RESUMEN

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals' immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1ß, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

Asunto(s)

Administración Intranasal , Citocinas , Mycobacterium tuberculosis , Streptomyces lividans , Tuberculosis , Animales , Ratones , Aerosoles , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/administración & dosificación , Citocinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/administración & dosificación , Streptomyces lividans/genética , Streptomyces lividans/inmunología , Tuberculosis/prevención & control , Tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética

RESUMEN

Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.

Asunto(s)

Vacunas contra la Tuberculosis , Tuberculosis , Adyuvantes Inmunológicos , Adyuvantes Farmacéuticos , Animales , Vacuna BCG/genética , Sistemas CRISPR-Cas , Escherichia coli , Ratones , Vacunas contra la Tuberculosis/genética

RESUMEN

Background: The fusion protein H65, composed of Mycobacterium tuberculosis (TB) ESX-secreted antigens, has improved the bacillus Calmette-Guerin-induced immune protection in a mouse model of bovine TB when formulated in the liposomal adjuvant CAF01. In this study, we aimed to evaluate the protective efficacy of an attenuated Mycobacterium bovis strain - a mutant in mce2 and phoP genes - combined with H65+CAF01 immunization. We evaluated the protection of MbΔmce2-phoP alone or combined with H65+CAF01 against M. bovis challenge in mice. Methods: Groups of BALBc mice were inoculated with the vaccine candidates or phosphate buffered saline (PBS), and 6 weeks after the last immunization, the animals were aerogenically challenged with virulent M. bovis. Bacterial load in organs was counted after 45 days of the challenge. One-way analysis of variance and Bonferroni's posttest were used for statistical analysis. Results: All vaccinated mice showed reduced bacterial loads in lungs compared to unvaccinated animals. However, the protection level was similar between vaccinated groups. Conclusions: The MbΔmce2-phoP strain combined with three doses of H65+CAF01 induced equivalent protection than the MbΔmce2-phoP strain alone. Thus, the use of combined vaccination strategies requires a careful analysis of the potential interactions of each of their components with the host's immune system.

Asunto(s)

Mycobacterium bovis , Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis Bovina , Tuberculosis , Animales , Vacuna BCG , Bovinos , Modelos Animales de Enfermedad , Humanos , Pulmón/microbiología , Ratones , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/genética , Tuberculosis Bovina/prevención & control , Vacunas Atenuadas

RESUMEN

Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 µg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes.

Asunto(s)

Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium bovis/inmunología , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis Bovina/prevención & control , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Bovinos , Clonación Molecular , Codón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Inmunogenicidad Vacunal , Interferón gamma/biosíntesis , Mycobacterium bovis/química , Mycobacterium bovis/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Bovina/inmunología , Tuberculosis Bovina/microbiología , Vacunación/métodos

RESUMEN

Despite the great increase in the understanding of the biology and pathogenesis of Mycobacterium tuberculosis achieved by the scientific community in recent decades, tuberculosis (TB) still represents one of the major threats to global human health. The only available vaccine (Mycobacterium bovis BCG) protects children from disseminated forms of TB but does not effectively protect adults from the respiratory form of the disease, making the development of new and more-efficacious vaccines against the pulmonary forms of TB a major goal for the improvement of global health. Among the different strategies being developed to reach this goal is the construction of attenuated strains more efficacious and safer than BCG. We recently showed that a sigE mutant of M. tuberculosis was more attenuated and more efficacious than BCG in a mouse model of infection. In this paper, we describe the construction and characterization of an M. tuberculosissigE fadD26 unmarked double mutant fulfilling the criteria of the Geneva Consensus for entering human clinical trials. The data presented suggest that this mutant is even more attenuated and slightly more efficacious than the previous sigE mutant in different mouse models of infection and is equivalent to BCG in a guinea pig model of infection.

