Comparative analysis of genomic characteristics and immune response between Mycobacterium tuberculosis strains cultured continuously for 25 years and H37Rv.

Tuberculosis (TB) continues to pose a significant global health challenge, emphasizing the critical need for effective preventive measures. Although many studies have tried to develop new attenuated vaccines, there is no effective TB vaccine. In this study, we report a novel attenuated Mycobacterium tuberculosis (M. tb) strain, CHVAC-25, cultured continuously for 25 years in the laboratory. CHVAC-25 exhibited significantly reduced virulence compared to both the virulent H37Rv strain in C57BL/6J and severe combined immunodeficiency disease mice. The comparative genomic analysis identified 93 potential absent genomic segments and 65 single nucleotide polymorphic sites across 47 coding genes. Notably, the deletion mutation of ppsC (Rv2933) involved in phthiocerol dimycocerosate synthesis likely contributes to CHVAC-25 virulence attenuation. Furthermore, the comparative analysis of immune responses between H37Rv- and CHVAC-25-infected macrophages showed that CHVAC-25 triggered a robust upregulation of 173 genes, particularly cytokines crucial for combating M. tb infection. Additionally, the survival of CHVAC-25 was significantly reduced compared to H37Rv in macrophages. These findings reiterate the possibility of obtaining attenuated M. tb strains through prolonged laboratory cultivation, echoing the initial conception of H37Ra nearly a century ago. Additionally, the similarity of CHVAC-25 to genotypes associated with attenuated M. tb vaccine positions it as a promising candidate for TB vaccine development.

Peptides-Based Vaccine MP3RT Induced Protective Immunity Against Mycobacterium Tuberculosis Infection in a Humanized Mouse Model.

Background: Tuberculosis (TB) is still a global infectious disease that seriously threatens human beings. The only licensed TB vaccine Bacille Calmette-Guérin (BCG)'s protective efficacy varies significantly among populations and regions. It is very urgent to develop more effective vaccines. Methods: In this study, eleven candidate proteins of Mycobacterium tuberculosis were selected to predict peptides with high-affinity binding capacity for the HLA-DRB1*01:01 molecule. The immunodominant peptides were identified with the enzyme-linked immunospot assay (ELISPOT) and linked in silico to result in a novel polypeptide vaccine in Escherichia coli cells. The vaccine's protective efficacy was evaluated in humanized and wild-type C57BL/6 mice. The potential immune protective mechanisms were explored with Enzyme-linked Immunosorbent Assay (ELISA), flow cytometry, and ELISPOT. Results: Six immunodominant peptides screened from 50 predicted peptides were used to construct a new polypeptide vaccine named MP3RT. After challenge with M. tuberculosis, the colony-forming units (CFUs), lung lesion area, and the number of inflammatory cells in humanized mice rather than wild-type mice vaccinated with MP3RT were significantly lower than these in mice immunized with PBS. The humanized mice vaccinated with MP3RT revealed significant increases in IFN-γ cytokine production, IFN-γ+ T lymphocytes, CD3+IFN-γ+ T lymphocytes, and the MP3RT-specific IgG antibody. Conclusions: Taken together, MP3RT is a promising peptides-based TB vaccine characterized by inducing high levels of IFN-γ and CD3+IFN-γ+ T lymphocytes in humanized mice. These new findings will lay a foundation for the development of peptides-based vaccines against TB.

[Construction and Evaluation of a Tuberculosis Multistage Vaccine Candidate Based on Recombinant Listeria ivanovii].

