TB Vaccine Research Articles

Immunogenicity assay of KatG protein from Mycobacterium tuberculosis in mice: preliminary screening of TB vaccine

the tuberculosis (tB) disease is still widely found even though BCg vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. objective of the research was to determine the immunogenicity of katg protein of M. tuberculosis clinical iso-late L19 in mice. the katg protein as antigen was prepared by expression of the katg gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the katg was vaccinated to mice to detect its immunogenicity. the expression of katg in e.coli BL21 could result in katg protein with molecular weight 80 kda in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of Igg1, Igg2a, Igg2b, Igg2c, Igg3, and IgM. the highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. the katg protein exhibited a high immunogenici ty in mice, so it is possible to develop as a vaccine candidate for tB. a clinical test should be performed in a future to ensure its safety as a therapeutic protein.

Comparison of pellicle and shake flask-grown BCG strains by quality control assays and protection studies

A global BCG vaccine shortage began in 2013 which impacted availability for infant vaccinations, as well as preclinical studies and clinical trials of new TB vaccines. Stakeholders met in 2015 at McGill University in Montreal to discuss the shortage and potential mitigation strategies. Manufacturing BCG through a more tractable liquid fermentation process instead of the traditional pellicle growth method was considered a potentially viable strategy. This pilot program compared pellicle-grown and shake flask-grown BCG strains (as a first step towards modeling fermenter-produced BCG vaccine) in selected quality control assays, as well as mouse and guinea pig protection studies. Conventional pellicle-grown, lyophilized BCG WHO Reference Reagents (Danish, Moreau, Russian, Tokyo strains) were obtained from the National Institute for Biological Standards and Control (NIBSC), UK. Strains were grown in shake flasks and glycerol stocks prepared. Shake flask-grown BCG culture preparations generally met the requirements of quality control testing at NIBSC. In mouse and guinea pig protection studies there were no significant differences in lung colony forming units (CFUs) between shake flask-grown and pellicle-grown preparations, with the exception of BCG Russian, where the shake flask-grown preparation protected better in mice (P = 0.0042), but the pellicle-grown preparation protected better in guinea pigs (P = 0.0015). Producing BCG vaccines by a more tractable liquid growth process could be a viable solution to the global BCG shortage.

Research and development of new tuberculosis vaccines: a review.

Tuberculosis kills more people worldwide than any other single infectious disease agent, a threat made more dire by the spread of drug-resistant strains of Mycobacterium tuberculosis (Mtb). Development of new vaccines capable of preventing TB disease and new Mtb infection are an essential component of the strategy to combat the TB epidemic. Accordingly, the WHO considers the development of new TB vaccines a major public health priority. In October 2017, the WHO convened a consultation with global leaders in the TB vaccine development field to emphasize the WHO commitment to this effort and to facilitate creative approaches to the discovery and development of TB vaccine candidates. This review summarizes the presentations at this consultation, updated with scientific literature references, and includes discussions of the public health need for a TB vaccine; the status of efforts to develop vaccines to replace or potentiate BCG in infants and develop new TB vaccines for adolescents and adults; strategies being employed to diversify vaccine platforms; and new animal models being developed to facilitate TB vaccine development. A perspective on the status of these efforts from the major funders and organizational contributors also is included. This presentation highlights the extraordinary progress being made to develop new TB vaccines and provided a clear picture of the exciting development pathways that are being explored.

Designing and construction of a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis

Background and Aim : Novel TB vaccines that aim to boost and/or replace Bacillus Calmette-Guerin (BCG) are currently in development. DNA vaccines can stimulate both humoral and cell-mediated immunity in different animal models of TB and is thought to be a promising strategy in the development of new vaccines against TB. The aim of this study was to design and construct a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis . Materials and Methods: Tb10.4 fragment was amplified by PCR and the products were digested with restriction enzymes. Next, it was cloned into the pcDNA3.1 + plasmid. The ag85a gene and pcDNA3.1 + / tb10.4 plasmid were digested by EcoRI and BamH1 restriction enzymes. Eukaryotic cells were transfected with pcDNA3.1 + / tb10.4 - ag85a plasmid for confirming expression of tb10.4 - ag85a in these cells. Results: Using electrophoresis of PCR products, fragments 297 bp for tb10.4 and 1017 bp for ag85a were observed. Eukaryotic cells transfection with pcDNA3.1 + / tb10.4 - ag85a vector was confirmed with cDNA synthesis and existence of tb10.4-ag85a was confirmed with RT-PCR. Conclusion: In this study, we constructed a DNA vaccine encoding tb10.4-ag85a fusion fragment. It can be used for development of more new DNA vaccines in future studies .

