Oral immunization with attenuated vaccine strains of Vibrio cholerae expressing a dodecapeptide repeat of the serine-rich Entamoeba histolytica protein fused to the cholera toxin B subunit induces systemic and mucosal antiamebic and anti-V. cholerae antibody responses in mice.

Entamoeba histolytica is a significant cause of morbidity and mortality worldwide. The serine-rich E. histolytica protein (SREHP) is a surface-expressed trophozoite protein that includes multiple hydrophilic tandem repeats. A purified fusion protein between the dodecapeptide repeat of SREHP and cholera toxin B subunit (CTB) has previously been shown to be immunogenic in mice after oral inoculation when cholera toxin is coadministered as an immunoadjuvant. We engineered a live attenuated El Tor Vibrio cholerae vaccine strain, Peru2, to express the SREHP-12-CTB fusion protein to the supernatant from either a plasmid [Peru2 (pETR5.1)] or from a chromosomal insertion (ETR3). Vector strains were administered orally to germfree mice that were subsequently housed under nongermfree conditions; mice received one (day 0) or two (days 0 and 14) inoculations. No immunoadjuvant or cholera holotoxin was administered. Mice that received two inoculations of Peru2(pETR5.1) had the most pronounced antiamebic systemic and mucosal immunologic responses. Less marked, but significant, anti-SREHP serum immunoglobulin G antibody responses were also induced in mice that received either one or two oral inoculations of strain ETR3. Anti-V. cholerae responses were also induced, as measured by the induction of serum vibriocidal antibodies and by serum and mucosal anti-CTB antibody responses. These results suggest that V. cholerae vector strains can be successful delivery vehicles for the SREHP-12-CTB fusion protein, to induce mucosal and systemic antiamebic and anti-V. cholerae immune responses. The magnitude of these responses is proportional to the amount of SREHP-12-CTB produced by the vector strain.     

In vivo expression and immunoadjuvancy of a mutant of heat-labile enterotoxin of Escherichia coli in vaccine and vector strains of Vibrio cholerae

Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) of Escherichia coli, the latter when expressed in V. cholerae. Both toxins are also potent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed by E. coli have been developed. One such mutant LT molecule has the substitution of a glycine residue for arginine-192 [LT(R192G)]. Live attenuated strains of V. cholerae that have been used both as V. cholerae vaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed. In order to ascertain whether LT(R192G) can act as an immunoadjuvant when expressed in vivo by V. cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains of V. cholerae, Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous antigens in the chromosome of the vaccine vectors. We found that LT(R192G) was expressed from pCS95 in vitro by both E. coli and V. cholerae strains but that LT(R192G) was detectable in the supernatant fraction of V. cholerae cultures only. In order to assess potential immunoadjuvanticity, groups of germfree mice were inoculated with the three V. cholerae vaccine strains alone and compared to groups inoculated with the V. cholerae vaccine strains supplemented with purified CT as an oral immunoadjuvant or V. cholerae vaccine strains expressing LT(R192G) from pCS95. We found that mice continued to pass stool containing V. cholerae strains with pCS95 for at least 4 days after oral inoculation, the last day evaluated. We found that inoculation with V. cholerae vaccine strains containing pCS95 resulted in anti-LT(R192G) immune responses, confirming in vivo expression. We were unable to detect immune responses directed against the heterologous antigens expressed at low levels in any group of animals, including animals that received purified CT as an immunoadjuvant. We were, however, able to measure increased vibriocidal immune responses against vaccine strains in animals that received V. cholerae vaccine strains expressing LT(R192G) from pCS95 compared to the responses in animals that received V. cholerae vaccine strains alone. These results demonstrate that mutant LT molecules can be expressed in vivo by attenuated vaccine strains of V. cholerae and that such expression can result in an immunoadjuvant effect.     

