Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine.

Development and characterization of murine monoclonal antibody specific for the P1,4 Por A proteins from strain B: 4: P1,(7b),4, of Neisseria meningitidis / s. t

Neisseria meningitidis isolates are conventionally classified by serosubtyping that characterizes the reactivities of the PorA outer membrane protein variable-region epitopes with monoclonal antibodies. Porins are outer membrane proteins (OMPs) of N. meningitidis serogroup B and have attracted study principally for two reasons: their use in the classification of meningococcal isolates into serotype and subtype and as potential components of vaccines against this important pathogen. New murine hybridomas, secreting specific monoclonal antibodies against PorA serotype P1.4 of N. meningitidis serogroup B, were generated using conventional hybridoma procedures. The monoclonal antibodies obtained were characterized by Western blot and whole cell ELISA, using reference strains from different N. meningitidis serotypes and subtypes. All monoclonal antibodies belong to isotype IgG1. Others hybridomas producing MAbs against PorB and FrpB were also obtained(AU)

Los aislamientos de Neisseria meningitidis se clasifican convencionalmente por serosubtipos. Su reactividad se realiza entre el epítope de la región variable de la proteína de membrana externa PorA con anticuerpos monoclonales. Las porinas, proteínas de membrana externa de N. meningitidis del serogrupo B, son atractivas para su estudio principalmente por la clasificación en serotipo y subtipo de los aislamientos del meningococo y como posibles componentes de vacunas contra este importante agente patógeno. Se generaron nuevos hibridomas murinos secretores de anticuerpos monoclonales específicos contra la proteína PorA subtipo P1.4 de N. meningitidis del serogrupo B, mediante los procedimientos convencionales de hibridomas. Los anticuerpos monoclonales, pertenecientes al isotipo IgG1, fueron caracterizados mediante Western blot y ELISA de células enteras. Se utilizaron cepas de referencia de diferentes serotipos y subtipos de N. meningitidis y se obtuvieron hibridomas productores de anticuerpos monoclonales contra otras proteínas como PorB y FrpB(AU)

A new oral vaccine candidate based on the microencapsulation by spray-drying of inactivated Vibrio cholerae.

The aim of this work was to evaluate the microencapsulation by spray-drying of inactivated Vibrio cholerae, using methacrylic copolymers Eudragit® L30D-55 and FS30D. The microparticles obtained presented a particle size around 3.0 µm. The preparation temperature affected the morphology and the antigenicity of microparticles, but it did not affect the V. cholerae content. In vitro release studies showed that in acid medium less than 5% of bacteria was released, and in neutral medium, Eudragit® L30D-55 microparticles released 86% after 24 h, whereas FS30D released less than 30%. Rats inoculated with microparticles exhibited vibriocidal antibody titres. Microencapsulation by spray-drying of inactivated V. cholerae could be proposed as a method to obtain an oral vaccine which provides controlled release of the bacteria.

Purificación de inmunoglobulina A secretora a partir de calostro humano / Purification of secretory immunoglobulin A from human colostrum

La inmunoglobulina A (IgA) es el isotipo de anticuerpo más abundante en las secreciones de las superficies mucosales del tracto gastrointestinal, respiratorio y genitourinario y en secreciones externas como el calostro, la leche materna, las lágrimas y la saliva. En el presente trabajo se obtuvo inmunoglobulina A secretora a partir de calostro humano mediante una combinación de métodos cromatográficos, y su presencia fue demostrada mediante Dot blot. La fracción de IgA fue analizada mediante electroforesis en gel de poliacrilamida en presencia de dodecilsulfato de sodio (SDS-PAGE) bajo condiciones reductoras, obteniéndose una elevada pureza al identificarse la presencia del componente secretor, la cadena pesada y la cadena ligera de dicha inmunoglobulina, con un patrón de migración correspondiente a sus respectivas masas molares(AU)

Immunoglobulin A is the most abundant antibody isotype in secretions from mucosal surfaces of the gastrointestinal, respiratory, and genitourinary tracts and in external secretions such as colostrums, breast milk, tears and saliva. In this study, secretory immunoglobulin A was obtained from human colostrum by a combination of chromatographic methods which was demonstrated by Dot blot. IgA fraction was analyzed by SDS-PAGE electrophoresis under reducing conditions, obtaining a high purity product by identifying the presence of secretory component, heavy and light chains of the immunoglobulin, with a migration pattern corresponding to their respective molecular mass(AU)

Prophylactic effect of administration of human gamma globulins in a mouse model of tuberculosis.

