RESUMEN
The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of humans, and various species of animals and birds, in which it may induce a mild colitis and diarrhoea. The aim of the current study was to evaluate the use of putative oligopeptide-binding proteins of B. pilosicoli as vaccine components. A partial genome sequence of B. pilosicoli porcine strain 95/1000 was subjected to bioinformatics analysis, and six genes predicted to encode oligopeptide-binding proteins were selected. Following a PCR-based distribution study of the genes across different strains of the spirochaete, they were amplified from B. pilosicoli human strain WesB and cloned in Escherichia coli. The recombinant histidine-tagged proteins were purified and subjected to in vitro and in vivo immunogenicity analysis. Recombinant products (P-1 and P-3) from two genes that were immunogenic and recognized by sera from pigs that had recovered from B. pilosicoli infections were tested in a mouse model of intestinal spirochaetosis. For each recombinant protein, groups of 12 C3H/HeJ mice were vaccinated subcutaneously with 100 microg protein emulsified in Freund's incomplete adjuvant, twice with a 2 week interval. Two weeks later the vaccinated and non-vaccinated control animals were challenged orally with B. pilosicoli strain WesB. Both proteins induced systemic and local colonic IgG antibody responses, and, following experimental infection, the cumulative number of colonization days was significantly (P<0.001) less in both groups of vaccinated mice compared to the control mice. There were significantly (P=0.012) fewer mice colonized in the group vaccinated with P-1 than in the non-vaccinated control group. The results suggest that oligopeptide-binding proteins may have potential for use as components of vaccines for B. pilosicoli.
Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Brachyspira/inmunología , Brachyspira/aislamiento & purificación , Proteínas Portadoras/inmunología , Colitis/prevención & control , Diarrea/prevención & control , Infecciones por Bacterias Gramnegativas/prevención & control , Lipoproteínas/inmunología , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Brachyspira/genética , Proteínas Portadoras/genética , Colitis/microbiología , Recuento de Colonia Microbiana , ADN Bacteriano/química , ADN Bacteriano/genética , Diarrea/microbiología , Heces/microbiología , Femenino , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Mucosa Intestinal/química , Mucosa Intestinal/inmunología , Lipoproteínas/genética , Masculino , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de Secuencia de ADN , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
This article provides an overview of developments in approaches to identify novel bacterial components for use in recombinant subunit vaccines. In particular it describes the processes involved in "reverse vaccinology", and some associated complementary technologies such as proteomics that can be used in the identification of new and potentially useful vaccine antigens. Results obtained from the application of these new methods are forming a basis for a new generation of vaccines for use in the control of bacterial infections of humans and animals.
Asunto(s)
Antígenos Bacterianos/genética , Infecciones Bacterianas/prevención & control , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Genómica , Animales , Bacterias/genética , Bacterias/patogenicidad , Infecciones Bacterianas/veterinaria , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Investigación Biomédica , Genoma Bacteriano , Humanos , Proteómica , Vacunas de Subunidad , Vacunas SintéticasRESUMEN
Previously, a clpX gene encoding a predicted 67 kDa membrane-associated ATPase subunit of the Clp protease (ClpX) was identified in a porcine strain (95/1,000) of the intestinal spirochaete Brachyspira pilosicoli. In the current study, the distribution of this large clpX gene was investigated in a collection of strains representing all seven Brachyspira spp. Using PCR with internal primers, an 878 bp portion of the gene was detected in 29 of 35 strains (83 %) of B. pilosicoli, 6 of 24 strains (25 %) of Brachyspira hyodysenteriae, 14 of 16 strains (88 %) of Brachyspira intermedia, 6 of 17 strains (35 %) of Brachyspira innocens, 1 of 6 strains (17 %) of Brachyspira murdochii, 1 of 2 strains (50 %) of Brachyspira aalborgi and not in the single strain of Brachyspira alvinipulli. The whole gene was sequenced from 20 Brachyspira spp. strains and compared with the clpX gene from B. pilosicoli 95/1,000 (GenBank accession no. AY466377). The genes had 99.3-99.7 % nucleotide sequence similarity and the predicted products had 99.7-100 % amino acid sequence similarity. The clpX gene from WesB, a human strain of B. pilosicoli, was cloned and expressed as a histidine-tagged fusion protein in Escherichia coli BL21. The purified protein was used to vaccinate mice and their sera were found to recognize the expected approximately 67 kDa protein in whole-cell preparations of WesB. Sera from mice vaccinated with formalin-treated whole-cell proteins of WesB reacted with the recombinant protein. These results indicate that ClpX is both conserved and immunogenic and hence might be useful as a subunit vaccine component for Brachyspira spp. infections. Sera from humans with no known exposure to B. pilosicoli reacted with the recombinant ClpX protein, indicating that it is unlikely to be useful as a reagent for serological detection of Brachyspira spp. infections.