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1.
Proc Natl Acad Sci U S A ; 112(12): 3680-5, 2015 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-25775551

RESUMEN

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.


Asunto(s)
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/inmunología
2.
Infect Immun ; 81(8): 2851-60, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23716610

RESUMEN

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.


Asunto(s)
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/inmunología
3.
J Infect Dis ; 206(7): 1041-9, 2012 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-22829645

RESUMEN

Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models. Protection appeared to be associated with functional antibodies that were shown to mediate opsonophagocytosis, to be effective in passive transfer experiments, and to potentially block FhuD2-mediated siderophore uptake. Furthermore, the protein was found to be up-regulated in infected tissues and was required for staphylococcal dissemination and abscess formation. Herein we show that the staphylococcal iron-hydroxamate uptake system is important in invasive infection and functions as an efficacious vaccine target.


Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/metabolismo , Vacunación , Absceso/inmunología , Absceso/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Compuestos Férricos/metabolismo , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Células HL-60 , Humanos , Ácidos Hidroxámicos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Conejos , Sepsis/inmunología , Sepsis/prevención & control , Infecciones Estafilocócicas/inmunología , Vacunas Estafilocócicas/administración & dosificación , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/inmunología
4.
Clin Vaccine Immunol ; 23(6): 442-50, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27030589

RESUMEN

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.


Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/inmunología , Proteínas Hemolisinas/química , Proteínas Hemolisinas/inmunología , Imitación Molecular , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus , Proteína ADAM10/metabolismo , Animales , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Línea Celular , Citotoxinas , Epítopos/inmunología , Escherichia coli/genética , Proteínas Hemolisinas/administración & dosificación , Proteínas Hemolisinas/genética , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Modelos Moleculares , Ingeniería de Proteínas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Vacunación
5.
J Med Microbiol ; 62(Pt 9): 1444-1452, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23722432

RESUMEN

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.


Asunto(s)
Clostridioides difficile/genética , Genes Bacterianos , Sitios Genéticos , Variación Genética , Proteínas Bacterianas/genética , Secuencia de Bases , Clostridioides difficile/clasificación , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/microbiología , Secuencia Conservada , Genotipo , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Ribotipificación
6.
PLoS One ; 8(9): e74718, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24069334

RESUMEN

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.


Asunto(s)
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/genética
7.
Methods Mol Biol ; 799: 361-403, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-21993656

RESUMEN

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.


Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Biotecnología/métodos , Genómica/métodos , Meningitis Meningocócica/prevención & control , Neisseria meningitidis Serogrupo B/inmunología , Antígenos Bacterianos/aislamiento & purificación , Western Blotting , Cromatografía de Afinidad , Clonación Molecular , Biología Computacional/métodos , Cartilla de ADN/genética , Diseño de Fármacos , Electroforesis en Gel de Agar , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa
8.
OMICS ; 15(9): 545-66, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21682594

RESUMEN

Vaccine research and development are experiencing a renaissance of interest from the global scientific community. There are four major reasons for this: (1) the lack of efficacious treatment for many devastating infections; (2) the emergence of multidrug resistant bacteria; (3) the need for improving the safety of the more traditional licensed vaccines; and finally, (4) the great promise for innovative vaccine design and research with convergence of omics sciences, such as genomics, proteomics, immunomics, and vaccinology. Our first project based on omics was initiated in 2000 and was termed reverse vaccinology. At that time, antigen identification was mainly based on bioinformatic analysis of a singular genome. Since then, omics-guided approaches have been applied to its full potential in several proof-of-concept studies in the industry, with the first reverse vaccinology-derived vaccine now in late stage clinical trials and several vaccines developed by omics in preclinical studies. In the meantime, vaccine discovery and development has been further improved with the support of proteomics, functional genomics, comparative genomics, structural biology, and most recently vaccinomics. We illustrate in this review how omics biotechnologies and integrative biology are expected to accelerate the identification of vaccine candidates against difficult pathogens for which traditional vaccine development has thus far been failing, and how research will provide safer vaccines and improved formulations for immunocompromised patients in the near future. Finally, we present a discussion to situate omics-guided rational vaccine design in the broader context of global public health and how it can benefit citizens in both developed and developing countries.


Asunto(s)
Salud Global , Vacunas , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antígenos/genética , Antígenos/metabolismo , Investigación Biomédica/tendencias , Industria Farmacéutica/legislación & jurisprudencia , Perfilación de la Expresión Génica , Genómica , Humanos , Modelos Biológicos , Modelos Moleculares , Proteómica , Vacunas/inmunología
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