An approach to a cold chain free oral cholera vaccine: in vitro and in vivo characterization of Vibrio cholerae gastro-resistant microparticles.

The present work describes the formulation of Eudragit(®) L30 D-55 microparticles (MP) alone or with mucoadhesive agents, alginate or Carbopol(®), as an approach for the development of an oral cholera vaccine. In the first part, a spray drying technique was optimized for microparticle elaboration, obtaining a microparticle size ranging from 7 to 9 µm with high encapsulation efficiencies. Moreover, gastro resistant properties and Vibrio cholerae (VC) antigenicity were maintained, but for Eudragit(®)-Carbopol(®) microparticles which showed low antigenicity values, ≈25%. Next, a stability study was performed following ICH Q1 A (R2) guidelines, i.e. 25°C-60% relative humidity (RH) for 12 months, and 30°C-65% RH and 40°C-75% RH for 6 months. Upon storage, microparticle size changed slightly, 1 µm for Eudragit(®)-alginate MPs and 0.36 µm for Eudragit(®)MP. However, gastro resistance and antigenicity values were kept in an acceptance range. In the third stage of this work, in vivo experiments were performed. The immune response evoked was measured by means of vibriocidal titer quantification, observing that Eudragit(®)-alginate MPs were able to induce stronger immune responses, comparable to the free VC. Therefore, microencapsulation of VC by spray drying could be proposed as an approach to a cold chain free and effective oral cholera vaccine.

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.

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.

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.