Una vacuna de vesículas de membrana externa de Vibrio cholerae es aún una promesa: Caracterización proteómica / Outer membrane vesicle vaccine from Vibrio cholerae is still a promise: Proteomic characterization

Las epidemias de cólera afectan a un gran número de países africanos, asiáticos y del Caribe. Los cambios climatológicos y las constantes migraciones hacen que esta enfermedad se extienda, por lo que resulta necesario disponer de vacunas protectoras. En el presente trabajo se caracterizó una nueva vacuna de vesículas de membrana externa (VMEs) obtenidas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa cepa C7258, en el Instituto Finlay de vacunas (Cuba), a través de métodos proteómicos. Se identificaron 53 proteínas presentes en las VME (4 proteínas por banda electroforética) separadas por electroforesis unidimensional (1D) y digeridas con tripsina. Los fragmentos obtenidos fueron separados por cromatografía líquida de alta resolución (HPLC) acoplada a espectrometría de masa, secuenciados e identificados mediante bases de datos de proteínas Swiss-Prot y TrEMBL. El patrón proteico obtenido presentó algunas de las proteínas (12 proteínas citoplasmáticas y 5 proteínas de membrana externa) sugeridas dentro del proteoma de buena calidad para candidatos vacunales. Se estudiaron las mejores condiciones para la separación de las proteínas a través de electroforesis bidimensional. Las VME evaluadas cuentan con una composición fundamentada en proteínas necesarias para garantizar una respuesta inmune que proteja contra Vibrio cholerae O1 biotipo El Tor serotipo Ogawa.

Cholera epidemics affect a large number of African, Asian and Caribbean countries. The climate changes and the constant migrations cause this disease to spread, making it is necessary to obtain protective vaccines. In the present work, a new vaccine of outer membrane vesicles (OMV) from V. cholerae O1 El Tor biotype Ogawa serotype strain C7258 at Finlay Institute of vaccines (Cuba) was characterized by proteomic methods. A total of 53 proteins present in the OMV (approximate ratio of 4 proteins by electrophoresis band) were identified, separated by one dimension electrophoresis and digested by tripsin method. The fragments were separated by high performance liquid chromatography (HPLC) coupled to mass spectrometry, sequenced and identified, using Swiss-Prot and TrEMBL protein databases. The pattern showed some proteins (12 cytoplasmic proteins and 5 outer membrane proteins) suggested within the highest quality proteome for vaccine candidate. The best conditions for proteins separation by two dimension electrophoresis were studied. The OMV composition was based on proteins described to the immunity response and protection against V. cholerae O1 El Tor biotype Ogawa serotype.

As epidemias de cólera afetam um grande número de países africanos, asiáticos e caribenhos. As mudanças climáticas e as constantes migrações fazem com que esta doença se espalhe, portanto é necessário ter vacinas protectoras. No presente trabalho, uma nova vacina de vesículas de membrana externa (VMEs) obtidas de Vibrio cholerae 01 biotipo El Tor sorotipo Ogawa cepa C7258, no Instituto de Vacinas Finlay (Cuba), através de métodos proteômicos. Foram identificadas 53 proteínas presentes nas VME (4 proteínas por banda eletroforética) separadas por eletroforese unidimensional (1D) e digeridas com tripsina. Os fragmentos obtidos foram separados por cromatografia de alta resolução (HPLC) acoplada a espectrometria de massa, sequenciados e identificados usando bancos de dados de proteínas Swiss-Prot e TrEMBL. O padrão proteico obtido apresentou algumas das proteínas (12 proteínas citoplasmáticas e 5 proteínas de membrana externa) sugeridas dentro do proteoma de boa qualidade para candidatos vacinais. As melhores condições para a separação de proteínas através de eletroforese bidimensional foram estudadas. As VME avaliados possuem uma composição baseada em proteínas necessárias para garantir uma resposta imune que proteja contra Vibrio cholerae O1 biotipo El Tor sorotipo Ogawa.

Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine.

Preparation and evaluation of vibrio cholerae O1 EL Tor Ogawa lipopolysaccharide-tetanus toxoid conjugates.

The lipopolysaccharide (LPS) of Vibrio cholerae is considered one of the most important antigens from the point of view of immunogenicity in these bacteria. We have undertaken detoxification of this LPS by basic hydrolysis and the resultant amine groups were used for their conjugation to tetanus toxoid as carrier protein using carbodiimide-mediated coupling. The resulting conjugates were inoculated in Balb/c mice for immunogenicity studies. The anti-LPS IgG and vibriocidal antibodies were measured in serum. The antigenicity of this conjugated was evaluated by ELISA, with serums of humans vaccinated with a strain genetically modified. The conjugated elicited: high titers of IgG anti-LPS, high titers of vibriocidal antibodies and there was recognition of LPS by antibodies from cholerae immunised human serum. These results show that the conjugated LPS obtained by us, could be evaluated like a potential vaccine for human use.

