Expression in Saccharomyces boulardii of recombinant Toxin-coregulated pilus A subunit (TcpA) of Vibrio cholerae O1

Cholera is endemic in over 50 countries with an estimated mortality of 100,000-120,000. Vaccination is considered the complementary key to prevent and control cholera; therefore, alternative vaccine preparations are needed. Toxin Co-regulated Pilus is part of the toxR virulence regulon, which is necessary for colonization in the intestinal mucosa. In order to express Vibrio cholerae TcpA protein in Saccharomyces boulardii, the expression plasmid pYES2 was constructed by inserting tcpA gene isolated from local Vibrio cholerae Eltor Inaba isolates. The new construct was transferred into Saccharomyces boulardii cells and the expression of tcpA gene was induced from the GAL1 promoter by adding galactose to the medium. The SDS-PAGE and Western blot analysis showed the presence of TcpA in yeast. These results showed that Saccharomyces boulardii is a promising host to express Vibrio cholerae toxin TcpA as the first step in attempt to produce an oral Vibrio cholerae vaccine(AU)

El cólera es endémico en más de 50 países. Se estima una mortalidad entre 100.000 - 120.000 debido a esta enfermedad. La vacunación se considera una medida complementaria para prevenir y controlar el cólera, por lo tanto, se necesitan preparaciones vacunales alternativas a las existentes. El Pili corregulado con la toxina, es parte del regulón de virulencia toxR, y es necesario para la colonización en la mucosa intestinal. Para expresar la proteína tcpA de Vibrio cholerae en Saccharomyces boulardii, se construyó el plásmido de expresión pYES2 insertando el gen tcpA obtenido a partir de aislamientos locales de Vibrio cholerae El Tor Inaba. La nueva construcción se transfirió a las células de Saccharomyces boulardii y se indujo la expresión del gen tcpA a partir del promotor GAL1 mediante la adición de galactosa al medio. El análisis mediante SDS-PAGE y Western blot demostró la presencia de TcpA en levaduras. Los resultados demostraron que Saccharomyces boulardii es un hospedero prometedor para expresar el gen tcpA de Vibrio cholerae como el primer paso en el intento de producir una vacuna oral contra Vibrio cholerae(AU)

Cost Evaluation of a Government-Conducted Oral Cholera Vaccination Campaign-Haiti, 2013.

The devastating 2010 cholera epidemic in Haiti prompted the government to introduce oral cholera vaccine (OCV) in two high-risk areas of Haiti. We evaluated the direct costs associated with the government's first vaccine campaign implemented in August-September 2013. We analyzed data for major cost categories and assessed the efficiency of available campaign resources to vaccinate the target population. For a target population of 107,906 persons, campaign costs totaled $624,000 and 215,295 OCV doses were dispensed. The total vaccine and operational cost was $2.90 per dose; vaccine alone cost $1.85 per dose, vaccine delivery and administration $0.70 per dose, and vaccine storage and transport $0.35 per dose. Resources were greater than needed-our analyses suggested that approximately 2.5-6 times as many persons could have been vaccinated during this campaign without increasing the resources allocated for vaccine delivery and administration. These results can inform future OCV campaigns in Haiti.

Strategies to Prevent Cholera Introduction during International Personnel Deployments: A Computational Modeling Analysis Based on the 2010 Haiti Outbreak.

BACKGROUND: Introduction of Vibrio cholerae to Haiti during the deployment of United Nations (UN) peacekeepers in 2010 resulted in one of the largest cholera epidemics of the modern era. Following the outbreak, a UN-commissioned independent panel recommended three pre-deployment intervention strategies to minimize the risk of cholera introduction in future peacekeeping operations: screening for V. cholerae carriage, administering prophylactic antimicrobial chemotherapies, or immunizing with oral cholera vaccines. However, uncertainty regarding the effectiveness of these approaches has forestalled their implementation by the UN. We assessed how the interventions would have impacted the likelihood of the Haiti cholera epidemic. METHODS AND FINDINGS: We developed a stochastic model for cholera importation and transmission, fitted to reported cases during the first weeks of the 2010 outbreak in Haiti. Using this model, we estimated that diagnostic screening reduces the probability of cases occurring by 82% (95% credible interval: 75%, 85%); however, false-positive test outcomes may hamper this approach. Antimicrobial chemoprophylaxis at time of departure and oral cholera vaccination reduce the probability of cases by 50% (41%, 57%) and by up to 61% (58%, 63%), respectively. Chemoprophylaxis beginning 1 wk before departure confers a 91% (78%, 96%) reduction independently, and up to a 98% reduction (94%, 99%) if coupled with vaccination. These results are not sensitive to assumptions about the background cholera incidence rate in the endemic troop-sending country. Further research is needed to (1) validate the sensitivity and specificity of rapid test approaches for detecting asymptomatic carriage, (2) compare prophylactic efficacy across antimicrobial regimens, and (3) quantify the impact of oral cholera vaccine on transmission from asymptomatic carriers. CONCLUSIONS: Screening, chemoprophylaxis, and vaccination are all effective strategies to prevent cholera introduction during large-scale personnel deployments such as that precipitating the 2010 Haiti outbreak. Antimicrobial chemoprophylaxis was estimated to provide the greatest protection at the lowest cost among the approaches recently evaluated by the UN.