In vitro and in vivo analyses of constitutive and in vivo-induced promoters in attenuated vaccine and vector strains of Vibrio cholerae

The optimal promoter for in vivo expression of heterologous antigens by live, attenuated vaccine vector strains of Vibrio cholerae is unclear; in vitro analyses of promoter activity may not accurately predict expression of antigens in vivo. We therefore introduced plasmids expressing the B subunit of cholera toxin (CtxB) under the control of a number of promoters into V. cholerae vaccine strain Peru2. We evaluated the tac promoter, which is constitutively expressed in V. cholerae, as well as the in vivo-induced V. cholerae heat shock htpG promoter and the in vivo-induced V. cholerae iron-regulated irgA promoter. The functionality of all promoters was confirmed in vitro. In vitro antigenic expression was highest in vaccine strains expressing CtxB under the control of the tac promoter (2 to 5 microgram/ml/unit of optical density at 600 nm [OD(600)]) and, under low-iron conditions, in strains containing the irgA promoter (5 microgram/ml/OD(600)). We orally inoculated mice with the various vaccine strains and used anti-CtxB immune responses as a marker for in vivo expression of CtxB. The vaccine strain expressing CtxB under the control of the tac promoter elicited the most prominent specific anti-CtxB responses in vivo (serum immunoglobulin G [IgG], P 相似文献   

Optimizing the germfree mouse model for in vivo evaluation of oral Vibrio cholerae vaccine and vector strains

The germfree mouse model of Vibrio cholerae infection can be used to judge immune responses to V. cholerae vaccine and vector strains. In the original model, a single oral inoculation was administered on day 0, a booster oral inoculation was administered on day 14, and immune responses were analyzed with samples collected on day 28. Unfortunately, immune responses in this model frequently were low level, and interanimal variability occurred. In order to improve this model, we evaluated various primary and booster V. cholerae inoculation schedules. The most prominent systemic and mucosal antibody responses were measured in mice that received a multiple primary inoculation series on days 0, 2, 4, and 6 and booster inoculations on days 28 and 42. These modifications result in improved preliminary evaluation of V. cholerae vaccine and vector strains in mice.     

宋内痢疾菌无毒株S7表达霍乱弧菌O抗原及霍乱毒素B亚单位工程菌的构建

以宋内氏痢疾菌无毒株S7作为受体,将含有编码霍乱弧菌O抗原及霍乱毒素B亚单位基因的质粒转移入其中,构建成重组菌株S7COB。质粒电泳图谱显示重组株中存在被转移的外源质粒。重组株的生化特性和毒力表型与受体相同。菌体凝集和全菌体酶联免疫吸附试验(ELISA)证明在重组株的菌体表面同时表达了霍乱弧菌和宋内氏痢疾菌的O抗原。GM1-ELISA表明该重组株能在细胞内表达霍乱毒素B亚单位。以5亿、10亿的重组株免疫LIBP品系小鼠,免疫小鼠对宋内氏毒株S63攻击的保护率为70％～100％,对霍乱弧菌毒株569B攻击的保护率为30％～54％。     

Immunogenicity and protective role of an IgA reactive 31-kDa antigen of Vibrio cholerae O139

Immunoglobulin A (IgA) is important in protective immunity against infection by Vibrio cholerae. In this study, the immune response to and protective role of a 31-kDa antigen of V. cholerae O139, reacting with IgA antibodies present in the sera of cholera patients and common to V. cholerae strains O139 and O1 was evaluated in BALB/c mice. From the various antigens of V. cholerae O139 and V. cholerae O1 which reacted with IgA antibodies in sera of a cholera patient, a 31-kDa common antigen was selected and purified by DEAE-Sepharose CL 6B column chromatography. Oral administration of live V. cholerae O139 in BALB/c mice elicited an IgA response to the 31-kDa antigen in serum and intestinal fluid, and a proliferation of the splenic lymphocytes on stimulation with the same antigen. The cytokine profile of these splenic lymphocytes revealed a shift from a mixed Th1 and Th2 response--interleukin-10 (IL-10) and interferon-gamma--in the first week after infection to a Th2 type of response--IL-10--in the third week. In passive protection studies, hyperimmune serum to the 31-kDa antigen was able to protect infant mice against challenge with O139 and O1 strains. These results demonstrate the ability of the 31-kDa antigen of V. cholerae O139 to induce humoral and cellular immune responses in mice, and its immunoprotective nature.     

