RESUMO
No commercially live vaccine against cholera caused by Vibrio cholerae O139 serogroup is available and it is currently needed. Virulent O139 strain CRC266 was genetically modified by firstly deleting multiple copies of the filamentous phage CTXφ, further tagging by insertion of the endoglucanase A coding gene from Clostridium thermocellum into the hemagglutinin/protease gene and finally deleting the mshA gene, just to improve the vaccine biosafety. One of the derived strains designated as TLP01 showed full attenuation and good colonizing capacity in the infant mouse cholera model, as well as highly immunogenic properties in the adult rabbit and rat models. Since TLP01 lacks MSHA fimbriae, it is refractory to infection with another filamentous phage VGJφ and therefore protected of acquiring CTXφ from a recombinant hybrid VGJφ/CTXφ. This strategy could reduce the possibilities of stable reversion to virulence out of the human gut. Furthermore, this vaccine strain was impaired to produce biofilms under certain culture conditions, which might have implications for the strain survival in natural settings contributing to vaccine biosafety as well. The above results has encouraged us to consider TLP01 as a live attenuated vaccine strain having an adequate performance in animal models, in terms of attenuation and immunogenicity, so that it fulfills the requirements to be evaluated in human volunteers.
Assuntos
Vacinas contra Cólera/imunologia , Proteínas de Fímbrias/imunologia , Vibrio cholerae O139/imunologia , Administração Oral , Animais , Anticorpos Antibacterianos/imunologia , Derrame de Bactérias , Sequência de Bases , Biofilmes , Cólera/imunologia , Cólera/prevenção & controle , Vacinas contra Cólera/genética , Vacinas contra Cólera/farmacologia , Modelos Animais de Doenças , Fezes/microbiologia , Proteínas de Fímbrias/genética , Mucosa Intestinal/imunologia , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Dados de Sequência Molecular , Coelhos , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Deleção de Sequência/genética , Estatísticas não Paramétricas , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/farmacologia , Vibrio cholerae O139/genéticaRESUMO
Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler's diarrhea. The heat-labile (LT) and heat-stable (ST) toxins mediate ETEC induced diarrhea. ETEC strains may express LT, ST, or both LT and ST, with LT-expressing strains accounting for approximately 50-60% of ETEC-related traveler's diarrhea. Cholera toxin (CT) is >80% homologous to LT and vaccination with CT-B subunit (CT-B) -based vaccines elicit a protective immune response against LT-producing ETEC strains. Peru-15 is an oral, single-dose, live-attenuated cholera vaccine candidate that has been investigated in several clinical trials (n>400 subjects) and was proven well tolerated, immunogenic, and efficacious. Peru-15 was genetically engineered to express and secrete high levels of CT-B by cloning ctxB onto a glnA balanced-lethal plasmid under the transcriptional control of a strong constitutive promoter, resulting in Peru-15pCTB. In vitro characterization demonstrated that Peru-15pCTB secreted approximately 30-fold more CT-B than Peru-15 and CT-B was stably produced after 40 generations of growth and throughout simulated Seed Bank and FDP (Final Drug Product) production conditions. In preclinical studies, the geometric mean anti-CT-B IgG titer in the sera of mice inoculated intranasally with two doses of Peru-15pCTB was >32-fold higher than in mice inoculated with Peru-15. Similarly, rabbits orally inoculated with a single dose of Peru-15pCTB developed titers that were approximately 30-fold higher than rabbits inoculated with a single dose of Peru-15. Sera from Peru-15pCTB vaccinated mice and rabbits neutralized LT toxicity in an in vitro assay. Peru-15pCTB has several promising characteristics of an oral, single-dose, bivalent cholera/ETEC vaccine and is advancing towards a Phase 1 clinical trial.
Assuntos
Adjuvantes Imunológicos/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Vacinas contra Cólera/genética , Vacinas contra Cólera/imunologia , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/biossíntese , Especificidade de Anticorpos , Western Blotting , Vacinas contra Cólera/administração & dosagem , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plasmídeos/genética , Coelhos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vibrio cholerae/imunologiaRESUMO
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.
Assuntos
Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/imunologia , Cólera/prevenção & controle , Vibrio cholerae/imunologia , Administração Oral , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Bacteriófagos/genética , Celulase/genética , Vacinas contra Cólera/genética , Clostridium thermocellum , Método Duplo-Cego , Fezes/microbiologia , Deleção de Genes , Hemaglutininas/genética , Humanos , Masculino , Peptídeo Hidrolases/genética , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Vibrio cholerae/virologiaRESUMO
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.
Assuntos
Vacinas contra Cólera , Cólera/prevenção & controle , Vibrio cholerae/imunologia , Administração Oral , Análise de Variância , Animais , Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/efeitos adversos , Vacinas contra Cólera/genética , Vacinas contra Cólera/imunologia , Vacinas contra Cólera/isolamento & purificação , Interpretação Estatística de Dados , Doxiciclina/farmacologia , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Animais , Coelhos , Tetraciclina/farmacologia , Vacinas Atenuadas , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/ultraestruturaRESUMO
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.