Yinlan Bai

Recombinant Mycobacterium smegmatis expressing an ESAT6-CFP10 fusion protein induces anti-mycobacterial immune responses and protects against Mycobacterium tuberculosis challenge in mice.

The currently used vaccine against tuberculosis, Bacille Calmette‐Guérin (BCG), has variable efficacy, so new vaccine development is crucial. In this study, we evaluated a recombinant vaccine prepared from non‐pathogenic Mycobacterium smegmatis (rMS) that expresses a fusion of early secreted antigenic target 6‐kDa antigen (ESAT6) and culture filtrate protein 10 (CFP10). C57BL/6 mice were immunized with the rMS expressing the ESAT6‐CFP10 fusion protein (rM.S‐e6c10) or with BCG. The mice in the rM.S‐e6c10 group had a significantly higher titre of anti‐ESAT6‐CFP10 antibodies than did animals in the BCG or saline groups. Spleen cells from rM.S‐e6c10‐immunized mice exhibited a cytotoxic response to ESAT6 and CFP10‐expressed target cells, but spleen cells from animals in the other groups did not. Levels of IFN‐γ and IL‐2 production by purified T cells from spleens were significantly higher in rM.S‐e6c10 group than in BCG group. Finally, after M. tuberculosis (MTB)‐challenged mice, dramatic reduction in the numbers of MTB colony‐forming units (CFUs) in the lungs was observed for the mice immunized with the rMS. The protective efficacy of rM.S‐e6c10 and BCG vaccination was similar based on measures of MTB burden and lung pathology. Our data indicate that the recombinant M. smegmatis vaccine expressing the ESAT6‐CFP10 fusion protein has potential in clinic application.

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.

Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65(Hsp65) with human interleukin‐2 against Mycobacterium tuberculosis in BALB/c mice

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin‐2 (hIL‐2) in BALB/c mice. We showed that the DNA vaccine pcDNA‐Hsp65‐hIL‐2 could induce high levels of antigen‐specific antibody, IFN‐γ, CD4+ and CD8+ T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.

Immunotherapeutic efficacy of recombinant Mycobacterium smegmatis expressing Ag85B–ESAT6 fusion protein against persistent tuberculosis infection in mice

The application of immunotherapy in combination with chemotherapy is considered an effective treatment strategy against persistent Mycobacterium tuberculosis (Mtb) infection. In this study, we constructed a novel recombinant Mycobacterium smegmatis (rMS) strain that expresses Ag85B and ESAT6 fusion protein (AE–rMS). Immunization of C57BL/6 mice with AE–rMS generated mainly Th1-type immune responses by strongly stimulating IFN-γ- and IL-2-producing splenocytes and increasing antigen-specific cytotoxic T lymphocyte (CTL) activity. To test the immunotherapeutic efficacy of AE–rMS, a persistent tuberculosis infection (PTBI) model was established via tail-vein injection of C57BL/6 mice with 1 × 104 colony forming units (CFU) of Mtb strain H37Rv in combination with concurrent chemotherapy drugs isoniazid (INH) and pyrazinamide (PZA). PTBI mice immunized with AE–rMS showed high levels of IFN-γ secreted by splenocytes and decreased bacteria loads in lung. Treatment with only the anti-tuberculosis (anti-TB) drugs RFP and INH (RI), decreased bacteria loads to low levels, with the Th1-type immune response further attenuated. Moreover, AE–rMS, when combined with RI treatment, further reduced the bacteria load as well as the pathological tissue damage in lung. Together, these results demonstrated the essential roles of AE–rMS-induced Th1-type responses, providing an effective treatment strategy by combining AE–rMS and RI for persistent TB.

Construction and immunogenicity of the DNA vaccine of Mycobacterium Tuberculosis dormancy antigen rv1733c.

