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Tuberculosis represents the leading global cause of death from an infectious agent. Controlling the tuberculosis epidemic thus represents an urgent global public health priority. Epidemiological modelling suggests that, although drug treatments for tuberculosis continue to improve, WHO timelines to control the spread of the disease require a new vaccine capable of preventing tuberculosis, particularly in adolescents and adults. The spread of strains resistant to multiple drugs adds additional urgency to the vaccine development effort yet attempts to develop new vaccines with wider applicability and better, longer-lasting efficacy than BCG-the only tuberculosis vaccine licensed for use globally-have proven challenging. Results from clinical efficacy trials, particularly a completed, phase 2b trial for preventing tuberculosis disease in people infected with Mycobacterium tuberculosis using the adjuvanted protein subunit vaccine M72/AS01E give hope. We review the current status of tuberculosis vaccine candidates and outline the diversified vaccine development that are underway., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Clinical trials of novel tuberculosis (TB) vaccines are expensive, while global resources for TB vaccine development are limited. Therefore, there is a need for robust and predictive preclinical data to support advancement of candidate vaccines into clinical trials. Here, we provide a rationale for using the nonhuman primate as an essential component of these efforts, as well as guidance to the TB community for standardizing experimental design and aligning endpoints to facilitate development of new TB vaccines., (Copyright © 2018 American Society for Microbiology.)

Recently, we introduced a temperature sensitive Mycobacterium spp., Mycobacterium paragordonae (Mpg). Here, we checked its potential as a candidate for live vaccination against Mycobacterium tuberculosis and Mycobacterium abscessus. Intravenous infections of mice with Mpg led to lower colony forming units (CFUs) compared to infection with BCG, suggesting its usefulness as a live vaccine. The analyses of immune responses indicated that the highly protective immunity elicited by Mpg was dependent on effective dendritic maturation, shift of cytokine patterns and antibody production toward a Th1 phenotype, and enhanced cytotoxic T cell response. Compared to BCG, Mpg showed a more effective protective immune response in the vaccinated mice against challenges with 2 different mycobacterial strains, M. tuberculosis H37Ra or M. abscessus Asan 50594. Our data suggest that a temperature sensitive Mpg may be a potentially powerful candidate vaccine strain to induce enhanced protective immune responses against M. tuberculosis and M. abscessus.

Introduction: BCG remains the only vaccine against tuberculosis (TB) in use today and despite its impressive global coverage, the nature of BCG protection against the pulmonary forms of TB remains subject to ongoing debate. Because of the limitations of BCG, novel TB vaccine candidates have been developed and several have reached the clinical pipeline. One of these candidates is MTBVAC, the first and only TB vaccine in the clinical pipeline to date based on live-attenuated Mycobacterium tuberculosis that has successfully entered clinical evaluation, a historic milestone in human vaccinology. Areas covered: This review describes development of MTBVAC from discovery to clinical development in high burden TB-endemic countries. The preclinical experiments where MTBVAC has shown to confer improved safety and efficacy over BCG are presented and the clinical development plans for MTBVAC are revealed. The search of all supportive literature in this manuscript was carried out via Pubmed. Expert commentary: Small experimental medicine trials in humans and preclinical efficacy studies with a strong immunological component mimicking clinical trial design are considered essential by the scientific community to help identify reliable vaccine-specific correlates of protection in order to support and accelerate community-wide efficacy trials of new TB vaccines.