OBJECTIVE: To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model. METHODS: T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD 50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD 50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI- msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry. RESULTS: A recombinant strain named LI- msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD 50 value of LI- msv for C57BL/6 mice (i.v.) was 3.3×10 8 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ + CD4 + and IFN-γ + CD8 +T lymphocytes were successfully induced in LI -msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4 + and CD8 +T cells was significantly higher than those of vector control group and NS control group ( P<0.005). Additionally, the frequency of specific TNF-α + CD4 + T cell in LI -msv immunized group was significantly higher than that of vector control ( P<0.01) and NS control group ( P<0.005), and TNF-α + CD8 + T cell frequency obviously increased than NS control group ( P<0.005). CONCLUSIONS: A novel multistage TB vaccine candidate expressing TB multistage antigen based on LI was successfully constructed. This vaccine candidate is safe and can induce specific cellular immune response to some extent. It is promising to be further studied as a candidate vaccine against tuberculosis.

Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity.

Tuberculosis (TB) pathogenesis is characterized by inadequate immune cell activation and delayed T cell response in the host. Recent immunotherapeutic efforts have been directed at stimulating innate immunity and enhancing interactions between antigen presenting cells and T cells subsets to improve the protective immunity against TB. In this study, we investigated the immunostimulatory properties of bacterial ghosts (BG) as a novel approach to potentiate the host immunity against mycobacterial infection. BG are intact cytoplasm-free Escherichia coli envelopes and have been developed as bacterial vaccines and adjuvant/delivery system in cancer immunotherapy. However, BG have yet to be exploited as immunopotentiators in the context of infectious diseases. Here, we showed that BG are potent inducers of dendritic cells (DC), which led to enhanced T cell proliferation and differentiation into effector cells. BG also induced macrophage activation, which was associated with enhanced nitric oxide production, a key anti-mycobacterial weapon. We further demonstrated that the immunostimulatory capability of BG far exceeds that of LPS and involves both TLR4-dependent and independent pathways. Consistently, BG treatment, but not LPS treatment, reduced the bacterial burden in infected mice, which correlated with increased influx of innate and adaptive effector immune cells and increased production of key cytokines in the lungs. Finally and importantly, enhanced bacilli killing was seen in mice co-administered with BG and second-line TB drugs bedaquiline and delamanid. Overall, this work paves the way for BG as potent immunostimulators that may be harnessed to improve mycobacteria killing at the site of infection.

Heterologous prime-boost vaccination against tuberculosis with recombinant Sendai virus and DNA vaccines.

In an earlier study, a novel Sendai virus-vectored anti-tuberculosis vaccine encoding Ag85A and Ag85B (SeV85AB) was constructed and shown to elicit antigen-specific T cell responses and protection against Mycobacterium tuberculosis (Mtb) infection in a murine model. In this study, we evaluate whether the immune responses induced by this novel vaccine might be elevated by a recombinant DNA vaccine expressing the same antigen in a heterologous prime-boost vaccination strategy. The results showed that both SeV85AB prime-DNA boost (SeV85AB-DNA) and DNA prime-SeV85AB boost (DNA-SeV85AB) vaccination strategies significantly enhanced the antigen-specific T cell responses induced by the separate vaccines. The SeV85AB-DNA immunization regimen induced higher levels of recall T cell responses after Mtb infection and conferred better immune protection compared with DNA-SeV85AB or a single immunization. Collectively, our study lends strong evidence that a DNA vaccine boost might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb. KEY MESSAGES: A heterologous prime-boost regimen with a novel recombinant SeV85AB and a DNA vaccine increase the T cell responses above those from a single vaccine. The heterologous prime-boost regimen provided protection against Mtb infection. The DNA vaccine might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb.

Single-Dose Mucosal Immunotherapy With Chimpanzee Adenovirus-Based Vaccine Accelerates Tuberculosis Disease Control and Limits Its Rebound After Antibiotic Cessation.

