Experimental Study In A Tuberculosis DNA Vaccine Expressing A Fusion Protein Of Ag85B-Esat6-HspX

Caused by Mycobacterium tuberculosis (MTB). tuberculosis (TB) is a kind of serious infectious disease with a long history. The World Health Organization reported approximately one-third of the total population in the world has been infected with MTB. With the emergence of multi-drug resistant M.tuberculosis (MDR) and extensively drug resitant TB strains (XDR). population of AIDS and TB co-infection leads to an increasing number of active TB.Mycobacterium bovis bacillus Calmette-Guerin (BCG) is a unique vaccine against TB that is currently available BCG efficiently protects children against severe disease. but this vaccine does not offer consistent protection for adults, and it does not prevent reactivation of latent TB. Development of new TB vaccines is badly needed not only to prevent the infants and uninfected people against TB, but also to enhance the cellular immunity of those who had been infected but not active patients and eliminate the MTB faster.In this study, we constructed recombinant plasmid named pcDNA-Ag85B-East6-HspX which could be expressed in eukaryotic cells by molecular biological technology. In terms of this vaccine, we conducted not noly the evaluation of immunogenicity, but also the one of protective as well as therapeutic efficacy in mice.1. The construction and expression of Ag85B-East6-HspX fused DNA vaccineThe gene encoding Ag85B, ESAT6 and HspX protein was amplified by PCR from genome of Mycobacterium tuberculosis H37Rv strain, and was inserted into cloning vector pUC19-T. After sequence was confirmed, the fused gene were subcloned to eukaryotic expression vector pcDNA3.1(-). After identified by restrictive enzymatic digestion and verified by DNA sequencing again. the recombinant plasmid pcDNA-Ag85B-Esat6-HspX was transfected into 293T cells with MegaTran 1.0. Western blot was used to detect the expression of goal protein. A 65kDa protein was all detected with anti-Ag85B polyclonal antibody. anti-Esat6 monoclonal antibody and anti-HspX monoclonal antibody, respectively. The successful construction of eukaryotic expression vector pcDNA-Ag85B-Esat6-HspX would become the basis of late assessment on immunogenicity and protective efficacy. 2. Immunogenicity evaluation of the new DNA vaccineMice were vaccinated intramuscularly with 100μl DNA vaccine at one side of the quadricep three times two weeks apart. BALB/c mice were randomly divided into four groups:the pHspX plasmid group, the pAEH plasmid group, the BCG group and the pcDNA3.1 group. Total IgG levels were detected at 2.4 and 6 weeks after immunization. Two weeks after the final immunization, peripheral blood samples were obtained by removing the eye balls from individual mice and the frequency of CD4+,CD8+ andγδT cells was characterized by flow cytometry analysis using a FACScalibur instrument. Mice were sacrificed two weeks after the last immunization and their splenic mononuclear cells were prepared. The concentrations of serum antigen-specific immunoglobulin G (IgG), IgG1 and IgG2a in individual animals were analyzed by ELISA. Lymphocytes of spleen were stimulated in vitro by HspX antigen, and level of cells proliferation as well as counts of lymphocytes capable of secrecting antigen-specific IFN-γwere determined by ELISPOT using the mouse IFN-γprecoated ELISPOT kit. Also, the concentrations of IFN-γ. IL-2 and IL-4 in the supernatants were measured by ELISA using cytokine ELISA detection system.In terms of humoral immune responses, the levels of serum anti-HspX IgG and the ratios of IgG2a to IgG1 against HspX in the pAEH-vaccinated mice were significantly higher than that of other groups (P<0.05). The main antibody subtype was IgG2a. a characteristic of Th1 type of immune response. In the cellular immune responses, the index of splenocyte proliferation in fusion gene group was 2.33, which was higher than that of BCG group (1.26) (P<0.05). Vaccination with pAEH induced higher frequency of antigen-specific IFN-γ-secreted SFC response to HspX, Esat6 and Ag85B antigen than that of in