Live attenuated TB vaccines representing the three modern Mycobacterium tuberculosis lineages reveal that the Euro-American genetic background confers optimal vaccine potential.

BACKGROUND: Human tuberculosis (TB) is caused by a plethora of Mycobacterium tuberculosis complex (MTBC) strains belonging to seven phylogenetic branches. Lineages 2, 3 and 4 are considered "modern" branches of the MTBC responsible for the majority of worldwide TB. Since the current BCG vaccine confers variable protection against pulmonary TB, new candidates are investigated. MTBVAC is the unique live attenuated vaccine based on M. tuberculosis in human clinical trials. METHODS: MTBVAC was originally constructed by unmarked phoP and fadD26 deletions in a clinical isolate belonging to L4. Here we construct new vaccines based on isogenic gene deletions in clinical isolates of the L2 and L3 modern lineages. These three vaccine candidates were characterized at molecular level and also in animal experiments of protection and safety. FINDINGS: Safety studies in immunocompromised mice showed that MTBVAC-L2 was less attenuated than BCG Pasteur, while the original MTBVAC was found even more attenuated than BCG and MTBVAC-L3 showed an intermediate phenotype. The three MTBVAC candidates showed similar or superior protection compared to BCG in immunocompetent mice vaccinated with each MTBVAC candidate and challenged with three representative strains of the modern lineages. INTERPRETATION: MTBVAC vaccines, based on double phoP and fadD26 deletions, protect against TB independently of the phylogenetic linage used as template strain for their construction. Nevertheless, lineage L4 confers the best safety profile. FUNDING: European Commission (TBVAC2020, H2020-PHC-643381), Spanish Ministry of Science (RTI2018-097625-B-I00), Instituto de Salud Carlos III (PI18/0336), Gobierno de Aragón/Fondo Social Europeo and the French National Research Council (ANR-10-LABX-62-IBEID, ANR-16-CE35-0009, ANR-16-CE15-0003).

Genome wide approaches discover novel Mycobacterium tuberculosis antigens as correlates of infection, disease, immunity and targets for vaccination.

Every day approximately six thousand people die of Tuberculosis (TB). Its causative agent, Mycobacterium tuberculosis (Mtb), is an ancient pathogen that through its evolution developed complex mechanisms to evade immune surveillance and acquire the ability to establish persistent infection in its hosts. Currently, it is estimated that one-fourth of the human population is latently infected with Mtb and among those infected 3-10% are at risk of developing active TB disease during their lifetime. The currently available diagnostics are not able to detect this risk group for prophylactic treatment to prevent transmission. Anti-TB drugs are available but only as long regimens with considerable side effects, which could both be reduced if adequate tests were available to monitor the response of TB to treatment. New vaccines are also urgently needed to substitute or boost Bacille Calmette-Guérin (BCG), the only approved TB vaccine: although BCG prevents disseminated TB in infants, it fails to impact the incidence of pulmonary TB in adults, and therefore has little effect on TB transmission. To achieve TB eradication, the discovery of Mtb antigens that effectively correlate with the human response to infection, with the curative host response following TB treatment, and with natural as well as vaccine induced protection will be critical. Over the last decade, many new Mtb antigens have been found and proposed as TB biomarkers and vaccine candidates, but only a very small number of these is being used in commercial diagnostic tests or is being assessed as candidate TB vaccine antigens in human clinical trials, aiming to prevent infection, disease or disease recurrence following treatment. Most of these antigens were discovered decades ago, before the complete Mtb genome sequence became available, and thus did not harness the latest insights from post-genomic antigen discovery strategies and genome wide approaches. These have, for example, revealed critical phase variation in Mtb replication and accompanying gene -and therefore antigen- expression patterns. In this review, we present a brief overview of past methodologies, and subsequently focus on the most important recent Mtb antigen discovery studies which have mined the Mtb antigenome through "unbiased" genome wide approaches. We compare the results for these approaches -as far as we know for the first time-, highlight Mtb antigens that have been identified independently by different strategies and present a comprehensive overview of the Mtb antigens thus discovered.

