Bingdong Zhu

The immunology of tuberculosis: from bench to bedside.

Tuberculosis (TB) is an international public health priority and kills almost two million people annually. TB is out of control in Africa due to increasing poverty and HIV coinfection, and drug‐resistant TB threatens to destabilize TB control efforts in several regions of the world. Existing diagnostic tools and therapeutic interventions for TB are suboptimal. Thus, new vaccines, immunotherapeutic interventions and diagnostic tools are urgently required to facilitate TB control efforts. An improved understanding of the immunopathogenesis of TB can facilitate the identification of correlates of immune protection, the design of effective vaccines, the rational selection of immunotherapeutic agents, the evaluation of new drug candidates, and drive the development of new immunodiagnostic tools. Here we review the immunology of TB with a focus on aspects that are clinically and therapeutically relevant. An immunologically orientated approach to tackling TB can only succeed with concurrent efforts to alleviate poverty and reduce the global burden of HIV.

Evaluation of a recombinant BCG expressing antigen Ag85B and PPE protein Rv3425 from DNA segment RD11 of Mycobacterium tuberculosis in C57BL/6 mice

Antigen 85B (Ag85B) is an important immunodominant antigen of Mycobacterium tuberculosis, and is a very promising vaccine candidate molecule. Rv3425 is a member of the subgroup 3 of the PPE family, which does not exist in all BCG strains. In this study we constructed a new rBCG which included this united gene (Ag85B-Rv3425). The level of antigen-stimulated T cells expressing IFN-γ was significantly higher in the C57BL/6 mice vaccinated with rBCG::Ag85B-Rv3425 than with BCG. In addition, the sera from mice immunized with rBCG::Ag85B-Rv3425 revealed an increase in the specific immunoglobulin G titers than that from mice immunized with BCG. Antigen specific IgG subclass analysis showed that rBCG::Ag85B-Rv3425 tended to facilitate IgG2a production, suggesting enhancement of predominant Th1 response which in turn may facilitate increased production of protective IFN-γ. These results suggested that this rBCG::Ag85B-Rv3425 could be a strong vaccine candidate for further study.

Fusion protein Ag85B-MPT64190-198-Mtb8.4 has higher immunogenicity than Ag85B with capacity to boost BCG-primed immunity against Mycobacterium tuberculosis in mice

Tuberculosis (TB) remains a major infectious disease worldwide despite chemotherapy and BCG vaccine. The efficacy of the current TB vaccine BCG varies from 0 to 80%. New vaccines that have better protection than BCG or have the capability to boost BCG-primed immunity are urgently needed. We have previously constructed a fusion protein Ag85B-MPT64(190-198)-Mtb8.4 (AMM). In this study, we investigated the immunogenicity of the fusion protein AMM in a novel adjuvant of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA-BCG PSN), and its capacity to boost BCG-primed immunity. The anti-Ag85B antibodies IgG1 and IgG2a were determined using ELISA and the number of spleen cells secreting IFN-gamma was determined by ELISPOT. In addition, the ability of the subunit vaccine AMM to boost BCG-primed immunity against Mycobacterium tuberculosis was analyzed. The fusion protein AMM induced more effective humoral and cell-mediated immune responses in mice than Ag85B alone. Mice primed with BCG vaccination followed by boosting with AMM produced a stronger immune response and afforded a better protection against M. tuberculosis infection than mice immunized with BCG alone or BCG priming followed by boosting with Ag85B. These findings suggest that AMM is a promising candidate subunit vaccine to enhance the protective efficiency of BCG.

Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine Consisting of Antigen Ag85B and HspX against Mycobacterium tuberculosis Infection in Mice

Subunit vaccines have the potential advantage to boost Mycobacterium bovis Bacillus Calmette‐Guérin (BCG)‐primed immunity in adults. However, most candidates are antigens highly expressed in replicating bacilli but not in dormant or persisting bacilli, which exist during Mycobacterium tuberculosis infection. We constructed M. tuberculosis fusion protein Ag85B‐Mpt64190–198‐HspX (AMH) and Ag85B‐Mpt64190–198‐Mtb8.4 (AMM), which consist of Ag85B, the 190–198 peptide of Mpt64, HspX (Rv2031c) and Mtb8.4 (Rv1174c), respectively. AMH and/or AMM were mixed with adjuvants composed of dimethyl‐dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA‐BCG PSN) to construct subunit vaccines. Mice were immunized thrice with Ag85B, AMH and AMM vaccines and the immunogenicity of the fusion protein vaccines was determined. Then, mice were primed with BCG and boosted twice with Ag85B, AMH, AMM and AMM + AMH vaccines, respectively, followed by challenging with M. tuberculosis virulent strain H37Rv, and the immune responses and protective effects were measured. It was found that mice immunized with AMH vaccine generated high levels of antigen‐specific cell‐mediated responses. Compared with the group injected only with BCG, the mice boosted with AMM, AMH and AMM + AMH produced higher levels of Ag85B‐specific IgG1 and IgG2a and IFN‐γ‐secreting T cells upon Ag85B and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation. It is interesting that only mice boosted with AMM + AMH had significantly lower bacterial count in the lungs than those receiving BCG, whereas mice boosted with AMH or AMM did not. The results suggest that AMH consisting of HspX, the antigen highly expressed in dormant bacilli, could be combined with antigens from replicating bacilli to enhance BCG primed immunity so as to provide better protection against both growing and non‐growing bacteria that occur during the infection process.

Knockdown of astrocyte elevated gene-1 (AEG-1) in cervical cancer cells decreases their invasiveness, epithelial to mesenchymal transition, and chemoresistance

During cancer development, epithelial–mesenchymal transition (EMT) facilitates tumor dissemination and metastatic spread, which is characterized by morphologic changes from epithelial cells to fibroblast-like cells, disassembly of intercellular junction, and increased cell motility. Overexpression of astrocyte elevated gene-1(AEG-1) in various cancer cell lines and cancers has been found to be associated with aggressive tumor behavior. We found that AEG-1 expression was elevated in low differentiation cervical cancer specimens from patients. However, little is known about the AEG-1’s precise role in invasion and metastasis. Here we demonstrate that downregulation of AEG-1 by RNAi significantly decreased the invasion and migration of cervical cancer cells, suggesting that AEG-1 overexpression may enhance cancer cell motility by inducing EMT. Downregulation of AEG-1 also led to reduced expression of mesenchymal marker vimentin and the transcription factor Snail but upregulation of epithelial marker E-cadherin in HeLa cells. In addition, knockdown of AEG-1 decreased colony forming units and increased sensitivity to cancer drugs in vitro. Taken together, our results suggest that knockdown of AEG-1 could decrease EMT and chemoresistance in cervical cancer cells and attenuate their aggressive behavior.

Systems approach to tuberculosis vaccine development

Tuberculosis is both highly prevalent across the world and eludes our attempts to control it. The current bacillus Calmette–Guérin vaccine has unreliable protection against adult pulmonary tuberculosis. As a result, tuberculosis vaccine development has been an ongoing area of research for several decades. Only recently have research efforts resulted in the development of several vaccine candidates that are further along in clinical trials. The majority of the barriers surrounding tuberculosis vaccine development are related to the lack of defined biomarkers for tuberculosis protective immunity and the lack of understanding of the complex interactions between the host and pathogen in the human immune system. As a result, testing various antigens discovered through molecular biology techniques have been only with surrogates of protection and do not accurately predict protective immunity. This review will address new discoveries in latency antigens and new next‐generation candidate vaccines that promise the possibility of sterile eradication. Also discussed are the potentially important roles of systems biology and vaccinomics in shortening development of an efficacious tuberculosis vaccine through utilization of high‐throughput technology, computer modelling and integrative approaches.

