From cholera to enterotoxigenic Escherichia coli (ETEC) vaccine development.

It was shown earlier that immune responses against cholera toxin (CT) as well as Vibrio cholerae lipopolysaccharide (LPS) or whole bacterial cells (WC) were protective and that these different antibody specificities co-operated synergistically for protection against experimental cholera. Similarly, antibodies against the heat-labile toxin (LT) and major colonization factors (CFs) of enterotoxingenic Escherichia coli (ETEC) co-operated synergistically for protection against LT-producing ETEC expressing homologous CFs. Studies in humans revealed that repeated oral antigen administration was optimal in inducing intestinal immune responses. Based on these findings oral inactivated vaccines consisting of toxin antigen and whole cells, i.e. the licensed recombinant cholera B subunit (rCTB)-WC cholera vaccine Dukoral®, and candidate ETEC vaccines have been developed. In different trials the rCTB-WC cholera vaccine has provided very high (85-100%) short term protection, which was significantly higher than that induced by the WC component alone, whereas rCTB-WC and WC alone provided comparable (50-60%), long term protection. An oral ETEC vaccine consisting of rCTB and formalin-inactivated E. coli bacteria expressing major CFs was shown to be safe and immunogenic in adults and children in different countries. The vaccine also induced significant protection against non-mild ETEC diarrhoea, i.e. diarrhoea interfering with daily activity in American travellers but not against ETEC diarrhoea in young children in Egypt. Against this background, a modified ETEC vaccine consisting of recombinant E. coli strains overexpressing the major CFs and a more LT like hybrid toxoid (LCTBA) has been developed. This vaccine will be tested soon alone and together with a mucosal adjuvant, i.e. dmLT, in clinical trials.

Cholera toxin – A foe & a friend.

After De’s pivotal demonstration in 1959 of a diarrhoeogenic exo-enterotoxin in cell-free culture filtrates from Vibrio cholerae (of classical biotype), much insight has been gained about cholera toxin (CT), which is arguably now the best known of all microbial toxins. The subunit structure and function of CT, its receptor (the GM1 ganglioside), and its effects on the cyclic AMP system and on intestinal secretion were defined in the 1970s, and the essential aspects of the genetic organization in the 1980s. Recent findings have generated additional perspectives. The 3D-crystal structure of CT has been established, the CT-encoding operon has been shown to be carried by a non-lytic bacteriophage, and in depth knowledge has been gained on how the bacterium controls CT gene expression in response to cell density and various environmental signals. The mode of entry into target cells and the intracellular transport of CT are becoming clearer. CT has become the prototype enterotoxin and a widely used tool for elucidating important aspects of cell biology and physiology, e.g., cell membrane receptors, the cyclic AMP system, G proteins, as well as normal and pathological ion transport mechanisms. In immunology, CT has emerged as a potent, widely used experimental adjuvant, and the strong oral-mucosal immunogenicity of the non-toxic B-subunit (CTB) has led to the use of CTB as a protective antigen together with killed vibrios in a widely licensed oral cholera vaccine. CTB has also been shown to promote immunological tolerance against certain types of mucosally co-administered antigens, preferably tissue antigens linked to the CTB molecule; this has stimulated research and development to use CTB in this context for treatment of autoimmune and allergic diseases. In summary, in the 50 years after De’s discovery of CT, this molecule has emerged from being the cholera patient’s “foe” to also becoming a highly useful scientist’s “friend”.

Ensayo clínico de reto, para evaluar una cepa candidata a vacuna contra el cólera / Challenge clinical trial for evaluation of a vaccine candidate strain against cholera

INTRODUCCIÓN: la cepa atenuada 638 Vibrio cholerae O1 El Tor, Ogawa, ha demostrado ser bien tolerada e inmunogénica por vía oral en estudios realizados en voluntarios sanos. OBJETIVO: se evaluó la protección conferida contra el cólera, en un ensayo clínico de reto, para el escalado tecnológico y farmacéutico de este candidato vacunal como ingrediente activo a escala industrial. MÉTODOS: en el estudio participaron 21 voluntarios sanos, 12 de ellos recibieron el candidato vacunal, y 9 ingirieron un placebo; 28 d después, todos recibieron una dosis infectante de una cepa virulenta de V.cholerae. RESULTADOS: la diarrea se registró en 7 de los 9 placebos, mientras que ninguno de los voluntarios vacunados presentó diarrea. Los voluntarios placebos del grupo sanguíneo O, tuvieron diarreas con mayor frecuencia e intensidad. Todos los voluntarios en el grupo placebo excretaron V. cholerae mientras que solo 3 (25 por ciento) de los 12 vacunados la excretaron. CONCLUSIONES: en este modelo de ensayo de reto, la cepa 638 demostró proteger contra la diarrea producida por una cepa virulenta de V. cholerae.