Asunto(s)

Ligasas/deficiencia , Mycobacterium tuberculosis/inmunología , Factor sigma/deficiencia , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Animales , Proteínas Bacterianas , Modelos Animales de Enfermedad , Cobayas , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Vacunas contra la Tuberculosis/efectos adversos , Vacunas contra la Tuberculosis/genética , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Virulencia

RESUMEN

Tuberculosis (TB) remains a main public health concern and 10.4 million new cases occurred in 2015 around the world. BCG is the only approved vaccine against TB, but has variable efficacy and new vaccines are needed. We developed two new mTB vaccine candidates based on the recombinant fusion proteins, rCMX and rECMX formulated with Advax4, a new combination adjuvant combining delta inulin, CpG oligonucleotide and murabutide. BALB/c mice were immunized three times intramuscularly with these vaccine formulations. Injection of Advax4 alone increased the percentage of lymphatic endothelial cells and activated macrophages (F480/CD11b+) in the draining lymph nodes consistent with a chemotactic adjuvant effect. Advax4+CMX and Advax4+ECMX induced the highest levels of IgG1 and IgG2a antibodies against rCMX and rECMX, respectively. Immunized mice challenged with Mycobacterium tuberculosis (Mtb) had increased vaccine-specific Th1 responses in the lungs together with reduced Mtb - associated alveolar damage, although only the Advax4+ECMX vaccine demonstrated significant reduction of lung bacterial load. This study confirmed Advax4+ECMX as a potential TB vaccine candidate, with potential for further optimization and clinical development.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Inulina/análogos & derivados , Células TH1/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Carga Bacteriana , Femenino , Humanos , Inmunoglobulina G/sangre , Inulina/administración & dosificación , Pulmón/microbiología , Ganglios Linfáticos/inmunología , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Resultado del Tratamiento , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

This study was aimed at characterising the PPE7 protein from the PE/PPE protein family. The presence and transcription of the rv0354c gene in the Mycobacterium tuberculosis complex was determined and the subcellular localisation of the PPE7 protein on mycobacterial membrane was confirmed by immunoelectron microscope. Two peptides were identified as having high binding activity (HABPs) and were tested in vitro regarding the invasion of Mycobacterium tuberculosis H37Rv. HABP 39224 inhibited invasion in A549 epithelial cells and U937 macrophages by more than 50%, whilst HABP 39225 inhibited invasion by 40% in U937 cells. HABP 39224, located in the protein's C-terminal region, has a completely conserved amino acid sequence in M. tuberculosis complex species and could be selected as a base peptide when designing a subunit-based, anti-tuberculosis vaccine.

Asunto(s)

Proteínas Bacterianas , Membrana Celular , Mycobacterium tuberculosis , Células A549 , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/patología , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/ultraestructura , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/metabolismo , Células U937

Asunto(s)

Proteínas Bacterianas/inmunología , Eliminación de Gen , Mycobacterium bovis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Femenino , Pulmón/microbiología , Ratones Endogámicos C57BL , Mycobacterium bovis/genética , Análisis de Supervivencia , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología

RESUMEN

Tuberculosis (TB) is a chronic infectious disease, considered as the second leading cause of death worldwide, caused by Mycobacterium tuberculosis. The limited efficacy of the bacillus Calmette-Guérin (BCG) vaccine against pulmonary TB and the emergence of multidrug-resistant TB warrants the need for more efficacious vaccines. Reverse vaccinology uses the entire proteome of a pathogen to select the best vaccine antigens by in silico approaches. M. tuberculosis H37Rv proteome was analyzed with NERVE (New Enhanced Reverse Vaccinology Environment) prediction software to identify potential vaccine targets; these 331 proteins were further analyzed with VaxiJen for the determination of their antigenicity value. Only candidates with values ≥0.5 of antigenicity and 50% of adhesin probability and without homology with human proteins or transmembrane regions were selected, resulting in 73 antigens. These proteins were grouped by families in seven groups and analyzed by amino acid sequence alignments, selecting 16 representative proteins. For each candidate, a search of the literature and protein analysis with different bioinformatics tools, as well as a simulation of the immune response, was conducted. Finally, we selected six novel vaccine candidates, EsxL, PE26, PPE65, PE_PGRS49, PBP1, and Erp, from M. tuberculosis that can be used to improve or design new TB vaccines.

Asunto(s)

Antígenos Bacterianos/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Tuberculosis Pulmonar/inmunología , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/uso terapéutico , Vacuna BCG/inmunología , Biología Computacional , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Proteoma/genética , Proteoma/inmunología , Vacunas contra la Tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis Resistente a Múltiples Medicamentos/prevención & control , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/prevención & control

RESUMEN

In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as an antituberculosis experimental vaccine in animal models. The double mutant strain was significantly more attenuated than the wild type strain in inmunocompetent and inmunodeficient mice. Vaccination with the double mutant protected mice against challenge with a virulent M. bovis strain.