Using vaccine Immunostimulation/Immunodynamic modelling methods to inform vaccine dose decision-making

Unlike drug dose optimisation, mathematical modelling has not been applied to vaccine dose finding. We applied a novel Immunostimulation/Immunodynamic mathematical modelling framework to translate multi-dose TB vaccine immune responses from mice, to predict most immunogenic dose in humans. Data were previously collected on IFN-γ secreting CD4+ T cells over time for novel TB vaccines H56 and H1 adjuvanted with IC31 in mice (1 dose groups (0.1–1.5 and 15 μg H56 + IC31), 45 mice) and humans (1 dose (50 μg H56/H1 + IC31), 18 humans). A two-compartment mathematical model, describing the dynamics of the post-vaccination IFN-γ T cell response, was fitted to mouse and human data, separately, using nonlinear mixed effects methods. We used these fitted models and a vaccine dose allometric scaling assumption, to predict the most immunogenic human dose. Based on the changes in model parameters by mouse H56 + IC31 dose and by varying the H56 dose allometric scaling factor between mouse and humans, we established that, at a late time point (224 days) doses of 0.8–8 μg H56 + IC31 in humans may be the most immunogenic. A 0.8–8 μg of H-series TB vaccines in humans, may be as, or more, immunogenic, as larger doses. The Immunostimulation/Immunodynamic mathematical modelling framework is a novel, and potentially revolutionary tool, to predict most immunogenic vaccine doses, and accelerate vaccine development.

Addressing diversity in tuberculosis using multidimensional approaches.

Tuberculosis is a complex disease, which can affect many organs other than the lungs. Initial infection may be cleared without inducing immunological memory, or progress directly to primary disease. Alternatively, the infection may be controlled as latent TB infection, that may progress to active tuberculosis at a later stage. There is now a greater understanding that these infection states are part of a continuum, and studies using PET/CT imaging have shown that individual lung granulomas may respond to infection independently, in an un-synchronized manner. In addition, the Mycobacterium tuberculosis organisms themselves can exist in different states: as nonculturable forms, as 'persisters', as rapidly growing bacteria and a biofilm-forming cording phenotype. The 'omics' approaches of transcriptomics, metabolomics and proteomics can help reveal the mechanisms underlying these different infection states in the host, and identify biosignatures with diagnostic potential, that can predict the development of disease, in 'progressors' as early as 12-18months before it can be detected clinically, or that can monitor the success of anti-TB therapy. Further insights can be obtained from studies of BCG vaccination and new TB vaccines. For example, epigenetic changes associated with trained immunity and a stronger immune responses following BCG vaccination can be identified. These omics approaches may be particularly valuable when linked to studies of mycobacterial growth inhibition, as a direct read-out of the ability to control mycobacterial growth. The second generation of omics studies is identifying much smaller signatures based on as few as 3 or 4 genes. Thus, narrowing down omics-derived biosignatures to a manageable set of markers now opens the way to field-friendly point of care assays.

Interplay of strain and race/ethnicity in the innate immune response to M. tuberculosis.