The bacillus Calmette Guérin (BCG) vaccine, the only licensed vaccine against TB, displays partial and variable efficacy, thus making the exploitation of novel vaccination strategies a major priority. Most of the current vaccines in pre-clinical or clinical development are based on the induction of T cells recognizing protein antigens. However, a large number of T cells specific for mycobacterial lipids are induced during infection, suggesting that lipid-based vaccines might represent an important component of novel sub-unit vaccines. Here, we investigated whether immunization with defined mycobacterial lipid antigens induces protection in guinea pigs challenged with M. tuberculosis. Two purified mycobacterial lipid antigens, the diacylated sulfoglycolipids (Ac 2 SGL) and the phosphatidyl-myo-inositol dimannosides (PIM 2 ) were formulated in biophysically characterized liposomes made of dimethyl-dioctadecyl-ammonium (DDA) and synthetic trehalose 6,6'-dibehenate (TDB). In three protection trials, a reduction of bacterial load in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Moreover, a reduction in the number of lesions and severity of pathology was detected in the lungs and spleen of the lipid vaccine group compared to unvaccinated controls. As the degree of protection achieved is similar to that observed using protein antigens in the same guinea pig model, these promising results pave the way to future investigations of lipid antigens as subunit vaccines., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Traditional approaches to vaccine development have failed to identify better vaccines to replace or supplement BCG for the control of tuberculosis (TB). Subunit vaccines offer a safer and more reproducible alternative for the prevention of diseases. In this study, the immunogenicity of bacterially derived polyester beads displaying three different Rv antigens of Mycobacterium tuberculosis was evaluated. Polyester beads displaying the antigens Rv1626, Rv2032, Rv1789, respectively, were produced in an endotoxin-free Escherichia coli strain. Beads were formulated with the adjuvant DDA and subcutaneously administered to C57BL/6 mice. Cytokine responses were evaluated by CBA and antibody responses by ELISA. Specificity of the IgG response was assessed by immunoblotting cell lysates of the vaccine production strains using sera from the vaccinated mice. Mice vaccinated with beads displaying Rv1626 had significantly greater IgG1 responses compared to mice vaccinated with Rv1789 beads and greater IgG2 responses than the group vaccinated with Rv2032 beads (p<0.05). Immunoblotting of antisera from these mice indicated the antibody responses were Rv1626 antigen-specific and there was no detectable immune response to the polyester component of the vaccine. Overall, this study suggested that selected TB antigens derived from reverse vaccinology approaches can be displayed on polyester beads to produce antigen-specific immune responses potentially relevant to the prevention of TB., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Mycobacterium tuberculosis infects one third of the world's population. Due to variable efficacy of the Bacille Calmette Guerin (BCG) vaccine, development of novel TB vaccines remains a priority. Here, we demonstrate the protective efficacy of a novel multivalent DNA vaccine, which contains 15 synthetic antigens targeting the Mtb ESX secretion system.

In this article we present experimental Mycobacterium bovis infection models in domestic livestock species and how these models were applied to vaccine development, biomarker discovery, and the definition of specific antigens for the differential diagnosis of infected and vaccinated animals. In particular, we highlight synergies between human and bovine tuberculosis (TB) research approaches and data and propose that the application of bovine TB models could make a valuable contribution to human TB vaccine research and that close alignment of both research programs in a one health philosophy will lead to mutual and substantial benefits.

Mycobacterium tuberculosis (M.tb) has co-evolved with humans for thousands of years, to cause tuberculosis (TB). The success of M.tb as a pathogen is in part because of the ways in which M.tb evades and exploits different cell subsets, to persist and cause disease. M.tb expresses numerous molecules to prevent its recognition and destruction by immune cells. The only licensed vaccine against TB, Bacillle Calmette-Guerin (BCG), is effective at preventing disseminated disease in infants but confers highly variable efficacy against pulmonary TB in adults, particularly in the developing world. A greater understanding of the reasons for this variability, together with a better understanding of the early, innate, and non-antigen specific mechanisms of protection would facilitate the design and development of more effective vaccines.

Vaccines are critical for the control of tuberculosis (TB) affecting humans and animals worldwide. First-generation vaccines protect from active TB but new vaccines are required to protect against pulmonary disease and infection. Recent advances in post-genomics technologies have allowed the characterization of host-pathogen interactions to discover new protective antigens and mechanisms to develop more effective vaccines against TB. Studies in the wild boar model resulted in the identification of complement component 3 (C3) as a natural correlate of protection against TB. Oral immunization with heat-inactivated mycobacteria protected wild boar against TB and showed that C3 plays a central role in protection. These results point at C3 as a target to develop novel vaccine formulations for more effective protection against TB in humans and animals.

