Suraj B. Sable

Nanobead-based interventions for the treatment and prevention of tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most devastating bacterial diseases to affect humans. M. tuberculosis is a robust pathogen that has evolved the capacity to survive and grow inside macrophage phagosomes. A cocktail of antibiotics has long been successfully used against M. tuberculosis but is becoming less effective owing to the emergence of multidrug resistance. The only available preventive vaccine, using Mycobacterium bovis bacille Calmette–Guérin, is considered to be ineffective against adult pulmonary TB, the most prevalent form of the disease. Here, we review the potential use of biodegradable nanoparticle-based anti-TB drug delivery systems that have been shown to be more effective against M. tuberculosis in animal models than conventional antibiotic treatment regimens. This technology also has substantial potential for vaccination and other therapeutic strategies against TB and other infectious diseases.

Bacillus Calmette-Guérin vaccination using a microneedle patch

Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be a leading cause of mortality among bacterial diseases, and the bacillus Calmette-Guérin (BCG) is the only licensed vaccine for human use against this disease. TB prevention and control would benefit from an improved method of BCG vaccination that simplifies logistics and eliminates dangers posed by hypodermic needles without compromising immunogenicity. Here, we report the design and engineering of a BCG-coated microneedle vaccine patch for a simple and improved intradermal delivery of the vaccine. The microneedle vaccine patch induced a robust cell-mediated immune response in both the lungs and the spleen of guinea pigs. The response was comparable to the traditional hypodermic needle based intradermal BCG vaccination and was characterized by a strong antigen specific lymphocyte proliferation and IFN-γ levels with high frequencies of CD4(+)IFN-γ(+), CD4(+)TNF-α(+) and CD4(+)IFN-γ(+)TNF-α(+) T cells. The BCG-coated microneedle vaccine patch was highly immunogenic in guinea pigs and supports further exploration of this new technology as a simpler, safer, and compliant vaccination that could facilitate increased coverage, especially in developing countries that lack adequate healthcare infrastructure.

Peripheral Blood and Pleural Fluid Mononuclear Cell Responses to Low-Molecular-Mass Secretory Polypeptides of Mycobacterium tuberculosis in Human Models of Immunity to Tuberculosis

ABSTRACT A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H37Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a persons lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test‐negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage‐specific CD68+ epithelioid macrophages and small round CD3+ lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid‐fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead‐based assay using the Luminex 100 and were found to contain interleukin (IL)‐6, IL‐8, interferon‐γ and tumor necrosis factor‐α, cytokines known to be involved in human granuloma formation, in quantities from two‐fold to 7000‐fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.

Lung and blood mononuclear cell responses of tuberculosis patients to mycobacterial proteins.

The differences in specificity of human lung and peripheral lymphocytes for mycobacterial antigens (Ag) need to be evaluated in order to identify vaccine candidates against pulmonary tuberculosis (TB). Therefore, the present study examined the response to low molecular weight secretory proteins of Mycobacterium tuberculosis in bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMCs) from minimal pulmonary TB and non-TB patients. Ag85A, Ag85B, culture filtrate protein (CFP)-31, CFP-22.5, CFP-21, M. tuberculosis protein-64 and an as yet uncharacterised 19 kDa protein were found to be predominantly recognised by BAL cells of TB patients on the basis of lymphocyte proliferation and significant interferon-γ release. However, recognition of CFP-8, 6-kDa early secreted antigenic target, CFP-10, CFP-14.5, M. tuberculosis secretory protein-17 and five other as yet uncharacterised low molecular weight polypeptides was found to be high on the basis of lymphocyte proliferation at the level of PBMCs. Furthermore, BAL macrophages, and not blood monocytes, were found to produce nitric oxide (NO) in response to mycobacterial Ags. Among polypeptides predominantly recognised by BAL lymphocytes, only Ag85A and Ag85B were found to induce both NO and interleukin-12 (p40) by alveolar macrophages. In conclusion, the present results indicate heterogeneity in antigen recognition by bronchoalveolar lavage cells and peripheral mononuclear blood cells of minimal tuberculosis patients, and also suggest the utility of antigen 85 complex polypeptides for the development of a future mucosal antituberculous vaccine.