BACKGROUND: The development of strategies to accelerate disease resolution and shorten antibiotic therapy is imperative in curbing the global tuberculosis epidemic. Therapeutic application of novel vaccines adjunct to antibiotics represents such a strategy. METHODS: By using a murine model of pulmonary tuberculosis (TB), we have investigated whether a single respiratory mucosal therapeutic delivery of a novel chimpanzee adenovirus-vectored vaccine expressing Ag85A (AdCh68Ag85A) accelerates TB disease control in conjunction with antibiotics and restricts pulmonary disease rebound after premature (nonsterilizing) antibiotic cessation. RESULTS: We find that immunotherapy via the respiratory mucosal, but not parenteral, route significantly accelerates pulmonary mycobacterial clearance, limits lung pathology, and restricts disease rebound after premature antibiotic cessation. We further show that vaccine-activated antigen-specific T cells, particularly CD8 T cells, in the lung play an important role in immunotherapeutic effects. CONCLUSIONS: Our results indicate that a single-dose respiratory mucosal immunotherapy with AdCh68Ag85A adjunct to antibiotic therapy has the potential to significantly accelerate disease control and shorten the duration of conventional treatment. Our study provides the proof of principle to support therapeutic applications of viral-vectored vaccines via the respiratory route.

Enhanced delivery of Mycobacterium tuberculosis antigens to antigen presenting cells using RVG peptide.

Among the various strategies to improve vaccines against infectious diseases, targeting of antigens to dendritic cells (DCs), which are professional antigen presenting cells (APCs), has received increased attention in recent years. Here, we investigated whether a synthetic peptide region named RVG, originated from Rabies Virus Glycoprotein that binds to the α-7 subunit of the nicotinic acetylcholine receptors (AchR-α7) of APCs, could be used for the delivery of Mycobacterium tuberculosis (Mtb) peptide antigens to DCs and macrophages. Mouse bone marrow derived DCs (BMDCs) and human THP-1 macrophages stimulated with RVG fused peptide epitopes 85B241 and 85B96 (represent Ag85B241-256 and Ag85B96-111, respectively) from antigen 85B (Ag85B) of Mtb showed enhanced antigen presentation as compared to unfused peptide epitopes and BCG. Further, BMDCs stimulated with RVG fused 85B241 showed higher levels of IL-12 positive cells. Consistent with in vitro data, splenocytes of mice immunized with RVG-85B241 showed increased number of antigen specific IFN-γ, IL-2, and TNF-α producing cells in relation to splenocytes from mice immunized with 85B241 alone. These results suggest that RVG may be a promising tool to develop effective alternate vaccines against tuberculosis (TB).

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice.

BACKGROUND: Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world's population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0-80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed. RESULTS: A novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice's spleen, lung and GIT. CONCLUSIONS: This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.

Adjuvant Potential of Poly-α-l-Glutamine from the Cell Wall of Mycobacterium tuberculosis.

Novel adjuvants are in demand for improving the efficacy of human vaccines. The immunomodulatory properties of Mycobacterium tuberculosis cell wall components have been highlighted in the formulation of complete Freund's adjuvant (CFA). We have explored the adjuvant potential of poly-α-l-glutamine (PLG), a lesser-known constituent of the pathogenic mycobacterial cell wall. Immune parameters indicated that the adjuvant potency of PLG was statistically comparable to that of CFA and better than that of alum in the context of H1 antigen (Ag85B and ESAT-6 fusion). At 1 mg/dose, PLG augmented the immune response of Ag85B, BP26, and protective antigen (PA) by increasing serum antibodies and cytokines in the culture supernatant of antigen-stimulated splenocytes. PLG modulated the humoral response of vaccine candidate ESAT-6, eliciting significantly higher levels of total IgG and isotypes (IgG1, IgG2a, and IgG2b). Additionally, the splenocytes from PLG-adjuvanted mice displayed a robust increase in the Th1-specific gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), Th2-specific IL-6 and IL-10, and Th17-specific IL-17A cytokines upon antigenic stimulation. PLG improved the protective efficacy of ESAT-6 by reducing bacillary load in the lung and spleen as well as granuloma formation, and it helped in maintaining vital health parameters of mice challenged with M. tuberculosis The median survival time of PLG-adjuvanted mice was 205 days, compared to 146 days for dimethyl-dioctadecyl ammonium bromide-monophosphoryl lipid A (DDA-MPL)-vaccinated groups and 224 days for Mycobacterium bovis BCG-vaccinated groups. PLG enhanced the efficiency of the ESAT-6 vaccine to the level of BCG and better than that of DDA-MPL (P < 0.05), with no ill effect in C57BL/6J mice. Our results propose that PLG is a promising adjuvant candidate for advanced experimentation.