Optimizing the management of children with latent tuberculosis infection.

INTRODUCTION: The management of latent tuberculosis (LTBI) in children represents an important issue for paediatricians because of the disease burden, the lack of a gold standard for the diagnosis and the high annual risk of progression to active disease. Areas covered: A review of English language articles on LTBI in children, published between the 1st of January 2010 and the 1st of July 2016, was conducted using multiple keywords and standardized terminology in PubMed database. This review provides an updated overview of the available tests for LTBI diagnosis in children, management strategies and treatment options. Expert commentary: Two tests are available for LTBI diagnosis: tuberculin skin test and interferon-gamma release assays, both with a suboptimal specificity and sensitivity, and both with the lack of capability in distinguishing between infection and disease. Several new markers have been identified but further studies are needed. Among all treatment regimes, because of the high safety and efficacy profile showed and to avoid the poor completion rate, the treatment with a three-month course of isoniazid and rifampicin is currently recommended. New vaccines are needed because of the spread of the disease despite BCG vaccination in high risk countries. Currently, 15 new vaccines are in the pipeline.

Recombinant Bacillus subtilis spores for the delivery of Mycobacterium tuberculosis Ag85B-CFP10 secretory antigens.

Tuberculosis continues to be a great cause of morbidity and mortality in different parts of the world. Unfortunately, the current BCG vaccine being administered is not fully protective against tuberculosis; therefore, there is a great need for alternate vaccines. With an aim to develop such vaccines, we have analyzed the utility of Bacillus subtilis spores for the expression of two major immunodominant antigens of Mycobacterium tuberculosis, Ag85B and CFP10. We created three recombinant B. subtilis strains to express a truncated fusion of Ag85B191-325 and CFP101-70 antigens (T85BCFP), either on the spore coat (MTAG1 strain) or in the cytosol of B. subtilis (MTAG 2 and MTAG 3 strains). Examination of spores isolated from these strains revealed successful expression of T85BCFP antigens on the spore coat of MTAG1 as well as in the cytosol of vegetatively grown cells of MTAG2 and MTAG3, indicating that spores can indeed express M. tuberculosis antigens. In vitro antigen presentation assays with spore-infected mouse bone marrow derived macrophages (BMDM) showed that all three recombinant spores could deliver these antigens to antigen presenting cells (APCs). Mice immunized with recombinant spores displayed significantly higher levels of Ag85B specific IFN-γ producing cells in the spleen than in mice immunized with wild-type (non-recombinant) spores. In addition, these mice showed relatively higher levels of Ag85B specific IgG antibodies in the serum in comparison to mice immunized with non-recombinant spores, thus providing additional evidence that recombinant spores can deliver these antigens in vivo. These results suggest that B. subtilis spores are ideal vehicles for antigen delivery and have great potential in the development of primary and booster vaccines against tuberculosis.

Global Efforts in the Development of Vaccines for Tuberculosis: Requirements for Improved Vaccines Against Mycobacterium tuberculosis.

Currently, more than 9.0 million people develop acute pulmonary tuberculosis (TB) each year and about 1.5 million people worldwide die from this infection. Thus, developing vaccines to prevent active TB disease remains a priority. This article discusses recent progress in the development of new vaccines against TB and focusses on the main requirements for development of improved vaccines against Mycobacterium tuberculosis (M. tb). Over the last two decades, significant progress has been made in TB vaccine development, and some TB vaccine candidates have currently completed a phase III clinical trial. The potential public health benefits of these vaccines are possible, but it will need much more effort, including new global governance investment on this research. This investment would certainly be less than the annual global financial toll of TB treatment.

Construction and immunogenicity of a new Fc-based subunit vaccine candidate against Mycobacterium tuberculosis.