We aimed to construct the DNA vaccine encoding Mycobacterium Tuberculosis (Mtb) dormancy antigen Rv1733c and investigate its immunogenicity in mice. The recombinant plasmid pcDNA‐Rv1733c was transfected into P815 cells and its product was detected by indirect immunofluorescence. The mice were immunized once every 2 weeks by intramuscular injection of pcDNA‐Rv1733c plasmid for a total of three times. The specific antibodies in the serum of the immunized mice were detected by enzyme‐linked immunosorbent assay at the indicted time. Enzyme‐linked immunosorbent spot was applied to determine the levels of IFN‐γ, IL‐2 and IL‐4 secreted by splenic lymphocytes. Total cytotoxicity T lymphocyte (CTL) active of the splenic lymphocytes was detected by lactate dehydrogenase assay. Additionally, we analysed the percentages of CD4+ and CD8+ T cells in splenic lymphocytes using flow cytometry. The specific antibody was detected at 2 weeks after the first immunization, and the antibody titre was increased with time which was reached to 1:1600 at 8 weeks. The stimulation index of spleen lymphocytes and the levels of IFN‐γ, IL‐2 and IL‐4 of pcDNA‐Rv1733c‐immunized mice were both higher than those of saline‐immunized mice (P < 0.05). However, no difference was found in the percentages of CD4+ and CD8+ T cells and the activity of CTL between the pcDNA‐Rv1733c‐ and saline‐immunized mice (P > 0.05). So we got the conclusion that the plasmid pcDNA‐Rv1733c DNA could induce specific humoral and cellular immunity in mice. Improving the immune effect of Rv1733c by several strategies, such as choosing appropriate immunization route and adjuvant, would be significant for Rv1733c as new tuberculosis vaccine.

Expression, purification and characterization of Mycobacterium tuberculosis RpfE protein.

Resuscitation promoting factor E (RpfE) is one of the five Rpf-like proteins in Mycobacterium tuberculosis (M. tuberculosis). These Rpf-like proteins are secretory, which make them candidates for recognition by the host immune system. In this study, the RpfE gene was amplified from M. tuberculosis, cloned into the expression vectors pDE22 and pPRO EXHT, and were expressed in Mycobacterium vaccae (M. vaccae) and Escherichia coli DH5α, respectively. Both recombinant RpfE proteins were purified by Ni-Sepharose affinity chromatography, and were given to C57BL/6 mice. The RpfE proteins elicited T cell proliferation, and stimulated the production of gamma interferon (IFN-γ), interleukin-10 (IL-10) and IL-12. Our results indicated that the RpfE protein expressed in M. vaccae could more efficiently stimulate cellular immune response, making it a promising candidate as a subunit vaccine.

Immunological characteristics of Mycobacterium tuberculosis subunit vaccines immunized through different routes

Tuberculosis is chronic infectious disease caused by Mycobacterium tuberculosis (M.tb) that is prevalent worldwide. Several specific antigens, such as Antigen 85B (Ag85B) and 6 kDa early secretory antigenic target (ESAT-6) protein of M.tb, are listed as some of the candidate subunit vaccines against M.tb. ESAT-6, as a virulent factor and differential gene in M.tb, shows insufficient immunogenicity in animal model. In order to investigate the ways to improve the immunogenicity of ESAT-6, we immunized ESAT-6 by subcutaneous and intramuscular routes with different adjuvants. We found that ESAT-6 immunized alone did not induce significant humoral immunity in both immunization routes. However, subcutaneous immunization of ESAT-6 plus incomplete Freunds adjuvant can induce a significant humoral immune response, enhanced proliferation and elevated secretion of IFN-γ from splenocytes. Intramuscular immunization of ESAT-6 plus adjuvant aluminum salt or poly(I:C) did not enhance humoral and cellular immune responses. Therefore, it is concluded that immunization of ESAT-6 subcutaneously plus incomplete Freunds adjuvant induces stronger humoral and cellular immune responses, which can be considered of ESAT-6 as a subunit vaccine in further research against tuberculosis.

Phosphatidylethanolamine-binding protein 4 promotes the epithelial-to-mesenchymal transition in non-small cell lung cancer cells by activating the sonic hedgehog signaling pathway: JIAN et al.

