Determinants of immunogenicity and mechanisms of protection by virulent and mutant Vibrio cholerae O1 in rabbits.

The colonizing capacities of 16 Vibrio cholerae strains, including nine genetically and/or phenotypically defined parent-mutant pairs, were determined in unobstructed adult rabbit small bowel. There were marked interstrain differences in colonizing capacity, which was enhanced by bacterial motility and the production of cholera holotoxin but was unrelated to production of cholera toxin B-subunit or hemolysin or to bacterial serotype or biotype. The role of colonizing capacity and other bacterial features in determining the immunizing efficiency of live V. cholerae was studied by determining the efficiency with which graded inocula of each strain immunized against attempted recolonization of the ileum or induction of choleralike diarrhea by the RITARD (removable intestinal tie-adult rabbit diarrhea) challenge technique using standard inocula of virulent V. cholerae. Mucosal colonizing capacity was the only quantitative predictor of bacterial immunizing capacity; none of the other bacterial features cited above influenced bacterial immunogenicity against either type of challenge, except as they affected colonizing capacity. Live V. cholerae immunized much more efficiently than Formalin-killed bacteria; the former caused marked protection after a single inoculum of 10(2) CFU, whereas the latter gave only partial protection after three inoculations of 10(11) killed organisms. Protection induced by live bacteria was due largely to resistance to colonization and included marked inhibition of bacterial growth within the bowel lumen. These findings strongly suggest that an optimally efficient oral cholera vaccine would be composed of avirulent live V. cholerae selected for their capacity to colonize the small-bowel mucosa.     

Colonization of the rabbit small intestine by clinical and environmental isolates of non-O1 Vibrio cholerae and Vibrio mimicus.

We examined the capability of 12 isolates of non-cholera toxin-producing O1 and non-O1 Vibrio cholerae to colonize the small intestine of adult rabbits and cause diarrhea. Using the removable intestinal tie-adult rabbit diarrhea model, we found that eight environmental isolates that showed no or marginal biological activity in other diarrhea models (rabbit ileal loop, infant rabbit, and suckling mouse) appeared to be incapable of attaching to and colonizing, even transiently, the small intestinal mucosa of animals with normal clearance mechanisms. In contrast, three clinical isolates attached, proliferated rapidly, and colonized mucosal surfaces of the entire small intestine within 8 h of challenge. This led to diarrhea with strikingly high rates of mortality compared with that of rabbits given similar challenges doses with strains of O1 V. cholerae that produce cholera toxin and Vibrio mimicus, which produces a toxin similar to cholera toxin. We have further demonstrated that multiple exposures to enteric infection by these strains elicited local and serum antibodies that reacted strongly with cell surface antigens of the homologous strain and showed a high degree of cross-reactivity against the cell surface antigens of the two heterologous strains. The enteric infections appeared to engender protection against subsequent infection as well, as evidenced by reduced incidence of diarrhea and duration of fecal shedding of the challenge organism upon subsequent challenges.     

Vibrio cholerae O139 conjugate vaccines: synthesis and immunogenicity of V. cholerae O139 capsular polysaccharide conjugates with recombinant diphtheria toxin mutant in mice

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to < or =50. Treatment with 2-ME reduced the titers of 17 of 20 patients to < or =50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.     

Vibrio cholerae non-serogroup O1 cystitis.

We report a case of a patient who developed cystitis caused by non-serogroup O1 Vibrio cholerae after swimming in the Chesapeake Bay. Treatment was empirical, with complete symptomatic resolution. Genitourinary tract infections by Vibrio spp. are uncommon but should be considered when cystitis occurs after saltwater exposure in appropriate geographic regions.     

Vibrio cholerae O1 isolated in Kenya.

Biological and serological analyses of 272 isolates of Vibrio cholerae O1 from six epidemics and from a few sporadic cases in Kenya were carried out. All of the isolates were identified as V. cholerae biotype E1 Tor, and 210 out of 272 isolates were hemolytic as examined by Feeley's method.     

Construction of a recombinant live oral vaccine from a non-toxigenic strain of Vibrio cholerae O1 serotype inaba biotype E1 Tor and assessment of its reactogenicity and immunogenicity in the rabbit model.

The disease cholera is an important cause of mortality in many developing countries. Though it can be controlled through improved sanitation, this goal is not easily attainable in many countries. Development of an efficacious vaccine offers the best immediate solution. A new oral candidate vaccine has been constructed from a non-toxigenic strain of Vibrio cholerae E1 Tor, Inaba, which is not only devoid of the cholera toxin (CT) virulence cassette but also is completely non-reactogenic in rabbit ileal loop assay. The strain, however, had toxR and tcpA genes. Through a series of manipulations, the ctxB gene of V. cholerae, responsible for the production of the 'B' subunit of the cholera toxin (CTB) was introduced into the cryptic hemolysin locus of the strain. The resulting strain, named vaccine attempt 1.3 (VA1.3), was found to be able to produce copious amounts of CTB. In the RITARD model this strain was found to be non-reactogenic and provided full protection against the challenge doses of both V. cholerae O1, classical and E1 Tor. In the immunized rabbit it invoked significant levels of anti-bacterial and anti-toxin immunity.     

