Transcutaneous immunization with a Vibrio cholerae O1 Ogawa synthetic hexasaccharide conjugate following oral whole-cell cholera vaccination boosts vibriocidal responses and induces protective immunity in mice.

A shortcoming of currently available oral cholera vaccines is their induction of relatively short-term protection against cholera compared to that afforded by wild-type disease. We were interested in whether transcutaneous or subcutaneous boosting using a neoglycoconjugate vaccine made from a synthetic terminal hexasaccharide of the O-specific polysaccharide of Vibrio cholerae O1 (Ogawa) coupled to bovine serum albumin as a carrier (CHO-BSA) could boost lipopolysaccharide (LPS)-specific and vibriocidal antibody responses and result in protective immunity following oral priming immunization with whole-cell cholera vaccine. We found that boosting with CHO-BSA with immunoadjuvantative cholera toxin (CT) or Escherichia coli heat-labile toxin (LT) following oral priming with attenuated V. cholerae O1 vaccine strain O395-NT resulted in significant increases in serum anti-V. cholerae LPS IgG, IgM, and IgA (P < 0.01) responses as well as in anti-Ogawa (P < 0.01) and anti-Inaba (P < 0.05) vibriocidal titers in mice. The LPS-specific IgA responses in stool were induced by transcutaneous (P < 0.01) but not subcutaneous immunization. Immune responses following use of CT or LT as an adjuvant were comparable. In a neonatal mouse challenge assay, immune serum from boosted mice was associated with 79% protective efficacy against death. Our results suggest that transcutaneous and subcutaneous boosting with a neoglycoconjugate following oral cholera vaccination may be an effective strategy to prolong protective immune responses against V. cholerae.

Design and validation of an improved nonferrous smoking device for self-administration of smoked drugs with concurrent fMRI neuroimaging.

Several popularly abused drugs, such as nicotine (tobacco) and THC (delta9-tetrahydrocannabinol) (marihuana) are commonly self-administered by the smoked route. Although the neuronal substrates mediating the effect of smoked drugs are of interest, studies of their acute actions in living human brain has been difficult due to the unique constraints imposed by neuroimaging equipment and scanning environments. We have previously reported a device for the self-administration of smoked drugs with concurrent blood oxygen level dependent (BOLD) fMRI imaging. Here we report improvements to the device which result in improved drug delivery to the smoker. Gas chromatography/mass spectrometry (GCMS) analysis of nicotine recovered from filter extracts revealed that the amount of nicotine delivered to subjects smoking with our original device was reduced by approximately 44% compared to nicotine delivered by cigarettes smoked normally. Improvements were made to the smoke delivery component of our apparatus in an attempt to improve drug delivery, while not interfering with collection of MRI data. Nicotine plasma levels in 9 subjects smoking both with and without the improved smoking device in the laboratory were not significantly different. Similarly, the device produced no significant difference in either ratings of the subjective effects of nicotine, or changes in cardiovascular parameters in this experiment. The improved device does not interfere with typical drug effects produced by normal smoking. Phantom scans revealed that BOLD signal was not found to be altered by the (in-bore) installation and operation of the improved device. Preliminary data analysis of smoking induced changes in the BOLD response to visual stimulation suggest that this response is not affected by the improved device, the act of smoking, air puffing, nicotine, or other components of cigarette smoke. The improved device does not interfere with the collection of MRI neuroimaging data. Use of this device will facilitate investigations of the acute neuronal effects of smoked drugs.

A variant in long palate, lung and nasal epithelium clone 1 is associated with cholera in a Bangladeshi population.

Vibrio cholerae causes a dehydrating diarrheal illness that can be rapidly fatal in the absence of specific treatment. The organism is an historic scourge and, like similar infectious diseases, may have influenced the evolution of the human genome. We report here the results of the first candidate gene association study of cholera. In a family-based study of 76 pedigrees from Dhaka, Bangladesh, we evaluated the association between cholera and five candidate genes-the cystic fibrosis transmembrane receptor; lactoferrin; long palate, lung and nasal epithelium clone 1 (LPLUNC1); estrogen-related receptor alpha and calcium-activated chloride channel 1. We found a significant association with a marker in the promoter region of LPLUNC1 (rs11906665), a member of a family of evolutionarily conserved innate immunity proteins. An earlier microarray-based study of duodenal biopsies showed significantly increased expression of LPLUNC1 in cholera patients compared with healthy control subjects. Our results suggest that variation in host innate immune responses may influence the outcome of exposure to V. cholerae in an endemic setting.