Phosphatidylethanolamine‐binding protein 4 (PEBP4), a member of the PEBP family, has been reported to play a pivotal role in tumor progression. However, its role in epithelial‐to‐mesenchymal transition (EMT) in non–small cell lung cancer (NSCLC) cells remains unclear. Here, we investigated the effects and underlying mechanism of PEBP4 in NSCLC EMT. Three human NSCLC cell lines (A549, H1299, and H460) were transfected with pcDNA3.1‐PEBP4 or PEBP4‐targeting small interfering RNA. Then, cell proliferation was analyzed by the MTT assay, and cell migration and invasion were analyzed by the transwell chamber assay. Protein and messenger RNA expression of the related genes and proteins were assessed by Western blot analysis and quantitative real‐time polymerase chain reaction, respectively. Results showed that PEBP4 was highly expressed in the human lung cancer tissues and three human NSCLC cell lines. Pretreatment with pcDNA3.1‐PEBP4 promoted the proliferation, invasion, and migration of NSCLC cells and increased EMT in vitro and lung tumor metastasis in vivo. Whereas knockdown of PEBP4 suppressed NSCLC cell migration, PEBP4, and invasion with prevented EMT. Furthermore, PEBP4 overexpression significantly promoted the transcriptional activity of sonic hedgehog (Shh) signaling in NSCLC cells. Further analysis showed that using cyclopamine to inhibit Shh signaling significantly ameliorated the effect on cell proliferation, invasion, and migration, as well as EMT triggered by PEBP4 overexpression. Together, these results suggest that PEBP4 may promote tumorigenesis in NSCLC by regulating cell proliferation and EMT via activation of the Shh signaling pathway.

Antitumor effect of recombinant Mycobacterium smegmatis expressing MAGEA3 and SSX2 fusion proteins

Mycobacterium smegmatis (M. smegmatis), which is a nonpathogenic and fast-growing mycobacterium, is a potential vaccine vector capable of expressing heterologous antigens. Spontaneous humoral and cellular immune responses have been demonstrated against cancer/testis antigens (CTA), including melanoma-associated antigen A (MAGEA) and SSX. In the present study, recombinant plasmids expressing MAGEA3 and SSX2 were constructed. The recombinant plasmids were transferred into M. smegmatis to generate the novel antitumor DNA vaccine. As MAGEA3 and SSX2 were in different ligation sequences, the two DNA vaccines were recombinant M. smegmatis MAGEA3-SSX2 (rM.S-MS) and recombinant M. smegmatis SSX2-MAGEA3 (rM.S-SM), respectively. The expression levels of Fusion proteins were assessed by western blotting. BALB/c mice were immunized with rM.S and western blot analysis was used to determine whether antibodies against MAGEA3 or SSX2 were produced in immunized mice. EC9706 cells were inoculated into BALB/c nude mice and the mice were maintained until an obvious visible tumor appeared on the back. Subsequently, the blood from the rM.S immunized BALB/c mice was injected into the BALB/c nude mice via the tail vein. In order to evaluate the antitumor effect of the vaccines, tumor volume and weight were measured 5 to 21 days after injection. Mice were euthanized on day 21 of tumor growth, and the tumor was dissected and weighed. The two fusion proteins were expressed in the rM.S and the specific fusion protein antibodies were expressed in the blood of immunized BALB/c mice. The tumor volumes and weight in the recombinant M. smegmatis MAGEA3 (rM.S-M) and recombinant M. smegmatis SSX2 (rM.S-S) groups were significantly reduced compared with the control group. Furthermore, the decrease in tumor volumes and weight in the rM.S-MS and rM.S-SM groups was more severe than in the rM.S-M or rM.S-S groups. There was no significant difference in the antitumor effect of the rM.S-MS and rM.S-SM groups. The present findings suggest that this rM.S may be a potential candidate therapeutic vaccine for the treatment of cancer.

Expression of Mycobacterium tuberculosis Ferric Uptake Regulator A Gene in Escherichia coli and Generation of Monoclonal Antibodies to FurA

Ferric uptake regulator A of Mycobacterium tuberculosis (MTB), which belongs to the Fur superfamily, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the pro-drug isoniazid. The feature and role of FurA in oxidative stress contribute to research on the pathogenesis of mycobacteria. In this study, four novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurA protein as immunogen. The furA gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pRSET-A and effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as soluble form in cell lysates and was purified via Ni-NTA purification system. Using the fusion protein to immunize BALB/c mice, four monoclonal antibodies (H9H6, H9E12, H10H6, and H10H8) were produced. As shown by Western blot analysis and cell fluorescence microscopy assay, the four antibodies could recognize the FurA protein, respectively. Then we assessed the effect of iron on the expression of FurA in MTB H37Rv and we concluded that iron does not affect FurA expression. These results suggest that the antibodies against FurA may provide a powerful tool for elucidating FurA biofunctions and regulation mechanism in the pathogenesis of tuberculosis.