The protective effect of human gamma globulins on Mycobacterium tuberculosis infection was evaluated in a mouse model of intratracheal infection. Animals receiving human gamma globulins intranasally, 2h before intratracheal challenge showed a significant decrease in lung bacilli load compared to non-treated animals in different time intervals of up to 2 months after challenge. The same effect was obtained when M. tuberculosis was pre-incubated with the gamma globulin before challenge. The protective effect of the gamma-globulin formulation was abolished after pre-incubation with M. tuberculosis. These results suggest a potential role of specific antibodies in the defence against mycobacterial infections.

A proteoliposome based formulation administered by the nasal route produces vibriocidal antibodies against El Tor Ogawa Vibrio cholerae O1 in BALB/c mice.

A vaccine candidate against the enteric pathogen Vibrio cholerae was developed based on a proteoliposome (PL) formulation using a wild type strain C7258, V. cholerae O1, El Tor Ogawa as part of strategy to develop a combined formulation against enteric diseases preventable by the stimulation of the mucosal immune system. A detergent extraction method was applied to obtain the PL. Scanning electron microscopy and molecular exclusion chromatography showed the presence of two PL populations. Photon correlation spectroscopy studies were then carried out to evaluate the size (169.27+/-3.85nm), polydispersity (0.410) and zeta potential (-23.28+/-1.21mV) of the PL. SDS-PAGE and Western blot analysis revealed the presence of lipopolysaccharide (LPS), mannose-sensitive haemagglutinin (MSHA) and a range of outer membrane proteins, including OmpU. BALB/c mice were immunized intranasally with two doses of PL containing 25mug of LPS each 28 days apart. The mice showed high anti-LPS IgG titres (3.36+/-0.235) and vibriocidal antibodies (3.70+/-0.23) after two weeks from last dose. These results show for the first time that PL can be obtained from V. cholerae O1 and when administer by intranasal route has the potential to protect against this pathogen.

The effect of the administration of human gamma globulins in a model of BCG infection in mice.

The effect of the administration of a commercial preparation of human gamma globulins has been evaluated in a mouse model of intranasal infection with BCG. First, we demonstrated the passage of specific antibodies to saliva and lung lavage following the intranasal or intraperitoneal administration to mice of human gamma globulins. This treatment of mice inhibited BCG colonization of the lungs (p < 0.01). A similar inhibitory effect was observed after infection of mice with gamma globulin opsonized BCG organisms (p < 0.01). These results are relevant for the development of new strategies for the control and treatment of tuberculosis.

Desarrollo de biomodelos para la evaluación de la inmunidad secretora contra M. tuberculosis en ratones Babl/c / Development of Biomodels for the evaluation of the secretory immunity against M tuberculosis in Balb/c mice

Poco se ha estudiado acerca del papel de los anticuerpos específicos, presentes en las secreciones del aparato respiratorio, en la defensa contra patógenos intracelulares, como es el caso de las micobacterias causantes de la tuberculosis en el hombre: Micobacterium tuberculosis, bovis y africanum. Con el objetivo de desarrollar modelos adecuados para evaluar el posible papel de la inmunidad secretoria en la defensa contra la tuberculosis, se desarrollaron dos modelos animales con la utilización de un anticuerpo monoclonal IgA dirigido contra la proteína de 16 kD de M. tuberculosis y M. bovis. En el primer modelo se inocularon ratones Balb/c, por vía subcutánea al nivel de la nuca, con diferentes cantidades de células del hibridoma TBA61, productor de la IgA específica. En un segundo modelo, se inoculó por vía intraperitoneal líquido ascítico correspondiente a este hibridoma obtenido en ratón. En ambos casos se determinó, a diferentes tiempos, la concentración del monoclonal en saliva y sólo en suero para el segundo. En los dos modelos se demostró el paso del monoclonal a la saliva, donde alcanzó la máxima concentración: a los 21 días en los animales inoculados con el hibridoma, y a las 2 horas en saliva y suero en los animales inoculados con el líquido ascítico. Se sugiere, por su sencillez y mayor inocuidad, el uso del segundo modelo para la realización de estudios de reto por vía mucosal(AU)