Asunto(s)

Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium bovis/genética , Vacunas contra la Tuberculosis/genética , Tuberculosis Bovina/prevención & control , Animales , Bovinos , Recuento de Colonia Microbiana , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Ratones Endogámicos BALB C , Ratones Desnudos , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium bovis/patogenicidad , Células TH1/inmunología , Vacunas contra la Tuberculosis/inmunología , Vacunas contra la Tuberculosis/toxicidad , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/toxicidad , Virulencia/genética

RESUMEN

The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Asunto(s)

Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Portadores de Fármacos , Lactococcus lactis/genética , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Vacunas de ADN/administración & dosificación , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Clonación Molecular , Colon/inmunología , Heces/química , Inmunoglobulina A Secretora/análisis , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/genética , Plásmidos , Bazo/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/aislamiento & purificación , Vacunas de ADN/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación

RESUMEN

It is estimated that there are approximately eight million new cases of active tuberculosis (TB) worldwide annually. There is only 1 vaccine available for prevention: bacillus Calmette-Guérin (BCG). This has variable efficacy and is only protective for certain extrapulmonary TB cases in children, therefore new strategies for the creation of novel vaccines have emerged. One of the promising approaches is the DNA vaccine, used as a direct vaccination or as a prime-boost vaccine. This review describes the experimental data obtained during the design of DNA vaccines for TB.

Asunto(s)

Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis/prevención & control , Vacunas de ADN/administración & dosificación , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Humanos , Tuberculosis/inmunología , Vacunas contra la Tuberculosis/química , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Vacunas de ADN/química , Vacunas de ADN/genética , Vacunas de ADN/inmunología

RESUMEN

Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6'-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Antígenos Bacterianos/inmunología , Factores Cordón/administración & dosificación , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Vacunas de ADN/inmunología , Animales , Antígenos Bacterianos/genética , Carga Bacteriana , Modelos Animales de Enfermedad , Portadores de Fármacos/administración & dosificación , Femenino , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Microesferas , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética , Tuberculosis Pulmonar/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética

RESUMEN

The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis.

Asunto(s)

Bovinos/inmunología , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Bovina/inmunología , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis Bovina/prevención & control

RESUMEN

Of the hundreds of new tuberculosis (TB) vaccine candidates, some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65+CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Proteínas Bacterianas/inmunología , Chaperonina 60/inmunología , Sistemas de Liberación de Medicamentos , Microesferas , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Animales , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/genética , Chaperonina 60/administración & dosificación , Chaperonina 60/genética , Modelos Animales de Enfermedad , Femenino , Ácido Láctico/administración & dosificación , Ácido Láctico/metabolismo , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Ácido Poliglicólico/administración & dosificación , Ácido Poliglicólico/metabolismo , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

Immunodominant MPB83 antigen from Mycobacterium bovis was expressed as a chimeric protein fused to either ß-galactosidase, outer membrane lipoprotein OMP19 or periplasmic protein BP26 in gram-negative Brucella abortus S19, in all cases driven by each gene's own promoter. All fusion proteins were successfully expressed and localized in the expected subcellular fraction. Moreover, OMP19-MPB83 was processed as a lipoprotein when expressed in B. abortus. Splenocytes from BALB/c mice immunized with the recombinant S19 strains carrying the genes coding for the heterologous antigens in replicative plasmids, showed equally specific INF-γ production in response to MPB83 stimulation. Association to the lipid moiety of OMP19 presented no advantage in terms of immunogenicity for MPB83. In contrast, fusion to BP26, which was encoded by an integrative plasmid, resulted in a weaker immune response. None of the constructions affected the survival rate or the infection pattern of Brucella. We concluded that B. abortus S19 is an appropriate candidate for the expression of M. bovis antigens both associated to the membrane or cytosolic fraction and may provide the basis for a future combined vaccine for bovine brucellosis and tuberculosis.