Desarrollo de un ensayo de PCR para detectar los genes codificadores de la toxina del cólera (ctxAB) en preparaciones del candidato vacunal vivo atenuado CV638 contra el cólera / Development of a PCR assay to detect cholera toxin genes (ctxAB) in preparations of attenuated live vaccine candidate against cholera CV638

El candidato vacunal vivo oral atenuado CV638 se produce siguiendo los criterios de las guías de Buenas Prácticas de Producción específicas para vacunas. El ingrediente activo de este candidato vacunal es la cepa atenuada genéticamente Vibrio cholerae 638; desarrollada por investigadores del Centro Nacional de Investigaciones Científicas de Cuba (CNIC), a partir de la cepa toxigénica de V. cholerae serogrupo O1, biotipo El Tor C7258, (Perú, 1991), mediante la remoción de los genes que codifican la producción de la toxina del cólera (ctxAB). Dado que la cepa 638 carece de estos genes en su genoma, la presencia de ctxAB en las preparaciones vacunales estaría dada por una contaminación con una cepa toxigénica de V. cholerae. El presente estudio tuvo como objetivo desarrollar un PCR específico para detectar los genes ctxAB a partir de ADN aislado de preparaciones del candidato vacunal CV638 contaminado artificialmente con V. cholerae toxigénico. La sensibilidad del ensayo de PCR empleando como molde ADN de la cepa toxigénica fue de 1 picogramo (pg) de ADN genómico por reacción, correspondiente a ~200 copias del genoma de la bacteria. La sensibilidad del método de PCR para detectar cepas toxigénicas en preparaciones vacunales de la cepa 638, contaminadas con una cepa toxigénica fue de ~7 x 103 unidades formadoras de colonia (UFC) de la cepa toxigénica por dosis del candidato vacunal CV638. Este método, una vez validado, pudiera emplearse en el control de la calidad de la producción del candidato vacunal vivo CV638(AU)

The live oral vaccine candidate against cholera CV638 is produced under Good Manufacturing Practices. The active ingredient of this vaccine candidate is the genetically modified strain Vibrio cholerae 638; developed by researchers at the National Center of Scientific Research, from the strain V. cholerae serogroup O1 biotype El Tor C7258 (Peru, 1991), by removing cholera toxin genes (ctxAB). Since the strain 638 lacks these genes in its genome, the presence of ctxAB in vaccine preparations would be given by a contamination with a toxigenic V. cholerae strain. The present study aimed at designing a specific PCR to detect ctxAB genes in DNA isolated from preparations of the vaccine candidate CV638, artificially contaminated with toxigenic V. cholerae strain. Sensitivity of the optimized PCR assay was 1 pg of genomic DNA from toxigenic strain of V. cholerae C7258, corresponding to ~200 genomic copies. The sensitivity of the PCR method for detecting toxigenic strains in CV638 vaccine preparations contaminated with a toxigenic strain was ~7 x 103 colony forming units of the toxigenic strain per dose of CV638. Once validated, this method could be used in the quality control of the production of the live vaccine candidate CV638(AU)

Biomodelos preclínicos para vacunas contra Vibrio cholerae / Preclinical biomodels for Vibrio cholerae vaccines

Con respecto a los artículos publicados en la revista Vaccimonitor en el volumen 17 número 1 del año 2008 y en el volumen 23 número 3 del 2014 y en los que se describen la aplicación, utilidad y desarrollo de diferentes biomodelos para realizar investigaciones encaminadas a obtener candidatos vacunales contra el cólera humano, quisiera profundizar sobre algunos de los tópicos abordados, lo que pudiera contribuir a una mejor comprensión de los biomodelos usados y evaluar adecuadamente su alcance y limitaciones. Desde el descubrimiento del agente etiológico del cólera se realizan múltiples intentos para desarrollar biomodelos que permitan estudiar las interacciones entre V. cholerae y su hospedero humano. Hasta el momento ningún modelo animal reproduce todos los síntomas y signos de la enfermedad de forma natural. La utilización....