Nucleotide sequence encoding the mannose-fucose-resistant hemagglutinin of Vibrio cholerae O1 and construction of a mutant.

The region of DNA encoding the mannose-fucose-resistant hemagglutinin (MFRHA) of Vibrio cholerae O1 has been localized, and the nucleotide sequence has been determined. The region contains a single open reading frame encoding 230 amino acids, corresponding to a protein of 26.9 kDa. The N terminus of this protein is atypical for a protein localized in the outer membrane. A mutant lacking MFRHA activity has been constructed by allelic exchange after inactivation via the insertion of a kanamycin resistance gene cartridge. The MFRHA-negative mutant has been assessed for virulence in the infant mouse cholera model. This mutant shows a marked defect in its ability to persist in the infant mouse gut and is incapable of competing with the wild-type organism, even when given in 25-fold excess. This defect also leads to a > 100-fold increase in the 50% lethal dose. These data suggest that the MFRHA is an important colonization factor in the infant mouse model.     

Cell-associated hemagglutinin-deficient mutant of Vibrio cholerae.

Cell-associated hemagglutinin-negative mutants were derived from cholera enterotoxin-negative Vibrio cholerae JBK70 by Tn5 mutagenesis. One of the mutants identified, SB001, was characterized in greater detail. Its ability to colonize ilea of adult rabbits was determined by feeding approximately 10(8) V. cholerae to each animal. At 17 h after feeding, the numbers of viable vibrios in the ilea were determined. There was a significant, 4 log, decrease in the ability of the hemagglutinin-negative mutant to colonize ileal tissue compared with the parent strain JBK70. In addition, the higher levels of colonization attained by JBK70 and the wild-type parent of JBK70, N16961, were associated with intestinal fluid accumulation and death. Rabbits immunized orally with approximately 10(8) SB001, when challenged 3 weeks later with either homologous biotype and serotype El Tor Inaba N16961 or heterologous Classical Ogawa 395, were protected to the same extent as those animals immunized with either the challenge strain or JBK70. This was evidenced by decreases in both the number of animals showing detectable colonization and the level of colonization achieved. A hemagglutinin-negative mutant of V. cholerae may therefore be of potential use as a live oral vaccine against cholera.     

Comparative study of Vibrio cholerae non-O1 protease and soluble hemagglutinin with those of Vibrio cholerae O1.

Protease and soluble hemagglutinating activities produced by a non-O1 Vibrio cholerae strain isolated from a patient with diarrhea were compared with similar activities produced by V. cholerae O1. The soluble protease activities were indistinguishable in heat stability, immunodiffusion, inhibition by antiserum, and electrophoretic analysis. On the other hand, the soluble hemagglutinating activities of both strains were not completely identical. The hemagglutinating activity of the non-O1 V. cholerae strain was not inhibited by Zincov; it was more sensitive to inhibition by normal serum, and it had an unusual pattern of heat stability. Heating at 100 degrees C resulted in some recovery of activity of a sample previously inactivated by heating at 60 degrees C.     

Biotype-specific probe for Vibrio cholerae serogroup O1.

The O1 serogroup of Vibrio cholerae can be divided into two biotypes, El Tor and Classical. Current tests to distinguish between these biotypes are often difficult to interpret. On the basis of the difference in sequence of the hlyA gene in these biotypes, we have developed a simple probe that can easily and reliably differentiate between the two biotypes.     

Vibrio cholerae serogroup O1 in northeast Thailand

Strains of Vibrio cholerae serogroup O1 biotype El Tor that are susceptible to Mukerjee cholera bacteriophage group IV (S. Mukerjee, Bull. W.H.O. 28:333-336, 1963) were found. Cholera vibrios isolated from epidemics in northeast Thailand were characterized, and 57 of 60 strains isolated in 1986 were susceptible to cholera phage IV. However, all 113 strains isolated in 1988 were not susceptible to the phage. All isolates in both epidemics revealed behaviors typical of El Tor vibrios, except phage IV susceptibility in the 57 strains. Although the plaques of phage IV were generally translucent, plaques on some isolates looked transparent, just like those on classical vibrios. The organisms grown in the plaques were lysogenized. If this kind of strain is frequently isolated, the biotype of V. cholerae O1 should be reconsidered.     

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.     

Monoclonal antibodies to Vibrio cholerae O1 serotype inaba.

Eighteen monoclonal antibodies were generated from mice immunized with Vibrio cholerae O1 serotype Inaba. Reactivities of these antibodies were investigated by slide agglutination, microdilution agglutination, and passive hemagglutination tests. Although all antibodies reacted to the Inaba-type cells, reactivity to Ogawa cells showed some variation. These antibodies were roughly subdivided into three groups by slide agglutination tests and microdilution agglutination tests by using type-specific standard strains. The first group showed almost identical reactivities to both the Ogawa- and the Inaba-type cells, while the second group reacted to Inaba-type cells but only very weakly reacted to Ogawa-type cells. The third group reacted only to Inaba-type cells; no agglutination was observed for Ogawa-type cells in either the slide agglutination or the microdilution agglutination tests. Most of the third group showed cross-reactivity to Brucella abortus and Yersinia enterocolitica O9. Results of passive hemagglutination tests showed that all 18 antibodies were to the lipopolysaccharide of V. cholerae O1.     

