Repeated dose toxicity study of Vibrio cholerae-loaded gastro-resistant microparticles.

CONTEXT: Microencapsulation of antigens has been extensively studied over the last decades aiming at improving the immunogenicity of vaccine candidates. OBJECTIVE: Addressing microparticles (MPs) toxicity in rats. MATERIAL AND METHODS: Spray-dried Eudragit® L 30 D-55 MPs and Eudragit® L 30 D-55 alginate MPs were elaborated and characterized. MPs obtained were administered to rats, three groups were defined: G1, control group; G2, administered with Vibrio cholerae (VC)-loaded MPs; G3, receiving VC-loaded alginate MPs. Animals received three vaccine doses. Body weight, food and water intake were controlled during the study. Haematological parameters, vibriocidal titres, organ weight and histology in necropsy were also analyzed. RESULTS: All animals grew healthy. Body weight gain, food and water intake and haematological parameters remained within physiological values, showing no treatment-related differences. Moreover, organ weight changes were not detected and animals developed protective vibriocidal titres. CONCLUSION: VC-loaded MPs and VC-loaded alginate MPs have proved to be safe and effective in the assessed conditions.

Biodistribución de IgA secretora purificada de calostro humano en fluidos biológicos de ratón Balb/c / Biodistribution of the secretory IgA purified from human colostrum in biological fluids of Balb/c mice

La IgA secretora humana (IgAsh) es estructural y funcionalmente liberada por el ambiente mucosal, con la capacidad de neutralizar antígenos, participa en la aglutinación y exclusión de estos y previene la adherencia de patógenos a las superficies del epitelio mucosal. En este estudio se evaluó la biodistribución de la IgAsh purificada de calostro, después de su administración por vía intranasal, en el modelo de ratón Balb/c y se determinaron los niveles de esta inmunoglobulina en diferentes fluidos biológicos mediante ELISA. Los resultados mostraron la presencia del anticuerpo en saliva de los animales del grupo que recibió la IgAsh en todos los intervalos de tiempo estudiados. En las muestras de lavado tráqueo-bronquial se obtuvo solo la presencia del anticuerpo a las 2 y 3 h posteriores a la inoculación. A partir de los resultados obtenidos consideramos interesante en el futuro utilizar este modelo experimental para evaluar el papel protector de esta inmunoglobulina como candidato terapéutico frente a la infección por Mycobacterium tuberculosis(AU)

The human secretory IgA (IgAsh) is structural and functionally released by the mucosal environment, with the ability to neutralize antigens and participating in promoting the binding and exclusion of them and preventing the adhesion of pathogens to mucosal epithelial surfaces. In this study, the biodistribution of hsIgA purified from colostrum was evaluated, after being administered intranasally to BALB/c mice model and the levels of this immunoglobulin in several biological fluids were determined by ELISA. Results showed the presence of antibody in saliva samples from animals that received the IgAsh, which was significantly higher than levels found in samples from non-treated animals, at all time intervals. In samples of tracheobronchial lavage, high antibody levels were only obtained two and three hours after the inoculation of the animals of the group that received the IgAsh, compared to the non-treated group. According to the results obtained, we consider interesting to evaluate the protective role of this immunoglobulin as a therapeutic candidate against the infection by M. tuberculosis in a near future(AU)

Pharmacology and toxicology of an oral tablet whole cells inactivated cholera vaccine in Sprague Dawley rats.

Here we further investigate the pharmacological and toxicological properties of a cholera vaccine based on inactivated whole cells presented in either enteric coated (COA) or uncoated (U/C) tablet formulation from Vibrio cholerae C7258 strain. Tablets were dispersed in 2mL drinking water and administered orally to Sprague Dawley rats distributed in five groups (I COA7, II U/C7 immunized at 0, 7, 69days and III COA14, IV U/C14 immunized at 0, 14, 69days and V control group). Serum vibriocidal antibody response was measured after the administration of two doses with an interval of 7-14days. To further investigate the toxicological aspects a third dose was applied 10 weeks after the initial one. Animals were observed daily and water and food consumption was measured every other day. Periodic blood extractions were performed for hematology, biochemistry, and the titer of serum vibriocidal antibodies was determined. Anatomopathological analysis was performed at days 3 or 14 after the third dose. Results from clinical observations, as well as from water and food consumption and body weigh indicated no toxicity of the vaccine product. Meanwhile, no biological differences were found among different groups in hematological, hemo-chemistry, and anatomopathological studies. Moreover, enteric coated and uncoated tablets against human cholera were found to induce an immune response in rats.