Performance of Homologous and Heterologous Prime-Boost Immunization Regimens of Recombinant Adenovirus and Modified Vaccinia Virus Ankara Expressing an Ag85B-TB10.4 Fusion Protein against Mycobacterium tuberculosis.

Tuberculosis (TB) remains a serious health issue around the word. Adenovirus (Ad)-based vaccine and modified vaccinia virus Ankara (MVA)-based vaccine have emerged as two of the most promising immunization candidates over the past few years. However, the performance of the homologous and heterologous prime-boost immunization regimens of these two viral vector-based vaccines remains unclear. In the present study, we constructed recombinant Ad and MVA expressing an Ag85B-TB10.4 fusion protein (AdH4 and MVAH4) and evaluated the impact of their different immunization regimens on the humoral and cellular immune responses. We found that the viral vector-based vaccines could generate significantly higher levels of antigen-specific antibodies, IFN-γ-producing splenocytes, CD69⁺CD8⁺ T cells, and IFN-γ secretion when compared with bacillus Calmette-Guérin (BCG) in a mouse model. AdH4-containing immunization regimens (AdH4-AdH4, AdH4-MVAH4, and MVAH4-AdH4) induced significantly stronger antibody responses, much more IFN-γ-producing splenocytes and CD69⁺CD8⁺ T cells, and higher levels of IFN-γ secretion when compared with the MVAH4-MVAH4 immunization regimen. The number of IFN-γ-producing splenocytes sensitive to CD8⁺ T-cell restricted peptides of Ag85B (9-1p and 9-2p) and Th1-related cytokines (IFN-γ and TNF-α) in the AdH4-MVAH4 heterologous prime-boost regimen immunization group was significantly higher than that in the other viral vector-based vaccine- and BCG-immunized groups, respectively. These results indicate that an immunization regimen involving AdH4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that by regimens containing BCG or MVAH4 alone, and the AdH4-MVAH4 prime-boost regimen may generate an ideal protective effect.

A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

In this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ-chain fragment and the tailpiece from immunoglobulin µ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB-PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N-glycans linked to TB-PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low-affinity FcγRs. Immunization studies in mice indicated that TB-PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen-IgG γ-chain molecules with inherent functional characteristics that are desirable in vaccines.

Immunogenicity and protective efficacy of multistage vaccine candidates (Mtb8.4-HspX and HspX-Mtb8.4) against Mycobacterium tuberculosis infection in mice.

In this study, Mtb8.4 and HspX, which are expressed at proliferating and dormant stages of Mycobacterium tuberculosis (M. tuberculosis), respectively, were chosen to construct two fusion proteins, Mtb8.4-HspX (8.4H) and HspX-Mtb8.4 (H8.4), and we investigated whether the antigen dose and protein sequential order could impact the immunogenicity and protective efficacy of these fusion protein vaccines against M. tuberculosis. C57BL/6 mice were vaccinated with new constructions containing a fusion protein with adjuvant of N, N'-dimethyl-N, N'-dioctadecylammonium bromide (DDA) or a mixed adjuvant composed of DDA, polyribocytidylic acid and gelatin (DPG), and the antigen specific immune responses and protective efficacy against M. tuberculosis H37Rv were evaluated. The results showed that both antigens, Mtb8.4-HspX and HspX-Mtb8.4, could elicit strong human T cell responses. With the existing of DDA adjuvant, HspX-Mtb8.4 induced significantly higher secretion level of IFN-γ and TNF-α in spleen cells than Mtb8.4-HspX (p<0.05). In its protective efficacy study, the isolated bacterial Colony Form Unit (CFU) in H8.4-DPG group was significantly reduced compared to 8.4H-DPG group (p<0.05). Furthermore, with the stimulation of Mtb8.4 in vitro, the secretion of IFN-γ and TNF-α from mice immunized with 20µg of H8.4 exhibited relative higher level than the group immunized by 7µg of H8.4 (p<0.05), whereas, IL-2 secreting showed contrary result. The data suggest that the antigen sequential order and dose selection should be considered when a tuberculosis protein vaccine is to be constructed and its immune strategy is to be planned.