Antigenicidad e inmunogenicidad de una cepa inactivada de Vibrio cholerae O1 El Tor Inaba / Antigenicity and immunogenicity of inactivated Vibrio cholerae 01 El Tor Inaba

INTRODUCCIÓN: el cólera continúa siendo problema de salud, principalmente en las zonas más pobres. La mayoría de los aislamientos que se realizan al nivel mundial pertenecen a Vibrio cholerae O1 biotipo El Tor serotipo Ogawa, aunque se han reportado brotes causados por el serotipo Inaba, por lo que se aconseja que una vacuna efectiva contra la enfermedad deberá contener células o antígenos de ambos serotipos. OBJETIVO: describir las características de la cepa de V. cholerae O1 El Tor Inaba C6706 como posible cepa para la obtención de vacunas inactivadas contra el cólera. MÉTODOS: se elaboró un lote de siembra de trabajo crioconservado a - 70 ºC, se evaluó su identidad, pureza y viabilidad. La cepa fue cultivada en caldo triptona soya y su inactivación se realizó a 56 ºC durante 20 min. Se evaluó la capacidad antigénica del producto obtenido mediante western blot y ELISA de inhibición de lipopolisacárido. La inmunogenicidad en conejos, inoculados por vía intraduodenal, se determinó mediante la cinética de anticuerpos vibriocidas. RESULTADOS: el lote de siembra de trabajo mantuvo su identidad, pureza y viabilidad durante 18 meses de estudio. Se observó la presencia de los antígenos lipopolisacárido, hemaglutinina sensible a manosa y proteína de membrana externa U, en las suspensiones de células inactivadas de V. cholerae O1, no así de las subunidades A y B de la toxina colérica (CTA y CTB, respectivamente) o pilli corregulado con la toxina (TCP). Por otra parte, se obtuvieron títulos vibriocidas en los sueros de conejos, similares a la respuesta inducida por la cepa viva atenuada 638 de V. cholerae O1, candidata a vacuna oral. CONCLUSIONES: la cepa de V. cholerae O1 El Tor Inaba C6706 mostró características culturales, antigénicas e inmunogénicas que permiten considerarla como posible cepa para la obtención de vacunas inactivadas contra el cólera(AU)

INTRODUCTION: cholera continues being a serius health problem, mainly in the poorest areas. Most of the isolations worldwide belong to Vibrio cholerae O1 biotype El Tor serotype Ogawa, although there have been reported outbreaks caused by serotype Inaba, that is why it is recommended that an effective vaccine against the illness should contain cells or antigens of both serotypes. OBJECTIVE: to describe the characteristics of the strain C6706 of V. cholerae O1 El Tor Inaba as a likely strain for the obtaining of inactivated vaccines for cholera. METHODS: a criopreserved working culture batch was made up at - 70 ºC to evaluate identity, purity and viability. The strain was cultured in tryptone soy broth and was inactivated at 56º C for 20 minutes. Antigenicity of this preparation was tested by Western Blot and ELISA inhibition test. Immunogenicity in adult rabbits, inoculated intraduodenally, was determined by means of kinetics of vibriocidal antibodies. RESULTS: the working culture batch kept their identity, purity and viability during 18 months of study. The results confirmed the presence of relevant antigens as lipopolysaccharide (LPS), mannose-sensitive hemagglutinin (MSHA) and outer membrane protein U (OmpU) in the suspension of inactivated V. cholerae O1 cells, but not cholera toxin subunits (CTA, CTB) or toxin-coregulated pili (TCP).On the other hand, vibriocidal titers were found in rabbit sera, which were similar to those previously reported for the live V. cholerae O1 strain 638, an attenuated oral vaccinal candidate CONCLUSIONS: the V. cholerae O1 El Tor Inaba C6706 strain showed cultural, antigenic and immunogenic characteristics that allow us to consider it as a possible strain for the obtaining of inactivated vaccines for cholera(AU)