Different members of the Mycobacterium genus have evolved to cause tuberculosis in diverse human populations and in a variety of animal species. Our cumulative knowledge of mycobacterial genomes indicates that mutations in the PhoPR two-component virulence system were acquired not only during the natural evolution of mycobacterial species but also during in vitro subculture, which has given rise to the attenuated reference strain H37Ra or to different daughter strains of Mycobacterium bovis BCG. PhoPR is a well-known regulator of pathogenic phenotypes, including secretion of the virulence factor ESAT-6, biosynthesis of acyltrehalose-based lipids, and modulation of antigen export, in members of the Mycobacterium tuberculosis complex (MTBC). Evolutionarily conserved polymorphisms in PhoPR from Mycobacterium africanum, M. bovis, or M. tuberculosis H37Ra result in loss of functional phenotypes. Interestingly, some members of the MTBC have acquired compensatory mutations to counteract these polymorphisms and, probably, to maintain their pathogenic potential. Some of these compensatory mutations include the insertion of the IS6110 element upstream from phoPR in a particular M. bovis strain that is able to transmit between humans or polymorphisms in M. africanum and M. bovis that affect the regulatory region of the espACD operon, allowing PhoPR-independent ESAT-6 secretion. This review highlights the increasing knowledge of the significance of PhoPR in the evolution of the MTBC and its potential application in the construction of new attenuated vaccines based on phoPR inactivation. In this context, the live attenuated vaccine MTBVAC, based on a phoP fadD26 deletion mutant of M. tuberculosis, is the first vaccine of this kind to successfully enter into clinical development, representing a historic milestone in the field of human vaccinology., (Copyright © 2015 Broset et al.)

It is known that cellular immune response is relevant to fight against tuberculosis (TB); hence, identification of mycobacterial antigens that induce a protective immune cellular response is of great interest, especially for the development of effective TB vaccines. Genomic data have an impact on the identification of potential antigens as new vaccine targets. In this review, we summarize the current knowledge about the advances in new TB vaccine designs as well as the features reported for the pro-glu&#95;polymorphic GC-rich sequence (PE&#95;PGRS33) protein, considering this molecule as a prototype of the PE&#95;PGRS family to better understand the biological function of this protein family that could be considered an ideal target for future vaccine design.

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.

A new tuberculosis vaccine is needed to replace or enhance BCG, which induces variable protection against Mycobacterium tuberculosis pulmonary infections in adults. Development of new TB vaccine candidates is severely hampered by the lack of a correlate of immunity, unproven animal models, and limited funding opportunities. One candidate, MVA85A, recently failed to meet its efficacy endpoint goals despite promising early-phase trial data. As a result, some in the field believe we should now shift our focus away from product development and toward a research-oriented approach. Here, we outline our suggestions for this research-oriented strategy including diversification of the candidate pipeline, expanding measurements of immunity, improving pre-clinical animal models, and investing in combination pre-clinical/experimental medicine studies. As with any evolution, this change in strategy comes at a cost but may also represent an opportunity for advancing the field.

The development of more effective vaccines against Mycobacterium tuberculosis (Mtb) remains a major goal in the effort to reduce the enormous global burden of disease caused by this pathogen. Whole-cell vaccines based on live mycobacteria with attenuated virulence represent an appealing approach, providing broad antigen exposure and intrinsic adjuvant properties to prime durable immune responses. However, designing vaccine strains with an optimal balance between attenuation and immunogenicity has proven to be extremely challenging. Recent basic and clinical research efforts have broadened our understanding of Mtb pathogenesis and created numerous new vaccine candidates that have been designed to overcome different aspects of immune evasion by Mtb. In this review, we provide an overview of the current efforts to create improved vaccines against tuberculosis based on modifications of live attenuated mycobacteria. In addition, we discuss the use of such vaccine strains as vectors for stimulating protective immunity against other infectious diseases and cancers.

Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.

The present study describes a novel and simple vaccination strategy that involve culturing of M. tuberculosis in the macrophage cells. Isolation of phagosome from macrophage (cell line J774) infected with M. tuberculosis (H37) and M. bovis (BCG) at early and late phase of infection was done ensuing the identification and characterization of these phagosome. In vitro study of apoptosis induced by phagosome infected with (H37) and (BCG) was performed. The vaccine candidate with H1137 MOI- 1:10 at 3 h, MOI- 1:20 at 1, 1.5, 2.5 and 3 h and BCG MOI- 1:20 at 3.5 h showed percentage apoptosis as 38.64, 39.93, 34.66, 22.56,34.59 and 37.81% respectively. The results designates that macrophages provide cellular niche during infection and illustrate considerable immunogenic property. Novel antigens expressed or secreted by H37 in infected macrophages can provide evidence to be a successful vaccine candidate as it endures enhanced immune response than BCG.

