Evaluation of the potential of Mycobacterium smegmatis as vaccine Candidate against tuberculosis by in silico and in vivo studies

In this study, we scanned multiple published databases of gene expression in vivo of M. tuberculosis at different phases of infection in animals and humans, to select 38 proteins that are highly expressed in the active, latent and reactivation phases. The selected proteins were predicted for T and B epitopes. For each proteins, the regions containing T and B epitopes were selected at the same time to look for identical epitopes on M. smegmatis based on sequence alignments. Preliminary studies of humoral immunogenicity and cross-reactivity with M. tuberculosis in mice using two M. smegmatis-derived experimental vaccines were carried out, demonstrating the immunogenicity of M. smegmatis proteoliposomes and the recognition of M. tuberculosis proteins by the sera of animals immunized with this vaccine candidate. The conjunction of in silico and in vivo studies suggested the potential for future evaluation of M. smegmatis as vaccine candidate against tuberculosis using different strategie

Detección de proteínas de adhesión a fibronectina y colágeno presentes en Leptospira interrogans serovar Canicola

Como parte de los estudios encaminados a la obtención de una formulación vacunal por subunidades contra la leptospirosis humana, se describe la purificación y caracterización de las proteínas de unión a fibronectina y a colágeno en Leptospira interrogans. Las proteínas de la membrana externa fueron extraídas mediante la solubilización con Tritón X-114 y se aplicaron en una columna de afinidad de IgG AntiBSA-Sepharose 2B CL, para eliminar la BSA, contaminante principal del medio de cultivo en que crece el microorganismo. La muestra libre de BSA (no fijado) se aplicó a una columna de afinidad de fibronectina Sepharose 4B-CNBr, que permitió la separación y detección de una fracción que contenía una proteína de unión a fibronectina presente en la cepa 87 de Leptospira interrogans serovar Canicola, cuyo peso molecular fue estimado en 40 kDa. La proteína aislada demostró ser antigénica y conservada en los serovares Canicola, Copenhageni y Mozdok, en el ensayo de inmunodetección utilizado en este estudio (Dot blot). Para ello se utilizaron sueros específicos obtenidos en ratas infectadas experimentalmente con cada serovar y una mezcla de sueros de humanos convalecientes de leptospirosis. Las proteínas de membrana externa solubilizadas con Tritón X-114, libres de BSA, fueron aplicadas también a una columna de afinidad colágeno-Sepharosa 4B-CNBr, que permitió la purificación de una proteína de unión a colágeno con un peso molecular de aproximadamente 25 kDa, la cual resultó ser antigénica frente a sueros de humanos convalecientes de la enfermedad. Ambas proteínas seleccionadas (40 kD y 25 kD) podrían ser evaluadas como posibles inmunógenos en futuros estudios encaminados a la obtención de nuevos antígenos vacunales

The Importance of Animal Models in Tuberculosis Vaccine Development

Research, development, and production of vaccines are still highly dependent on the use of animal models in the various evaluation steps. Despite this fact, there are strong interests and ongoing efforts to reduce the use of animals in vaccine development. Tuberculosis vaccine development is one important example of the complexities involved in the use of animal models for the production of new vaccines. This review summarises some of the general aspects related with the use of animals in vaccine research and production, as well as achievements and challenges towards the rational use of animals, particularly in the case of tuberculosis vaccine development

Strategy for the purification of soluble fibronectin from human plasma, as ligand for affinity chromatography

El presente trabajo se desarrolló con el objetivo de purificar y caracterizar fibronectina soluble procedente de plasma para su posterior aplicación en la purificación de proteínas de adhesión. Se inmovilizó gelatina en Sefarosa 2BCL como soporte cromatográfico para la preparación de una columna. Se estudiaron diferentes alternativas de elución con arginina 0.5 M y 1M, con glicina -NaCl, acetato de sodio - NaCl y urea - NaCl, lo cual confirmó las ventajas en el uso de la arginina como agente eluyente. Se incorporó una segunda etapa de purificación en una columna de Heparina - Sefarosa 4B CNBr a partir de la que se obtuvo fibronectina con alto grado de pureza en estado nativo, comprobado por SDS-PAGE. La proteína purificada se inmovilizó en una matriz de Sefarosa con un grado de sustitución de 0,98 mg fibronectina/mL de gel lo cual permitió una capacidad de adsorción de gelatina libre con una recuperación de 8 mg (0,64 mg gelatina/mL de gel). La utilización de esta matriz permitirá su aplicación preliminar en la purificación de proteínas de adhesión que podrían ser usadas como antígenos en vacunas contra leptospirosis.Soluble fibronectin from human plasma was purified and characterized in order to be applied in the purification of adhesion protein. Gelatin was immobilized in a chromatographic bed as Sepharose 2B CL to prepare a column. Different alternatives of elution with 0.5 M and 1 M arginin, glicin- NaCl, sodium acetate - NaCl and urea - NaCl buffers, confirmed the advantages of the arginin as elution solution in this column. Second step of purification in a column of Heparin - Sepharose 4B CNBr was included to obtain the native fibronectin with high purity degree, verified by SDS-PAGE. Purified protein was immobilized in a matrix of Sepharose with a substitution grade of 0.98 mg fibronectin/mL of gel that permited an adsorption capacity of free gelatin to the column of 8 mg (0.64 mg gelatin/mL of gel). This matrix will allow its preliminary application in the purification of adhesion proteins which could be used as antigens in vaccines againt leptospirosis.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Large-scale application of highly-diluted bacteria for Leptospirosis epidemic control

