Evaluation of humoral and cellular immune responses against Vibrio cholerae using oral immunization by multi-epitope-phage-based vaccine.

INTRODUCTION: Cholera is a severe gastrointestinal disease that manifests with rapid onset of diarrhea, vomiting, and high mortality rates. Due to its widespread occurrence in impoverished communities with poor water sanitation, there is an urgent demand for a cost-effective and highly efficient vaccine. Multi-epitope vaccines containing dominant immunological epitopes and adjuvant compounds have demonstrated potential in boosting the immune response. MATERIAL AND METHODS: B and T epitopes of OMPU, OMPW, TCPA, CTXA, and CTXB proteins were predicted using bioinformatics methods. Subsequently, highly antigenic multi-epitopes that are non-allergenic and non-toxic were synthesized. These multi-epitopes were then cloned into the pCOMB phagemid. A plasmid M13KO7ΔpIII containing all helper phage proteins except pIII was created to produce the recombinant phage. Female Balb/c mice were divided into three groups and immunized accordingly. The mice received the helper phage, recombinant phage or PBS via gavage feeding thrice within two weeks. Serum samples were collected before and after immunization for the ELISA test as well as evaluating immune system induction through ELISpot testing of spleen lymphocytes. RESULTS: The titer of the recombinant phage was determined to be 1011 PFU/ml. The presence of the recombinant phage was confirmed through differences in optical density between sample and control groups in the ELISA phage technique, as well as by observing transduction activity, which demonstrated successful production of a recombinant phage displaying the Vibrio multi-epitope on M13 phage pIII. ELISA results revealed significant differences in phage antibodies before and after inoculation, particularly notable in the negative control mice. Mice treated with multi-epitope phages exhibited antibodies against Vibrio cholerae lysate. Additionally, ELISpot results indicated activation of cellular immunity in mice receiving both Vibrio and helper phage. CONCLUSION: This study emphasizes the potential of multi-epitope on phage to enhance both cellular and humoral immunity in mice, demonstrating how phages can be used as adjuvants to stimulate mucosal immunity and act as promising candidates for oral vaccination.

Plasmid targeting and destruction by the DdmDE bacterial defence system.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

Comparative analysis of cholera serum vibriocidal antibodies from Convalescent and vaccinated adults in Zambia.

Cholera is responsible for 1.3 to 4.0 million cholera cases globally and poses a significant threat, with Zambia reporting 17,169 cases as of 4th February 2024. Recognizing the crucial link between natural cholera infections and vaccine protection, this study aimed to assess immune responses post cholera infection and vaccination. This was a comparative study consisting of 50 participants enrolled during a cholera outbreak in Zambia's Eastern Province and an additional 56 participants who received oral cholera vaccinations in Zambia's Central Province. Vibriocidal antibodies were plotted as geometric mean titres in the naturally infected and vaccinated individuals. A significant difference (p < 0.047) emerged when comparing naturally infected to fully vaccinated individuals (2 doses) on day 28 against V. cholerae Ogawa. Those who received two doses of the oral cholera vaccine had higher antibody titres than those who were naturally infected. Notably, the lowest titres occurred between 0-9 days post onset, contrasting with peak responses at 10-19 days. This study addresses a critical knowledge gap in understanding cholera immunity dynamics, emphasizing the potential superiority of vaccination-induced immune responses. We recommend post infection vaccination after 40 days for sustained immunity and prolonged protection, especially in cholera hotspots.

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.

Desarrollo de vacunas basadas en la atenuación racional de bacterias: la experiencia con salmonella typhi y vibro cholerae

Una de las observaciones pilares en el control de enfermedades infecciosas, es el hecho de que muchas cepas microbianas atenuadas, es decir, debilitadas en su capacidad de producir enfermedad, conservan su habilidad de inducir una respuesta inmune protectora. Esto las convierte en excelentes candidatos para ser utilizadas como vacunas. El conocimiento más profundo de los mecanismos de virulencia y patogenicidad microbianos, sumado al rápido desarrollo de técnicas de manipulación genética de las últimas decádas nos han provisto con las herramientas necesarias para llevar acabo una atenuación más precisa de microorganismos. Al mismo tiempo han garantizado un acercamiento más cauteloso al desarrollo de vacunas, en el que el riesgo de reversión al fenotipo silvestre, ha sido dramáticamente minimizado.Este artículo quiere dar, a través de dos ejemplos, una idea de como la atenuación racional puede conducir al desarrollo de vacunas más efectivas y seguras contra enfermedades infecciosas de alta incidencia en Latinoamerica, tales como cólera y fiebre tifoidea

Análise dos anticorpos vibriocidas e aglutinantes na população urbana do Município do Manacapura/AM (1992/1993) / Vibriocidal and agglutinating antibodies in the urban population in the municipality of Manacapuru/AM (1992-1993)

Foi realizado um estudo sorológico em 1.196 indivíduos residentes na área urbana do município de Manacapuru/AM, visando analisar o perfil dos anticorpos vibriocidas e aglutinantes. Empregou-se o procedimento de amostragem aleatória sistemática na obtenção da amostra populacional. Um ano após, obteve-se uma 2ª amostra de soro de 120 indivíduos (10% da amostra inicial), escolhidos aleatoriamente entre os participantes do inquérito, com o objetivo de avaliar o comportamento dos anticorpos nesse intervalo de tempo. Foram empregados os métodos de microtitulação de anticorpos vibriocidas e de soroaglutinação em tubos. A associação entre os anticorpos estudados foi determinada pelo coeficiente de correlação, calculado com base na distribuição de freqüência dos títulos detectados. A análise dos resultados revelou forte correlação positiva entre os anticorpos (r = 1,0) e queda nos títulos em grande proporção das amostras após um ano.