Selección de cepas atenuadas de Vibrio cholerae para la obtención de candidatos vacunales atenuados orales contra el cólera / Selection of attenuated Vibrio cholerae strains to obtain oral attenuated candidate vaccines against cholera

Se desarrolló una metodología para la selección de cepas de Vibrio cholerae O1 y O139 modificadas genéticamente, con el objetivo de obtener candidatos vacunales atenuados orales contra el cólera. A las cepas modificadas se les realizó caracterización microbiológica, susceptibilidad bacteriana y diferentes pruebas biológicas (dosis letal media, capacidad colonizadora, adherencia en ratones, intestino ligado e inoculación intraduodenal en conejos como pruebas de virulencia y potencia). Las cepas 81, 638, 638T y 1333 fueron evaluadas en ensayos clínicos para determinar su reactogenicidad e inmunogenicidad. Todas las cepas fueron sensibles a la tetraciclina y doxiclicina y mostraron su atenuación e inmunogenicidad en modelos animales, resultando las cepas 638 y 1333 inmunogénicas y no reactogénicas en voluntarios

The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers.

Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXPhi prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 10(9) CFU of freshly harvested 638 buffered with 1.3% NaHCO(3), while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 10(9) CFU of DeltaCTXPhi attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 x 10(5) CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO(3). Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (10(9) CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.

[Selection of attenuated Vibrio cholerae strains to obtain oral attenuated candidate vaccines against cholera]. / Selección de cepas atenuadas de Vibrio cholerae para la obtención de candidatos vacunales atenuados orales contra el cólera.

A methodology was developed for the selection of genetically modified strains of Vibrio cholerae 01 and 0139 aimed at obtaining oral attenuated candidate vaccines against cholera. The modified strains underwent microbiological characterization, bacterial susceptibility and different biological tests (mean lethal dose, colonizing capacity, adherence in mice, ligated intestine and intraduodenal inoculation in rabbits as virulence and potency tests. The strains 81, 638, 638T and 1333 were evaluated in clinical trials to determine their reactogenicity and immunogenicity. All the strains were sensitive to tetracycline and doxoclycine. They showed their attenuation and immunogenicity in animal models. The strains 638 and 1333 proved to be immunogenic and non reactogenic in volunteers.

Construction and characterisation of O139 cholera vaccine candidates.

The hemagglutinin/protease (HA/P) seems to be an attractive locus for the insertion of heterologous tags in live cholera vaccine strains. A deltaCTXphi spontaneous mutant derived from a pathogenic strain of O139 Vibrio cholerae was sequentially manipulated to obtain hapA Colon, two colons celA derivatives which were later improved in their environmental safety by means of a thyA mutation. All the strains here obtained showed similar phenotypes in traits known to be remarkable for live cholera vaccines irrespective of their motility phenotypes, although the hapA mutants had a 10-fold decrease in their colonisation capacity compared with their parental strains in the infant mouse cholera model. However, the subsequent thyA mutation did not affect their colonisation properties in the same model. These preliminary results pave the way for further clinical assays to confirm the possibilities of these vaccine prototypes as safe and effective tools for the prevention of O139 cholera.

[Helicobacter pylori CagA antibodies in various gastroduodenal diseases from 2 different populations]. / Anticuerpos frente a la citotoxina CagA de Helicobacter pylori en diversas enfermedades gastroduodenales en dos poblaciones diferentes.

BACKGROUND: We aimed at studying the prevalence of infection by H. pylori along with the CagA status of the strain in two populations (Spain and Cuba) and the relationship with several gastroduodenal lesions. We also studied the role of the test-and-scope strategy in the decrease of unnecessary gastroscopies. PATIENTS AND METHOD: 100 dyspeptic patients from Spain and 100 from Cuba were included. At endoscopy, antrum biopsies were obtained and H. pylori status was evaluated by rapid urease test. CagA status of the strain was assessed by Western Blot. The test-and-scope strategy was evaluated according to H. pylori infection and CagA status. RESULTS: Mean age of Spanish and Cuban patients was 45 (16) and 46 (15) years, respectively. Dyspeptic symptoms were similar in both groups. Prevalence of infection by H. pylori was higher in Cuban (73%) than in Spaniards (40%) (p < 0.01). Prevalence of CagA+ strains was also higher in Cuban (81 vs. 27%) (p < 0.01). Among CagA+ Spanish patients, 11% had a duodenal ulcer, whereas this lesion was not found in any CagA patient (p < 0.05). Duodenal ulcer prevalence in CagA+ and CagA Cuban patients was 31 and 0%, respectively (p < 0.05). The test-and-scope strategy would have avoided endoscopy in only 24% Spanish and 15% Cuban patients. CONCLUSIONS: The prevalence of H. pylori infection is higher in Cuban than in Spanish dyspeptic patients. H. pylori strains of Cuba seem to be more virulent than those of Spain. CagA protein is a marker of peptic ulcer in both populations. These differences could partly explain the variations in the prevalence of different gastroduodenal disorders between both countries. The test-and-scope strategy appears to avoid a low number of endoscopies.