GI-19007, a Novel Saccharomyces cerevisiae-Based Therapeutic Vaccine against Tuberculosis.

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

Mycobacterium tuberculosis MymA is a TLR2 agonist that activate macrophages and a TH1 response.

Cell wall of Mycobacterium tuberculosis (M.tb) is a major source of immunogenic proteins that can be tested as vaccine candidates. MymA (Rv3083), a 55 kDa M.tb flavin containing monooxygenase, is involved in modification of mycolic acids during acidic shock following M.tb internalization in macrophage. In this study, we have investigated the role of this cell wall associated protein in activation of macrophages by toll like receptor (TLRs) engagement and subsequent signaling. Our results showed that MymA stimulation of THP1 cells and human monocyte derived macrophages (MDM) lead to upregulation of TLR2 and co-stimulatory molecules CD40, CD80, CD86 and HLA-DR. This upregulation is partially reduced by TLR2 blocking antibodies. The activation of macrophage following MymA stimulation also resulted in release of proinflammatory cytokines, TNF-α and IL-12. Moreover, MymA also polarized the immune response towards TH1 as shown by an increased IFN-γ level in the supernatant of stimulated peripheral blood mononuclear cells (PBMC). In consensus with the TLR2 signaling involving MyD88 and NF-κB, we also observed several fold increase in mRNA for TLR2, MyD88 and NF-κB on MymA induction of THP-1 and MDM by qRT-PCR. The increased production of NF-κB following recognition of MymA by TLR2 was further confirmed by HEK-TLR2 reporter cell line colorimetric assay. In conclusion, immunological evaluation revealed that MymA is a TLR2 agonist that upregulates signaling via MyD88 and NF-κB in macrophages to stimulate the release of proinflammatory cytokines. The MymA protein should be investigated further for expression in recombinant BCG as a pre-exposure vaccine or as a post-exposure subunit vaccine candidate.

H1:IC31 vaccination is safe and induces long-lived TNF-α+IL-2+CD4 T cell responses in M. tuberculosis infected and uninfected adolescents: A randomized trial.

BACKGROUND: Control of the tuberculosis epidemic requires a novel vaccine that is effective in preventing tuberculosis in adolescents, a key target population for vaccination against TB. METHODS: Healthy adolescents, stratified by M. tuberculosis-infection status, were enrolled into this observer-blinded phase II clinical trial of the protein-subunit vaccine candidate, H1:IC31, comprising a fusion protein (H1) of Ag85B and ESAT-6, formulated with the IC31 adjuvant. Local and systemic adverse events and induced T cell responses were measured after one or two administrations of either 15µg or 50µg of the H1 protein. RESULTS: Two hundred and forty participants were recruited and followed up for 224days. No notable safety events were observed regardless of H1 dose or vaccination schedule. H1:IC31 vaccination induced antigen-specific CD4 T cells, co-expressing IFN-γ, TNF-α and/or IL-2. H1:IC31 vaccination of M.tb-uninfected individuals preferentially drove the emergence of Ag85B and ESAT-6 specific TNF-α+IL-2+CD4 T cells, while H1:IC31 vaccination of M.tb-infected individuals resulted in the expansion of Ag85B-specific but not ESAT-6-specific TNF-α+IL-2+CD4 T cells. CONCLUSIONS: H1:IC31 was safe and immunogenic in uninfected and M.tb-infected adolescents. Two administrations of the 15µg H1:IC31 dose induced the greatest magnitude immune response, and was considered optimal (South African National Clinical Trials Register, DoH-27-0612-3947; Pan African Clinical Trial Registry, PACTR201403000464306).

