Jiuling Wang

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a potential B-cell antigen used for serological diagnosis to distinguish vaccinated controls from tuberculosis patients

Proteins encoded by a 9.5-kb DNA segment, termed the region of difference (RD), of Mycobacterium tuberculosis have been demonstrated to be important in bacterial virulence, vaccine development and the design of diagnostic reagents. This study evaluated the immunogenic properties of Rv3425, a member of the PPE family of proteins, encoded by an open reading frame found in RD11 of M. tuberculosis, in comparison with two other well-known antigens, the early secreted antigen target 6 (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). RT-PCR demonstrated that Rv3425 mRNA is expressed in liquid culture by M. tuberculosis H37Rv. When tested in a conventional ELISA in the form of a His-tagged recombinant protein, Rv3425 revealed a statistically significant antigenic distinction between healthy bacille Calmette-Guérin (BCG)-vaccinated controls and tuberculosis (TB) patients (p <0.0001). The anti-IgG response to recombinant Rv3425 was almost equal to that for CFP-10, and was higher than that for ESAT-6. The results highlight the immunosensitive and immunospecific nature of Rv3425, which shows promise for use in the serodiagnosis of TB.

Novel recombinant RD2- and RD11-encoded Mycobacterium tuberculosis antigens are potential candidates for diagnosis of tuberculosis infections in BCG-vaccinated individuals.

Proteins encoded by region of deletions (RD) of Mycobacterium tuberculosis are useful in development of vaccines and diagnostic reagents. In the present study, six M. tuberculosis genes from RD2 and RD11, rv1978, nrdf1, mpt64, cfp-21, ppe57 and ppe59, were cloned and overexpressed in Escherichia coli. All six purified recombinant proteins could distinguish tuberculosis (TB) patients and latent TB infected subjects (LTBI), or called subclinical TB infection, from BCG-vaccinated healthy controls by T-cell IFN-gamma releasing ELISPOT. ELISPOT of Rv1978, NrdF1, Mpt64, CFP-21, Ppe57 and Ppe59 achieved sensitivities of 59%, 60%, 82%, 48%, 59% and 47% respectively in the detection of active TB and specificities of 94%, 90%, 76%, 93%, 100% and 93% respectively in BCG-vaccinated healthy controls. Combination of Ppe57 or NrdF1 with early secreted antigen target 6 (ESAT-6) or 10-kDa culture filtrate protein (CFP-10) in the IFN-gamma releasing ESLIPOT assay could increase the sensitivities in detecting active TB, for ESAT-6 from 82.1% to 85.7% or 92.9% (P=0.5 or 0.03, respectively) and for CFP-10 from 67.9% to 78.6% or 83.9%, respectively (both P<0.05). The high sensitivities, specificities and promising antigenic combination of NrdF1 and Ppe57 in detection of TB in BCG-vaccinated controls suggest their potential application in TB diagnosis.

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.

Different effects of tacrolimus on innate and adaptive immune cells in the allograft transplantation

While tacrolimus (FK506) is currently used as immunosuppression therapy in transplant recipient, the immunological mechanism remains unknown. Herein, the immunoregulatory effects of FK506 were investigated in the physiological status and allogeneic skin transplantation. FK506 cannot significantly alter the functions of innate immune cells (macrophages and neutrophils) and adaptive immune cells (T cells) in the physiological status. However, it can effectively delay allogeneic skin‐graft rejection through ameliorating the T cell responses, but not myeloid‐derived innate immune cell responses. Importantly, it did not affect the allograft recipient macrophage innate immune defence capacity to bacteria. In clinics, FK506 treatment can significantly control the cytokine production in T cells, but not non‐T cells. This study shows targeting calcineurin signalling, FK506, to be essential in inducing allograft tolerance, but not to damage the innate defence capacity, validating the immune cell phenotypes as a potential marker in transplantation following FK506 treatment.

Protective efficacy of a recombinant BCG secreting antigen 85B/Rv3425 fusion protein against Mycobacterium tuberculosis infection in mice.

In this study, the protective efficacy of a novel recombinant BCG strain co-expressing Ag85B and Rv3425 against Mycobacterium tuberculosis H37Rv was evaluated in mice. This rBCG::Ag85B-Rv3425 strain could provide similar or even better protective efficacy against M. tuberculosis challenge compared with BCG, as shown by no weight loss, significantly reduced lung:body weight ratios and lung bacteria load only at early time of infection. The results suggest that rBCG::Ag85B-Rv3425 could be a potential tuberculosis vaccine candidate for further study.