BackgroundThe roles of host and pathogen factors in determining innate immune responses to M. tuberculosis are not fully understood. In this study, we examined host macrophage immune responses of 3 race/ethnic groups to 3 genetically and geographically diverse M. tuberculosis lineages.MethodsMonocyte-derived macrophages from healthy Filipinos, Chinese and non-Hispanic White study participants (approximately 45 individuals/group) were challenged with M. tuberculosis whole cell lysates of clinical strains Beijing HN878 (lineage 2), Manila T31 (lineage 1), CDC1551 (lineage 4), the reference strain H37Rv (lineage 4), as well as with Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA) and TLR4 agonist lipopolysaccharide (TLR4/LPS). Following overnight incubation, multiplex assays for nine cytokines: IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα, and GM-CSF, were batch applied to supernatants.ResultsFilipino macrophages produced less IL-1, IL-6, and more IL-8, compared to macrophages from Chinese and Whites. Race/ethnicity had only subtle effects or no impact on the levels of IL-10, IL-12p70, TNFα and GM-CSF. In response to the Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA), Filipino macrophages again had lower IL-1 and IL-6 responses and a higher IL-8 response, compared to Chinese and Whites. The TLR2/LTA-stimulated Filipino macrophages also produced lower amounts of IL-10, TNFα and GM-CSF. Race/ethnicity had no impact on IL-12p70 levels released in response to TLR2/LTA. The responses to TLR4 agonist lipopolysaccharide (TLR4/LPS) were similar to the TLR2/LTA responses, for IL-1, IL-6, IL-8, and IL-10. However, TLR4/LPS triggered the release of less IL-12p70 from Filipino macrophages, and less TNFα from White macrophages.ConclusionsBoth host race/ethnicity and pathogen strain influence the innate immune response. Such variation may have implications for the development of new tools across TB therapeutics, immunodiagnostics and vaccines.

Use of GLA-nanoalum as an effective adjuvant for a therapeutic ID93 TB vaccine

Abstract Tuberculosis (TB) caused by the intracellular bacterium Mycobacterium tuberculosis (Mtb) reportedly killed 1.3 million people in 2016 and is the leading cause of death caused by a single infectious organism. Increasingly worrisome is the ability of Mtb to develop extensive drug resistance. According to the 2017 WHO global TB report, there were 600,000 new rifampicin-resistant cases in 2016, and almost half a million cases with multiple drug resistant (MDR) TB. The development of new host-targeted therapeutic strategies that prevent the outgrowth of resistant mutants and/or modulate the immune response to combat Mtb infection and/or reduce disease pathology is one solution to prevent the generation of antibacterial resistance and treat drug resistant (DR)-TB. Here we evaluate a novel nanoalum adjuvant formulation containing a synthetic TLR4 agonist, glucopyranosyl lipid adjuvant (GLA), with our clinical ID93 protein as an immunotherapeutic vaccine. We show that immunotherapy with ID93+GLA-nanoalum is effective against Mtb when given as an adjunct to drug treatment in a mouse TB therapy model.

Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study.

The appearance of Mycobacterium tuberculosis strains leading to drug resistance has caused new problems in TB treatment in various parts of the world and forces WHO to declare TB as a global emergency. With the increase of TB drug resistance, it is convinced that a more effective vaccine development will stop the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT83, have been examined as TB vaccine candidate. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of this research is to conduct amplification of MPT83 antigen cloning, and expression of its antigen on E. coli bacteria. From the result of the research, it is expected that raw material to produce TB vaccine as well as a high-quality antigen can be obtained. The band of DNA in PCR product is 660 bp, while the one in pGEMT-Easy-Mpt83 recombinant plasmid is 3678 bp. This is expressed in E. coli BL21 strain and produces 48 kDa protein as well as GST-MPT83 fusion protein.

Mycobacterium tuberculosis protein Rv2220 induces maturation and activation of dendritic cells

Tuberculosis remains a serious health problem worldwide. Characterization of the dendritic cell (DC)-activating mycobacterial proteins has driven the development of effective TB vaccine candidates besides improving the understanding of immune responses. Some studies have emphasized the essential role of protein Rv2220 from M. tuberculosis in mycobacterial growth. Nonetheless, little is known about cellular immune responses to Rv2220. In this study, our aim was to test whether protein Rv2220 induces maturation and activation of DCs. Rv2220-activated DCs appeared to be in a mature state with elevated expression of relevant surface molecules and proinflammatory cytokines. DC maturation caused by Rv2220 was mediated by MAPK and NF-κB signaling pathways. Specifically, Rv2220-matured DCs induced the expansion of memory CD62LlowCD44highCD4+ T cells in the spleen of mycobacteria-infected mice. Our results suggest that Rv2220 regulates host immune responses through maturation of DCs, a finding that points to a new vaccine candidate against tuberculosis.

Measuring autophagy level along with vaccine reactive IFN-+CD4+ Th1 cells may be a promising approach to understand effi cacy of anti TB vaccine(s)

Autophagy is a homeostatic process in the eukaryotic cells which contributes towards degradation of unwanted cellular constituents, killing of the invading intracellular microbes and generation of cell mediated immunity (CMI).

