RESUMEN
BACKGROUND: Bovine Tuberculosis is a respiratory disease caused by the pathogen Mycobacterium bovis (M. bovis) that infects cattle. Though rare, this disease can also affect humans, as well as domestic and wild animals, making it a serious concern. Therefore, searching for alternative and new vaccines with high efficiency and safety is the main goal in bovine tuberculosis prophylaxis. New vaccines, known as vector vaccines, have the potential to become safe and effective alternatives to the traditional BCG vaccine. In this study, two major immunodominant proteins of M. bovis Esat-6 and TB10.4 were utilized to create a vector vaccine for bovine tuberculosis. METHODS: The Esat-6 and TB10.4 genes were amplified by PCR. The amplified and purified PCR products were sequenced by the Sanger method. Assembly and multiple alignments of amplicon nucleotides were carried out in the MEGA 11 software. RESULT: Two genes of the local strain 0078-M. bovis-8/RIBSP were sequenced. The nucleotide sequences were deposited in the GenBank database. Comparative analysis of the nucleotide sequences of the ESAT-6 and TB10.4 genes established 100% identity of the compared strains of Mycobacterium. CONCLUSION: Through the use of phylogenetic analysis, it has been confirmed that the amplified genes are related to the mycobacteria genus. This discovery allows the development of a vector vaccine against bovine tuberculosis utilising these genes.
RESUMEN
BCG is the only licensed vaccine against Mycobacterium tuberculosis (M.tb) infection. Due to its intramuscular administration route, BCG is unable to induce a local protective immune response in the respiratory system. Moreover, BCG has a diminished ability to induce long-lived memory T-cells which are indispensable for antituberculosis protection. Recently we described the protective efficacy of new mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing TB10.4 and HspX proteins of M.tb within an NS1 influenza protein open reading frame. In the present work, the innate and adaptive immune response to immunization with the Flu/THSP and the immunological properties of vaccine candidate in the BCG-prime â Flu/THSP vector boost vaccination scheme are studied in mice. It was shown that the mucosal administration of Flu/THSP induces the incoming of interstitial macrophages in the lung tissue and stimulates the expression of co-stimulatory CD86 and CD83 molecules on antigen-presenting cells. The T-cellular immune response to Flu/THSP vector was mediated predominantly by the IFNγ-producing CD8+ lymphocytes. BCG-prime â Flu/THSP vector boost immunization scheme was shown to protect mice from severe lung injury caused by M.tb infection due to the enhanced T-cellular immune response, mediated by antigen-specific effector and central memory CD4+ and CD8+ T-lymphocytes.
RESUMEN
New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1-124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime â Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.
RESUMEN
Tuberculosis (TB) remains a serious health issue around the word. Adenovirus (Ad)-based vaccine and modified vaccinia virus Ankara (MVA)-based vaccine have emerged as two of the most promising immunization candidates over the past few years. However, the performance of the homologous and heterologous prime-boost immunization regimens of these two viral vector-based vaccines remains unclear. In the present study, we constructed recombinant Ad and MVA expressing an Ag85B-TB10.4 fusion protein (AdH4 and MVAH4) and evaluated the impact of their different immunization regimens on the humoral and cellular immune responses. We found that the viral vector-based vaccines could generate significantly higher levels of antigen-specific antibodies, IFN-γ-producing splenocytes, CD69âºCD8⺠T cells, and IFN-γ secretion when compared with bacillus Calmette-Guérin (BCG) in a mouse model. AdH4-containing immunization regimens (AdH4-AdH4, AdH4-MVAH4, and MVAH4-AdH4) induced significantly stronger antibody responses, much more IFN-γ-producing splenocytes and CD69âºCD8⺠T cells, and higher levels of IFN-γ secretion when compared with the MVAH4-MVAH4 immunization regimen. The number of IFN-γ-producing splenocytes sensitive to CD8⺠T-cell restricted peptides of Ag85B (9-1p and 9-2p) and Th1-related cytokines (IFN-γ and TNF-α) in the AdH4-MVAH4 heterologous prime-boost regimen immunization group was significantly higher than that in the other viral vector-based vaccine- and BCG-immunized groups, respectively. These results indicate that an immunization regimen involving AdH4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that by regimens containing BCG or MVAH4 alone, and the AdH4-MVAH4 prime-boost regimen may generate an ideal protective effect.
Asunto(s)
Aciltransferasas/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Inmunización/métodos , Mycobacterium tuberculosis/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas contra la Tuberculosis/inmunología , Aciltransferasas/genética , Adenoviridae/genética , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Linfocitos T CD8-positivos/inmunología , Citocinas/metabolismo , Portadores de Fármacos , Vectores Genéticos , Inmunidad Celular , Inmunidad Humoral , Ratones , Mycobacterium tuberculosis/genética , Proteínas Recombinantes de Fusión/genética , Resultado del Tratamiento , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virus Vaccinia/genéticaRESUMEN
Tuberculosis (TB) remains a serious health problem worldwide, and the only available vaccine, bacillus Calmette-Guérin (BCG), has shown highly variable efficacy in adults against TB. New vaccines are urgently needed, and the modified vaccinia virus Ankara (MVA)-based vaccine has emerged as one of the most promising candidates based on many preclinical and early clinical studies over the past few years. However, the maximum tolerable dose and strength of induced immune responses have limited the protective effect of MVA-based prophylactic vaccines. To improve the immunogenicity of MVA-based vaccines, we introduced the tPA signal sequence in order to increase the antigen expression and secretion. Two recombinant MVA vectors expressing the Ag85B-TB10.4 fusion protein with or without tPA signal sequence were constructed and verified. Following the homologous prime-boost administration regimen in mice, levels of antigen-specific antibodies and cytokines (e.g., IFN-γ, TNF-α, IL-5, IL-6) and the percent of activated T cells were found to be significantly increased by the tPA signal sequence. However, the mean IgG2a/IgG1 ratios in the two recombinant MVA immunization groups were similar. Our present study demonstrated that the tPA signal sequence could enhance the immunogenicity of an MVA-based vaccine against TB without changing the balance of Th1 and Th2 immune responses. Thus, the tPA signal sequence may be applied to MVA-vector based vaccines for providing a better immune effect.