Local production of anti-vibrio cholerae mucosal antibody in reproductive tract tissues after cholera.

To investigate whether intestinal presentation of an antigen by Vibrio cholerae, a noninvasive organism, could induce an anatomically distant mucosal immune response in reproductive tract tissues, the endocervical immune responses of women in Bangladesh were evaluated after cholera. Endocervical secretions were analyzed for secretory IgA (sIgA) antibody against the B subunit of cholera toxin (CtxB) in 9 women with cholera and 8 women with diarrhea caused by neither V. cholerae nor heat labile enterotoxin-producing Escherichia coli. Women infected with V. cholerae developed significant sIgA anti-CtxB responses in endocervical samples (P< or =.02). Antibody subtype analysis of endocervical IgA was consistent with local mucosal production (P< or =.001). Women with cholera did not develop sIgA anti-CtxB responses in serum. The ability to generate specific mucosal immune responses in reproductive tract tissues after intestinal presentation of antigen could facilitate development of vaccines effective against reproductive tract pathogens.

Clostridium difficile--Associated diarrhea: A review.

Clostridium difficile causes 300 000 to 3 000 000 cases of diarrhea and colitis in the United States every year. Antibiotics most frequently associated with the infection are clindamycin, ampicillin, amoxicillin, and cephalosporins, but all antibiotics may predispose patients to C difficile infection. The clinical presentation varies from asymptomatic colonization to mild diarrhea to severe debilitating disease, with high fever, severe abdominal pain, paralytic ileus, colonic dilation (or megacolon), or even perforation. The most sensitive and specific test available for diagnosis of C difficile infection is a tissue culture assay for the cytotoxicity of toxin B. However, this test takes 1 to 3 days to complete and requires tissue culture facilities. Detection of C difficile toxin by means of enzyme-linked immunoassay is more rapid and inexpensive. A minority of patients may require more than 1 stool assay to detect toxin. Oral metronidazole or oral vancomycin hydrochloride for 10 to 14 days are equally effective at resolving clinical symptoms; oral metronidazole is preferred in most cases because of lowered cost and less selective pressure for vancomycin-resistant organisms. Approximately 15% of patients experience relapse after initial therapy and require retreatment, sometimes with an extended, tapering regimen. Immunity appears to be incomplete and predominantly mediated by serum IgG to toxin A. Measures for preventing the spread of the pathogen, appropriate diagnostic testing, and treatment may avert morbidity and mortality due to C difficile-associated diarrhea.

Cholera vaccines.

Cholera causes significant morbidity and mortality worldwide. For travelers, the risk of developing cholera per month of stay in a developing country is approximately 0.001%-0.01%, and cholera may present as traveler's diarrhea. In the United States, only a poorly tolerated, marginally effective, parenterally administered, phenol-inactivated vaccine is available. Outside the United States, 2 additional vaccines are commercially available: an oral killed whole cell-cholera toxin recombinant B subunit vaccine (WC-rBS) and an oral live attenuated Vibrio cholerae vaccine (CVD 103-HgR). These oral vaccines are well tolerated. In field trials, WC-rBS provides 80%-85% protection from cholera caused by V. cholerae serogroup O1 for at least 6 months. In volunteer studies, CVD 103-HgR provides 62%-100% protection against cholera caused by V. cholerae for at least 6 months. No commercially available cholera vaccine protects against disease caused by V. cholerae serogroup O139. New cholera vaccines are being developed.

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

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.

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

Mortality, morbidity, and microbiology of endemic cholera among hospitalized patients in Dhaka, Bangladesh.