Construction and characterization of a potential live oral carrier-based vaccine against Vibrio cholerae O139.

The rfb region from Vibrio cholerae O139 strain MO45 was cloned from cosmid gene banks established in Escherichia coli HB101, using an immunoblot assay for screening of the correct clones. Immunoblot analysis of lipopolysaccharide (LPS) preparations revealed the presence of two types of positive clones: (i) those expressing only a short core-linked O polysaccharide (SOPS) and (ii) those also expressing a highly polymerized capsular polysaccharide (CPS) not bound to the E. coli K-12 LPS core. In addition, the latter clones appear to contain a locus which may encode a putative regulator of SOPS and CPS chain length. Further characterization in E. coli showed that CPS constitutes a barrier against large particles such as the bacteriophage Ffm but not against bacteriophage lambda or P1. In addition, a portion of the K-12 LPS core may not be substituted with SOPS. Loci associated with the two clonal types were transferred into V. cholerae CH19, an rfbAB deletion mutant of CVD103-HgR deficient in the production of the homologous Inaba O polysaccharide. This resulted in the stable expression of SOPS, alone or together with CPS, that was indistinguishable from that of wild-type V. cholerae O139. Strains CH25 and CH26, which correspond to CH19 bearing the V. cholerae O139 rfb region integrated into the chromosome, were found to be genetically stable and essentially identical to the parent CVD103-HgR with respect to physiological properties such as cell motility, mercury resistance, toxicity, and production of the cholera toxin B subunit. Rabbits immunized with CH25 elicited high titers of anti-O139 SOPS- and CPS-specific serum antibodies. These strains possess characteristics desirable in candidate live oral vaccines against V. cholerae O139.     

Electron microscopic study of Vibrio cholerae O1 adherence to the mucus coat and villus surface in the human small intestine.

Vibrio cholerae O1, irrespective of the biotype or serotype, adhered to and was entrapped in the mucus coat covering the mucosal surface of isolated human ileal segments. The evidence for such mucus coat adherence was obtained by treatment of the ileal segments with 10% Formalin. In any case, adherence to the mucus coat was much more prominent than adherence to the epithelial cell surface of the small intestinal villi. Mucus coat adherence was affected by sugars and by the growth phase of the bacterial culture and was diminished by the heating of V. cholerae O1. We conclude that the small intestinal mucus coat is a primary adherence target for V. cholerae O1 in human infection and that the cell-associated hemagglutinin of V. cholerae O1 plays a role, at least in part, in adherence.     

Enteropathogenicity of non-toxigenic Vibrio cholerae O1 for adult mice.

The enteropathogenic potential of 32 Vibrio cholerae O1 isolates that do not produce cholera toxin was examined in the orally inoculated, sealed adult mouse model. Live cultures (2 x 10(10) cfu/ml) of 7/16 clinical and 6/16 environmental isolates produced a positive intestinal fluid accumulation (FA) ratio that reached near maximum at approximately 5 h post-inoculation. Colony hybridization did not detect genes for cholera toxin, Escherichia coli heat-labile and heat-stable toxins, or shiga-like toxins. FA activity did not correlate precisely with cytotoxic activities on Chinese hamster ovary (28/32 positive), Vero (29/32) or HeLa (25/32) cells. Certain clinical and environmental isolates of non-toxigenic V. cholerae O1 appear to be enteropathogenic for the mouse, providing evidence that they may have pathogenic potential for humans through an as yet undefined mechanism(s).     

Safety and immunogenicity of a booster dose of Vibrio cholerae CVD 103-HgR live oral cholera vaccine in Swiss adults.

Adult volunteers received a booster dose (4 x 10(8) CFU) of attenuated Vibrio cholerae CVD 103-HgR oral vaccine 15 or 24 months after primary immunization. The immune response was modest, presumably due to rapid clearance of the vaccine strain by a primed immune system.     

Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.

Vibrio cholerae serogroup O139, now considered to be the second organism capable of causing epidemic severe dehydrating cholera, contains a capsular polysaccharide which makes it difficult for it to be used in the conventional vibriocidal antibody assay optimized for V. cholerae O1. After modification of the procedure, which involved the use of specific bacterial strains, a lower bacterial inoculum, and increased amounts of complement, the vibriocidal antibody responses to V. cholerae O139 were measured in acute- and convalescent-phase sera from 33 V. cholerae O139-infected and 18 V. cholerae O1-infected patients and in single serum samples from 20 healthy control subjects. The responses in these individuals to V. cholerae O1 strains were also determined. Significant elevations in the homologous antibody response were found only in the convalescent-phase sera from both groups of patients with cholera. These findings may explain the basis for the lack of heterologous protection between the two serogroups of V. cholerae. Healthy controls had higher background levels of vibriocidal antibody to V. cholerae O1 than to V. cholerae O139.