Asunto(s)
Aciltransferasas/genética , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium tuberculosis/inmunología , Señales de Clasificación de Proteína/genética , Linfocitos T/inmunología , Activador de Tejido Plasminógeno/genética , Tuberculosis/inmunología , Vacunas Sintéticas/inmunología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Vacunas Virales/inmunología , Animales , Células Cultivadas , Citocinas/metabolismo , Femenino , Humanos , Activación de Linfocitos , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos C57BL , Mycobacterium bovis/inmunología , Vacunación , Vacunas de ADN , Vacunas Sintéticas/genéticaRESUMEN
BACKGROUND: Tuberculosis (TB) remains as a major cause of death. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1(+) plasmid and evaluation of its expression in eukaryotic cells. METHODS: Firstly, tb10.4 fragment was amplified by PCR and the PCR product was digested with restriction enzymes. Next, it was cloned into pcDNA3.1(+) plasmid. Following that, pcDNA3.1(+)/tb10.4 recombinant plasmid was transfected into eukaryotic cells. RESULTS: 5700 bp band for pcDNA3.1(+)/tb10.4 recombinant plasmid and 297 bp fragment for tb10.4 were observed. Cloning and transfection were successful. CONCLUSION: Successful cloning provides a basis for the development of new DNA vaccines against tuberculosis.
RESUMEN
The TB10.4 antigen of Mycobacterium bovis/Mycobacterium tuberculosis induces a strong Th1 CD4+ T-cell response. Thus, it is currently under intensive study as a possible vaccine candidate. However, how TB10.4 activates innate immune cells is unclear. How TB10.4 interacts with toll-like receptors (TLRs) and signaling pathways responsible for active inflammation have also not been fully elucidated. Here, as stimulated RAW264.7 cells with recombinant TB10.4 (rTB10.4), derived from M. bovis, increased TNF-α, IL-6 and IL-12 p40 secretin in a dose-dependent manner. Blocking assays showed that TLR2-, but not TLR4-neutralizing antibody reduced expression of TNF-α, IL-6 and IL-12 p40 in RAW264.7 cells. rTB10.4 stimulation activated p38 kinase (p38) and extracellular-regulated kinase (ERK) was TLR2-dependent, whereas inhibition of p38 and ERK activity significantly reduced TNF-α, IL-6 and IL-12 p40 production. Furthermore, rTB10.4 stimulation of RAW264.7 cells resulted in TLR2-mediated activation of NF-κB and induced translocation of NF-κB p65 from the cytoplasm to the nucleus via IκBα degradation. rTB10.4-induced TNF-α, IL-6 and IL-12 p40 release was attenuated by the specific IκB phosphorylation inhibitor, BAY 11-7082. These findings indicate that the M. bovis-derived rTB10.4 induced production of TNF-α, IL-6 and IL-12 p40 involves p38, ERK and NF-κB via the TLR2 pathway.
Asunto(s)
Antígenos Bacterianos/inmunología , Citocinas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Receptor Toll-Like 2/fisiología , Animales , Antígenos Bacterianos/farmacología , Células Cultivadas , Activación Enzimática , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , FN-kappa B/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Relatively few MHC class I epitopes have been identified from Mycobacterium tuberculosis, but during the late stage of infection, CD8(+) T-cell responses to these epitopes are often primed at an extraordinary high frequency. Although clearly available for recognition during infection, their role in resistance to mycobacterial infections still remain unclear. As an alternative to DNA and viral vaccination platforms, we have exploited a novel CD8(+) T-cell-inducing adjuvant, cationic adjuvant formulation 05 (dimethyldioctadecylammonium/trehalose dibehenate/poly (inositic:cytidylic) acid), to prime high-frequency CD8 responses to the immunodominant H2-K(b) -restricted IMYNYPAM epitope contained in the vaccine Ag tuberculosis (TB)10.4/Rv0288/ESX-H (where ESX is mycobacterial type VII secretion system). We report that the amino acid C-terminal to this minimal epitope plays a decisive role in proteasomal cleavage and epitope priming. The primary structure of TB10.4 is suboptimal for proteasomal processing of the epitope and amino acid substitutions in the flanking region markedly increased epitope-specific CD8(+) T-cell responses. One of the optimized sequences was contained in the closely related TB10.3/Rv3019c/ESX-R Ag and when recombinantly expressed and administered in the cationic adjuvant formulation 05 adjuvant, this Ag promoted very high CD8(+) T-cell responses. This abundant T-cell response was functionally active but provided no protection against challenge, suggesting that CD8(+) T cells play a limited role in protection against M. tuberculosis in the mouse model.