Tuberculosis (TB) is an air-born, transmissible disease, having an estimated 9.4 million new TB cases worldwide in 2009. Eventual control of this disease by developing a safe and efficient new vaccine able to detain its spread will have an enormous impact on public health policy. Selecting potential antigens to be included in a multi-epitope, minimal subunit-based, chemically-synthesized vaccine containing the minimum sequences needed for blocking mycobacterial interaction with host cells is a complex task due to the multiple mechanisms involved in M. tuberculosis infection and the mycobacterium's immune evasion mechanisms. Our methodology, described here takes into account a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-TB vaccine; it has been based on identifying short mycobacterial protein fragments using synthetic peptides having high affinity interaction with alveolar epithelial cells (A549) and monocyte-derived macrophages (U937) which are able to block the microorganism's entry to target cells in in vitro assays. This manuscript presents a review of the results obtained with some of the MTB H37Rv proteins studied to date, aimed at using these high activity binding peptides (HABPs) as platforms to be included in a minimal subunit-based, multiepitope, chemically-synthesized, antituberculosis vaccine.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a major threat to global public health. A new TB vaccine affording superior immune protection to M. bovis Bacillus Calmette-Guérin (BCG) is imperative. The advantage of a live attenuated vaccine is that it can mimic the bona fide pathogen, elicit immune responses similar to natural infection, and potentially provide more protection than other vaccines. BCG, the only vaccine and a live attenuated vaccine that is the result of cumulative mutations by serial passage of M. bovis, has provided clues for the construction of novel improved vaccines. A strategy is put forward for identifying a new live attenuated TB vaccine generated by cumulative mutation based on M.tb. Given the important role of the M.tb signaling network consisting of a two-component system, eukaryotic-like Ser/Thr protein kinase system and sigma factor system based on comparisons among M.tb H37Rv, M. bovis, and BCG, we have put a premium on this signaling circuit as the starting point for the generation of an attenuated TB vaccine.

Substantial efforts have been made over the past decade to develop vaccines against tuberculosis. We review recent developments in tuberculosis vaccines in the global portfolio, including those designed for use in a prophylactic setting, either alone or as boosts to Bacille Calmette-Guérin, and therapeutic vaccines designed to improve chemotherapy. While there is no doubt that progress is still being made, there are limitations to our animal model screening processes, which are further amplified by the lack of understanding of the immunological responses involved and the precise type of long-lived immunity that new vaccines need to induce. The challenge ahead is to optimize the planning for advanced clinical trials in poor endemic settings, which could be greatly facilitated by identifying correlates of protection.

Previous experiments for the identification of novel diagnostic or vaccine candidates for bovine tuberculosis have followed a targeted approach, wherein specific groups of proteins suspected to contain likely candidates are prioritized for immunological assessment (for example, with in silico approaches). However, a disadvantage of this approach is that the sets of proteins analyzed are restricted by the initial selection criteria. In this paper, we describe a series of experiments to evaluate a nonbiased approach to antigen mining by utilizing a Gateway clone set for Mycobacterium tuberculosis, which constitutes a library of clones expressing 3,294 M. tuberculosis proteins. Although whole-blood culture experiments using Mycobacterium bovis-infected animals and M. bovis BCG-vaccinated controls did not reveal proteins capable of differential diagnosis, several novel immunogenic proteins were identified and prioritized for efficacy studies in a murine vaccination/challenge model. These results demonstrate that Rv3329-immunized mice had lower bacterial cell counts in their spleens following challenge with M. bovis. In conclusion, we demonstrate that this nonbiased approach to antigen mining is a useful tool for identifying and prioritizing novel proteins for further assessment as vaccine antigens.

