Boosting with recombinant MVA expressing M. tuberculosis α-crystallin antigen augments the protection imparted by BCG against tuberculosis in guinea pigs.

Tuberculosis (TB) is one of the major causes of mortality all over the globe. BCG, the only vaccine available against this disease has been successful in preventing the severe forms of childhood TB. However, the unsatisfactory performance of BCG in controlling the adult pulmonary tuberculosis has made the development of an effective vaccine against M. tuberculosis a prime objective of the TB research. In this study, a genetically stable, marker-free recombinant MVA expressing α-crystallin of M. tuberculosis (rMVA.acr) was generated which was further evaluated for its ability to impart protection as a booster vaccine against tuberculosis in a heterologous prime boost approach. Our results demonstrated that intradermal delivery of rMVA.acr was able to efficiently boost the BCG induced protection against M. tuberculosis infection in guinea pigs by significantly reducing the pulmonary bacillary load (1.27 log10 fewer bacilli) in comparison to BCG vaccination alone. In addition, boosting BCG vaccinated animals with intramuscular delivery of rMVA.acr resulted in significantly superior protective efficacy in both lungs and spleen with 0.83 log10 and 0.74 log10 CFU fewer bacilli, respectively, when compared to animals vaccinated with BCG only. These findings establish the promise of this prime-boost strategy involving rMVA.acr in enhancing the efficacy of BCG.

bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs.

Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette-Guérin (BCG) vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in de novo biotin biosynthesis. In this study, we demonstrate that a bioA mutant of Mycobacterium tuberculosis (MtbΔbioA) is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔbioA conferred significant protection in guinea pigs against an aerosol challenge with virulent M. tuberculosis, when compared with the unvaccinated animals. Booster immunization with MtbΔbioA offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated M. tuberculosis bioA mutant against tuberculosis.

Differential Roles of Iron Storage Proteins in Maintaining the Iron Homeostasis in Mycobacterium tuberculosis.

Ferritins and bacterioferritins are iron storage proteins that represent key players in iron homeostasis. Several organisms possess both forms of ferritins, however, their relative physiological roles are less understood. Mycobacterium tuberculosis possesses both ferritin (BfrB) and bacterioferritin (BfrA), playing an essential role in its pathogenesis as reported by us earlier. This study provides insights into the role of these two proteins in iron homeostasis by employing M. tuberculosis bfr mutants. Our data suggests that BfrA is required for efficient utilization of stored iron under low iron conditions while BfrB plays a crucial role as the major defense protein under excessive iron conditions. We show that these two proteins provide protection against oxidative stress and hypoxia. Iron incorporation study showed that BfrB has higher capacity for storing iron than BfrA, which augurs well for efficient iron quenching under iron excess conditions. Moreover, iron release assay demonstrated that BfrA has 3 times superior ability to release stored iron emphasizing its requirement for efficient iron release under low iron conditions, facilitated by the presence of heme. Thus, for the first time, our observations suggest that the importance of BfrA or BfrB separately might vary depending upon the iron situation faced by the cell.

Unique residues at the 3-fold and 4-fold axis of mycobacterial ferritin are involved in oligomer switching.

To identify the crucial residues involved in the self-assembly and function of BfrB, one of the important iron storage proteins of Mycobacterium tuberculosis, we constructed various mutants by employing site-directed mutagenesis. The analysis of mutants led to the identification of "interface hot-spot residues" (R69, L129, and F159) that act as "switch points" for BfrB oligomerization, and our observations show the importance of 4-fold axis residues in assembly formation. Moreover, we demonstrate that single-point mutations Q51A, Q126A, and E135A can enhance the thermal stability of the protein without affecting its assembly. Importantly, a comparative analysis of various mutations revealed that the function of various homologous positions in different ferritins could be at variance; hence, predicting the function of a residue just based on sequence-structure comparisons may not be appropriate. Thus, we report the identification of novel residues in the assembly formation and function of BfrB and show that single-point mutations have a remarkable potential for alteration of multiple properties of ferritins. Besides, "switch residues" or "interface hot spots" identified in this study could also prove to be helpful for the rational design of interfacial inhibitors.

Ferritin structure from Mycobacterium tuberculosis: comparative study with homologues identifies extended C-terminus involved in ferroxidase activity.

Ferritins are recognized as key players in the iron storage and detoxification processes. Iron acquisition in the case of pathogenic bacteria has long been established as an important virulence mechanism. Here, we report a 3.0 Å crystal structure of a ferritin, annotated as Bacterioferritin B (BfrB), from Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis that continues to be one of the world's deadliest diseases. Similar to the other members of ferritin family, the Mtb BfrB subunit exhibits the characteristic fold of a four-helical bundle that possesses the ferroxidase catalytic centre. We compare the structure of Mtb BfrB with representatives of the ferritin family belonging to the archaea, eubacteria and eukarya. Unlike most other ferritins, Mtb BfrB has an extended C-terminus. To dissect the role of this extended C-terminus, truncated Mtb BfrB was purified and biochemical studies implicate this region in ferroxidase activity and iron release in addition to providing stability to the protein. Functionally important regions in a protein of known 3D-structure can be determined by estimating the degree of conservation of the amino-acid sites with its close homologues. Based on the comparative studies, we identify the slowly evolving conserved sites as well as the rapidly evolving variable sites and analyze their role in relation to structure and function of Mtb BfrB. Further, electrostatic computations demonstrate that although the electrostatic environment of catalytic residues is preserved within the family, extensive variability is exhibited by residues defining the channels and pores, in all likelihood keeping up with the diverse functions executed by these ferritins in varied environments.

Development of vaccines against tuberculosis.

Development of an effective vaccine against tuberculosis (TB) hinges on an improved understanding of the human immune responses to Mycobacterium tuberculosis. A successful vaccination strategy should be able to stimulate the appropriate arm of the immune system with concomitant generation of the memory cells. In the absence of a perfect strategy, while long term efforts of TB researchers continue to resolve the nature of protective immunity against TB and other related issues, the current approach, dictated by the urgency of a TB vaccine, employs available knowledge and technology to develop new TB vaccines and channel the promising ones to clinical trials. While Indian scientists have contributed in several areas towards the development of a TB vaccine, this review is an attempt to summarize their contributions mainly pertaining to the discovery of new antigens, immune responses elicited by antigens against TB and development of new vaccines and their evaluation in animal models.