As an ancient disease, tuberculosis (TB) is a major global health threat. Therefore, there is an urgent need for an effective and safe anti-TB vaccine. In the current study, a delivery system of Fc domain of mouse IgG2a and early secreted antigenic target protein 6 (ESAT-6) was evaluated for the selective uptake of antigens by antigen-presenting cells (APCs). Thus, it was based on the immunogenicity of a fusion protein. The study was initiated by the transfer of recombinant expression vectors of pPICZαA-ESAT-6:Fcγ2a and pPICZαA-ESAT-6: His into Pichia pastoris (P. pastoris). Recombinant proteins were assessed for immunogenicity following the immunoblotting analysis. High levels of IFN-γ and IL-12 were produced to induce Th1-type cellular responses through vaccination with both recombinant proteins [ESAT-6:Fcγ2a (EF) and ESAT-6:His (EH)]. The Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low increment in IL-4 compared to PBS, BCG, and EH groups. Although in all the immunized groups, the ratio of IFN-γ/IL-4 was in favor of Th1 responses, the highest Th1/Th2 balance was observed in EF immunized group. Fc fragment of mouse IgG2a may induce a selective uptake of APCs towards the cross-presentation and formation of Th1 responses in favor of an appropriate protective anti-tuberculosis reaction. Thus, further research on Fc-fusion proteins is required to develop Fc-based TB vaccines.

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-vectored tuberculosis vaccine candidate.

Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials.

Understanding and overcoming the barriers to T cell-mediated immunity against tuberculosis.

Despite the overwhelming success of immunization in reducing, and even eliminating, the global threats posed by a wide spectrum of infectious diseases, attempts to do the same for tuberculosis (TB) have failed to date. While most effective vaccines act by eliciting neutralizing antibodies, T cells are the primary mediators of adaptive immunity against TB. Unfortunately, the onset of the T cell response after aerosol infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes TB, is exceedingly slow, and systemically administered vaccines only modestly accelerate the recruitment of effector T cells to the lungs. This delay seems to be orchestrated by Mtb itself to prolong the period of unrestricted bacterial replication in the lung that characterizes the innate phase of the response. When T cells finally arrive at the site of infection, multiple layers of regulation have been established that limit the ability of T cells to control or eradicate Mtb. From this understanding, emerges a strategy for achieving immunity. Lung resident memory T cells may recognize Mtb-infected cells shortly after infection and confer protection before regulatory networks are allowed to develop. Early studies using vaccines that elicit lung resident T cells by targeting the lung mucosa have been promising, but many questions remain. Due to the fundamental nature of these questions, and the need to understand and manipulate the early events in the lung after aerosol infection, only coordinated approaches that utilize tractable animal models to inform human TB vaccine trials will move the field toward its goal.

Engineering bacteria to manufacture functionalized polyester beads.

The ability to generate tailor-made, functionalized polyester (polyhydroxyalkanoate, PHA) beads in bacteria by harnessing their natural carbon-storage granule production system is an exciting recent development. Proteins that naturally attach to the polyester granule core were rationally engineered to enable in vivo production of PHA beads which are applicable in bioseparation, protein purification, enzyme immobilization and diagnostics and which show advantageous properties toward the development of safe and efficient particulate vaccines. These beads are recombinantly produced as fully functional, insoluble polyester inclusions that can be easily separated from the cell. This simple one-step production of functionalized beads provides a tantalizing alternative to current commercial functional beads, for which proteins must be expressed, purified and then chemically attached to solid supports. The recent success in generating antigen-displaying PHA granules in the food-grade bacterium Lactococcus lactis capable of mediating protective immunity against Mycobacterium tuberculosis infection highlights the promise and flexibility of this new technology.

A novel recombinant Mycobacterium bovis bacillus Calmette-Guerin strain expressing human granulocyte macrophage colony-stimulating factor and Mycobacterium tuberculosis early secretory antigenic target 6 complex augments Th1 immunity.

