A New Adjuvant MTOM Mediates Mycobacterium tuberculosis Subunit Vaccine to Enhance Th1-Type T Cell Immune Responses and IL-2+ T Cells

The only licensed vaccine Mycobacterium bovis Bacillus Calmette–Guérin (BCG) cannot prevent the prevalence of tuberculosis (TB), which remains a major public health problem worldwide. A more effective TB vaccine than BCG is urgently needed. Subunit vaccine is a promising strategy, and suitable adjuvants will benefit the development of effective TB subunit vaccines. MTO, consisting of monophosphoryl lipid A (MPLA), trehalose-6,6′-dibehenate (TDB), and MF59, was developed as an adjuvant of TB vaccine because of its ability to evoke the Th1-type T cell responses, while it is insufficient to induce single and multifunctional IL-2+ T cells and has a limited ability to confer protection against Mycobacterium tuberculosis infection. Heat-killed Mycobacterium vaccae (Mv), which can evoke cytotoxic CD8+ and CD4+ T cell responses and has adjuvanticity, was, in this study, combined with MTO to produce a new adjuvant, called MTOM. The TB fusion protein Rv3407-PhoY2-Ag85A-Rv2626c-RpfB (WH121) was mixed with MTO, Mv, and MTOM to produce three subunit vaccines, and the protective efficacy and immune responses were compared in C57BL/6 mice. WH121/MTOM provided better protection against TB than the other two vaccines, matching the performance of BCG vaccine. MTOM showed stronger ability to increase single and multifunctional IL-2+ T cells and induce Th1-type responses than MTO or Mv. Therefore, MTOM might be a promising adjuvant that could contribute to the development of TB subunit vaccines

Immunogenicity and Protective Efficacy against Murine Tuberculosis of a Prime-Boost Regimen with BCG and a DNA Vaccine Expressing ESAT-6 and Ag85A Fusion Protein

Heterologous prime-boost regimens utilizing BCG as a prime vaccine probably represent the best hope for the development of novel tuberculosis (TB) vaccines. In this study, we examined the immunogenicity and protective efficacy of DNA vaccine (pcD685A) expressing the fusion protein of Ag85A and ESAT-6 (r685A) and its booster effects in BCG-immunized mice. The recombinant r685A fusion protein stimulated higher level of antigen-specific IFN-γ release in tuberculin skin test- (TST-) positive healthy household contacts of active pulmonary TB patients than that in TST-negative population. Vaccination of C57BL/6 mice with pcD685A resulted in significant protection against challenge with virulent Mycobacterium tuberculosis H37Rv when compared with the control group. Most importantly, pcD685A could act as a BCG booster and amplify Th1-type cell-mediated immunity in the lung of BCG-vaccinated mice as shown the increased expression of IFN-γ. The most significant reduction in bacterial load of both spleen and lung was obtained in mice vaccinated with BCG prime and pcD685A DNA booster when compared with BCG or pcD685A alone. Thus, our study indicates that pcD685A may be an efficient booster vaccine against TB with a strong ability to enhance prior BCG immunity

Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium Tuberculosis-Induced Apoptosis in Mice

Background: Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive. Methodology/Principal Findings: We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-α, IL-1β, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration. Conclusions/Significance: These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection

Heterologous Boost Following Mycobacterium bovis BCG Reduces the Late Persistent, Rather Than the Early Stage of Intranasal Tuberculosis Challenge Infection

Adults are the leading population affected by tuberculosis (TB) epidemic and death. Developing an effective vaccine against adult TB is urgently needed. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) prime-heterologous boost strategy has been explored extensively to protect adults against primary TB infection, but the majority of experimental regimens have not improved the protection primed by the BCG vaccine. The reason attributed to the failure remains unknown. In this study, CTT3H-based vaccines, namely DMT adjuvanted CTT3H subunit or DNA vaccine (pCTT3H-DMT), and recombinant adenovirus rAdCTT3H were constructed. Protective efficacy and immunogenicity of BCG prime-CTT3H based boosters were compared in C57BL/c mice models of primary or late persistent TB infection. Similar protective efficacy against early intranasal infection was provided by different CTT3H-based vaccines alone in vaccinated mice, and their protection was inferior to that of the BCG vaccine. In addition, CTT3H-based heterologous boosters did not enhance the protection conferred by the BCG vaccine against primary infection. However, all of these three boosters provided stronger protection against late persistent TB infection than BCG alone, regardless of vaccine types. Although BCG prime-boosters elicited Th1-biased responses to the antigen CTT3H, the number of CTT3H-sepcific IFN-γ-expressing TEM (CD62LloCD44hi) and IL-2-expressing TCM (CD62LhiCD44hi) cells in the spleen was not improved before exposure to Mycobacterium tuberculosis infection. In contrast, IFN-γ+ TEM and IL-2+ TCM cells in spleens, especially in lungs were significantly increased in BCG prime-boosters after exposure vaccination. Our results indicate that BCG prime-boost strategy might be a promising measure for the prevention against late persistent TB infection by induction of IFN-γ+ TEM and IL-2+ TCM cells in the lung, which can be used as alternative biomarkers for guiding the clinical practice and future development of TB vaccine for adults

Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium tuberculosis–Induced Apoptosis in Mice

Background: Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive. Methodology/Principal Findings: We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-alpha, IL-1 beta, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3 beta in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3 beta in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration. Conclusions/Significance: These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000274923700001&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Multidisciplinary SciencesSCI(E)11ARTICLE2null

Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection

Despite the vaccine Mycobacterium bovis Bacillus Calmette–Guérin is used worldwide, tuberculosis (TB) remains the first killer among infectious diseases. An effective vaccine is urgently required. DNA vaccine has shown prophylactic as well as therapeutic effects against TB, while its weak immunogenicity hinders the application. As a strong inducer of Th1-biased immune response, DMT, consisting of dimethyldioctadecylammonium (DDA) and two pattern recognition receptor agonists monophosphoryl lipid A and trehalose 6,6′-dibehenate (TDB), was a newly developed liposomal adjuvant. To elucidate the action mechanism of DMT and improve immunological effects induced by DNA vaccine, a new recombinant eukaryotic expression plasmid pCMFO that secretes the fusion of four multistage antigens (Rv2875, Rv3044, Rv2073c, and Rv0577) of Mycobacterium tuberculosis was constructed. pCMFO/DDA and pCMFO/DMT complexes were then prepared and their physicochemical properties were analyzed. The immunogenicity and protection against M. tuberculosis infection in vaccinated C57BL/6 mice were compared. Formulation of DNA and two agonists into the DDA liposome decreased zeta potential but increased the stability of storage, which resulted in a slower and longer-lasting release of DNA from the DNA–DMT complex than the DNA–DDA liposome. Besides Th1-biased responses, pCMFO/DMT vaccinated mice elicited more significantly CFMO-specific IL2+ TCM cell responses in the spleen and provided an enhanced and persistent protection against M. tuberculosis aerosol infection, compared to pCMFO/DDA and pCMFO groups. Therefore, the adjuvant DMT can release DNA and agonists slowly, which might attribute to the improved protection of DMT adjuvanted vaccines. pCMFO/DMT, a very promising TB vaccine, warrants for further preclinical and clinical trials

An Improved Whole-Blood Gamma Interferon Assay Based on the CFP21-MPT64 Fusion Protein▿

Differentiation of latent tuberculosis infection (LTBI) from a healthy, unexposed population plays a vital role in the strategy of controlling and eliminating tuberculosis (TB). Both CFP21 and MPT64, antigens encoded by the RD2 region which are restricted in the Mycobacterium tuberculosis complex, are TB-specific diagnostic candidate antigens. In this study, we designed a fusion protein by linking both CFP21 and MPT64 with a 15-amino-acid peptide, (G4S1)3, and overexpressed the fusion protein in Escherichia coli. A new whole-blood gamma interferon assay based on the recombinant fusion protein, CFP21-MPT64 (rCM-WBIA), was developed and compared with the tuberculin skin test (TST) for screening of LTBI in household contacts of patients with sputum-positive TB. rCM-WBIA had a slightly higher sensitivity (66.7%; 24/36 contacts) than that of the TST (61.1%; 22/36 contacts) for household contacts. We found that rCM-WBIA had a very high sensitivity (90.9%) and specificity (71.4%) for LTBI detection compared with TST. The overall agreement between rCM-WBIA and TST was 83.3% (k = 0.64); rCM-WBIA positivity was associated with a larger TST induration. These results suggest that rCM-WBIA, based on the recombinant fusion protein CFP21-MPT64, is a promising alternative diagnostic tool for detection of LTBI

Immunogenicity and Protective Efficacy of a Novel Recombinant BCG Strain Overexpressing Antigens Ag85A and Ag85B

Recombinant Bacillus Calmette-Guérin (rBCG) strain is the promising vaccine candidate for tuberculosis (TB) prevention, which aims at providing more enduring and enhanced protection than the parental BCG vaccine. In this study, three rBCG strains overexpressing immunodominant antigens Ag85B (rBCG::85B), Ag85A (rBCG::85A), or both (rBCG::AB) of Mycobacterium tuberculosis were constructed, respectively. rBCG strains showed higher level of overexpression of Ag85A and/or Ag85B proteins than BCG containing empty vector pMV261(rBCG::261), which had low levels of endogenous expression of both proteins as expected. rBCG::AB strain could provide the strongest short-term and long-term protection in the lung against intravenous infection with virulent M. tuberculosis than rBCG::261 control and other two rBCG strains overexpressing single antigen. The stronger and longer-lasting protection provided by rBCG::AB than rBCG::261 was correlated with systemic in vitro antigen-specific IFN-γ responses. Therefore, our results indicate that rBCG::AB could be a very promising TB vaccine candidate and should be further evaluated for the preclinical test

Differential Immunogenicity and Protective Efficacy Elicited by MTO- and DMT-Adjuvanted CMFO Subunit Vaccines against Mycobacterium tuberculosis Infection

Tuberculosis (TB) remains a major and global problem of public health. An effective TB subunit vaccine is urgently needed. Proper selection of the delivery system for the vaccine is crucial for inducing an appropriate immune response tailored to control the target pathogen. In this study, we compared the immunogenicity and protective efficacy of CMFO subunit vaccines against primary progressive TB in two different adjuvant systems: the MTO oil-in-water (O/W) emulsion composed of monophosphoryl lipid A (MPL), trehalose-6,60-dibehenate (TDB), and oil in water emulsion MF59 and the DMT liposome containing dimethyldioctadecylammonium bromide (DDA), monophosphoryl lipid A (MPL), and trehalose-6,60-dibehenate (TDB). Our results demonstrated that the DMT-adjuvanted CMFO could confer more significant protection against M. tuberculosis infection than the CMFO/MTO did in mice. In particular, the adjuvant DMT showed a stronger ability than the O/W emulsion to adjuvant CMFO subunit vaccine and enhanced protection, attributed to elicit Th1-biased responses, strong Th1/Th17 cytokine responses, and IFN-γ+ or IL-2+ T cell responses. Therefore, our findings demonstrate that the liposome delivery system shows more effectiveness to adjuvant TB subunit vaccine than O/W emulsion and highlight the importance of adjuvant formulation for the better efficacy of a protein vaccine