Repeated dose toxicity evaluation of a cold chain-free, live, attenuated oral cholera vaccine in Sprague Dawley rats.

The repeated dose toxicity of a prototype cold chain-free, live, attenuated oral cholera vaccine containing 5 × 106 CFU/mL of the VCUSM14P strain was evaluated in Sprague Dawley (SD) rats (single dose administered daily for 30 days) to ascertain its safety for clinical use. Repeated dose toxicity studies for cholera vaccines in the literature have administered 2 or 3 fixed doses at 7, 14, 21 or 69 day intervals. The present study reports an evaluation of 30 repeated doses of cholera vaccine administered at three different concentrations (Group II (1.25 × 106 CFU), Group III (2.5 × 106 CFU) and Group IV (5 × 106 CFU)) in SD rats. The liquid vaccine was administered orally to the rats with the respective dose every day, and normal saline was administered to the control group (Group I). No significant difference (P > 0.05) was observed in the body weights and biochemical parameters of the rats after 15 and 30 repeated doses compared to those of the control group. However, compared to those of Group I, a significant increase (P < 0.05) in the organ to body weight ratios of the lungs, ureter, liver, kidney, heart and spleen was found in G-II, G-III and G-IV. In the haematological analysis, a significant increase in the WBC was observed in G-II and G-IV compared to that in G-I. The histopathological findings indicated mild to moderate degeneration in the liver, kidney, heart and spleen in the treated rats. Mild to moderate lymphocytic infiltration in the lungs was observed in the G-II and G-III rats, and severe infiltration was observed in the G-IV rats. These histopathological findings may be attributed to the 30 doses of vaccine given in daily succession without an interval. In the acute toxicity study, a single dose of vaccine up to 10 × 106 CFU did not cause any adverse effects and lethality in SD rats.

Retinoic acid pre-treatment down regulates V. cholerae outer membrane vesicles induced acute inflammation and enhances mucosal immunity.

Bacterial outer membrane vesicles have been extensively investigated and considered as a next generation vaccine. Recently, we have demonstrated that the cholera pentavalent outer membrane vesicles (CPMVs) immunogen induced adaptive immunity and had a strong protective efficacy against the circulating V. cholerae strains in a mouse model. In this present study, we are mainly focusing on reducing outer membrane vesicle (OMV) -mediated toxicity without altering its antigenic property. Therefore, we have selected All-trans Retinoic Acid (ATRA), active metabolites of vitamin A, which have both anti-inflammatory and mucosal adjuvant properties. Pre-treatment of ATRA significantly reduced CPMVs induced TLR2 mediated pro-inflammatory responses in vitro and in vivo. Furthermore, we also found ATRA pre-treatment significantly induced mucosal immune response and protective efficacy after two doses of oral immunization with CPMVs (75µg). This study can help to reduce OMV based vaccine toxicity and induce better protective immunity where children and men suffered from malnutrition mainly in developing countries.

[The outer membranes of Vibrio cholerae as a potential component in a chemical vaccine]. / Naruzhnye membrany kholernogo vibriona kak potentsial'nyi komponent khimicheskoi vaktsiny.

The results of the study of the preparation of V. cholerae eltor membrane, obtained by the lysis and inactivation of microbial cells with urea and the subsequent differential centrifugation and nuclease treatment. As revealed in this study, the outer membrane preparation, when introduced parenterally and orally to mice, induced pronounced immunity to experimental cholera infection and the production of vibriocidal antibodies in high titers. The treatment of V. cholerae eltor membranes with trypsin led to further increase of the immunogenic potency of the preparation. The protective action of V. cholerae eltor outer membranes considerably exceeded the protective effect of currently used whole-cell eltor vaccine. This opens prospects for using the above-mentioned preparation for the improvement of chemical vaccine as a component ensuring the formation of antibacterial immunity.

[An oral chemical vaccine from the hypertoxigenic strains of the causative agent of cholera KM-76 Inaba and KM-68 Ogawa]. / Oral'naia khimicheskaia vaktsina iz gipertoksigennykh shtammov KM-76 Inaba i KM-68 Ogava vozbuditel'a kholery.

The material on the development of chemical vaccine, prepared from two newly formed strains (KM-76 Inaba and KM-68 Ogawa) and intended for oral administration, is presented. The conditions for the submerged cultivation of these strains have been established, which makes it possible to increase the production of choleragen 8- to 10-fold and O-antigen 3- to 4-fold in comparison with V. cholerae natural strain 569B. The maximum accumulation of neuraminidase, protease, phospholipase, along with choleragen, has been registered in the logarithmic phase and that of O-antigen, in the stationary phase of growth. The use of strains KM-76 and KM-68 has led to the fourfold increase of the specific activity of the main immunogens, thus permitting the respective increase of the yield of the oral vaccine without changes in its high capacity for the formation of specific antibodies and its low residual toxigenicity.

