Formulation in tablets of a cholera whole cells inactivated vaccine candidate.

Licensed as well as candidate cholera vaccines available at the present requires the dose preparation (included buffer) at the moment of application. The aim of this work was to evaluate the presentation in oral tablets of an inactivated cholera vaccine to avoid that inconveniences during application. We have therefore compared inactivated cultures of Vibrio cholerae with tablets formulation vaccine. We obtained that antigenic activity (ELISA) and immunogenicity in animal model (ELISA and vibriocidal tests) of V. cholerae inactivated cell remained unaltered in the final tablet formulation. The results suggest that the oral tablet formulation could be a useful pharmaceutical form in order to produce a new and affordable cholera vaccine.

Process development for a Cuban cholera vaccine based on the attenuated strain Vibrio cholerae 638.

Genetically modified Vibrio cholerae strain 638 (biotype El Tor, serotype Ogawa) has previously been shown to be immunogenic in animal models and in human trials. Our objective in the work reported herein was to describe the process development methods for the production of the 638 attenuated cholera vaccine. Cell seed bank, culture of biomass, lyophilization and final formulation were processes were developed. The results show kinetics of culture that fulfils a logistical model. The microbiological properties, colonizing capability, immunogenicity and non-toxigenicity of the final product were indistinguishable from the properties of the working seed lot. We conclude that the non-reactogenic, immunogenic and protective strain 638 is robust and can withstand the fermentation processes required for large-scale production of a vaccine.

Production and characterization of monoclonal antibodies to E1 Tor toxin co-regulated pilus of Vibrio cholerae.

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.

Anticuerpos IgA anti-lipopolisacárido inducidos por candidato vacunal oral 638 vivo / Mucosal IgA anti-lipopolysaccharide antibodies induced by 638 oral live attenuated candidate vaccine

La cepa de Cólera atenuada 638 ha inducido una buena respuesta en modelos animales y en un estudio piloto humano ha probado ser segura e inmunogénica. Sin embargo, no ha sido evaluada la IgA específica en mucosas ni tampoco se ha comparado la respuesta inducida por la cepa 638 con aquélla inducida por la conocida cepa reactogénica JBK70. Por ello, fueron evaluadas las células secretoras de anticuerpos (ASC) anti-lipopolisacárido (LPS) sanguíneas y los anticuerpos antiLPS en saliva como indicadores de respuestas mucosas de voluntarios inoculados con las cepas 638 o JBK70. Con vistas a determinar la producción local o no de la IgA específica, la cinética de los anticuerpos IgA anti-LPS séricos y salivares fueron comparados. La respuesta vibriocida sérica fue también medida. Tres grupos con 638 (109, 108 y 107 unidades form adoras de colonias, CFU), uno con JBK70 (109 CFU) y otro con placebo fueron enrolados. La respuesta sérica de ASC IgA+ fue mayor que la de ASC IgG+.La IgA anti-LPS en saliva tuvo valores máximos a los 9 días y decayó hasta valores negativos en el día 14 después de la inoculación. La IgA anti-LPS sérica permanece elevada entre los 7 y 28 días después de la inoculación lo que sugiere que la IgA en saliva es localmente y transitoriamente producida. La respuesta vibriocida sérica fue incrementada después de la inoculación. Respuestas similares fueron obtenidas con las cepas 638 y JBK70 (AU)

Construction and characterization of a nonproliferative El Tor cholera vaccine candidate derived from strain 638.

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.

Passive protection of serum from volunteers inoculated with attenuated strain 638 of Vibrio cholerae O1 in animal models.

As part of the studies to obtain an oral vaccine against cholera disease, the protective effect of serum from volunteers inoculated in a controlled trial with a candidate live attenuated vaccine of Vibrio cholerae O1, El Tor Ogawa (638; CTXφ mutant, hap::celA), was tested. It was confirmed that the serum, as well as the purified IgG and IgA from the volunteers had a protective effect in both of the animal models used, although the purified antibodies needed the presence of complement to be protective. These results emphasize the expectations about the protective potential of the candidate in challenge studies in humans to be conducted very soon.

Preliminary assessment of the safety and immunogenicity of a new CTXPhi-negative, hemagglutinin/protease-defective El Tor strain as a cholera vaccine candidate.

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.

Production and characterization of a monoclonal antibody against mannose-sensitive hemagglutinin of Vibrio cholerae.

We have generated murine monoclonal antibodies (MAb) against Vibrio cholerae mannose-sensitive hemagglutinin (MSHA) using conventional hybridoma procedures. Seven hybridomas were obtained and one characterized. Hybridoma 2F12/F1 secreted an antibody of the IgG3 type that reacted with a 17-kDa antigen corresponding to the product of the mshA gene. This MAb inhibited mannose-sensitive agglutination of chicken erythrocytes by EL tor and O139 vibrios. Vibrios expressing MSHA activity inhibited binding of the antibody secreted by 2F12/F1 to MSHA-coated microtiter plates.