Asunto(s)

Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/inmunología , Brucella abortus/genética , Vectores Genéticos , Inmunidad Celular , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/inmunología , Mycobacterium bovis/inmunología , Animales , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Vacuna contra la Brucelosis/genética , Vacuna contra la Brucelosis/inmunología , Brucella abortus/inmunología , Bovinos , Femenino , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Lipoproteínas/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Mycobacterium bovis/genética , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Bazo/inmunología , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , beta-Galactosidasa/genética

RESUMEN

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination.

Asunto(s)

Formación de Anticuerpos , ADN/química , Ácidos Grasos Monoinsaturados/química , Liposomas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Compuestos de Amonio Cuaternario/química , Vacunas contra la Tuberculosis/química , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Cationes , Muerte Celular , Chaperonina 60/genética , Chaperonina 60/inmunología , ADN/genética , Sondas de ADN/química , Portadores de Fármacos , Ensayo de Cambio de Movilidad Electroforética , Fluorescencia , Ratones , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Transición de Fase , Plásmidos/genética , Temperatura de Transición , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Vacunas de ADN/química , Vacunas de ADN/genética , Vacunas de ADN/inmunología

RESUMEN

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.

Asunto(s)

Proteínas Bacterianas/inmunología , Proteínas de la Membrana/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Línea Celular , Genes Bacterianos , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Péptidos/genética , Péptidos/inmunología , Conejos , Vacunas contra la Tuberculosis/genética

RESUMEN

Given the variable protective efficacy provided by Mycobacterium bovis BCG (Bacillus Calmette-Guérin), there is a concerted effort worldwide to develop better vaccines that could be used to reduce the burden of tuberculosis. Recombinant BCG (rBCG) are vaccine candidates that offer some potential in this area. In this paper, we will discuss the molecular methods used to generate rBCG, and the results obtained with some of these new vaccines as compared with the conventional BCG vaccine in diverse animal models. Tuberculosis vaccine candidates based on rBCG are promising candidates, and some of them are now being tested in clinical trials.

Asunto(s)

Mycobacterium bovis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/inmunología , Vacunas Sintéticas/inmunología , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Humanos , Mycobacterium bovis/genética , Tuberculosis/genética , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/genética , Vacunas Sintéticas/genética

RESUMEN

O presente estudo foi proposto para investigar as características e a eficácia da resposta imune induzida pela vacina gênica para tuberculose em camundongos neonatos. A construção genética empregada continha o gene da hsp65 de M.Leprae inserida no vetor pVax. Foram objetivos específicos deste trabalho: avaliar a imunização no período neonatal induz tolerância; determinar a presença de mensagem para hsp65, através de PCR, nos vários tecidos; caracterizar a resposta imune induzida por vacinação integral com DNAhsp65 no período neonatal avaliar as características da resposta imune induzida pela estratégia BCG no período neonatal seguida de boosters com a vacina gênica no período adulto e avaliar a eficácia protetora desta estratégia. Os protocolos experimentais incluíram imunização integral (3 doses) no período neonatal (5, 12 e 19 dias de idade) com 50miug de DNA/ dose por via IM; 1 dose de DNA no quinto dia seguida de 1 ou 2 doses no período adulto (100miug/ via IM) ou 1 dose de BCG no quinto dia seguida de 2 doses de DNA no período adulto. As avaliações foram feitas com células dos camundongos normais, injetados com PBS ou previamente imunizados e estimuladas ex vivo com S. aureus, LPS ou ConA. Os sobrenadantes das culturas foram coletados após 48 h de incubação e as concentrações de IFN-gama, IL-4, IL-5 e IL-10 foram determinadas por ELlSA. A produção de anticorpos (lgG1 e IgG2a anti-hsp65) foi avaliada no soro dos animais também pelo método de ELlSA. A detecção de mensagem para hsp65 nos diferentes tecidos foi feita por RT -PCR. Trinta dias após a infecção por via intratraqueal, os pulmões dos animais previamente imunizados com o protocolo BCG/DNA, foram analisados quanto ao número de unidades formadoras de colônias (UFC), quantidade de IFN-gama e aspectos histopatológicos...

Asunto(s)

Animales , Recién Nacido , Ratones , Tuberculosis , Vacunas contra la Tuberculosis/genética , Ratones