Development of a DeltaglnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae

We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a DeltaglnA derivative of V. cholerae vaccine strain Peru2. This mutant strain, Peru2DeltaglnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1. 8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus. Strain Peru2DeltaglnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2DeltaglnA, Peru2DeltaglnA(pKEK71-NotI), Peru2(pTIC5), Peru2DeltaglnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2DeltaglnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals. However, specific anti-CtxB immune responses in serum (P 相似文献   

Vibrio cholerae O1 infection induces proinflammatory CD4+ T-cell responses in blood and intestinal mucosa of infected humans

Vibrio cholerae O1 is a noninvasive enteric pathogen and serves as a model for studies of mucosal immunity. Although symptomatic V. cholerae infection induces durable protection against subsequent disease, vaccination with oral killed whole-cell V. cholerae stimulates less long-lasting protection against cholera. In this study, we demonstrated that cholera induces an early proinflammatory cellular immune response that results in priming of Th1- and Th17-type cytokine responses to ex vivo antigenic stimulation and an increase in the ratio of Th1 to Th2 CD4(+) T-cell responses. Comparable priming of Th1 and Th17 responses, with an increased ratio of Th1 to Th2 CD4(+) T-cell responses, was not observed in subjects who received two doses of the oral cholera vaccine Dukoral (a whole-cell cholera toxin B subunit containing [WC-CTB] vaccine). These findings suggest that natural V. cholerae infection induces an early, proinflammatory cellular immune response, despite the apparent lack of clinical signs of inflammation. The failure of the WC-CTB vaccine to activate equivalent, CD4(+) T-cell responses is a potential explanation for the shorter duration of protection following immunization with this vaccine. Additional studies are needed to determine whether these early T-cell-mediated events predict the subsequent duration of immunologic memory.     

Comparison of memory B cell, antibody-secreting cell, and plasma antibody responses in young children, older children, and adults with infection caused by Vibrio cholerae O1 El Tor Ogawa in Bangladesh

Children bear a large component of the global burden of cholera. Despite this, little is known about immune responses to cholera in children, especially those under 5 years of age. Cholera vaccine studies have demonstrated lower long-term protective efficacy in young children than in older children and adults. Memory B cell (MBC) responses may correlate with duration of protection following infection and vaccination. Here we report a comparison of immune responses in young children (3 to 5 years of age; n = 17), older children (6 to 17 years of age; n = 17), and adults (18 to 60 years of age; n = 68) hospitalized with cholera in Dhaka, Bangladesh. We found that young children had lower baseline vibriocidal antibody titers and higher fold increases in titer between day 2 and day 7 than adults. Young children had higher baseline IgG plasma antibody levels to Vibrio cholerae antigens, although the magnitudes of responses at days 7 and 30 were similar across age groups. As a surrogate marker for mucosal immune responses, we assessed day 7 antibody-secreting cell (ASC) responses. These were comparable across age groups, although there was a trend for older age groups to have higher levels of lipopolysaccharide-specific IgA ASC responses. All age groups developed comparable MBC responses to V. cholerae lipopolysaccharide and cholera toxin B subunit at day 30. These findings suggest that young children are able to mount robust vibriocidal, plasma antibody, ASC, and MBC responses against V. cholerae O1, suggesting that under an optimal vaccination strategy, young children could achieve protective efficacy comparable to that induced in adults.     

Transcutaneous immunization with toxin-coregulated pilin A induces protective immunity against Vibrio cholerae O1 El Tor challenge in mice

Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 mug of TcpA or TcpA with an immunoadjuvant (cholera toxin [CT], 50 mug) on days 0, 19, and 40. Mice immunized with TcpA alone did not develop anti-TcpA responses. Mice that received transcutaneously applied TcpA and CT developed prominent anti-TcpA immunoglobulin G (IgG) serum responses but minimal anti-TcpA IgA. Transcutaneous immunization with CT induced prominent IgG and IgA anti-CT serum responses. In an infant mouse model, offspring born to dams transcutaneously immunized either with TcpA and CT or with CT alone were challenged with 10(6) CFU (one 50% lethal dose) wild-type V. cholerae O1 El Tor strain N16961. At 48 h, mice born to females transcutaneously immunized with CT alone had 36% +/- 10% (mean +/- standard error of the mean) survival, while mice born to females transcutaneously immunized with TcpA and CT had 69% +/- 6% survival (P < 0.001). Our results suggest that transcutaneous immunization with TcpA and an immunoadjuvant induces protective anti-TcpA immune responses. Anti-TcpA responses may contribute to an optimal cholera vaccine.     