other three groups (P<0.01). The fusion gene vaccine could induce the proliferation of CD4+ T cells and CD8+ T cells in splenolymphocytes of mice significantly compared with the pcDNA control group (P<0.05). The percentages of them were 27.58% and 8.64% respectively. However, there was no significant difference compared with BCG groups (P>0.05). As for the percentages ofγδT cells, we did not observe the differences among four groups. Antigen-specific IFN-γand IL-2 levels in the pAEH group were all significantly higher than those of in other three groups (P<0.05). The amounts of them were 1005.72±67.12 pg/ml and 84.63±8.76 pg/ml respectively. But we did not detect the contents of IL-4 among four groups. 3. Protective and therapeutic efficacy evaluation of the new DNA vaccineThe protective efficacy of the DNA vaccine enconding fusion protein against MTB infection in miceTwo weeks after the final immunization. different groups of mice were challenged intravenously at tail vein with 5×105 CFU of MTb H37Rv strain. Six weeks later. their spleens and lungs were dissected out aseptically and counted the bacteria numbers. The bacteria loads were expressed as log10CFU. The results showed the bacteria loads of spleens and lungs in mock group were 5.48±0.11 log10 and 5.40±0.09 log10 respectively, while those of in pAEH group were 4.97±0.05 log10 and 4.65±0.22 log10 respectively. Thus. the fusion gene vaccine could significantly reduce the bacteria numbers in spleens and lungs compared with the mock group (P<0.05). It demonstrated that the DNA vaccine was able to protect the mice against MTB infection. Nevertheless, the protective ability was not super to that of BCG (P>0.05). namely the bacteria loads in BCG group were 4.88±0.13 log10 and 4.46±0.17 log10 respectively.The therapeutic efficacy of the DNA vaccine encoding the fusion protein in MTB infected miceBALB/c mice were infected intravenously with 5×105 CFU of MTb and four weeks later. individual mice were randomly injected with pcDNA as controls or treated intramuscularly with 100μg of pHspX or pAEH, respectively. The vaccinated mice were boosted with the same dose of plasmid every two weeks for two times. In this study. we didn't set a positive control (BCG group) because the model in this study was a post exposure model and BCG had poor protection efficacy especially for the population who has been infected by tuberculosis. Six weeks after the final DNA treatment, the mice were sacrificed and the MTb loads in the spleens and lungs were determined. The result revealed that the bacteria loads of spleen and lungs in pcDNA control group were 5.43±.11 log10 and 5.46±0.08 log10 respectively. While the bacteria loads in spleen and lungs in the pAEH group were 4.88±0.18 log10 and 4.92±0.10 log10 respectively, the fusion gene vaccine could significantly decrease the bacteria numbers in TB mice compared with the mock group (P<0.05). It implied that the new DNA vaccine had therapeutic effect on TB mice.4. ConclusionIn this study, we firstly combined the protective antigen of Mtb Ag85b and secreted antigen ESAT6 with latent antigen HspX. Then we successfully constructed the eukaryotic expression plasmid named pcDNA-Ag85B-Esat6-HspX which could express a fusion protein. Next. we evaluated the immunogenicity and protective efficacy of this new vaccine. The results showed that the vaccine could induce strong cell immunity and humoral immunity in mice. On one hand. by the way of infection after the immunization. this protective effect of vaccine was assessed indicating that the fusion gene DNA vaccine could significantly reduce the bacteria loads in spleen and lungs of infected mice. but this protective role did not exceed the BCG On the other hand. by the way of treatment after infection. we evaluated the therapeutic efficacy of the vaccine as well. The results demonstrated that the DMA vaccine had therapeutic effect on TB mice. Thus. compared with BCG, the new DNA vaccine not noly induced a strong immune response in Thl type cells, but also had a therapeutic effect. which for the prevention and treatment of TB is very meaningful.