Human immune recognition-based multicomponent subunit vaccines against tuberculosis

The cell-mediated immune response, with its shift in favour of type-1 over type-2 T-helper cell immune response, is generally regarded as essential to protection against mycobacterial infections. The aim of this study was to evaluate the protective potential of two multicomponent subunit vaccines (MSV-1 and MSV-2) against tuberculosis (TB) based on human immune recognition. MSV-1 consisted of five immunodominant antigens (TB10.4, early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-8, CFP-10 and CFP-15) selected from a group of polypeptides, which induced a predominant T-cell response in immune human subjects, whereas MSV-2 consisted of antigens (CFP-11, CFP-21, CFP-22.5, Mycobacterium tuberculosis protein (MPT)-64 and CFP-31) selected from a group of polypeptides which induced a subdominant T-cell response along with the antibody response. Both of these sets of polypeptides were extensively recognised in healthy individuals with significant interferon gamma release compared to the diseased population. In C57BL/6J mice, at the level of the lungs, the order of protective efficacy for the test vaccines was: bacille Calmette–Guérin (BCG)>MSV-2>MSV-1. The protective efficacy of MSV-1 was found to be significantly less than that of MSV-2 and BCG at the level of spleen, whereas that of MSV-2 was comparable to that of BCG. The results of this study indicate that high T-helper cell type 1 response-inducing polypeptides selected on the basis of human immune recognition do not necessarily impart protection during vaccination experiments.

Cellular Immune Responses to Nine Mycobacterium tuberculosis Vaccine Candidates following Intranasal Vaccination

Background The identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis. Methods and Principal Findings In this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study. Conclusion and Significance Overall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis.

O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Programmed Death 1 Lives Up to Its Reputation in Active Tuberculosis

Programmed death 1 (PD-1; CD279), also known as programmed cell death protein 1 (PDCD-1), is a cell surface receptor of the immunoglobulin superfamily found on immune effector cells. It belongs to the extended CD28/CTLA-4 family of T-cell regulators and is expressed by a range of immune cells, including B cells, natural killer cells, and monocytes. Binding of PD-1 to one of its ligands, PD ligand 1 (PD-L1) or PD-L2, on antigen-presenting cells (APCs) is known to negatively regulate T-cell receptor signaling and inhibit T-cell activation [1]. In this issue of the Journal, Singh et al [2] demonstrate that the coinhibitory receptor PD-1 and the PD ligands are expressed on higher percentages of peripheral blood mononuclear cells (PBMCs) of patients with active tuberculosis than those of healthy controls in an area of tuberculosis endemicity. The expression of PD-1 on T cells is upregulated following in vitro stimulation with Mycobacterium tuberculosis antigens, and the PD-1 blockade in vitro enhances interferon γ (IFN-γ) and interleukin 2 production by specific T cells and rescues them from undergoing apoptosis. Of particular significance, the authors demonstrate during a 1-year follow-up period a significant decrease in the frequency of PD-1–expressing T cells after successful antituberculosis treatment that led to restoration of the M. tuberculosis–specific T-cell cytokine response in vitro. These data substantiate the role of PD-1–PD-L pathways in the inhibition of T-cell responses in patients with active tuberculosis. Optimal T-cell activation during infection requires 2 signals. The first signal is generated by T-cell–receptor recognition ofpeptide–majorhistocompatibilitycom

Boosting BCG-primed responses with a subunit Apa vaccine during the waning phase improves immunity and imparts protection against Mycobacterium tuberculosis

Heterologous prime–boosting has emerged as a powerful vaccination approach against tuberculosis. However, optimal timing to boost BCG-immunity using subunit vaccines remains unclear in clinical trials. Here, we followed the adhesin Apa-specific T-cell responses in BCG-primed mice and investigated its BCG-booster potential. The Apa-specific T-cell response peaked 32–52 weeks after parenteral or mucosal BCG-priming but waned significantly by 78 weeks. A subunit-Apa-boost during the contraction-phase of BCG-response had a greater effect on the magnitude and functional quality of specific cellular and humoral responses compared to a boost at the peak of BCG-response. The cellular response increased following mucosal BCG-prime–Apa-subunit-boost strategy compared to Apa-subunit-prime–BCG-boost approach. However, parenteral BCG-prime–Apa-subunit-boost by a homologous route was the most effective strategy in-terms of enhancing specific T-cell responses during waning in the lung and spleen. Two Apa-boosters markedly improved waning BCG-immunity and significantly reduced Mycobacterium tuberculosis burdens post-challenge. Our results highlight the challenges of optimization of prime–boost regimens in mice where BCG drives persistent immune-activation and suggest that boosting with a heterologous vaccine may be ideal once the specific persisting effector responses are contracted. Our results have important implications for design of prime–boost regimens against tuberculosis in humans.