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis : a novel immunodominant antigen of Mycobacterium tuberculosis induces humoral and cellular immune responses in mice

Subtractive DNA hybridization of pathogenic M. bovis and BCG, and comparative genome‐wide DNA microarray analysis of M. tuberculosis H37Rv and BCG identified several RD, designated as RD1 to RD16, between M. tuberculosis and M. bovis on the one hand and BCG on the other. These regions cover 108 ORF of M. tuberculosis H37Rv, and are deleted from all 13 BCG sub‐strains currently used as anti‐tuberculosis vaccines in different parts of the world. In this study, we evaluated cellular and humoral immune response in C57BL/6 mice immunized with the PPE protein Rv3425, encoded by an ORF found in RD11 of M. tuberculosis. Rv3425 protein induced an increased Th1/Th2 type immune response in mice, characterized by an elevated concentration of IFN‐γ in antigen stimulated splenocyte culture and a strong IgG1 antibody response. These results provide evidence on the immunogenicity of the PPE protein Rv3425 which, together with its reported immunodominant characteristics, imply that it may be a candidate for development of a vaccine for the control of TB.

More Vaccine Efficacy Studies on the Recombinant Bacille Calmette-Guerin Co-expressing Ag85B, Mpt64190–198 and Mtb8.4

The immunogenicity of the recombinant Bacille Calmette‐Guerin: rBCG‐Ag85B‐Mpt64190–198‐Mtb8.4 (rBCG‐AMM) was evaluated in our previous study. This paper compares the protective efficacy of rBCG‐AMM, rBCG‐A which overexpresses Ag85B and BCG in C57BL/6 mice. There was no significant difference in proliferation characteristics among rBCG‐AMM, rBCG‐A and BCG. The growth characteristics of rBCG‐AMM in host tissue were identical to control BCG, suggesting the improved protective efficacy was directly related to the expression of the Ag85B‐Mpt64190–198‐Mtb8.4 fusion protein. The protective experiment demonstrated that rBCG‐AMM could confer similar or even better protective efficacy against Mycobacterium tuberculosis infection compared with BCG or rBCG‐A as evaluated by bacterial organ loads, lung histopathology and net weight gain or loss. The results suggested that the recombinant BCG: rBCG‐Ag85B‐Mpt64190–198‐Mtb8.4 is a potential vaccine candidate for further study.

Chimaeric protein improved immunogenicity compared with fusion protein of Ag85B and ESAT-6 antigens of Mycobacterium tuberculosis

Antigen 85B (Ag85B) and ESAT‐6 are important immunodominant antigens of Mycobacterium tuberculosis, and both are very promising vaccine candidate molecules. In this study, we relied on the T‐cell epitopes of Ag85B and ESAT‐6 to design a chimaeric protein by inserting ESAT‐6 into Ag85B from the amino acids 167–182. We found the ratio of IgG2b/IgG1 and the secretion of interferon (IFN)‐γ in the mice vaccinated with the new protein with adjuvant MPL and TDM were higher than the mice immunized with fusion protein Ag85B‐ESAT‐6, which have been reported and could induce levels of protective immunity similar to BCG in the mouse model of tuberculosis (TB) infection. These results suggest that the chimaeric protein Ag85BN‐ESAT‐6‐Ag85BC is a strong candidate for further study and the T‐cell epitopes of the antigens should be considered when we design the subunit vaccine.

Evaluation of a new Recombinant BCG which Contains Mycobacterial Antigen ag85B–mpt64190–198–mtb8.4 in C57/BL6 Mice

Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be one of the major public health problems in the world. The eventual control of this disease will require the development of a safe and effective vaccine. Bacille Calmette‐Guerin (BCG), the only vaccine against TB, is not perfect for its limited ability to protect against the adult form of TB. Some improvements of TB vaccines relied to strengthening the immunogenicity and/or persistence of genetically modified recombinant BCG (rBCG) strain. Antigen 85B (Ag85B) and Mtb8.4 are importantly immunodominant antigens of M. tuberculosis, and both are very promising vaccine candidate molecules. MPT64190–198, is presented to CD8+ T cells during mycobacterial infections. In this study, we combined these above genes into one recombinant gene of ag85B–mpt64190–198–mtb8.4. Then we constructed the new rBCG containing this united gene. This rBCG can induce an increased Th1‐type immune response in mice, characterized by an elevated level of interferon‐γ in antigen‐stimulated splenocyte culture and a strong IgG2a antibody response. Also, it can elicit longer immune responses than BCG. The results show that this rBCG is a promising candidate for further study.