Vibrio factors cause rapid fluid accumulation in suckling mice.

Non-O-1 and O-1 Vibrio cholerae and Vibrio fluvialis isolated from clinical and environmental sources were examined for virulence factor production in 3-day-old suckling mice and in Y-1 tissue culture. The responses of the suckling mice to intragastrically administered bacterial cultures were measured by intestinal fluid accumulation (FA), diarrhea, and mortality. Regardless of the O-serovar, source of isolation, or ability to produce cholera toxin, all strains of V. cholerae stimulated increased FA, which was measurable in the mice at 4 h post-inoculation. The factor(s) causing these symptoms was found to be distinct from cholera toxin by the kinetics of FA and serological difference from cholera toxin based on in vivo neutralization tests. In most instances, FA was followed by high rates of mortality. Y-1 mouse adrenal tumor cell assays also showed that many V. cholerae produced extracellular heat-labile cytotoxic factor(s), and many cholera toxin-negative strains also caused a cytotonic-like morphological response. The majority of V. fluvialis strains produced smaller amounts of cytotoxic factor(s) but no cytotoxic reactions. The factor which stimulates rapid FA in suckling mice could be one of several virulence-associated factors contributing to diarrheal disease by nontoxigenic vibrios, but this is not verified at present.     

Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems.

Shiga-like toxin I (SLT-I) B-subunit expression was examined by using the trc promoter in two different constructs, pSBC32 and pSBC54, in which 710 bp of DNA downstream of the B subunit in pSBC32 was deleted. The trc promoter in pSBC54 was replaced also with the SLT-I iron-regulated promoter to create a third plasmid, pSBC61. SLT-I B-subunit expression was examined from all three plasmids following transfer into Escherichia coli JM105 and the cholera toxin A-subunit gene deletion mutant Vibrio cholerae 0395-N1. The SLT-I B subunit was expressed from all constructs. pSBC61 was regulated by elemental iron and produced equivalent amounts of SLT-I B subunit from both E. coli and V. cholerae. In contrast to the cholera toxin B subunit, virtually all released into the medium, the SLT-I B subunit was predominantly cell associated in the pSBC61 constructs. Both pSBC32 and pSBC54 were inducible with isopropyl-beta-D-thiogalactopyranoside (IPTG) in the E. coli background but not the V. cholerae background; however, when E. coli cultures were allowed to grow for 24 h, the yield of SLT-I B subunit was not increased by IPTG induction. Both pSBC32 and -54 expressed more SLT-I B subunit in the V. cholerae host than in the E. coli host. Scale-up to a 9.9-liter fermentor culture of V. cholerae 0395 N1 (pSBC32) resulted in the isolation of 220 mg of SLT-I B. The purified B subunit was identical, in terms of binding to Vero cells, stoichiometry after chemical cross-linking, and ability to inhibit cytotoxicity of intact Shiga toxin, to native SLT-I B subunit from E. coli O157:H7.     

Immunological responses of rabbits to various somatic and secreted antigens of Vibrio cholerae after intra-duodenal inoculation

The immunological responses of rabbits after intra-duodenal immunisation with live Vibrio cholerae organisms were studied in various body fluids. Serum, bile and intestinal samples were collected from rabbits at different times (1-8 weeks) after immunisation. Three different extracts from the small intestine were prepared. At first the small intestine was washed with saline (method A). Later, ultrasonic lysates were prepared from epithelial cells separated with citrate (method B) and of mucosal tissue above the muscularis layer (method C). All had agglutinating activities against V. cholerae strains belonging to both biotypes (classical and el tor) and both serotypes (Ogawa and Inaba). Levels in intestinal extracts, sera and bile of antibodies to somatic (lipopolysaccharide and cell-surface proteins) and secreted (cholera toxin and neuraminidase) antigens of V. cholerae were determined by an enzyme-linked immunosorbent assay. All contained antibodies to these antigens; although both IgA and IgG were present, IgA predominated. Serum and bile samples contained mainly IgG and IgA respectively. Immunoblotting studies demonstrated that the antisera contained antibodies to most cell-surface proteins and to cholera toxin. Cell-surface proteins appeared to be the major cross-reacting somatic antigens of V. cholerae.     

