RESUMEN

This report describes the immunogenicity and protective efficacy of Escherichia coli-expressed recombinant protective antigen (rPA) in New Zealand White rabbits and Rhesus Macaques against an aerosol challenge with Bacillus anthracis spores (IVRI strain, tox+cap+). A dose-ranging study was performed in which it became evident that the level of anti-PA IgG and toxin-neutralizing antibody titer was directly proportional to the dose of rPA administered. However, the onset time of primary and secondary immune response was not dependent on the dosage. Revaccination of primed animals with the same threshold dose yielded a robust and rapid secondary response. Quantitative differences in peak titers were obtained for both the animal models, in addition to qualitative differences in the immune kinetics. In spite of a weak priming response, the secondary response in rabbits peaked earlier than that in macaques once the booster dose was administered. However, evaluation of the post-challenge quantitative anti-rPA ELISA titer measurements indicated higher titers for non-human primates as compared to the lagomorphs. Importantly, 100% protection was seen for the dosage groups that received > or = 25 microg rPA, following a challenge against a target dose of 1000 LD(50) of aerosolized spores of Bacillus anthracis.

Asunto(s)

Vacunas contra el Carbunco/uso terapéutico , Carbunco/prevención & control , Antígenos Bacterianos/química , Bacillus anthracis/inmunología , Toxinas Bacterianas/química , Esporas Bacterianas/inmunología , Vacunas Sintéticas/uso terapéutico , Aerosoles , Animales , Vacunas contra el Carbunco/química , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Cinética , Macaca mulatta , Pruebas de Neutralización , Conejos , Factores de Tiempo , Vacunas Sintéticas/química

RESUMEN

Studies have demonstrated that lipid rafts ultimately regulate the endocytosis of anthrax toxin via clathrin dependent pathway. Interestingly, GPI-anchored protein rich rafts have also been shown to be transported down to the endocytic pathway to reducing late endosomes. Taking advantage of this parallelism, we tried translating the anthrax toxin natural intoxication mechanism by administering a DNA chimera that encoded protective antigen attached to a mammalian GPI-anchor sequence at its C-terminus (pGPI-PA63). We also designed a chimera that had an additional N-terminal TPA leader sequence (pTPA.GPI-PA63) with an aim to target GPI-PA63 to ER where new CD1 molecules are synthesized. Analysis of antibody titers demonstrated successful priming and potential IgG titers after the first boost. In vitro cell proliferation studies in the presence of GPI-attached PA63 peptides revealed that there was a clonal expansion of CD4(+) NK1.1(+) helper T cell population which rapidly produced IL-4 in response to T cell receptor ligation. These cells provided direct B cell help that aided IgG formation. Effector responses generated by NKT cells were found to be MHC II-independent and CD1d-restricted. In addition, the group pTPA.GPI-PA63 also displayed low magnitude MHC-II restricted (CD1d-independent) NKT cell and CD4(+) T cell helper responses in response to non-GPI attached PA63 peptides which overall resulted in the heightened responses seen for this group. Importantly, DNA vaccination mediated the generation of high avidity neutralizing antibodies that mediated protection against lethal toxin challenge.

Asunto(s)

Vacunas contra el Carbunco/uso terapéutico , Anticuerpos Antivirales/biosíntesis , Antígenos CD1/inmunología , Antígenos Virales/inmunología , Genes MHC Clase II/inmunología , Glicosilfosfatidilinositoles/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Vacunas de ADN/uso terapéutico , Animales , Vacunas contra el Carbunco/inmunología , Anticuerpos Antivirales/análisis , Afinidad de Anticuerpos , Linfocitos B/inmunología , Western Blotting , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Interleucina-4/biosíntesis , Ratones , Pruebas de Neutralización , Plásmidos/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Transfección , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunología

RESUMEN

Based on the hypothesis that immune outcome can be influenced by the form of antigen administered and its ability to access various antigen-processing pathways, we targeted the 63 kDa fragment of protective antigen (PA) of Bacillus anthracis to various subcellular locations by DNA chimeras bearing a set of signal sequences. These targeting signals, namely, lysosome-associated membrane protein 1 (LAMP1), tissue plasminogen activator (TPA) and ubiquitin, encoded various forms of PA viz. lysosomal, secreted and cytosolic, respectively. Examination of IgG subclass distribution arising as a result of DNA vaccination indicated a higher IgG1:IgG2a ratio whenever the groups were immunized with chimeras bearing TPA, LAMP1 signals alone or when combined together. Importantly, high end-point titers of IgG antibodies were maintained until 24 wk. It was paralleled by high avidity toxin neutralizing antibodies (TNA) and effective cellular adaptive immunity in the systemic compartment. Anti-PA and TNA titers of approximately 10(5) and approximately 10(3), respectively, provided protection to approximately 90% of vaccinated animals in the group pTPA-PA63-LAMP1. A significant correlation was found between survival percentage and post-challenge anti-PA titers and TNA titers. Overall, immune kinetics pointed that differential processing through various compartments gave rise to qualitative differences in the immune response generated by various chimeras.

Asunto(s)

Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Vacunas de ADN/inmunología , Animales , Vacunas contra el Carbunco/administración & dosificación , Vacunas contra el Carbunco/genética , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Biolística , Citocinas/inmunología , Femenino , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/inmunología , Ratones , Ratones Endogámicos BALB C , Linfocitos T Citotóxicos/inmunología , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/inmunología , Ubiquitina/genética , Ubiquitina/inmunología , Vacunación , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética

RESUMEN

Vaccination is an important tool for handling healthcare programs both in developed and developing countries. The current global scenario calls for a more-efficacious, acceptable, cost-effective and reliable method of immunization for many fatal diseases. It is hoped that the adoption of oral vaccines will help to provide an effective vaccination strategy, especially in developing countries. Mucosal immunity generated by oral vaccines can serve as a strong first line of defense against most of the pathogens infecting through the mucosal lining. Advances in elucidating the mechanism of action of oral vaccines will facilitate the design of more effective, new generation vaccines. There are promising developments in the use of different agents to effectively deliver the vaccine candidate. It is hoped that ongoing research may be able to set another cardinal point, after polio vaccine, in eradicating infectious diseases.

Asunto(s)

Vacunación , Vacunas , Administración Oral , Enfermedades Transmisibles/inmunología , Países en Desarrollo , Salud Global , Necesidades y Demandas de Servicios de Salud , Humanos , Inmunidad Mucosa/fisiología , Programas de Inmunización , Vacunación/economía , Vacunas/administración & dosificación , Vacunas/economía , Vacunas/inmunología

RESUMEN

We have investigated the efficiency of N-terminal 1-260 residues of Edema factor (EFn) as a delivery system for ESAT-6, an antigenic protein of Mycobacterium tuberculosis H(37)R(v), into the cytosol of mammalian cells. The EFn.ESAT-6 recombinant protein was obtained by genetic fusion of EFn and ESAT-6 DNA. Our data shows that in the presence of PA, EFn.ESAT-6 fusion protein is internalized into the cytosol of antigen presenting cells, and the splenocytes produced both Th1 and Th2 cytokines in vitro. Further, EFn.ESAT-6 elicited effective cytotoxic T lymphocyte (CTL) response in an in vitro CTL assay. This study for the first time demonstrates that EFn can be used as a vehicle to deliver heterologous proteins of therapeutic importance.

Asunto(s)

Adenilil Ciclasas/inmunología , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Sistemas de Liberación de Medicamentos/métodos , Inmunoterapia/métodos , Macrófagos/inmunología , Adenilil Ciclasas/administración & dosificación , Adenilil Ciclasas/genética , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas , Línea Celular , Femenino , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine.

Asunto(s)

Células Presentadoras de Antígenos/inmunología , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Sistemas de Liberación de Medicamentos/métodos , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Toxinas Bacterianas/administración & dosificación , Células Cultivadas , Citosol/inmunología , Citosol/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos

RESUMEN

Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with l-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of l-arginine, N(omega)-hydroxy-l-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein from a pathogenic bacterium opens up avenues for further studies in understanding the importance of this protein in pathogenicity.

Asunto(s)

Bacillus anthracis/enzimología , Óxido Nítrico Sintasa/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Modelos Moleculares , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/aislamiento & purificación , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Análisis Espectral