PLGA particulate subunit tuberculosis vaccines promote humoral and Th17 responses but do not enhance control of Mycobacterium tuberculosis infection.

Tuberculosis places a staggering burden on human health globally. The new World Health Organisation End-TB Strategy has highlighted the urgent need for more effective TB vaccines to improve control of the disease. Protein-based subunit vaccines offer potential as safe and effective generators of protective immunity, and the use of particulate vaccine formulation and delivery by the pulmonary route may enhance local immunogenicity. In this study, novel particulate subunit vaccines were developed utilising biodegradable poly(lactic-co-glycolic acid) (PLGA) slow-release particles as carriers for the Mycobacterium tuberculosis lipoprotein MPT83, together with the adjuvants trehalose-dibehenate (TDB) or Monophosphoryl lipid A (MPL). Following delivery by the pulmonary or subcutaneous routes, the immunogenicity and protective efficacy of these vaccines were assessed in a murine model of M. tuberculosis infection. When delivered peripherally, these vaccines induced modest, antigen-specific Th1 and Th17 responses, but strong anti-MPT83 antibody responses. Mucosal delivery of the PLGA(MPT83) vaccine, with or without TDB, increased antigen-specific Th17 responses in the lungs, however, PLGA-encapsulated vaccines did not provide protection against M. tuberculosis challenge. By contrast, peripheral delivery of DDA liposomes containing MPT83 and TDB or MPL, stimulated both Th1 and Th17 responses and generated protection against M. tuberculosis challenge. Therefore, PLGA-formulated vaccines primarily stimulate strong humoral immunity, or Th17 responses if used mucosally, and may be a suitable carrier for vaccines against extracellular pathogens. This study emphasises the critical nature of the vaccine carrier, adjuvant and route of delivery for optimising vaccine efficacy against TB.

The influence of interferon-β supplemented human dendritic cells on BCG immunogenicity

IntroductionTuberculosis (TB) remains a huge worldwide burden, despite extensive vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is inadequate to protect the human population against TB. This underscore the critical necessitate to develop an improved TB vaccine, based on a better understanding of host-pathogen interactions and immune responses during mycobacterial infection. Aim of the workTo examine whether the exogenous addition of IFN-β could improve dendritic cell (DC) response to Mycobacterium bovis (M. bovis) and to evaluate the effect induced by the infection of human DCs with M. bovis (with and without IFN-β) and Mycobacterium tuberculosis (Mtb) on DC viability as well as to compare the ability of BCG and Mtb to provide DCs with a Th1-polarizing capacity through the assessment of the immunoregulatory cytokines interleukin (IL)-12, IL-10 and interferon-gamma (IFN-γ). MethodsImmature DCs (iDCs) were generated in vitro using peripheral blood monocytes separated by anti-CD14–conjugated microbeads in the presence of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and IL-4, cultured cells were analyzed using flow cytometry, then we tested DC viability after inoculation with M. bovis (with and without IFN-β pretreatment) and Mtb using light microscopic examination and trypan blue exclusion method. Additionally, supernatants from infected-DCs cultures were analyzed for IFN-γ, IL-12 and IL-10 by ELISA. ResultsThe viability of BCG-infected DCs was significantly higher than that of Mtb-infected DCs (61.55% vs 52.10%). BCG-infected DC produced significantly more IL-12 (p = 0.02) and less IL-10 (p = 0.01) compared with Mtb-infected cells. IFN-β-pretreated BCG-infected DCs produced significantly larger amounts of IL-12 than did BCG-infected DCs (p = 0.03) and Mtb-infected cells (p < 0.001). ConclusionIFN-β improves DC functions following BCG infection, thus assuming that IFN-β could be used as a vaccine adjuvant.

Dectin-1-Syk-CARD9 Signaling Pathway in TB Immunity.

One of the first steps toward mounting an effective immune response to Mycobacterium tuberculosis (Mtb) is recognition of the pathogen through pattern-recognition receptors (PRRs) expressed by innate immune cells. Activation of the PRR Dectin-1 by an unknown mycobacterial ligand triggers an intracellular signaling cascade involving numerous proteins, including spleen tyrosine kinase, protein kinase C-delta, and caspase recruitment domain family member 9, some of which have been shown to influence host immune response to TB infection. Here, we review the role of Dectin-1 signaling pathway in anti-mycobacterial immunity and discuss its contribution in the control of Mtb infection, and potential applications in TB vaccine adjuvanticity.