Biomodelo para la evaluación de cepas atenuadas como candidatosvacunales contra el cólera humano I: estudio de la virulencia, capacidadde colonización y adherencia a la mucosa intestinal / Biomodel for evaluating attenuated Vibrio cholerae strains as human cholera vaccine candidates I: virulence, colonizing capacity and adherence to the intestinal mucosa

El cólera continúa siendo en muchos países un problema para la salud humana, manteniéndose como una enfermedad epidémica o endémica que afecta tanto a niños como adultos y causa la muerte en casos no tratados. Una vacuna viva oral contra esta enfermedad puede ser la solución. En el presente trabajo se seleccionó y aplicó un biomodelo para la evaluación de cepas atenuadas genéticamente de Vibrio cholerae como candidatas vacunales contra el cólera. La virulencia, capacidad de colonización y adherencia a la mucosa intestinal de las cepas fueron evaluadas mediante el usode ratones neonatos de 2 a 4 días de nacidos de la línea Balb/c, con un peso entre 1,5-2 g. Los resultados obtenidos con este biomodelo demostraron que las cepas atenuadas genéticamente son no virulentas, colonizan y se adhieren a lamucosa intestinal. Se concluye que el biomodelo utilizado permite la evaluación y selección de cepas candidatas para vacunas vivas orales contra el cólera(AU)

Cholera is still a human health problem in many countries. It is an epidemic or endemic disease affecting both children and adults that causes death of untreated cases. A live oral vaccine could be the solution against this disease. In the present study a biomodel was selected and applied for the evaluation of genetically attenuated Vibrio cholerae strains as vaccinecandidates. The virulence, colonizing capacity and adherence to the intestinal mucosa of the strains were evaluated using 2-4 day-old neonatal Balb/c mice, weighing from 1.5-2 g. The results obtained with this biomodel showed that genetically attenuated strains are not virulent, colonize and adhere to the intestinal mucosa. The conclusion was that the biomodel used allows the evaluation and selection of candidate strains for live oral cholera vaccines(AU)

Peru-1 5 (AVANT).

Peru-15 is a single dose, recombinant cholera vaccine under development by AVANT Immunotherapeutics for the potential prevention of cholera. A phase II trial of Peru-15 was ongoing in June 2003, and as of September 2003 AVANT was planning a phase III trial in a developing country, and phase IIb and phase III challenge studies in travelers.

Cholera.

Intestinal infection with Vibrio cholerae results in the loss of large volumes of watery stool, leading to severe and rapidly progressing dehydration and shock. Without adequate and appropriate rehydration therapy, severe cholera kills about half of affected individuals. Cholera toxin, a potent stimulator of adenylate cyclase, causes the intestine to secrete watery fluid rich in sodium, bicarbonate, and potassium, in volumes far exceeding the intestinal absorptive capacity. Cholera has spread from the Indian subcontinent where it is endemic to involve nearly the whole world seven times during the past 185 years. V cholerae serogroup O1, biotype El Tor, has moved from Asia to cause pandemic disease in Africa and South America during the past 35 years. A new serogroup, O139, appeared in south Asia in 1992, has become endemic there, and threatens to start the next pandemic. Research on case management of cholera led to the development of rehydration therapy for dehydrating diarrhoea in general, including the proper use of intravenous and oral rehydration solutions. Appropriate case management has reduced deaths from diarrhoeal disease by an estimated 3 million per year compared with 20 years ago. Vaccination was thought to have no role for cholera, but new oral vaccines are showing great promise.

Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1.

Cholera vaccine candidate Peru-15 was derived from a Vibrio cholerae O1 El Tor Inaba strain by deleting the cholera toxin genetic element, introducing the gene encoding cholera toxin B subunit into recA, and screening for nonmotility. In a controlled study, Peru-15 (2 x 10(8) cfu) was administered to 11 volunteers. No vaccinee developed diarrhea, and 10 of 11 had > 4-fold rises in vibriocidal antibody titers. One month later, 5 vaccinees and 5 control volunteers were challenged with wild type V. cholerae O1. Four of 5 controls developed diarrhea (mean, 1.9 L). Two Peru-15 vaccinees developed diarrhea, 1 with < 0.3 L and 1 with approximately 1.0 L; this latter volunteer had not developed a significant vibriocidal immune response to vaccination. Peru-15 shows promise as a single-dose, oral cholera vaccine that is safe, immunogenic, and protective.