Recombinant bacille Calmette-Guerin coexpressing Ag85b, CFP10, and interleukin-12 elicits effective protection against Mycobacterium tuberculosis.

BACKGROUND: The tuberculosis (TB) pandemic remains a leading cause of human morbidity and mortality, despite widespread use of the only licensed anti-TB vaccine, bacille Calmette-Guerin (BCG). The protective efficacy of BCG in preventing pulmonary TB is highly variable; therefore, an effective new vaccine is urgently required. METHODS: In the present study, we assessed the ability of novel recombinant BCG vaccine (rBCG) against Mycobacterium tuberculosis by using modern immunological methods. RESULTS: Enzyme-linked immunospot assays demonstrated that the rBCG vaccine, which coexpresses two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 (rBCG2) elicits greater interferon-γ (IFN-γ) release in the mouse lung and spleen, compared to the parental BCG. In addition, rBCG2 triggers a Th1-polarized response. Our results also showed that rBCG2 vaccination significantly limits M. tuberculosis H37Rv multiplication in macrophages. The rBCG2 vaccine surprisingly induces significantly higher tumor necrosis factor-α (TNF-α) production by peripheral blood mononuclear cells that were exposed to a nonmycobacterial stimulus, compared to the parental BCG. CONCLUSION: In this study, we demonstrated that the novel rBCG2 vaccine may be a promising candidate vaccine against M. tuberculosis infection.

Gene-based neonatal immune priming potentiates a mucosal adenoviral vaccine encoding mycobacterial Ag85B.

Tuberculosis remains a major public health hazard worldwide, with neonates and young infants potentially more susceptible to infection than adults. BCG, the only vaccine currently available, provides some protection against tuberculous meningitis in children but variable efficacy in adults, and is not safe to use in immune compromised individuals. A safe and effective vaccine that could be given early in life, and that could also potentiate subsequent booster immunization, would represent a significant advance. To test this proposition, we have generated gene-based vaccine vectors expressing Ag85B from Mycobacterium tuberculosis (Mtb) and designed experiments to test their immunogenicity and protective efficacy particularly when given in heterologous prime-boost combination, with the initial DNA vaccine component given soon after birth. Intradermal delivery of DNA vaccines elicited Th1-based immune responses against Ag85B in neonatal mice but did not protect them from subsequent aerosol challenge with virulent Mtb H37Rv. Recombinant adenovirus vectors encoding Ag85B, given via the intranasal route at six weeks of age, generated moderate immune responses and were poorly protective. However, neonatal DNA priming following by mucosal boosting with recombinant adenovirus generated strong immune responses, as evidenced by strong Ag85B-specific CD4+ and CD8+ T cell responses, both in the lung-associated lymph nodes and the spleen, by the quality of these responding cells (assessed by their capacity to secrete multiple antimicrobial factors), and by improved protection, as indicated by reduced bacterial burden in the lungs following pulmonary TB challenge. These results suggest that neonatal immunization with gene-based vaccines may create a favorable immunological environment that potentiates the pulmonary mucosal boosting effects of a subsequent heterologous vector vaccine encoding the same antigen. Our data indicate that immunization early in life with mycobacterial antigens in an appropriate vaccine setting can prime for protective immunity against Mtb.

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.

Induction of an Immune-Protective T-Cell Repertoire With Diverse Genetic Coverage by a Novel Viral-Vectored Tuberculosis Vaccine in Humans.

