Multiple genetic loci influence vaccine-induced protection against Mycobacterium tuberculosis in genetically diverse mice.

Mycobacterium tuberculosis (M.tb.) infection leads to over 1.5 million deaths annually, despite widespread vaccination with BCG at birth. Causes for the ongoing tuberculosis endemic are complex and include the failure of BCG to protect many against progressive pulmonary disease. Host genetics is one of the known factors implicated in susceptibility to primary tuberculosis, but less is known about the role that host genetics plays in controlling host responses to vaccination against M.tb. Here, we addressed this gap by utilizing Diversity Outbred (DO) mice as a small animal model to query genetic drivers of vaccine-induced protection against M.tb. DO mice are a highly genetically and phenotypically diverse outbred population that is well suited for fine genetic mapping. Similar to outcomes in people, our previous studies demonstrated that DO mice have a wide range of disease outcomes following BCG vaccination and M.tb. challenge. In the current study, we used a large population of BCG-vaccinated/M.tb.-challenged mice to perform quantitative trait loci mapping of complex infection traits; these included lung and spleen M.tb. burdens, as well as lung cytokines measured at necropsy. We found sixteen chromosomal loci associated with complex infection traits and cytokine production. QTL associated with bacterial burdens included a region encoding major histocompatibility antigens that are known to affect susceptibility to tuberculosis, supporting validity of the approach. Most of the other QTL represent novel associations with immune responses to M.tb. and novel pathways of cytokine regulation. Most importantly, we discovered that protection induced by BCG is a multigenic trait, in which genetic loci harboring functionally-distinct candidate genes influence different aspects of immune responses that are crucial collectively for successful protection. These data provide exciting new avenues to explore and exploit in developing new vaccines against M.tb.

Immunogenic profiling of Mycobacterium tuberculosis Rv1513 reveals its ability to switch on Th1 based immunity.

Tuberculosis (TB) is one of the infectious diseases caused by the pathogen Mycobacterium tuberculosis that continuously threatens the global human health. Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine that has been used clinically to prevent tuberculosis in recent centuries, but its limitations in preventing latent infection and reactivation of tuberculosis do not provide full protection. In this study, we selected the membrane-associated antigen Rv1513 of Mycobacterium. In order to achieve stable expression and function of the target gene, the prokaryotic expression recombinant vector pET30b-Rv1513 was constructed and expressed and purified its protein. Detection of IFN- γ levels in the peripheral blood of TB patients stimulated by whole blood interferon release assay (WBIA) and multi-microsphere flow immunofluorescence luminescence (MFCIA) revealed that the induced production of cytokines, such as IFN-γ and IL-6, was significantly higher than that in the healthy group. Rv1513 combined with adjuvant DMT (adjuvant system liposomes containing dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL), and trehalose-660-dibenzoic acid (TDB)) was used to detect serum specific antibodies, cytokine secretion from splenic suprasplenic cell supernatants, and multifunctional T-cell levels in splenocytes in immunised mice. The levels of IFN-γ, TNF-α, and IL-2 secreted by mouse splenocytes were found in the Rv1513+DMT group and the BCG+Rv1513+DMT group. The serum levels of IgG and its subclasses and the number of IFN-γ+T cells, TNF-α+T and IFN-γ+TNF-α+T cells in the induced CD4+/CD8+T cells in mice were significantly higher than those in the BCG group, and the highest levels were found in the BCG+Rv1513+DMT group. These findings suggest that Rv1513/DMT may serve as a potential subunit vaccine candidate that may be effective as a booster vaccine after the first BCG vaccination.

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

Immune Response to the Recombinant Apa Protein from Mycobacterium tuberculosis Expressed in Streptomyces lividans After Intranasal Administration in Mice. Induction of Protective Response to Tubercle Bacillus Aerosols Exposure.

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals' immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1ß, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

Synthesis of plant-based, self-adjuvanted, dual antigen specific to Mycobacterium tuberculosis as a novel tuberculosis subunit vaccine that elicits immunogenicity in rabbit.