Background: Leptospirosis is a zoonotic disease of major importance in the tropics where the incidence peaks in rainy seasons. Natural disasters represent a big challenge to Leptospirosis prevention strategies especially in endemic regions. Vaccination is an effective option but of reduced effectiveness in emergency situations. Homeoprophylactic interventions might help to control epidemics by using highly-diluted pathogens to induce protection in a short time scale. We report the results of a very large-scale homeoprophylaxis (HP) intervention against Leptospirosis in a dangerous epidemic situation in three provinces of Cuba in 2007. Methods: Forecast models were used to estimate possible trends of disease incidence. A homeoprophylactic formulation was prepared from dilutions of four circulating strains of Leptospirosis. This formulation was administered orally to 2.3 million persons at high risk in an epidemic in a region affected by natural disasters. The data from surveillance were used to measure the impact of the intervention by comparing with historical trends and non-intervention regions. Results: After the homeoprophylactic intervention a significant decrease of the disease incidence was observed in the intervention regions. No such modifications were observed in non-intervention regions. In the intervention region the incidence of Leptospirosis fell below the historic median. This observation was independent of rainfall. Conclusions: The homeoprophylactic approach was associated with a large reduction of disease incidence and control of the epidemic. The results suggest the use of HP as a feasible tool for epidemic control, further research is warranted. Homeopathy (2010) 99, 156e166

Specific and cross-reactive immune response against Mycobacterium tuberculosis antigens in mice immunized with proteoliposomes from Mycobacterium bovis BCG

Objective: To characterize the immunogenicity and the induction of cross-reactive responses against Mycobacterium tuberculosis (M. tuberculosis) of a proteoliposome (PL) from Mycobacterium bovis Bacillus Calmette–Guérin (BCG) with and without alum hydroxide (AL) as adjuvant (PLBCG-AL and PLBCG, respectively) in BALB/c mice. Methods: BALB/c mice were inoculated with phosphate buffer solution, BCG, PLBCG and PLBCG-AL. The humoral immunogenicity was determined by ELISA [immunoglobulin G (IgG), IgG1 and IgG2a] and the cellular immunogenicity was evaluated in vivo by delayed type hypersensitivity. The humoral cross-reactive response against M. tuberculosis was determined by Western blot. Results: Sera from animals immunized with PLBCG-AL and PLBCG showed significant increase in specific total IgG and IgG1 antibodies and the presence of cross-reactive antibodies against M. tuberculosis antigens, which were more intense with the use of alum as adjuvant. Mice immunized with PLBCG and PLBCG-AL also showed a specific cellular response in vivo. Conclusions: The cellular and humoral immunogenicity of PLBCG and the capacity to induce cross-reactive responses against M. tuberculosis is in agreement with the protective capacity previously demonstrated by this vaccine candidate and supports the continuation of its evaluation in further stages

Protective Effect of a Lipid-Based Preparation from Mycobacterium smegmatis in a Murine Model of Progressive Pulmonary Tuberculosis

A more effective vaccine against tuberculosis (TB) is urgently needed. Based on its high genetic homology with Mycobacterium tuberculosis (Mtb), the nonpathogenic mycobacteria, Mycobacterium smegmatis (Ms), could be an attractive source of potential antigens to be included in such a vaccine. We evaluated the capability of lipid-based preparations obtained from Ms to provide a protective response in Balb/c mice after challenge with Mtb H37Rv strain. The intratracheal model of progressive pulmonary TB was used to assess the level of protection in terms of bacterial load as well as the pathological changes in the lungs of immunized Balb/c mice following challenge with Mtb. Mice immunized with the lipid-based preparation from Ms either adjuvanted with Alum (LMs-AL) or nonadjuvanted (LMs) showed significant reductions in bacterial load (P<0.01) compared to the negative control group (animals immunized with phosphate buffered saline (PBS)). Both lipid formulations showed the same level of protection as Bacille Calmette and Guerin (BCG). Regarding the pathologic changes in the lungs, mice immunized with both lipid formulations showed less pneumonic area when compared with the PBS group (P<0.01) and showed similar results compared with the BCG group. These findings suggest the potential of LMs as a promising vaccine candidate against TB

Passive administration of purified secretory IgA from human colostrum induces protection against <it>Mycobacterium tuberculosis</it> in a murine model of progressive pulmonary infection

<p>Abstract</p> <p>Background</p> <p>Immunoglobulin A is the most abundant isotype in secretions from mucosal surfaces of the gastrointestinal, respiratory and genitourinary tracts and in external secretions such as colostrum, breast milk, tears and saliva. The high concentration of human secretory IgA (hsIgA) in human colostrum strongly suggests that it should play an important role in the passive immune protection against gastrointestinal and respiratory infections.</p> <p>Materials and methods</p> <p>Human secretory IgA was purified from colostrum. The reactivity of hsIgA against mycobacterial antigens and its protective capacity against mycobacterial infection was evaluated.</p> <p>Results</p> <p>The passive administration of hsIgA reduces the pneumonic area before challenge with <it>M.</it> tuberculosis. The intratracheal administration of <it>M. tuberculosis</it> preincubated with hsIgA to mice greatly reduced the bacterial load in the lungs and diminished lung tissue injury.</p> <p>Conclusions</p> <p>HsIgA purified from colostrum protects against <it>M. tuberculosis</it> infection in an experimental mouse model.</p