Ensayo de inmunogenicidad y toxicidad local del cocleato de Neisseriameningitidis en ratas Sprague Dawley / Study of immunogenicity and local toxicity of the Neisseria meningitidis cochleate in Sprague Dawley rats

Los estudios de tolerancia local para productos vacunales resultan fundamentales como requisitos regulatorios para los estudios preclínicos. En este ensayo se utilizaron 48 ratas Sprague Dawley, de ambos sexos, con un peso corporal de 180-220 g, suministradas por el Centro Nacional para la Producción de Animales de Laboratorio, y una duración de 17 días. Las ratas fueron distribuidas en 5 grupos: 3 con diferentes concentraciones de cocleatos, uno sin inocular y otro que recibió el diluente del producto en prueba. A estas se les realizaron las observaciones clínicas necesarias, tales como: mediciones de incremento de peso, consumo de alimentos y agua, así como la evaluación anatomopatológica en los dos tiempos de sacrificio, uno a los 12 y otro a los 17 días; se enfatizó en el estudio histopatológico de encéfalo y las fosas nasales en tres niveles de su extensión. Se hicieron investigaciones inmunológicas consistentes en la determinación de IgG en suero, saliva y líquido cefalorraquídeo. Los resultados arrojaron un incremento del peso. En el consumo de agua y alimento no se observaron alteraciones clínicas, la anatomopatología evidenció discretos cambios inflamatorios que guardaron una relación directa con el aumento de las concentraciones del producto, los títulos de IgG en la saliva y suero de las ratas inoculadas con los cocleatos difirieron de forma significativa (P < 0,05) respecto a los grupos controles, el líquido cefalorraquídeo resultó negativo a la presencia de anticuerpos específicos IgG. Se concluye que los cocleatos aplicados por esta vía en ratas resultaron inmunogénicos e inocuos para el referido producto(AU)

Local Tolerance studies for vaccine products are a main link in the chain of Regulatory Requirements for toxicological preclinical studies. Mainly considering that the use of mucosal road seems to be a current tendency due to the advantages it offers. Sprague Dawley rats of both sexes weighing 100-120 g (reception weight) provided by CENPALAB with the corresponding certificates of sanitary and genetic quality were used in this test. Local tolerance study lasted 17 days. Rats were distributed in 5 groups (3 groups with different cochleate concentrations, one without inoculation and another one that received the diluents of the product in test. Measurement of weight increase, food and water consumption, as well as the anatomopathologic studies in the two times of sacrifice, at 12 and 17 days were performed. Main histopathological studies were carried out in brain and nasal nostrils in three levels of their extension (anterior, medium and posterior areas). In addition, immunological assays such as the determination of IgG in serum, saliva and cerebrospinal fluid (CSF) were carried out. Results showed a sustained weight increase. There were no clinical alterations during water and food consumption, while the existence of discreet acute inflammatory changes was found by anatomopathological studies, which were directly related to the increase of the product concentration. The presence of IgG antibodies in saliva and in sera of the rats inoculated with the cochleates showed significant differences (P <0.05) compared to the non inoculated control group and to the animals inoculated with the diluent. CSF studies resulted negative regarding the presence of specific IgG antibodies. The existence of a direct relation among the concentrations of the product in test and the inflammatory processes in the medium and posterior levels of the nasal nostril was proven, therefore it is considered that cochleates applied by this route and in the concentrations ...(AU)

Evaluación de la toxicidad por dosis única de la vacuna antitetánica vax-TET en ratas Sprague Dawley / Evaluation of toxicity by single dose of the tetanus vaccine vax-TET in Sprague Dawley rats

La vacuna vax-TET® está indicada en la prevención del tétanos y es efectiva si se logra un completo y apropiado esquema de inmunización. Para investigar el potencial tóxico de este producto se realizó una prueba toxicológica con una dosis única, por vía intramuscular, en un volumen de 0,2 mL en ratas Sprague Dawley. La composición de la vacuna de ensayo probada fue la misma de la vacuna comercial. Los animales fueron observados diariamente en buscade síntomas locales y sistémicos de toxicidad. Se realizaron mediciones del consumo de agua y alimento, así como del peso corporal. Dos semanas después de la inoculación las ratas fueron sacrificadas por métodos de eutanasia sin dolor y sometidas a necropsia. No se observaron muertes ni síntomas de toxicidad en los animales estudiados. Tampoco se encontraron diferencias de interés toxicológico entre los grupos experimentales en cuanto a las variables medidas. El estudio anatomopatológico reveló la presencia de formaciones granulomatosas de tipo macrofágico asociadas, fundamentalmente, al hidróxido de aluminio. Estos resultados permitieron concluir que, bajo las condiciones delestudio y según los criterios establecidos, esta vacuna no produce efectos adversos en el modelo animal usado, por lo que se considera potencialmente no tóxica para humanos(AU)