Mortality and morbidity associated with cholera acquired in a modern endemic setting have not been well defined. In Dhaka, Bangladesh from 1986 to 1996, we found that causative agents of cholera shifted over time, varying by serogroup, biotype, and serotype. At the International Centre for Diarrhoeal Disease Research (ICDDR,B: Centre for Health and Population Research) in 1996, 19,100 cholera patients were treated, 887 (4.6%) were admitted, and 33 died (mortality rate = 3.7% of cholera inpatients, 0.14% of all cholera patients). When cholera inpatients who were discharged improved were compared with those who died, bacteremia (odds ratio [OR] = 10.5, 95% confidence interval [CI] = 2.9-37.9), radiographic evidence of pneumonia (OR = 3.1, 95% CI = 1.2-7.7), and acidosis as estimated by the serum bicarbonate value (OR = 0.893, 95% CI = 0.825-0.963) were independently associated with death by multivariate analysis. Pneumonia was the leading cause of death and accounted for two-thirds of all deaths among individuals with cholera in this study. Death in hospitalized patients with cholera acquired in a modern endemic setting is, therefore, extremely rare, and most frequently due to concomitant infection, especially pneumonia.

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.

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.

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.

Coexpression of the B subunit of Shiga toxin 1 and EaeA from enterohemorrhagic Escherichia coli in Vibrio cholerae vaccine strains.

A promoterless gene for the Shiga toxin 1 B subunit (stxB1) has been placed under transcriptional control of the Vibrio cholerae heat shock gene htpG. A chromosomal enterohemorrhagic Escherichia coli fragment containing eaeA and 400 bp of upstream DNA was added to the construct, downstream of stxB1; no transcription terminators were located between the two genes. The plasmid construct was confirmed by DNA sequencing; in vitro transcription-translation studies demonstrated expression of EaeA from the plasmid. The htpGp-->stxB1, eaeA construct was inserted into lacZ on the chromosome of Peru2, an El Tor V. cholerae strain with both attRS1 sequences and the entire cholera toxin genetic element deleted, and into lacZ in JRB10, a Peru2 derivative that has a second copy of htpGp-->stxB1 also inserted in the V. cholerae virulence gene irgA. Two plasmid constructs, one containing stxB1 under the control of the tac promoter and another containing htpGp-->stxB1,eaeA, were transformed into Peru2. Expression of StxB1 by these constructs was quantified by enzyme-linked immunosorbent assay and was highest in the plasmid construct with stxB1 under the control of the tac promoter. Localization of EaeA to the outer membrane of the vector strains was demonstrated both by Western blotting and by immunofluorescence with an anti-EaeA antibody. A rabbit model for colonization by V. cholerae was used to compare the immune responses to the two heterologous antigens, StxB1 and EaeA, expressed by these strains. Rabbits immunized with Peru2 transformed with a plasmid carrying tac-->stxB1 developed neutralizing serum anti-StxB1 immunoglobulin G antibody responses. One of two rabbits immunized with a strain carrying a chromosomal copy of eaeA developed a marked immune response against EaeA. The plasmid construct containing htpGp-->stxB1,eaeA was unstable, producing low levels of StxB1 in vitro and not evoking anti-EaeA antibody responses in vivo following oral immunization. Chromosomal insertion of eaeA may be preferred for future expression of this antigen in V. cholerae vaccine constructs.

Development of a germfree mouse model of Vibrio cholerae infection.

A mouse model of Vibrio cholerae infection was successfully developed with germfree mice. Three- to four-week-old germfree mice were orally inoculated with strains of V. cholerae to be tested and then moved to normal housing after inoculation. Stool culture, measurement of serum vibriocidal antibody titers, and determination of immune responses to the cholera toxin B subunit demonstrated that germfree mice are readily colonized by V cholerae and develop systemic and mucosal immune responses to antigens expressed by these organisms. Immune responses to the B subunit of Shiga toxin 1, which was expressed from a V. cholerae vaccine vector, were less pronounced. This model should be valuable for studying immune responses to V. cholerae infection and immunization, including responses to heterologous antigens expressed by cholera vector strains.