OBJECTIVES: The only approved vaccine, Bacillus Calmette Guérin (BCG) used in global tuberculosis (TB) immunization programmes has been very effective in childhood TB but not in adult pulmonary and latent TB. Moreover, the emergence of multi-drug resistance-TB cases demands either to increase efficiency of BCG or replace it with the one with improved efficacy. RESULTS: A novel combination of two most effective secreted protein antigens specific for Mycobacterium tuberculosis (Mtb), ESAT-6 and MPT-64 (but not present in BCG strains) fused with a cholera toxin B subunit (CTB) and tagged with 6xHis was expressed for the first time in Escherichia coli as well as in transgenic cucumber plants developed using Agrobacterium tumefaciens-mediated transformation. The recombinant fusion protein (His6x.CTB-ESAT6-MPT64) expressed in E. coli was purified by a single-step affinity chromatography and used to produce polyclonal antibodies in rabbit. The transgenic cucumber lines were confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcriptase PCR (RT-PCR), real-time PCR (qRT-PCR) and expression of recombinant fusion protein by western blot analysis and its quantification by enzyme-linked immunosorbent assay (ELISA). A maximum value of the fusion protein, 478 ng.g-1 (0.030% of the total soluble protein) was obtained in a transgenic cucumber line. Rabbit immunized orally showed a significant increase in serum IgG levels against the fusion protein as compared to the non-immunized rabbit. CONCLUSIONS: Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.

Genomic characteristics of two most widely used BCG vaccine strains: Danish 1331 and Pasteur 1173P2.

BACKGROUND: Bacillus Calmette-Guérin (BCG) refers to a group of vaccine strains with unique genetic characteristics. BCG is the only available vaccine for preventing tuberculosis (TB). Genetic and biochemical variations among the BCG vaccine strains have been considered as one of the significant parameters affecting the variable protective efficacy of the vaccine against pulmonary tuberculosis. To track genetic variations, here two vaccine strains (Danish 1331 and Pasteur 1173P2) popularly used according to the BCG World Atlas were subjected to a comparative analysis against the Mycobacterium tuberculosis H37Rv, Mycobacterium bovis AF2122/97, and Mycobacterium tuberculosis variant bovis BCG str. Pasteur 1173P2 reference genomes. Besides, the presence or absence of the experimentally verified human T cell epitopes was examined. RESULTS: Only two variants were identified in BCG Danish 1331 that have not been reported previously in any BCG strains with the complete submitted genome yet. Furthermore, we identified a DU1-like 14,577 bp region in BCG Danish 1331; The duplication which was previously seemed to be exclusive to the BCG Pasteur. We also found that 35% of the T cell epitopes are absent from both strains, and epitope sequences are more conserved than the rest of the genome. CONCLUSIONS: We provided a comprehensive catalog of single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels) in BCG Danish 1331 and BCG Pasteur 1173P2. These findings may help determine the effect of genetic variations on the variable protective efficacy of BCG vaccine strains.

Protective efficacy of an attenuated Mtb ΔLprG vaccine in mice.

Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis, is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c (ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4+ T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4+ T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy.

Recent developments in systems biology and genetic engineering toward design of vaccines for TB.

Tuberculosis (TB) is one of the most prevalent diseases worldwide. The currently available Bacillus Calmette-Guérin vaccine is not sufficient in protecting against pulmonary TB. Although many vaccines have been evaluated in clinical trials, but none of them yet has proven to be more successful. Thus, new strategies are urgently needed to design more effective TB vaccines. The emergence of new technologies will undoubtedly accelerate the process of vaccine development. This review summarizes the potential and validated applications of emerging technologies, including: systems biology (genomics, proteomics, and transcriptomics), genetic engineering, and other computational tools to discover and develop novel vaccines against TB. It also discussed that the significant implementation of these approaches will play crucial roles in the development of novel vaccines to cure and control TB.

Mycobacterium tuberculosis extracellular vesicles: exploitation for vaccine technology and diagnostic methods.