Biophysical and immunological characterization of the ESX-4 system ESAT-6 family proteins Rv3444c and Rv3445c from Mycobacterium tuberculosis H37Rv

The ESAT-6 family proteins of Mycobacterium tuberculosis are regarded as the key mediators in mycobacterial virulence and are largely considered as antigens that can improve TB vaccines and diagnostics. We have characterized Rv3444c and Rv3445c proteins of the ESX-4 system of ESAT-6 family of M. tuberculosis H37Rv, and have experimentally established that these two proteins interact to form a heterodimeric complex. Complex formation resulted in induction of α-helical conformation and stability against chemical denaturation. To evaluate the immunogenic potential, we have immunized mice with Rv3444c or Rv3445c along with Freund's incomplete adjuvant (FIA). Immunization with Rv3444c-FIA or Rv3445c-FIA resulted in long term humoral responses. Re-stimulation of splenocytes from immunized mice resulted in significant lymphocyte proliferation with induction of TNF-α and IL-6. Further, the humoral responses to Rv3444c and Rv3445c antigens in Indian patients with active pulmonary TB (n = 44), and healthy individuals (n = 20), were investigated. Compared to healthy individuals, high levels of IgG against Rv3444c and Rv3445c were observed in TB patient's sera, indicating that these proteins are actively produced during the active phase of TB. Cellular immune responses to these proteins in active pulmonary TB patients (n = 5) were also investigated using peripheral blood mononuclear cells (PBMCs). Both the proteins induce significant lymphocyte proliferation and up-regulate the induction of TNF-α and IL-6 in TB patients.

Immunization with an adenovirus-vectored TB vaccine containing Ag85A-Mtb32 effectively alleviates allergic asthma

Current treatments for allergic asthma primarily ameliorate symptoms rather than inhibit disease progression. Regulating the excessive T helper type 2 (Th2) responses may prevent asthma exacerbation. In this study, we investigated the protective effects of Ad5-gsgAM, an adenovirus vector carrying two mycobacterial antigens Ag85A and Mtb32, against allergic asthma. Using an ovalbumin (OVA)-induced asthmatic mouse model, we found that Ad5-gsgAM elicited much more Th1-biased CD4+T and CD8+T cells than bacillus Calmette-Guérin (BCG). After OVA challenge, Ad5-gsgAM-immunized mice showed significantly lowered airway inflammation in comparison with mice immunized with or without BCG. Total serum immunoglobulin E and pulmonary inducible-nitric-oxide-synthase were efficiently reduced. The cytokine profiles in bronchial-alveolar-lavage-fluids (BALFs) were also modulated, as evidenced by the increased level of interferon-γ (IFN-γ) and the decreased level of interleukin (IL)-4, IL-5, and IL-13. Anti-inflammatory cytokine IL-10 was sharply increased, whereas pro-inflammatory cytokine IL-33 was significantly decreased. Importantly, exogenous IL-33 abrogated the protective effects of Ad5-gsgAM, revealing that the suppression of IL-33/ST2 axis substantially contributed to protection against allergic inflammation. Moreover, regulatory T cells were essential for regulating aberrant Th2 responses as well as IL-33/ST2 axis. These results suggested that modulating the IL-33/ST2 axis via adenovirus-vectored mycobacterial antigen vaccination may provide clinical benefits in allergic inflammatory airways disease.Key messages•Ad5-gsgAM elicits Th1 responses and suppresses Th2-mediated allergic asthma in mice.•Ad5-gsgAM inhibits IL-33/ST2 axis by reducing IL-33 secretion but not ILC2 recruiting.•Treg is essential for modulating Th2 responses and IL-33/ST2 axis by Ad5-gsgAM.

Mycobacterium tuberculosis Protein Rv3841 Activates Dendritic Cells and Contributes to a T Helper 1 Immune Response.