INTRODUCTION: live attenuated oral Vibrio cholerae O1 El Tor, Ogawa strain 638 has demonstrated to be well tolerated and immunogenic when administrated orally in studies carried out in healthy volunteers. OBJECTIVE: to evaluate the protection against cholera infection in a challenge clinical trial, for the technological and pharmaceutical scale-up of this vaccinal candidate as active ingredient at industrial level. METHOD: a total of 21 healthy volunteers were involved in this trial; the vaccine candidate was administered to 12 of them and the remaining nine were given the placebo. Twenty eight days later, all of them received an infective dose of a V. cholerae virulent strain. RESULTS: diarrheas were observed in 7 out of 9 placebos whereas not a single vaccinated volunteer showed diarrheas. More frequent and intense loose stools were found in the placebo volunteers with O-blood group. All volunteers in he placebo group excreted V. cholerae, but only three (25 percent) out of the 12 vaccinated volunteers did so. CONCLUSION: in this challenge clinical trial model, the 638 strain proved to protect people against the diarrhea caused by a virulent V. cholerae strain.

Traffic of antibody-secreting cells after immunization with a liposome-associated, CpG-ODN-adjuvanted oral cholera vaccine.

An oral cholera vaccine made up of heat-treated recombinant cholera toxin (rCT), V. cholerae lipopolysaccharide (LPS), and recombinant toxin-co-regulated pili subunit A (rTcpA), entrapped in liposomes in the presence of unmethylated bacterial CpG-DNA (ODN#1826) was used to orally immunize a group of eight week old rats. A booster dose was given 14 days later. Control rats received placebo (vaccine diluent). The kinetics of the immune response were investigated by enumerating the antigen specific-antibody secreting cells (ASC) in the blood circulation and intestinal lamina propria using the ELISPOT assay and a histo-immunofluorescence assay (IFA), respectively. ASC of all antigenic specificities were detected in the blood of the vaccinated rats as early as two days after the booster dose. The numbers of LPS-ASC and TcpA-ASC in the blood were at their peak at day 3 post booster while the number of CT-ASC was highest at day 4 after the booster immunization. At day 13 post immunization, no ASC were detected in the blood. A several fold increase in the number of ASC of all antigenic specificities in the lamina propria above the background numbers of the control animals were found in all vaccinated rats at days 6 and 13 post booster (earlier and later time points were not studied). Vibriocidal antibody and specific antibodies to CT, LPS and TcpA were detected in 57.1% and 52.4%, 14.3%, and 19.0% of the orally vaccinated rats, respectively. The data indicated that rats orally primed with the vaccine could produce a rapid anamnestic response after re-exposure to the V. cholerae antigens. Thus, a single dose of the vaccine is expected to elicit a similar anamnestic immune response in people from cholera endemic areas who have been naturally primed to V. cholerae antigens, while two doses at a 14 day interval should be adequate for a traveler to a disease endemicarea.

Estudio del patrón de excresión y la capacidad protectora en conejos inmunizados de forma oral con cepas atenuadas de Vibrio cholerae O1 biotipo El Tor

Con el fin de estudiar los patrones de excreción, colonización y la capacidad protectora de cepas vivas atenuadas de Vibrio cholerae O1El Tor, se inmunizaron conejos Nueva Zelandia con estas cepas y su correspondiente parental, con 2 dosis por el modelo de inoculación oral en conejos adultos. Fueron retados 2 semanas después de la segunda dosis por el modelo de intestino ligado, con cepas altamente virulentas de V. choleraeO1 serotipos Ogawa e Inaba y serogrupo O139. Se comprobó que las cepas manupuladas de forma genética no afectan los patrones de excreción, cuando se compara con su parental. Se observó en el reto una disminución en los niveles de colonización de las cepas virulentas de ambos serotipos; tanto en los conejos inmunizados con las cepas atenuadas como con la parental en comparación con animales controles inmunizados con la cepa Escherichia coli K-12, lo que indica que hubo cierto grado de protección. En el caso de los animales retados con la cepa 0139 se demostró que la protección es específica para cada serogrupo pues en este caso no se observó disminución de la colonización.