[An experimental study of the safety of a chemical monovalent tableted cholera vaccine in enteral administration]. / Eksperimental'noe izuchenie bezvrednosti kholernoi khimicheskoi monovalentnoi tabletirovannoi vaktsiny pri énteral'nom vvedenii.

The safety of experimental chemical cholera monovalent vaccine in tablets, produced by the institute "Microbe" (Saratov, USSR), has been studied. The study has shown that the vaccine, administered to adult rabbits and germ-free suckling rabbits by the enteral route, retains residual toxicity, mainly due to the presence of O-antigen. One or two administrations of 1-2 human doses of this preparation to adult rabbits induce minimal structural changes admissible from the viewpoint of safety. After immunization made in two administrations immunobiological transformation develops more rapidly and is more pronounced than after immunization in a single administration.

Construction and characterization of a Vibrio cholerae serogroup O139 vaccine candidate by genetic engineering.

The present study aimed to construct and evaluate the live attenuated Vibrio cholerae serogroup O139 vaccine candidate, in which genes encoding protective antigens were integrated into the chromosomal DNA. Using the initial strain, O139-ZJ9693, the toxin-linked cryptic (TLC) and cholera toxin (CTX) genetic elements and repeats in the toxin (RTX) gene cluster were deleted from its chromosomal DNA, and the cholera toxin genes, ctxB and rstR, were transferred into the chromosome to construct the candidate vaccine strain. The expression of ctxB and the vaccine virulence were then examined. Polymerase chain reaction (PCR), enzymatic digestion and electrophoresis were performed to confirm that TLC, CTX and RTX were deleted, and that ctxB and rstR were transferred into the vaccine candidate DNA. According to the preliminary evaluation, the ctxB gene exhibited cholera toxin subunit B expression, and no enterotoxigenic or cytotoxic effects were observed in this strain. In conclusion, a recombinant strain containing genes encoding protective antigens that replaced virulence-associated genes was successfully constructed in the present study; this candidate strain may have the potential to be utilized to further evaluate the immune response.

Pharmacology and toxicology of an oral tablet whole cells inactivated cholera vaccine in Sprague Dawley rats.

Here we further investigate the pharmacological and toxicological properties of a cholera vaccine based on inactivated whole cells presented in either enteric coated (COA) or uncoated (U/C) tablet formulation from Vibrio cholerae C7258 strain. Tablets were dispersed in 2mL drinking water and administered orally to Sprague Dawley rats distributed in five groups (I COA7, II U/C7 immunized at 0, 7, 69days and III COA14, IV U/C14 immunized at 0, 14, 69days and V control group). Serum vibriocidal antibody response was measured after the administration of two doses with an interval of 7-14days. To further investigate the toxicological aspects a third dose was applied 10 weeks after the initial one. Animals were observed daily and water and food consumption was measured every other day. Periodic blood extractions were performed for hematology, biochemistry, and the titer of serum vibriocidal antibodies was determined. Anatomopathological analysis was performed at days 3 or 14 after the third dose. Results from clinical observations, as well as from water and food consumption and body weigh indicated no toxicity of the vaccine product. Meanwhile, no biological differences were found among different groups in hematological, hemo-chemistry, and anatomopathological studies. Moreover, enteric coated and uncoated tablets against human cholera were found to induce an immune response in rats.

Repeated dose toxicity study of a live attenuated oral cholera vaccine in Sprague Dawley rats.

BACKGROUND AND AIMS: A live attenuated vaccine candidate against human cholera has been developed from the genetically modified Vibrio cholerae O1, biotype El Tor, serotype Ogawa, 638 strain. Previous single dose toxicity and local tolerance studies have demonstrated that the product is innocuous in Sprague Dawley rats by oral route and single dose. The present paper describes a repeated dose toxicity study using a further dose compared to the proposed clinical schedule. METHODS: Sprague Dawley rats (140-180g) were treated with two doses of the vaccine candidate with a dedicated placebo formulation or were not treated at all (controls). The test products were inoculated at a 21-day interval. Animals were observed daily, body weight was determined weekly and food and water intakes were measured every other day. Three and 14 days after the last inoculation, groups of rats were humanely sacrificed, bled and macroscopically examined. Blood samples were taken for hematology, serum biochemistry and to determine the vibriocide antibody titers. A comprehensive list of tissue and organ samples was taken for microscopic studies. RESULTS: There was no mortality and no animal showed any clinical symptoms. Food and water intake, body weight, and hematological and biochemical variables did not show differences of toxicological and/or statistical relevance among the experimental groups. Macroscopic examination did not demonstrate any alterations and there were no histological findings of toxicological significance. CONCLUSIONS: The vaccine was considered potentially safe for human use as indicated by the results in Sprague Dawley rats.