Live attenuated oral cholera vaccines

Live, orally administered, attenuated vaccine strains of Vibrio cholerae have many theoretical advantages over killed vaccines. A single oral inoculation could result in intestinal colonization and rapid immune responses, obviating the need for repetitive dosing. Live V. cholerae organisms can also respond to the intestinal environment and immunological exposure to in vivo expressed bacterial products, which could result in improved immunological protection against wild-type V. cholerae infection. The concern remains that live oral cholera vaccines may be less effective among partially immune individuals in cholera endemic areas as pre-existing antibodies can inhibit live organisms and decrease colonization of the gut. A number of live oral cholera vaccines have been developed to protect against cholera caused by the classical and El Tor serotypes of V. cholerae O1, including CVD 103-HgR, Peru-15 and V. cholerae 638. A number of live oral cholera vaccines have also been similarly developed to protect against cholera caused by V. cholerae O139, including CVD 112 and Bengal-15. Live, orally administered, attenuated cholera vaccines are in various stages of development and evaluation.     

Mucosal immunologic responses in cholera patients in Bangladesh

Vibrio cholerae O1 causes dehydrating diarrhea with a high mortality rate if untreated. The infection also elicits long-term protective immunity. Since V. cholerae is noninvasive, mucosal immunity is likely important for protection. In this study, we compared humoral immune responses in the duodenal mucosa and blood of cholera patients at different time points after the onset of disease and compared them with those of healthy controls (HCs). Immune responses to lipopolysaccharide (LPS) and the recombinant cholera toxin B subunit (rCTB) were assessed by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Significant increases in V. cholerae LPS-specific IgA and IgG antibody levels were seen in duodenal extracts on day 30, but the levels decreased to baseline by day 180; plasma V. cholerae LPS-specific IgA levels remained elevated longer. Levels of mucosal CTB antibodies also peaked on day 30, but the increase reached statistical significance only for IgG. A significant correlation was found between the CTB antibody-secreting cell (ASC) response in the circulatory system on day 7 and subsequent CTB-specific IgA levels in duodenal extracts on day 30 and the numbers of CTB-specific IgA ASCs in duodenal tissues on day 180. The proportion (0.07%) of mucosal V. cholerae LPS IgA ASCs peaked on day 30 and remained elevated through day 180 compared to that of HCs (P = 0.03). These results suggest that protective immunity against V. cholerae is not likely mediated by the constitutive secretion of antibodies at the mucosal surface; our results are consistent with those of other studies that suggest instead that anamnestic immune responses of mucosal lymphocytes may play a major role in protection against cholera.     

Immunogenicity of Vibrio cholerae O1 toxin-coregulated pili in experimental and clinical cholera.

A functional tcpA gene, encoding the major subunit of toxin-coregulated pili (TCP), is necessary for Vibrio cholerae O1 Ogawa strain 395 to colonize the human intestine and confer protective immunity to virulent challenge. The immunogenicity of TCP and other antigens in experimental and naturally acquired cholera was determined. Seroconversion to cholera toxin (CT), whole cell preparations, and to Ogawa lipopolysaccharide but not to purified native TCP or to a TcpA mimiotope was found in volunteers. Local intestinal secretory immunoglobulin A from volunteers showed conversions to cholera toxin and lipopolysaccharide but not to TCP. Cholera patients in Indonesia showed a seroconversion rate to TCP of 3 of 6 and a seroconversion to a TcpA mimiotope of 1 of 6. Volunteer and patient sera showed similar vibriocidal seroconversions when assayed against either TCP-positive and TCP-negative V. cholerae O1 strains, suggesting that TCP do not contribute demonstrably to the vibriocidal antigen. We conclude that although seroconversion to TCP can occur in naturally acquired cholera, solid long-term protection can be engendered in the absence of a detectable anti-TCP immune response.     

Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic.

