Efficacy Of Tuberculosis Multi-epitope DNA Vaccine To Drug-resistant Tuberculosis In Mice Model

Objective To study and compare the immunogenicity of multi-epitope DNAvaccine of Mycobacterium tuberculosis with BCG and therapeutic effects of the vaccinescombined with chemotherapy in a mouse model infected with multi-drug resistant (MDR)Mycobacterium tuberculosis.Method Animal immunization90mice were randomly separated into PBS controlgroup (A), BCG group (group B) and multi-epitope DNA vaccine group (Group C), with30mice in every group. Mice in group of A, B and C were treated with PBS, BCG andmulti-epitope vaccine with a dosage of0.3mL per mouse at1,3,5weeks, respectively.After three times immunization, eye blood was collected and serum was separated. IgGantibodies was determined by conventional indirect ELISA method. IL2and IFN-γ wasmeasured by sandwich ELISA. The mice were executed after three times immunizationand the mouse spleen were taken. The spleen cells were dispersed washed and celldensity was adjusted. TB-PPD was added to spleen cells and cultured for72h. spleenlymphocyte proliferation index was detected in a conventional MTT assay. Tuberculosisthat dual resistance to isoniazid and rifampicin isolated newly in clinical were routinelycultured. Then the bacterial concentration was adjusted to1×104cfu suspension andinjected with a dose of0.2mL per mouse in mice via the tail vein.5mice were randomlytaken4weeks after the infection. Smear and staining of blood, lungs and spleen wereperformed. Mice infected by M. tuberculosis were curtained by detection of TB undermicroscopy. model mice were randomly divided into model group (group D),chemotherapy alone group (E group), combined treatment group (F group)，with20mice per group. Group D received no treatment, E was treated with RFP and INH, groupF with RFP, INH and multi-epitope DNA vaccine. Isoniazid at0.03mg/(g d) and rifampicin0.02mg/(g d) was intragastric administration once daily for10weeks;while multi-epitope DNA vaccine in F group was administered by intramuscularinjection, with once every two weeks for10weeks. The mice were weighed and executedat the end of treatment. Mice lung, liver and spleen were dissected and weighted. Organindex of the lungs, liver, spleen were calculated. lungs and spleen were sampled, grindedand diluted in100-fold with sterile broth. Finally,0.5mL of the dilution was taken toinoculate on Lowenstein-Jensen medium dish of chicken egg. colony count wereperformed at37℃after4weeks. SPSS16.0statistical software was used to processdata, P≤0.05was considered statistically significant.Results tuberculosis-specific IgG antibodies, IL2and IFN-γ levels in TBmulti-epitope DNA vaccine and BCG group were significantly higher than control group(P <0.05); Tuberculosis-specific IgG antibodies, IL2and IFN-γ levels in Mycobacteriummulti-epitope DNA Vaccine group were higher than in BCG group, and the differencewas statistically significant (P <0.05). After immunization, lymphocyte proliferationindex in group of tuberculosis multi-epitope DNA vaccine and in group of BCG werehigher than controls (P <0.05), respectively. lymphocyte proliferation index in group ofmulti-epitope DNA vaccine group were higher than in BCG group, and the differencewas statistically significant (P <0.05). Chemotherapy alone had no significant effect oncolony counts of control group (P>0.05). colony counts in chemotherapy combined withmulti-epitope DNA vaccine group was significantly less than the colony counts in thecontrol group and the chemotherapy group, the difference was statistically significance(P <0.05). There is no significant difference between chemotherapy group and the controlgroup (P>0.05), organ index of the vaccine combination therapy were lower than thecontrol group and the chemotherapy group, respectively, and the difference wasstatistically significant (P <0.05).Conclusion Multi-epitope DNA vaccine consisted of Hsp70, Ag85Aand ESAT-6ofmycobacterium tuberculosis induced strong specific immune response in mice, whichcould produce a high level of specific IgG antibody, stimulate secretion of IL2and IFN-γ, proliferation of specific lymphocyte and significantly improve the efficacy ofanti-tuberculosis to drug resistant tuberculosis in mice.