Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae.

Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10(6) and 10(8) CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.

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.

Differential interleukin-8 response of intestinal epithelial cell line to reactogenic and nonreactogenic candidate vaccine strains of Vibrio cholerae.

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.

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.

Genetic manipulation of Vibrio cholerae for vaccine development: construction of live attenuated El Tor candidate vaccine strains.

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.