Tuberculosis remains a major global health challenge despite extensive vaccination schemes with the current live vaccine, Bacillus Calmette-Guérin. Tuberculosis vaccine research has been hampered by a scarcity of animal models which replicate human disease and are suitable for large-scale studies. We have shown recently that Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, causes an infection resembling human tuberculosis in adult zebrafish (Danio rerio). In the present study we use this model to show that BCG vaccination as well as DNA vaccination with selected mycobacterial antigens (Ag85B, CFP-10 and ESAT-6) protects adult zebrafish from mycobacterial infection. Using a low-dose (∼20-30 bacteria) intraperitoneal M. marinum infection, both the number of granulomas and the amount of infected organs were reduced in the DNA vaccinated fish. Likewise, when infecting with a lethal infection dose (∼20,000-27,000 bacteria), vaccination significantly reduced both mortality and bacterial counts in a manner dependent on the adaptive immune response. Protective effects of vaccination were associated with enhanced expression of interferon gamma. Our results indicate that the zebrafish is a promising new model for preclinical tuberculosis vaccine research., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Accruing data strongly support the possible role of CD8+ T cells in immunity against tuberculosis (TB). Multivalent vaccines against Mycobacterium tuberculosis (Mtb) that incorporate CD8+ T cell antigens with those that elicit CD4+ T cells are therefore highly desirable. To screen for potential CD8+ T cell antigens that are produced by Mtb during infection, we isolated pathogen-derived peptides that bound to MHC Class I molecules expressed in adherent splenocytes obtained from Mtb-infected mice. Mass spectroscopy analysis revealed the following four nonamer peptides that had 100% homology with Mtb proteins: DGYVGAPAH (MT&#95;0401), TTMPLFAD (MT&#95;1164), RSGAATPVR (MT&#95;2160.1) and LAAVVGVVL (MT&#95;0078). The gene MT&#95;0401 codes the protein 5'-phosphoribosylglycinamide transformylase 2 and the other three genes code for hypothetical proteins with unknown function. The NCBI/Blast analysis showed that among the four peptides DGYVGAPAH had the highest maximum alignment score and lowest E value (number of alignments expected by chance). Therefore, we assessed whether MT&#95;0401 expressed in two genetic vaccine formulations was capable of stimulating CD8+ T cell response that is specific to DGYVGAPAH peptide. When mice were immunized with a recombinant plasmid DNA and an E1/E3-deleted Adenovirus 5 expressing MT0401 protein, using both homologous and heterologous prime-boost protocols, they developed strong DGYVGAPAH-specific CD8+ T cell response as well as antibody and CD4+ specific T cell response to the full length MT0401 protein. Equally important was the observation that mice infected with Mtb developed DGYVGAPAH-specific CD8+ T cell responses in both spleen and lungs. These results demonstrate that Mtb antigens that are processed and presented via MHC Class I machinery can be readily identified by the described approach and may be useful candidate antigens to stimulate specific CD8+ T cell responses in vaccine development programs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The control of TB is a global health priority. Over the last decade, considerable progress has been made in the field of TB vaccines with numerous vaccine candidates entering the clinic and two candidates now in Phase IIb efficacy trials. Nevertheless, the lack of predictive animal models and biomarkers of TB vaccine efficacy prevents rational vaccine down-selection and necessitates prolonged and expensive clinical efficacy trials in target populations. Advances in molecular technology and progress in the development of human as well as animal mycobacterial challenge models make the identification of one or more immune correlates of protection a genuine prospect over the next decade. Moreover, the increasing pace, extent and coordination of global research efforts in TB promises to broaden understanding and inform the next generation of vaccine candidates against TB as well as related globally important pathogens.

The only vaccine available against tuberculosis (TB), the Bacille Calmette-Guerin (BCG), does not provide effective protection against the most common forms of adult TB and in recent years efforts have been made to develop a new and improved vaccine.  Among the strategies implemented, the generation of a new live attenuated mycobacterial strain is seen as one of the most promising and feasible, for scientific, ethical and practical reasons.  The new understanding of the biology of the tubercle bacilli and of host-pathogen interaction processes, coupled with the possibility to engineer BCG or M. tuberculosis, opened new avenues to design "intelligent" vaccines, capable of eliciting the immune response associated with protection while avoiding the induction of the host immune response associated with immunopathology.  The complex and highly immunogenic mycobacterial cell wall can shape the general and antigen specific immune response elicited following immunization, and the possibility to exploit this knowledge may lead to the development of new vaccines that could help conquer this ancient human disease.