The tetanus vaccine vax-TET® is indicated for the prevention of tetanus and it is effective if a complete and adequate immunization scheme is achieved. In order to investigate the toxic potential of this product, a toxicological test at single dose(0,2 ml), by intramuscular route was developed in Sprague Dawley rats using the same substance concentrations suggested by the manufacturer. The animals were daily observed looking for local and systemic symptoms of toxicity. Water and food consumption and body weight were also monitored during the experiment. Two weeks after inoculation, rats were slaughteredand submitted to necropsy studies. No deaths or toxicity symptoms were observed in the animals studied. No differences of toxicological interest were found among experimental groups regarding the variables measured. The anatomic-pathological study showed the presence of granulomatous formations mainly associated to aluminium hydroxide in the formulation. It wasconcluded that under the study conditions and following the established criteria, this vaccine does not produce any adverse reactions in the animal model used, suggesting a low potential toxicity in humans(AU)

Identification by genomic immunization of a pool of DNA vaccine candidates that confer protective immunity in mice against Neisseria meningitidis serogroup B.

We have shown previously that expression library immunization is viable alternative approach to induce protective immunity against Neisseria meningitidis serogroup B. In this study we report that few rounds of library screening allow identification of protective pools of defined antigens. A previously reported protective meningococcal library (L8, with 600 clones) was screened and two sub-libraries of 95 clones each were selected based on the induction of bactericidal and protective antibodies in BALB/c mice. After sequence analysis of each clone within these sub-libraries, we identified a pool of 20 individual antigens that induced protective immune responses in mice against N. meningitidis infection, and the observed protection was associated with the induction of bactericidal antibodies. Our studies demonstrate for the first time that ELI combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in a meningococcal vaccine.

Immunization of mice with a Mycobacterium tuberculosis genomic expression library results in lower bacterial load in lungs after challenge with BCG.

Tuberculosis is a serious infectious disease in many developing countries. The lack of an effective vaccine for preventing this disease has stimulated the search for new vaccine candidates against Mycobacterium tuberculosis. In the present work, the construction of a genomic expression library of M. tuberculosis in a eukaryotic expression vector was carried out. Immunization of Balb/c mice with a plasmid DNA pool from this library (containing 8360 clones) induced a significant IgG antibody response. Immunized mice were challenged by intratracheal route with 10(5) cfu of non-pathogenic Mycobacterium bovis BCG and were sacrificed 21 days post-challenge. Mice immunized with the genomic expression library showed a significant reduction of viable bacteria in lungs and less pulmonary tissue damage. Granulomas were not observed and the lungs had a more discrete perivascular inflammatory cell infiltrate compared to control mice. Results suggest that the genomic expression library contains genes encoding proteins that are protective against M. tuberculosis infection.

Evaluación de la toxicidad por dosis única de la vacuna antidiftérica-antitetánica en ratas Sprague-Dawley / Evaluation of toxicity by single dose of the Diphtheria-Tetanus Vaccine in SpragueDawley rats

En el presente ensayo se evaluó la toxicidad por dosis única de la vacuna antitetánica antidiftérica (VA-DIFTET®) en ratas Sprague-Dawley. Para ello se utilizó la vía intramuscular por ser la propuesta a usar en humanos. Se administraron 0,3 mL de la vacuna o sus componentes a un total de 20 animales por grupo (10 de cada sexo). Los mismos se observaron diariamente: se midió el consumo de agua y alimento en días alternos y se determinó el peso corporal con intervalos semanales. Se incluyó el análisis anatomopatológico de los animales al final del estudio. Ninguno murió durante el ensayo ni se observaron síntomas clínicos. No hubo diferencias estadísticamente significativas (p>0,1) en cuanto al consumo de agua, alimentos y el peso corporal. En el estudio anatomopatológico se observaron lesiones granulomatosas macrofágicas en las ratas tratadas con la vacuna o con el placebo, hallazgo este característico delas vacunas adyuvadas con hidróxido de aluminio. Los resultados sugirieron que la vacuna VADIFTETes potencialmente inocua al administrar la dosis única por vía intramuscular(AU)