In order to study the excretion patterns, colonization and protective capacity of live sttenuated strains of Vibrio cholerae O1. E1 Tor, rabbits were immunized in New Zealand with these strains and their corresponding parental strains. 2 doses were administered by the model of oral inoculation in adult rabbits. Rabbits were rotated 2 weeks after the second dose by the model of ligated intestine with highly virulent strains of V. cholerae O1 Ogawa and Inaba serotypes and O139 serogroup. It was proved that the genetically manipulated strains do not effect the excretion patterns when they are compared with their parental strains. It was observed in the challenge a decrease in the levels of colonization of virulent strains of both serotypes, not only among the rabbits immunized with the attenuated strains, but also among those immunizedwith the parental strains in comparison with control animals immunized with the strain of Escherichia coli K-12, which means that there was certain degree of protection. In the case of the animals challenged with the O139 strain it was demonstrated that the protection is specific for each serogroup, since in this case there was no reduction of the colonization.

Strategies for production of a potential candidate vaccine for cholera.

First attempt at cholera vaccination was made by Jaime Ferran in 1884. Since then, a variety of strategies and methods have been evolved to create a safe, efficacious vaccine against cholera. For the first few years emphasis was on the development of parenteral vaccines. However, as a result of accumulation of a tremendous amount of knowledge, not only on Vibrio cholerae-the causative agent, but also on its interaction with the host, emphasis has shifted towards the development of oral vaccines. Two such vaccines, one killed, a whole cell/B subunit combination vaccine and the other a live attenuated one, have shown promise. The combination vaccine in its present state of development confers only a transient protection in young children, while the live attenuated one produces adverse reaction. To combat these, various strategies are being evolved. In one attempt, a potential candidate vaccine strain has been constructed from a non-reactogenic clinical isolate of V. cholerae, which is devoid of all known major virulence genes and is also a good colonizer. In animal studies this construct has shown considerable promise. This review discusses the various strategies that have been employed so far in the quest for an ideal cholera vaccine.

Seguridad e inmunogenicidad de la vacuna oral anticólera CVD-103 HgR: estudio en 29 voluntarios / Safety and immunogenicity of the oral anti-cholera vaccine CVD-103 HgR: study in twenty nine volunteers

Con el fin de determinar si la vacuna CVD-103 HgR era segura e inmunogénica, se administró una dosis a 29 voluntarios sanos, en el Laboratorio de Microbiología de Microbiología del Instituo Nacional de Salud. Se anotaron los efectos secundarios durante siete días después de la ingestion de la vacuna. Se tomaron 5 muestras de sangre los días 0,7, 10, 14 y 28 después de la toma de la dosis. Se determinaron los títulos de anticuerpos antibacterianos por medio de la técnica vibriocida y anticuerpos antitoxina IgG e IgA por medio de la técnica de elisa. Nueve voluntarios (31 por ciento) manifestaron efectos secundarios leves (náuseas, dolor abdominal o cefalea) los cuales fueron más frecuentes durante el primero y segundo días después de haber tomado la vacuna. El mayor porcentaje de seroconversión en anticuerpos vibriocidas fue de 93 por ciento a los 7 días, en anticuerpos antitoxina IgG fue de 71,4 por ciento a los 14 días y para antitoxina IgA en igual porcentaje a los 10 días. La mayor media geométrica de anticuerpos vibriocidas fue de 1538 (1.064-2.223) a los 10 días, la de anticuerpos antitoxina IgG fue de 251 (212-297) a los 14 días y para anticuerpos antitoxina IgA fue de 48 (40-57) a los 10 días. Se concluyó que la vacuna era segura e inmunogénica en esta población

Seguridad e inmunogenicidad de la vacuna oral contra el cólera: estudio en 20 voluntarios / Safety and immunogenicity of the oral anti-cholera vaccine againts the cholera: Study in 20 volunteers

Se realizó un estudio de seguridad e inmunogenicidad de la vacuna de células enteras de Vibrio cholerae 01 más la subunidad B recombinante de la toxina (CE/sBr) en 20 voluntarios sanos, en el Laboratorio de Microbiología del Instituto Nacional de Salud. Se suministraron tres dosis de la vacuna con intervalos de dos semanas entre cada una y se recolectaron muestras de sangre el día de la primera dosis y semanalmente durante ocho semanas. Se anotaron los efectos secundarios durante los cinco días siguientes a la ingestión de cada una de las dosis. En los sueros, se determinaron los niveles de anticuerpos antibacterianos empleando la técnica vibriocida y los de anticuerpos antitoxina de las clases IgG e IgA empleando la técnica de ELISA. Se presentaron efectos secundarios leves como náuseas, cefalea y malestar abdominal después de cada una de las dosis en 4, 5 y 2 de los voluntarios. El porcentaje de seroconversión más alto para títulos vibriocida fue del 50 por ciento y la mayor media geométrica fue de 21,38 (15,3-29,85) ambos eventos en la tercera semana. El porcentaje máximo de seroconversión para los anticuerpos antitoxina fue del 84 por ciento en la octava semana para IgG y del 89 por ciento en la sexta semana para IgA y la media geométrica fue de 245,5 (198-305) en la sexta semana para IgG y de 74,13 (60-98) en la tercera semana para IgA. Con estos datos podemos concluir que la vacuna CE/sBr fue segura e inmunogénica en los voluntarios estudiados