Tuberculosis (TB) is a fatal epidemic disease usually caused by Mycobacterium tuberculosis (Mtb). Pervasive latent infection, multidrug- and extensively drug-resistant tuberculosis (MDR- and XDR-TB), and TB/HIV co-infection make TB a global health problem, which emphasises the design and development of efficient vaccines and diagnostic biomarkers. Extracellular vesicles (EVs) secretion is a conserved phenomenon in all the domains of life. Various cargos such as nucleic acids, toxins, lipoproteins, and enzymes have been recognised in these nano-sized vesicles that may be involved in bacterial physiology and pathogenesis. The intrinsic adjuvant effect, native immunogenic cargo, sensing by host immune cells, circulation in all body fluids, and comprehensive distribution of antigens introduce EVs as a promising tool for designing novel vaccines, diagnostic biomarkers, and drug delivery systems. Genetic engineering of the EV-producing bacteria and the subsequent production of proper EVs could facilitate the development of the EV-based therapeutic applications. Recently, it was demonstrated that thick-walled mycobacteria release EVs, which contain immunodominant cargos such as lipoglycans and lipoproteins. The present article is a comprehensive review on the recent findings of Mtb EVs biology and the exploitation of EVs for the vaccine technology and diagnostic methods.

Vaccine efficacy of a Mycobacterium tuberculosis Beijing-specific proline-glutamic acid (PE) antigen against highly virulent outbreak isolates.

Bacillus Calmette-Guerin vaccine confers insufficient pulmonary protection against tuberculosis (TB), particularly the Mycobacterium tuberculosis (Mtb) Beijing strain infection. Identification of vaccine antigens (Ags) by considering Mtb genetic diversity is crucial for the development of improved TB vaccine. MTBK_20640, a new Beijing genotype-specific proline-glutamic acid-family Ag, was identified by comparative genomic analysis. Its immunologic features were characterized by evaluating interactions with dendritic cells (DCs), and immunogenicity and vaccine efficacy were determined against highly virulent Mtb Beijing outbreak Korean Beijing (K) strain and HN878 strain in murine infection model. MTBK_20640 induced DCs via TLR2 and downstream MAPK and NF-κB signaling pathways, effectively promoting naive CD4-positive (CD4+) T-cell proliferation and IFN-γ production. Different IFN-γ response was observed in mice infected with Mtb K or reference H37Rv strain. Significant induction of T helper type 1 cell-polarized Ag-specific multifunctional CD4+ T cells and a marked Ag-specific IgG2c response were observed in mice immunized with MTBK_20640/glucopyranosyl lipid adjuvant-stable emulsion. The immunization conferred long-term protection against 2 Mtb Beijing outbreak strains, as evidenced by a significant reduction in colony-forming units in the lung and spleen and reduced lung inflammation. MTBK_20640 vaccination conferred long-term protection against highly virulent Mtb Beijing strains. MTBK_20640 may be developed into a novel Ag component in multisubunit TB vaccines in the future.-Kwon, K. W., Choi, H.-H., Han, S. J., Kim, J.-S., Kim, W. S., Kim, H., Kim, L.-H., Kang, S. M., Park, J., Shin, S. J. Vaccine efficacy of a Mycobacterium tuberculosis Beijing-specific proline-glutamic acid (PE) antigen against highly virulent outbreak isolates.

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.

[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.

Construction and Characterization of the Mycobacterium tuberculosis sigE fadD26 Unmarked Double Mutant as a Vaccine Candidate.

Despite the great increase in the understanding of the biology and pathogenesis of Mycobacterium tuberculosis achieved by the scientific community in recent decades, tuberculosis (TB) still represents one of the major threats to global human health. The only available vaccine (Mycobacterium bovis BCG) protects children from disseminated forms of TB but does not effectively protect adults from the respiratory form of the disease, making the development of new and more-efficacious vaccines against the pulmonary forms of TB a major goal for the improvement of global health. Among the different strategies being developed to reach this goal is the construction of attenuated strains more efficacious and safer than BCG. We recently showed that a sigE mutant of M. tuberculosis was more attenuated and more efficacious than BCG in a mouse model of infection. In this paper, we describe the construction and characterization of an M. tuberculosissigE fadD26 unmarked double mutant fulfilling the criteria of the Geneva Consensus for entering human clinical trials. The data presented suggest that this mutant is even more attenuated and slightly more efficacious than the previous sigE mutant in different mouse models of infection and is equivalent to BCG in a guinea pig model of infection.

Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall-anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-laurylsarcosine).

BACKGROUND: The current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, overexpression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated. RESULTS: Expression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days). CONCLUSION: The presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli.

Recombinant BCG Overexpressing phoP-phoR Confers Enhanced Protection against Tuberculosis.

The live tuberculosis vaccine Mycobacterium bovis BCG (Bacille Calmette-Guérin) comprises a number of genetically distinct substrains. In BCG-Prague, phoP of the PhoP-PhoR two-component system is a pseudogene due to a single insertion mutation. We hypothesized that this mutation partially accounts for the low immunogenicity of BCG-Prague observed in the 1970s. In this study, we showed that complementation with the M. bovis allele of phoP restored BCG-Prague's immunogenicity. Furthermore, we showed that overexpression of the M. bovis allele of phoP-phoR in BCG-Japan, a strain already containing a copy of phoP-phoR, further enhanced immunogenicity and protective efficacy. Vaccination of C57BL/6 mice with the recombinant strain rBCG-Japan/PhoPR induced higher levels of interferon-γ (IFN-γ) production by CD4+ T cells than that with the parental BCG. Guinea pigs vaccinated with rBCG-Japan/PhoPR were better protected against challenge with Mycobacterium tuberculosis than those immunized with the parental BCG, showing significantly longer survival time, reduced bacterial burdens, and less severe pathology. Taken together, our study has identified a genetic modification that could be generally applied to generate new recombinant BCG vaccines.

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.

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.

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.

Immunological consequences of intragenus conservation of Mycobacterium tuberculosis T-cell epitopes.

A previous unbiased genome-wide analysis of CD4 Mycobacterium tuberculosis (MTB) recognition using peripheral blood mononuclear cells from individuals with latent MTB infection (LTBI) or nonexposed healthy controls (HCs) revealed that certain MTB sequences were unexpectedly recognized by HCs. In the present study, it was found that, based on their pattern of reactivity, epitopes could be divided into LTBI-specific, mixed reactivity, and HC-specific categories. This pattern corresponded to sequence conservation in nontuberculous mycobacteria (NTMs), suggesting environmental exposure as an underlying cause of differential reactivity. LTBI-specific epitopes were found to be hyperconserved, as previously reported, whereas the opposite was true for NTM conserved epitopes, suggesting that intragenus conservation also influences host pathogen adaptation. The biological relevance of this observation was demonstrated further by several observations. First, the T cells elicited by MTB/NTM cross-reactive epitopes in HCs were found mainly in a CCR6(+)CXCR3(+) memory subset, similar to findings in LTBI individuals. Thus, both MTB and NTM appear to elicit a phenotypically similar T-cell response. Second, T cells reactive to MTB/NTM-conserved epitopes responded to naturally processed epitopes from MTB and NTMs, whereas T cells reactive to MTB-specific epitopes responded only to MTB. Third, cross-reactivity could be translated to antigen recognition. Several MTB candidate vaccine antigens were cross-reactive, but others were MTB-specific. Finally, NTM-specific epitopes that elicit T cells that recognize NTMs but not MTB were identified. These epitopes can be used to characterize T-cell responses to NTMs, eliminating the confounding factor of MTB cross-recognition and providing insights into vaccine design and evaluation.

Molecular characterization of novel immunodominant molybdenum cofactor biosynthesis protein C1 (Rv3111) from Mycobacterium tuberculosis H37Rv.

BACKGROUND: In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature. METHODS: We have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques. RESULTS: We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. CONCLUSION: Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. GENERAL SIGNIFICANCE: This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.