obtain a detoxified form of streptolysin O amenable to human vaccine formulation, and to investigate the role of streptolysin O-specific antibodies in protection against group A Streptococcus infection. On the basis of in silico structural predictions, we introduced two amino acid substitutions, one in the proline-rich domain 1 and the other in the conserved undecapeptide loop in domain 4. The resulting streptolysin O derivative showed no toxicity, was highly impaired in binding to eukaryotic cells, and was unable to form organized oligomeric structures on the cell surface. However, it was fully capable of conferring consistent protection in a murine model of group A Streptococcus infection. When we engineered a streptococcal strain to express the double-mutated streptolysin O, a drastic reduction in virulence as well as a diminished capacity to kill immune cells recruited at the infection site was observed. Furthermore, when mice immunized with the toxoid were challenged with the wild-type and mutant strains, protection only against the wild-type strain, not against the strain expressing the double-mutated streptolysin O, was obtained. We conclude that protection occurs by antibody-mediated neutralization of active toxin. IMPORTANCE: We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.
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Sustitución de Aminoácidos , Streptococcus pyogenes/patogenicidad , Estreptolisinas/genética , Estreptolisinas/toxicidad , Factores de Virulencia/genética , Factores de Virulencia/toxicidad , Animales , Anticuerpos Antibacterianos/sangre , Antitoxinas/sangre , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/toxicidad , Modelos Animales de Enfermedad , Ratones , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Proteínas Mutantes/toxicidad , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/patología , Infecciones Estreptocócicas/prevención & control , Streptococcus pyogenes/genética , Streptococcus pyogenes/inmunología , Estreptolisinas/inmunología , Análisis de Supervivencia , Virulencia , Factores de Virulencia/inmunologíaRESUMEN
We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Infecciones Estreptocócicas/prevención & control , Streptococcus pyogenes/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Análisis por Conglomerados , Femenino , Citometría de Flujo , Hemólisis , Humanos , Ratones , Faringitis/sangre , Faringitis/inmunología , Faringitis/microbiología , Análisis por Matrices de Proteínas , Proteoma/inmunología , Proteoma/metabolismo , Ovinos , Infecciones Estreptocócicas/sangre , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/metabolismo , VacunaciónRESUMEN
Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an approximately 120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.