Vacunas contra el cólera / Vaccines against cholera

El cólera es una enfermedad aguda e infecciosa que fue descrita antes de la época de Hipócrates en el siglo V AC. Se describieron varias epidemias de esta enfermedad en Asia entre los siglos XV y XVIII. A mediados del siglo XIX John Snow en Inglaterra fue el primero en describir las medidas de prevención de la enfermedad a raíz de una epidemia ocurrida en Londres. En 1883, Robert Koch realizó el descubrimiento del agente causal, Vibrio cholerae, un bacilo curvo de gran movilidad. Durante los siglos XIX y XX han ocurrido siete pandemias de cólera; en la actualidad ocurre la transmisión de la séptima. El cólera es una de las causas más importantes de morbilidad y mortalidad de algunos países de Asia y Africa y desde 1991 también en Latinoamérica. Desde principios del siglo se ha empleado una vacuna parenteral elaborada con una cepa de V. cholerae 01, inactivada con calor, la cual únicamente induce 50 por ciento de protección en jóvenes y adultos, durante un período de aproximadamente 6 meses. El empleo de adyuvantes no ha tenido influencia en su eficiencia, sino por el contrario incrementa las reacciones colaterales. Las perspectivas para el desarrollo de una vacuna eficaz contra el cólera se basan en el hecho de que más del 90 por ciento de los sujetos infectados en forma natural quedan protegidos para una segunda reinfección. El avance del desarrollo de las vacunas del cólera se ha podido efectuar gracias a un mejor conocimiento de los mecanismos de patogenicidad y antigenicidad del agente etiológico, aunque persisten incógnitas importantes. La vacuna ideal contra el cólera debería ser tan eficaz como la infección natural, sin riesgo de causar enfermedad infecciosa, de fácil administración, de bajo costo, de una sola dosis, inocua, que proteja contra la infección y obviamente contra la enfermedad grave, con protección de larga duración y probablemente de administración oral

Immunological studies on some bacterial and parasitic diseases in Thailand.

Over the past two decades a wide range of immunological studies have been carried out on organisms responsible for causing serious bacterial and parasitic diseases in Thailand. Particular emphasis has been placed on cholera, where investigations have been directed towards vaccine development and rapid diagnosis and on diagnosis of typhoid and enterotoxigenic Escherichia coli. In the area of parasitic diseases emphasis has been directed especially to immunodiagnosis of helminthic diseases, including gnathostomiasis, paragonimiasis, opisthorchiasis, strongyloidiasis and trichinellosis. These studies are reviewed in this report.

Relationship of vibriocidal antibodies to history of cholera vaccination in Thai adult volunteers.

Vibriocidal antibodies were determined by microtechnique in 5 groups of Thai adult volunteers who had never received or had received cholera vaccination within one year, more than one to five years ago, more than five to ten years ago and more than ten years ago respectively. Detailed questionnaires about socioeconomic status, educational levels and environmental factors were presented to every volunteer. There were no differences statistically in incomes, educational levels and environmental factors among the groups. It was found that the reciprocal geometric mean titers of antibodies in volunteers who had never received cholera vaccination was generally low. The reciprocal geometric mean titers of the volunteers who had received cholera vaccination within one year were statistically different from other groups (p = 0.05). There was no correlation between blood groups of volunteers and vibriocidal antibodies.

Assessment of antibody responses and protective immunity in cholera vaccinated subjects.

Cross-reactive antibody responses were assessed in volunteers vaccinated with classical Inaba and Ogawa cholera vaccines. The El Tor, Ogawa vibrios, the most often biotype, and serotype found to be the causative agent of cholera in Thailand, or their product were used throughout the in vitro and in vivo tests. The test involved were the passive hemagglutination test, vibriocidal tests and the mouse protection test. Classes of specific immunoglobulins produced in the volunteers were determined using anti-immunoglobulin enhancement of hemagglutination. It was found that the levels of hemagglutinating and vibriocidal antibodies reached their peaks on day 7 after the vaccination and were statistically constant for 3 months. Significant decrease was observed thereafter. The mouse protective antibody titer was highest at 1 month after the vaccination then declined significantly at the 6th month. Classes of specific immunoglobulins were found to be either IgM or IgG alone or mixture of both.