The attenuated vaccine Mycobacterium bovis BCG (Bacille Calmette Guerin) has limited protective efficacy against TB. The development of more effective TB vaccines has focused on the mycobacterial antigens that cause strong T helper 1 (Th1) responses. Mtb protein Rv3841 (bacterioferritin B; BfrB) is known to play a crucial role in the growth of Mtb. Nonetheless, it is unclear whether Rv3841 can induce protective immunity against Mtb. Here, we studied the action of Rv3841 in maturation of dendritic cells (DCs) and its engagement in the development of T-cell immunity. We found that Rv3841 functionally activated DCs by upregulating costimulatory molecules and increased secretion of proinflammatory cytokines. Activation of DCs by Rv3841 was mediated by Toll-like receptor 4 (TLR4), followed by triggering of mitogen-activated protein kinase and nuclear factor-κB signaling pathways. In addition, Rv3841-matured DCs effectively proliferated and polarized Th1 immune response of naïve CD4+ and CD8+ T-cells. Moreover, Rv3841 specifically caused the expansion of CD4+CD44highCD62Llow T-cells from Mtb-infected mice; besides, the T-cells activated by Rv3841-matured DCs inhibited intracellular mycobacterial growth. Our data suggest that Rv3841 induces DC maturation and protective immune responses, a finding that may provide candidate of effective TB vaccines.

A Critical View of "On TB Vaccines, Patients' Demands, and Modern Printed Media in Times of Biomedical Uncertainties: Buenos Aires, 1920-1950".

The putative Pueyo's vaccine was a commercial venture that obtained marketing authorization in 1946, a turbulent period of Argentine history. After a few months, health authorities withdrew financial support from the state to buy the vaccine and required patients to sign a written consent to receive that product. An independent investigation did not find any evidence of benefit in non-clinical and clinical evaluation of the putative vaccine.

The patterns of in vitro cell-death and inflammatory cytokines induced by distinct BCG vaccine strains are differentially induced in human mononuclear cells

ABSTRACTTuberculosis (TB) remains among the world's leading cause of mortality. For its control, studies of TB vaccines are needed. Since live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently in use, studies on the protective role of BCG are required. In this study, we analyzed host cells purified directly from whole blood of human immunodeficiency virus (HIV)-negative volunteers, comprising adult healthy donors (HD) and neonates (umbilical cord bloods, UCB), with the aim to directly compare in vitro immune responses with distinct BCG strains in human mononuclear cells. The Moreau, Pasteur, and Danish BCG strains were used to infect mononuclear cells in vitro for 48 h; bacilli viability and cell-death were subsequently detected by flow cytometry. In addition, cell culture supernatants were used in cytokine detection assays. Overall, the Moreau BCG strain induced higher levels of apoptosis than the Pasteur and Danish BCG strains in both the HD and UCB groups (p-value < 0.05), and a human monocytic cell-line mirrored those cell-death patterns after BCG infection. The Moreau BCG strain, exclusively, induced Th1 cytokines at the highest levels in cells from adults (p-value < 0.05) when compared with both Pasteur and Danish BCG strains, whereas TGF-β1 levels were reduced significantly (p-value < 0.01) in the HD group when cells were infected with the Moreau BCG vaccine. As expected, eight out of 22 pro-inflammatory cytokines were secreted at significant levels (p-value < 0.05) above the baseline rates in all BCG-infected cell cultures, in the HD group only. When analyzing these results, we excluded confounding factors related to storage and viability of the BCG strains used. These findings suggest that Moreau BCG is a more potent immunostimulating agent than the Pasteur and Danish BCG strains. Clinical trials will be needed to confirm these findings.

In silico identification of cytotoxic T lymphocyte epitopes encoded by RD5 region of Mycobacterium tuberculosis.

While CD8+ T cells (cytotoxic T lymphocytes, CTLs) play important roles in immunity against Mycobacterium tuberculosis, only a small number of human leukocyte differentiation antigen (HLA) class I-restricted CTL epitopes for TB have been identified. The current study evaluates CTL epitopes of Rv3117 and Rv3120 proteins, two newly found M. tuberculosis region-diffference-5 (RD5)-encoded antigens, and their population coverage. The amino acid sequences of the two proteins were subjected to epitope analysis under HLA-A2, A3 and B7 supertype restriction using NetCTL, SYFPEITHI, BIMAS, NetCTLPan, IEDB, NetMHC and NetMHCPan prediction online servers. Eight RD5-encoded CTL epitopes were identified in the two proteins and the average population coverage of these epitopes was 87.2% among populations worldwide. These CTL epitopes that were identified in silico and may have potential use for CD8+ T cell-mediated TB vaccine design.