RESUMO
Murine monoclonal antibodies (MAbs) against Vibrio cholerae toxin co-regulated pilus (TCP) were generated using conventional hybridoma procedures. Four hybridomas were obtained and two characterized. Hybridomas 10E10E1 and 4D6F9 secreted antibodies of the IgG2a and IgG1 isotypes, respectively, that reacted with a 24-kDa antigen corresponding to the product of the El Tor tcpA gene fused to a six Histidine tail. Additionally, MAbs produced by 4D6F9 selectively recognized the major pilin subunit (TcpA) of El Tor and O139 vibrios in western immunoblot, while MAbs from 10E10E1 also cross-reacted with classical TcpA. Furthermore, vibrios expressing TCP on their surface selectively inhibited binding of the antibodies secreted by both hybridomas to TcpA-coated microtiter plates. Thus, the MAbs reported in this work detected the structural subunit of the pilus either denatured or assembled on the bacterial surface.
Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas de Fímbrias/imunologia , Vibrio cholerae/imunologia , Animais , Anticorpos Monoclonais/química , Meios de Cultura , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/imunologia , Hibridomas , Camundongos , Camundongos Endogâmicos BALB CRESUMO
In this study, we analyzed whether attachment of Vibrio cholerae vaccine strains to human intestinal epithelial cells can induce an interleukin-8 (IL-8) response. The IL-8 transcripts were detected by PCR amplification of reverse-transcribed mRNA, and the gene product secretion was measured by an enzyme-linked immunosorbent assay. Infection of monolayers of the undifferentiated HT29-18N2 cell line with reactogenic (JBK70 and 81) and nonreactogenic (CVD103HgR and 638) vaccine strains of V. cholerae resulted in markedly higher IL-8 expression by epithelial cells exposed to reactogenic strains than by cells exposed to the nonreactogenic strains. Additionally, epithelial cells produced IL-8 transcripts following stimulation with cholera vaccine strains in a concentration-dependent manner. These results represent a new insight into the inflammatory component of reactogenicity and could be used as a predictive marker of vaccine reactogenicity prior to human testing.
Assuntos
Vacinas contra Cólera , Interleucina-8/genética , Mucosa Intestinal/metabolismo , Vibrio cholerae/fisiologia , Aderência Bacteriana , Células HT29 , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In recent clinical assays, our cholera vaccine candidate strain, Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the thyA gene from V. cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the celA marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.
Assuntos
Vacinas contra Cólera/imunologia , Timidilato Sintase/genética , Vibrio cholerae/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Coelhos , Vacinas Atenuadas/imunologia , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
Vibrio cholerae 638 (El Tor, Ogawa), a new CTXPhi-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers. In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638. Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools. The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers. V. cholerae 638, at doses ranging from 4 x 10(7) to 2 x 10(9) vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.
Assuntos
Toxina da Cólera/imunologia , Vacinas contra Cólera/imunologia , Metaloendopeptidases/imunologia , Vacinas Sintéticas/imunologia , Vibrio cholerae/imunologia , Adulto , Animais , Bacteriófagos/genética , Vacinas contra Cólera/efeitos adversos , Humanos , Masculino , Metaloendopeptidases/genética , Mutagênese , Coelhos , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vibrio cholerae/virologiaRESUMO
The recent spread of El Tor cholera to America augments the need for an effective, safe and economical vaccine. In the present paper we describe the construction of live attenuated V. Cholerae strains by specifically deleting the genes encoding cholera toxin and other putative toxins from the bacterial chromosome. To maximize the likelihood of exposing protective antigens relevant to currently circulating vibrios we selected for genetic manipulation recent epidemic V. cholerae isolates from Peru. The mutant strains did not produce cholera toxin in vitro and in vivo. Deletion of the virulence cassette was accompanied by marked attenuation in the infant mouse cholera model. A selected El Tor Ogawa candidate vaccine strain was refractory to acquisition of foreign genes by conjugation with toxigenic vibrios.