A serological study was carried out involving 1,196 individuals residents in the urban area of Manacapuru--Amazonas, to evaluate the behavior of vibriocidal and agglutinating antibodies. A systematic random sampling procedure was employed to obtain the sample. A year later a 2nd sample of serum was obtained from 120 individuals selected among the participants of the survey. Vibriocidal antibodies microtitulation and seroagglutination in tubes were employed. The correspondence between the studied antibodies was determined by the correlation coefficient calculated according to the frequency of the titles detected. The analysis of the results revealed positive correlation between the antibodies (r = 1.0) and a decrease in titles in a large proportion of the positive samples one year later.

Relaciones fenotípicas y moleculares entre cepas de vibrio cholerae 01 aisladas en Chile, Perú y Bolivia: comparación con cepas de reservorios ambientales / Phenotypical and molecular relation between vibrio cholerae 01 strains, isolated in Chile, Perú and Bolivia: comparison with strains from environmental reservoirs

The phenotype, biotype and susceptibility to nine antimicrobials was determined for each isolated strain. Also, the genes of cholera and termolabile toxins were determined using DNA probes and a chromosomal restriction profile was done using HindIII, EcoRI and NotI enzymes. Features studied were similar in the 53 strains isolated from patients. Those isolated from environmental reservoirs had different antimicrobial susceptibility, showing ampicillin resistance and the GT gene was detected in one of 20 strains, compared to clinical samples were it was present in all. Strains isolated from patients and envirinment had similar chromosomal restriction profiles. The chromosomal restriction profile gives an image of bacterial genome and it is a useful and reliable tool for the epidemiological surveillance of cholera

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

Caracterización de una cepa de vibrio cholerae 01 multirresistente, aislada de un caso de cólera en Chile / Characterization of a multiresistant vibrio cholerae 01 strain, isolated from a cholera case in Chile

This report characterizes a multiresistant Vibrio Cholerae 01 strain, isolated from a patient with cholera and investigates the mechanism of resistance. The analyzed strain was resistant to tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. The resistance was mediated by a 101 megadalton plasmid that was transferred to the resultant of a conjugation assay between the multiresistant V. Cholerae strain and E. coli C-600 used as receptor strain, that acquired the triple resistance of the parental strain. The resistant V. Cholerae strain had a Ogawa serotype, El Tor biotype and toxigenic capacity, demonstrated by ELISA and latex agglutination techniques. The biochemical features of the strain were identical to those of susceptible strains, except for the resistance to 10 and 150 ug o 129 vibriostatic factor. The emergence of plasmid mediated resistance to drugs of choice in the treatment of cholera must alert Chilean and Latin American health authorities, considering the cholera will continue affecting the region

La inmunología del cólera y la biología molecular de la toxina colérica: progresos recientes y perspectivas futuras / The immunology of the cholera and th molecular biology of toxins

Recientemente Vibrio cholerae ha llamado mucho la atención de los investigadores por ser un inmunógeno muy potente y, al mismo tiempo, un coadyuvante inmunomodulador de la respuesta inmunitaria en la mucosa intestinal, tanto para los antígenos que se administran mezclados como los ligados covalentemente a la toxina colérica. La inmunopatogenia del cólera es un fenómeno complejo. En este artículo se comunican los resultados preliminares de experimentos realizados con ratas de laboratorio para conocer la respuesta intestinal de la IgA en los roedores y los humanos

Relación entre grupo sanguíneo ABO y enfermedad diarreica aguda severa causada por Vibrio cholerae / Relationship of ABO blood groups and severe acute diarrhea by cholera

Hemos comparado las prevalencias de grupos sanguíneos entre los pacientes que acudieron al Servicio de Emergencia del Hospital Nacional Cayetano Heredia (Lima, Perú) durante el mes de abril de 1991 con diarrea aguda severa con aislamiento de Vibrio cholerae en los coprocultivos, con la de un grupo control conformado por los donantes del Banco de Sangre del mismo hospital, apareados por distritos de residencia. La prevalencia de grupo sanguíneo O entre los 136 pacientes con coprocultivo positivo (todos a V. cholerae o1 biotipo El Tor serotipo Inaba) fue de 94.9 por ciento mientras que en los 544 controles fue de 79.2 por ciento (similar a la reportada en otros estudios sobre prevalencia de grupos sanguíneos en nuestra población). Aunque el diseño del estudio tiende a subestimar el real riesgo relativo, encontramos un riesgo relativo estimado de 4.832 (IC95=2.196, 10.628) de presentar diarrea aguda severa por V.cholerae entre las persona con grupo sanguíneo O (p<0.00002). Queda por descubrir la base fisiopatológica de esta asociación y el impacto que puede tener en la morbilidad y mortalidad de la presente epidemia de cólera en el Perú, país con alta prevalencia de grupo sanguíneo O