Gut mucosal, salivary and serum antitoxic and antibacterial antibody responses in Swedes after oral immunization with B subunit-whole cell cholera vaccine.

Gut mucosal, salivary and serum antibody responses to a new oral cholera vaccine, consisting of B subunit and whole cell vaccine (WCV), were studied in Swedish volunteers. A single immunization with a 0.5 mg dose of B subunit together with WCV (5 X 10(10) killed cholera vibrios) induced a local intestinal immunoglobulin A (IgA) antitoxin response in 4/6 (67%) vaccine recipients as evident from specific antibody titre rises in intestinal lavage fluid. A second administration of vaccine did not further enhance the intestinal immune response beyond the peak level induced by the initial immunization. Different doses of B subunit (2.5 and 0.5 mg) given together with 5 X 10(10) killed vibrios (WCV) induced antitoxin antibody responses in serum in about the same frequency, 10/13 (77%) responders versus 13/14 (93%), as well as in saliva, 7/13 (54%) versus 9/14 (64%), and a single immunization was almost as efficient as two vaccine administrations. Single or repeated oral vaccination only irregularly resulted in modest antibacterial titre rises in serum (9/27 = 33%) or saliva (12/27 = 44%), but stimulated a significant mucosal antibacterial response in intestine of 5/6 (83%) examined volunteers.

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.

Overexpression of a mutant B subunit in toxigenic Vibrio cholerae diminishes production of active cholera toxin in vivo.

A mutant cholera toxin B subunit containing a G33E substitution was constructed and expressed in V. cholerae. The G33E amino acid substitution did not affect the amount of recombinant CTB secreted to the culture medium. The overexpression of the mutant B subunits in wild-type toxigenic cholera vibrios led to an 80% decrease in production of active cholera toxin in vitro and in vivo. Overexpression of BG33E subunits could be instrumental in the increase of the biosafety of live attenuated cholera candidate vaccine strains.

Diarrheagenicity evaluation of attenuated Vibrio cholerae O1 and O139 strains in the human intestine ex vivo.

The recent spread of El Tor cholera in Latin America highlights the need for a safe and economical vaccine. The main approach for developing live recombinant vaccines has been to disarm known pathogenic strains of cholera toxin leaving intact antigens involved in protection. These recombinant vaccine candidates do not cause severe diarrhea, but they are too reactogenic for wide scale usage. We describe here a test capable of determining the diarrheagenic potential of attenuated V. cholerae strains. The functional test consists in the simultaneous recording of net water movement, electrical potential difference and short-circuit current across the human intestine ex vivo. We found that human tissues incubated with supernatants from the attenuated 638, 413 and 251a V. cholerae strains caused no changes in the ion conductances and water absorption in ileal and colon tissues allowing them to be assayed in volunteers.

Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains.

Vibrio cholerae may be a particularly effective organism for use in delivering heterologous antigens to stimulate a common mucosal immune response. A live attenuated vaccine strain of V. cholerae was constructed from the ctxA deletion mutant 0395-N1, containing the B subunit of Shiga-like toxin I under the transcriptional control of the iron-regulated irgA promoter. The B subunit of Shiga-like toxin I is identical to the B subunit of Shiga toxin (StxB). irgA encodes the major iron-regulated outer membrane protein of V. cholerae, which is a known virulence factor for this organism. Clones of the structural gene irgA from the classical V. cholerae strain 0395, with the gene for the Shiga-like toxin I B subunit inserted under the control of the irgA promoter, were used to introduce an internal deletion of irgA into the chromosome of 0395-N1 by in vivo marker exchange, using the suicide vector plasmid pCVD442. This plasmid contains the sacB gene from Bacillus subtilis, which allowed positive selection for loss of plasmid sequences on exposure to sucrose. The construction of vaccine strains was confirmed by Southern hybridization studies and outer membrane protein analysis. The expression of StxB in the vaccine strain VAC2 following growth in high- or low-iron conditions was shown to be tightly iron-regulated by Western blot analysis and by quantification of StxB using a sandwich enzyme-linked immunosorbent assay. The production of StxB by VAC2 under low-iron conditions was greater than that of the reference strain Shigella dysenteriae 60R.(ABSTRACT TRUNCATED AT 250 WORDS)