Since Mycobacterium bovis bacillus Calmette-Guerin strain (BCG) fails to protect adults from pulmonary tuberculosis (TB), there is an urgent need for developing a new vaccine. In this study, we constructed a novel recombinant BCG strain (rBCG) expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and the 6 kDa early secretory antigenic target (ESAT6) of Mycobacterium tuberculosis, named rBCG:GE (expressing GMCSF-ESAT6 complex), and evaluated the immunogenicity of the construct in BALB/c mice. Our results indicated that the rBCG:GE was able to induce higher titer of antibody than the conventional BCG, the rBCG:G (expressing GM-CSF) and the rBCG:E (expressing ESAT6). Moreover, the rBCG:GE also elicited a longer-lasting and stronger Th1 cellular immune responses than the other groups, which was confirmed by the incremental proliferation of splenocytes, the increased percentages of CD4(+) and CD8(+) T cells of spleen, the elevated level of interferon-γ in splenocyte culture after tuberculin-purified protein derivative stimulation, and the increased concentration of GM-CSF in serum. The data presented here suggested the possibility that the recombinant BCG:GE might be a good vaccine candidate to TB.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Development of plant-based mucosal vaccines against widespread infectious diseases.

Mucosal vaccination is a perspective for the control of infectious diseases, since it is capable of inducing humoral and cell-mediated responses. In addition, the delivery of vaccines to mucosal surfaces makes immunization practice safe and acceptable, and eliminates needle-associated risks. Transgenic plants can be used as bioreactors for the production of mucosally delivered protective antigens. This technology shows great promise to simplify and decrease the cost of vaccine delivery. Herein, we review the development of mucosally administered vaccines expressed in transgenic plants. In particular, we evaluate the advantages and disadvantages of using plants for the production of mucosal vaccines against widespread infectious diseases such as HIV, hepatitis B and TB.

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model.

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.

Comparison of immune responses induced in mice by vaccination with DNA vaccine constructs expressing mycobacterial antigen 85A and interleukin-21 and Bacillus Galmette-Guérin.

In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.

Superexpression of tuberculosis antigens in plant leaves.

Recent developments in genetic engineering allow the employment of plants as factories for 1/foreign protein production. Thus, tuberculosis (TB) ESAT6 antigen was expressed in different plant systems, but the level of vaccine protein accumulation was extremely low. We describe the technology for superexpression of TB vaccine proteins (Ag85B, ESAT6, and ESAT6:Ag85B fusion) in plant leaves which involves: (i) construction of tobacco mosaic virus-based vectors with the coat protein genes substituted by those for TB antigens; (ii) Agrobacterium-mediated delivery to plant leaf tissues of binary vectors containing the cDNA copy of the vector virus genome; and (iii) replication of virus vectors in plant cells under conditions suppressing the virus-induced gene silencing. This technology enables efficient production of the TB vaccine proteins in plants; in particular, the level of Ag85B antigen accumulation was not less than 800 mg/kg of fresh leaves. Expression of TB antigens in plant cells as His(6)-tagged proteins promoted their isolation and purification by Ni-NTA affinity chromatography. Deletion of transmembrane domains from Ag85B caused a dramatic increase in its intracellular stability. We propose that the strategy of TB antigens superproduction in a plant might be used as a basis for the creation of prophylactic and therapeutic vaccine against TB.

Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis.

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.

Oral immunogenicity of a plant-made, subunit, tuberculosis vaccine.

Transgenic plants are a novel way to produce and deliver oral vaccines. Arabidopsis thaliana material shown previously to express the tuberculosis (TB) antigen ESAT-6 fused to the B subunit of Escherichia coli heat-labile enterotoxin (LTB) was fed to mice and the resulting immune response investigated. The plant-made LTB-ESAT-6 fusion protein induced antigen-specific responses from CD4+ cells and increased IFN-gamma production, indicating a Th1 response. In addition, a Th2 response was induced in the Peyer's patch. This is the first report of an orally delivered, subunit, tuberculosis vaccine priming an antigen-specific, Th1 response.

[Construction of the recombinant integrating shuttle plasmid with cfpl0-esat6 fusion gene of Mycobacterium tuberculosis and its expression in BCG].

This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.