Antibiotics and vaccines are the two most successful medical countermeasures that humans have created against a number of pathogens. However a select few e.g., Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) have evaded eradication by vaccines and therapeutic approaches. TB is a global public health problem that kills 1.4 million people per year. The past decade has seen significant progress in developing new vaccine candidates, but the most fundamental questions in understanding disease progression and protective host responses that are responsible for controlling Mtb infection still remain poorly resolved. Current TB treatment requires intense chemotherapy with several antimicrobials, while the only approved vaccine is the classical viable whole-cell based Bacille-Calmette-Guerin (BCG) that protects children from severe forms of TB, but fails to protect adults. Taken together, there is a growing need to conduct basic and applied research to develop novel vaccine strategies against TB. This review is focused on the discussion surrounding current strategies and innovations being explored to discover new protective antigens, adjuvants, and delivery systems in the hopes of creating an efficacious TB vaccine.

Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70(359-610)), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model., (Copyright © 2011 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

Despite the clear need to control tuberculosis, the diagnosis and prevention of this serious disease are poorly developed and have remained fundamentally unchanged for more than 50 years. Here, we introduce an innovative approach to directly identify Mycobacterium tuberculosis antigens produced in vivo in humans with tuberculosis. We combined reversed phase high performance liquid chromatography and mass spectrometry and categorize four distinct M. tuberculosis proteins produced presumably in lung lesions and excreted in the urine of patients with pulmonary tuberculosis. The genes (MT&#95;1721, MT&#95;1694, MT&#95;2462 and MT&#95;3444) coding for these proteins were cloned and the recombinant molecules were produced in Escherichia coli. The proteins were recognized by immunoglobulin G antibodies from tuberculosis patients but not from non-diseased subjects. In addition, the recombinant proteins were recognized strongly by peripheral blood mononuclear cells from healthy purified protein derivative of tuberculin-positive individuals and to a lesser extent from patients with tuberculosis. These molecules are the only proteins reported to date that are derived directly from bodily fluids of tuberculosis patients, therefore are interesting candidate antigens for the development of vaccine and/or antigen detection assay for accurate diagnosis of active tuberculosis.

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.

In 1996, an independent scientific committee chaired by Professor John Krebs, tasked to review the problem of bovine tuberculosis (TB) in GB, concluded that vaccination of cattle offered the best long-term solution for controlling the disease in the National Herd. This view has been re-affirmed recently in the House of Commons Environment, Food and Rural Affairs Committee's report on Bovine TB (2004) and by the findings of the Independent Scientific Group Vaccine Scoping Sub-committee. Significant progress in developing TB vaccines for cattle has been made over the last 5 years. Specifically: (i) DNA or protein subunit vaccines used in combination with BCG have been shown to give superior protection against experimental challenge in cattle than BCG (heterologous prime-boost); (ii) prototype reagents that allow discrimination between vaccinated and infected animals have been developed; and (iii) and correlates of disease severity have been identified that can predict the success or failure of vaccination. These significant advances are detailed in this review with a summary of future directions that TB vaccine development for cattle is likely to take.

Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6 x His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.

The tuberculosis vaccine field has blossomed in the past 10 years, with over a hundred new candidates going through animal model testing, and several now entering or approaching clinical trial evaluation. In this brief review the current animal screening models are discussed, as are the various types of new vaccines that have been developed. New approaches, especially in the area of BCG boosting in various prime/boost protocols, are starting to show considerable promise. More sophisticated readouts, including imaging approaches such as magnetic resonance imaging, and better definition of the immunopathology of the lung disease process, should help accelerate vaccine development even further in the next decade.

Transgenic plants are potentially safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with carrier proteins could increase the effectiveness of the antigen. This paper reports the ability of transgenic Arabidopsis thaliana plants to produce a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin and a 6 kDa tuberculosis antigen, the early secretory antigenic target ESAT-6. Both components of the fusion protein were detected using GM1-ganglioside-dependent enzyme-linked immunosorbant assay. This suggested the fusion protein retained both its native antigenicity and the ability to form pentamers.