HspX-mediated protection against tuberculosis depends on its chaperoning of a mycobacterial molecule

Department of Microbiology, Mycobacteria Research Laboratories, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA.
Immunology and Cell Biology (Impact Factor: 4.15). 07/2012; 90(10). DOI: 10.1038/icb.2012.34
Source: PubMed


New approaches consisting of 'multistage' vaccines against (TB) are emerging that combine early antigenic proteins with latency-associated antigens. In this study, HspX was tested for its potential to elicit both short- and long-term protective immune responses. HspX is a logical component in vaccine strategies targeting protective immune responses against primary infection, as well as against reactivation of latent infection, because as previously shown, it is produced during latency, and as our studies show, it elicits protection within 30 days of infection. Recent studies have shown that the current TB vaccine, bacilli Calmette-Guerin (BCG), does not induce strong interferon-γ T-cell responses to latency-associated antigens like HspX, which may be in part why BCG fails to protect against reactivation disease. We therefore tested HspX protein alone as a prophylactic vaccine and as a boost to BCG vaccination, and found that HspX purified from M. tuberculosis cell lysates protected mice against aerosol challenge and improved the protective efficacy of BCG when used as a booster vaccine. Native HspX was highly immunogenic and protective, in a dose-dependent manner, in both short- and long-term infection models. Based on these promising findings, HspX was produced as a recombinant protein in E. coli, as this would enable facile purification; however, recombinant HspX (rHspX) alone consistently failed to protect against aerosol challenge. Incubation of rHspX with mycobacterial cell lysate and re-purification following incubation restored the capacity of the protein to confer protection. These data suggest the possibility that the native form may chaperone an immunogenic and protective antigen that is mycobacteria-specific.Immunology and Cell Biology advance online publication, 17 July 2012; doi:10.1038/icb.2012.34.

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    • "It shows strong immunogenicity and can induce a potent Th1 immunity in Mtb-exposed patients. This protein as an antigen is mainly secreted by non-replicating bacilli that could induce a stronger immune response in latently infected persons than in existing TB patients (Taylor et al. 2012). Studies have demonstrated that targeting immunogens to Fcγ receptors (FcγR) on antigen-presenting cells (APCs) such as myeloid and plasmacytoid dendritic cells (DCs), monocytes, and macrophages can enhance the immune response in vitro and in vivo (Loureiro et al. 2010; Konduru et al. 2011; Lu et al. 2011; Kaplan 2012). "
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    ABSTRACT: Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore, the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine, a combination of an early and a dormant immunogenic protein, ESAT6 and HspX, fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6, a linker, and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6,6-dibehenate(TDB) adjuvant, and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1, Th2, Th17, and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ, 910 pg/mL, and IL-12, 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-β (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake, inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances, which is important for protection and prevention of damage.
    Applied Microbiology and Biotechnology 09/2015; DOI:10.1007/s00253-015-6952-z · 3.34 Impact Factor
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    • "HspX (-crystallin or 16-kDa antigen ) is a protein highly expressed by Mtb under stress conditions, including hypoxia, nutrient scarcity, and high levels of nitric oxide produced by macrophages [19]. This antigen has been shown to induce a strong immune response in mice and individuals with LTB [20] [21] [22] [23] [24] [25]. Few subunit vaccines composed of HspX have been tested in animal models. "
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    ABSTRACT: Background: Tuberculosis is a disease affecting millions of people throughout the world. One of the main problems in controlling the disease is the low efficacy of the Bacillus Calmette-Guérin (BCG) vaccine in protecting young adults. The development of new vaccines that induce a long-lasting immune response or that stimulate the immunity induced by BCG may improve the control of tuberculosis. Methods: The use of microstructured liposomes containing HspX, with or without MPL or CpG DNA adjuvants, as vaccines for tuberculosis was evaluated. The HspX-specific humoral and cellular immune responses to the different vaccine formulations were compared. Results: All vaccines containing liposome microparticles and HspX were immunogenic. Vaccines formulated with CpG DNA and HspX induced the strongest humoral and cellular immune responses, mainly by inducing interferon-γ and tumor necrosis factor-α expression by both CD4(+) and CD8(+) T cells. HspX and MPL mainly induced CD8(+) T-cell activation and specific humoral responses. When evaluated the protective efficacy of the formulations against Mycobacterium tuberculosis challenge, the microstructured liposome containing L-HspX and L-HspX-CPG DNA reduced both lung inflammatory lesions and the bacterial load. Conclusion: We have thus demonstrated, for the first time, the use of microstructured liposomes as an adjuvant and delivery system for a vaccine formulation against tuberculosis.
    Vaccine 06/2014; 32(34). DOI:10.1016/j.vaccine.2014.06.037 · 3.62 Impact Factor
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    • "These studies also demonstrated that while native HspX purified from Mtb culture lysates (nHspX) conferred protection , the recombinant protein expressed in and purified from E. coli (rHspX) did not, unless the purified recombinant protein was incubated with Mtb lysate and re-purified from this lysate (rHspX-PD) prior to vaccination, suggesting that HspX mediated protection was driven in part, by other products co-chaperoned with HspX. This finding was further supported by evidence that nHspX and rHspX-PD could protect mice against challenge with an HspX deletion strain of Mtb (X4-19; DHspX; Taylor et al., 2012). "
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    ABSTRACT: Mycobacterium tuberculosis (Mtb) currently infects billions of people; many of whom are latently infection and at risk for reactivation. Mycobacterium bovis Bacille Calmette-Guerin (BCG) while approved as a vaccine, is unable to prevent reactivation of latent tuberculosis infection (LTBI). Subunit vaccines boosting BCG or given alone are being tested for efficacy in LTBI models. Alpha-crystallin (Acr, HspX), is a latency associated protein and subunit vaccine candidate. In this report, three HspX formulas (native and two recombinant variants) were used as vaccines in the guinea pig model of tuberculosis; none were protective during challenge with WT Mtb. However, recombinant HspX was protective in animals challenged with a strain of Mtb lacking hspX (X4-19), indicating protection was driven by molecules co-purifying with HspX or an adjuvant effect of recombinant HspX in this system. Mtb X4-19 was significantly less virulent than WT Mtb. Quantitative PCR and whole genome sequencing identified several genes (Rv2030c-Rv2032, Rv1062, Rv1771, Rv1907, and Rv3479) with altered expression that may contribute to loss of virulence. Physiological differences required for the establishment of Mtb infection in different hosts may affect the potential of subunit vaccines to elicit protection, supporting the need for rigorous biochemical and modeling analyses when developing tuberculosis vaccines.
    Pathogens and Disease 02/2014; 71(3). DOI:10.1111/2049-632X.12147 · 2.40 Impact Factor
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