In January 1991, an outbreak of cholera started in Peru and spread throughout most of Latin America within 8 months. As of March 1992, over 450,000 cases and approximately 4,000 deaths have been reported to the Pan American Health Organization. The causative organism is toxigenic Vibrio cholerae O1 of the El Tor biotype and is distinct from the U.S. Gulf Coast strains. A polymerase chain reaction (PCR) that amplifies a 564-bp fragment of the cholera toxin A subunit gene (ctxA) was used to identify toxigenic V. cholerae O1 strains. A total of 150 V. cholerae O1 isolates were tested. They were of unknown toxin status, were associated with recent outbreaks, and were isolated from patients, food, and water. One hundred forty isolates were found to be toxigenic both by PCR and the routine diagnostic enzyme-linked immunosorbent assay. Thirty-eight known toxigenic strains isolated worldwide from 1921 to 1991 were also positive in the PCR. A collection of 18 nontoxigenic V. cholerae O1 strains, 35 Escherichia coli heat-labile-enterotoxin-I-producing strains, 26 Campylobacter strains, and 8 strains of Aeromonas hydrophila, previously reported to produce cholera toxin-like toxin, were all negative in the ctxA PCR. We conclude that this PCR is a diagnostic method that specifically detects toxin genes in V. cholerae O1 strains in a reference laboratory. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.     

Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain.

Clostridium difficile causes pseudomembranous colitis through the action of Rho-modifying proteins, toxins A and B. Antibodies directed against C. difficile toxin A prevent or limit C. difficile-induced colitis. We engineered plasmid pETR14, containing the hlyB and hlyD genes of the Escherichia coli hemolysin operon, to express a fusion protein containing 720 amino acid residues from the nontoxic, receptor-binding, carboxy terminus of C. difficile toxin A and the secretion signal of E. coli hemolysin A. We introduced pETR14 into Vibrio cholerae and found that the toxin A-HlyA fusion protein was secreted by a number of V. cholerae strains and recognized by both monoclonal and polyclonal anti-C. difficile toxin A antibodies. We introduced pETR14 into an attenuated V. cholerae strain, O395-NT, and inoculated rabbits orally with this construct. Colonization studies disclosed that the V. cholerae vector containing pETR14 was recoverable from rabbit ilea up to 5 days after oral inoculation. Vaccination produced significant systemic anti-C. difficile toxin A immunoglobulin G and anti-V. cholerae vibriocidal antibody responses. Vaccination also produced significant protection against toxin A in an ileal loop challenge assay, as assessed by determination of both fluid secretion and histological changes. These results suggest that the hemolysin system of E. coli can be used successfully in V. cholerae vector strains to effect secretion of large heterologous antigens and that a V. cholerae vector strain secreting a nontoxic, immunogenic portion of C. difficile toxin A fused to the secretion signal of E. coli HlyA induces protective systemic and mucosal immunity against this toxin.     

Characterization of Aeromonas trota strains that cross-react with Vibrio cholerae O139 Bengal.

It has previously been shown that Vibrio cholerae O139 Bengal shares antigens with V. cholerae serogroups O22 and O155. We detected six surface water isolates of Aeromonas trota that agglutinated in polyclonal antisera to V. cholerae O139 and V. cholerae O22 but not in antiserum to V. cholerae O155. On the basis of agglutinin-absorption studies, the antigenic relationship between the cross-reacting bacteria were found to be in an a,b-a,c fashion, where a is the common antigenic epitope and b and c are unique epitopes. The antigen sharing between A. trota strains and V. cholerae O139 was confirmed in immunoblot studies. However, A. trota strains did not react with two monoclonal antibodies specific for V. cholerae O139 and, consequently, tested negative in the Bengal SMART rapid diagnostic test for V. cholerae O139 which uses one of the monoclonal antibodies. A polyclonal antiserum to a cross-reacting A. trota strain cross-protected infant mice against cholera on challenge with virulent V. cholerae O139. All A. trota strains were cytotoxic for HeLa cells, positive for adherence to HEp-2 cells, and weakly invasive for HEp-2 cells; one strain was heat-stable toxin positive in the suckling mouse assay; however, all strains were negative for cholera toxin-like enterotoxin. Studies on bacteria that share somatic antigen with V. cholerae O139 may shed further light on the genesis of V. cholerae O139.