BACKGROUND: Whether a candidate tuberculosis vaccine induces clinically relevant protective T-cell repertoires in humans will not be known until the completion of costly efficacy clinical trials. METHODS: We have developed an integrated immunologic approach to investigate the clinical relevance of T cells induced by a novel tuberculosis vaccine in a phase 1 trial. This approach consists of screening for likely dominant T-cell epitopes, establishing antigen-specific memory T-cell lines for identifying CD8+ and CD4+ T-cell epitopes, determining the ability of vaccine-induced T cells to inhibit mycobacterial growth in infected cells, and examining the genetic diversity of HLA recognition and the clinical relevance of identified T-cell epitopes. RESULTS: A single-dose immunization in BCG-primed adults with an adenovirus-based tuberculosis vaccine elicits a repertoire of memory T cells capable of recognizing multiple Ag85A epitopes. These T cells are polyfunctional and cytotoxic and can inhibit mycobacterial growth in infected target cells. Some identified T-cell epitopes are promiscuous and recognizable by the common HLA alleles. These epitopes are clinically relevant to the epitopes identified in people with latent Mycobacterium tuberculosis infection and treated patients with tuberculosis. CONCLUSIONS: These data support further clinical development of this candidate vaccine. Our approach helps fill the gap in clinical tuberculosis vaccine development.

Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines.

UNLABELLED: Tumor necrosis factor alpha (TNF) plays a critical role in the control of Mycobacterium tuberculosis, in part by augmenting T cell responses through promoting macrophage phagolysosomal fusion (thereby optimizing CD4(+) T cell immunity by enhancing antigen presentation) and apoptosis (a process that can lead to cross-priming of CD8(+) T cells). M. tuberculosis can evade antituberculosis (anti-TB) immunity by inhibiting host cell TNF production via expression of specific mycobacterial components. We hypothesized that M. tuberculosis mutants with an increased capacity to induce host cell TNF production (TNF-enhancing mutants) and thus with enhanced immunogenicity can be useful for vaccine development. To identify mycobacterial genes that regulate host cell TNF production, we used a TNF reporter macrophage clone to screen an H37Rv M. tuberculosis cosmid library constructed in M. smegmatis The screen has identified a set of TNF-downregulating mycobacterial genes that, when deleted in H37Rv, generate TNF-enhancing mutants. Analysis of mutants disrupted for a subset of TNF-downregulating genes, annotated to code for triacylglycerol synthases and fatty acyl-coenzyme A (acyl-CoA) synthetase, enzymes that concern lipid biosynthesis and metabolism, has revealed that these strains can promote macrophage phagolysosomal fusion and apoptosis better than wild-type (WT) bacilli. Immunization of mice with the TNF-enhancing M. tuberculosis mutants elicits CD4(+) and CD8(+) T cell responses that are superior to those engendered by WT H37Rv. The results suggest that TNF-upregulating M. tuberculosis genes can be targeted to enhance the immunogenicity of mycobacterial strains that can serve as the substrates for the development of novel anti-TB vaccines. IMPORTANCE: One way to control tuberculosis (TB), which remains a major global public health burden, is by immunization with an effective vaccine. The efficacy of Mycobacterium bovis BCG, the only currently approved TB vaccine, is inconsistent. Tumor necrosis factor alpha (TNF) is a cytokine that plays an important role in controlling TB. M. tuberculosis, the causative agent of TB, can counter this TNF-based defense by decreasing host cell TNF production. This study identified M. tuberculosis genes that can mediate inhibition of TNF production by macrophage (an immune cell critical to the control of TB). We have knocked out a number of these genes to generate M. tuberculosis mutants that can enhance macrophage TNF production. Immunization with these mutants in mice triggered a T cell response stronger than that elicited by the parental bacillus. Since T cell immunity is pivotal in controlling M. tuberculosis, the TNF-enhancing mutants can be used to develop novel TB vaccines.