| W541 protein is encoded by a novel multi-antigen epitope-tandem DNA vaccine composed of MTB proliferation antigens Ag85 A and Ag85 B,and LTBI-related antigens Rv3407 and Rv1733 c.1.In this study,the bioinformatics methods were used to predict and analyzed the structure and function of MTB W541 protein;then the mouse LTBI model was developed,and preventively treated with W541 DNA vaccine,and injected by hormone to reactivate the latent MTB,comprehensively evaluated preventive effect of the vaccine on inhibiting reactivation of latent MTB.Bioinformatics analysis of MTB multi-antigen epitope-tandem protein W541.The amino acid sequence of the protein W541 encoded by the new MTB DNA vaccine constructed in our laboratory was analyzed the physicochemical properties,the hydrophilicity(hydrophobicity),transmembrane helix,secondary and tertiary structures,subcellular localization,signal peptide,glycosylation,phosphorylation sites,B cell,helper T(Th),and cytotoxic T lymphocyte(CTL)epitopes,the protein interaction network,the homology between W541 and human proteins using bioinformatics softwares Prot Param,Protscale,TMHMM,SOPMA,SWISS-MODEL,PSORT,Signal P,Net NGlyc,Net Phos,SYFPEITHI,RANKPEP,IEDB,Net MHC,Net CTL,STRING,and EXPASY,respectively.In the secondary structure,irregular curl is dominant(42.47%),and it also exists α-Spiral β-Folding and β-Corner(26.99%,19.03% and 11.51%respectively);The results showed that W541 protein consisted of 704 amino acids with the molecular formula of C3329H5035N923O993S24,was a hydrophilic unstable membrane protein with its instability index 45.37 and without transmembrane helical regions and signal peptide.In its secondary structure,irregular curl was dominant(42.47%),and it also hadα-helix,β-fold,and β-corner(26.99%,19.03%,and 11.51%,respectively).W541 protein had6 glycosylation sites,62 phosphorylation sites,including 15 threonine,35 serine,and 12 tyrosine phosphorylation sites,respectively.It hadmultiple potential T-cell and B-cell epitopes.W541 protein interacted with 10 proteins.The amino acid sequenceof W541 proteinhad less than3.3% homology with the human protein.2.Preventive effect of M.tuberculosis W541 DNA vaccine on mouse model with latent tuberculosis infection.one hundred and one female BALB/c mice aged 42-62 days and weighing 16g-18 g were randomly assigned to the following 11 groups:(1)the 4-week infection group(5 mice);(2)the 16-week infection group(8mice);(3)the 29-week infection group(10 mice);(4)the chemotherapy for the 12-week group(8 mice);(5)the chemotherapy withdrawal for the13-week group(10 mice);(6)normal saline group(i.e.,the hormone treatment discontinuation for the 3-week group,10 mice);(7)p VAX1 vector group(10 mice);(8)Vaccae vaccine group(10 mice);(9)ag85ab plasmid DNA group(10 mice);(10)W541 plasmid DNA group(10 mice);(11)normal control group(10 mice).Then,the mouse LTBI model was developed and treated as follow:(1)91 mice were injected with 0.4ml MTB standard strain H37 Rv suspension -forming units(CFUs)through the tail vein to develop mouse infection model;(2)After the MTB infection for 4 weeks,mice drank water containing 8 g/L pyrazinamide and 0.12 g/L isoniazid every day for 12 weeks to establish the mouse LTBI model.(3)At 16 weeks after MTB infection,the mouse LTBI model was immunized perspectively with saline(100μl/mouse),p VAX1 vector(100μg/100μl/mouse),Vaccae vaccine(22.5μg/100μl/mouse)and ag85 ab plasmid DNA(100μg/100μl),W541 plasmid DNA(100μg/100μl/mouse)by intramuscular injection once every 2 weeks for 3 times.(4)At 3 weeks after the third immunization,mice were injected intramuscularly with hydrocortisone sodium succinate(0.5mg/0.1ml/mouse),3 times a week for3 weeks,to develop the mouse LTBI-reactivation model;(5)At 4 weeks after stopping hormone injection,all mice were sacrificed,and then calculated the organ weight index,the number of IFN-γ-secreting T lymphocyte spots in splenic lymphocytes,and the proportion of Fox P3 regulatory T cells(Treg).The levels of Th1-and Th2-cytokines in mouse splenic lymphocyte culture supernatants were detected by flow cytometry microsphere microarray(CBA).The viable bacterium counts in the lungs and livers were calculated,and the pathological changes of lung tissues were observed to analyze the therapeutic effect of the vaccine.The above research results showed as follow: the lung weight index of the 29-week infection group and 16-week infection group were significantly higher than those of the 4-week infection group,the chemotherapy for the 12-week group,and the chemotherapy withdrawal forthe13-week group(P < 0.0001);the 4-week infection group and chemotherapy for the 12-week group were significantly higher than that of the chemotherapy drug withdrawal for the 13-week group(P<0.05).The liver weight index of the 29-week infection group and the 16-week infection group were significantly higher than those of the 4-week infection group,and the chemotherapy drug withdrawal for the 13-weekgroup(P <0.0001);the chemotherapy for the 12-week group was significantly higher than those of the 4-week infection group and the chemotherapy drug withdrawal for the 13-week group(P < 0.0001);the 4-week infection group was significantly higher than that of the chemotherapy drug withdrawal for the 13-week group(P < 0.05).The spleen weight index of the 29-week infection group and the 16–week infection group were significantly higher than those of the 4-week infection group,chemotherapy for the 12-week group,and the chemotherapy withdrawal for the 13-week group(P <0.0001);the 4-week infection group was significantly higher than that of the chemotherapy withdrawal for the 13-week group(P < 0.05).There was no significant difference in the weight index of lung,liver,and spleen among the treatment groups of the mouse LTBI model(P > 0.05).The mouse LTBI model has stopped the treatment with Hormone for 3weeks,a small number of W541 protein-specific IFN-γ-secreting T lymphocytes could be observedin the normal control group,saline group,p VAX-1 vector group,and Vaccae vaccine group.The moderate number of W541 protein-specific IFN-γ-secreting T lymphocytes could be observed in the ag85 ab plasmid DNA group and W541 plasmid DNA group.The number of T lymphocytes secreting IFN-γin ag85 ab plasmid DNA group and W541 plasmid DNA group increased significantly compared with the normal control group,saline group,p VAX-1 vector group,and Vaccae vaccine group(P<0.0001 or P<0.01);that in ag85 ab DNA group was significantly higher than that in W541 DNA group(P<0.05);those in the saline group,p VAX-1 vector group,and the Vaccae vaccine group were significantly higher than that in the normal group(P<0.0001 or P<0.05);that in the saline group was significantly higher than that in p VAX-1 vector group(P<0.01).The percentage of Fox P3Treg(CD4+CD25+FOXP3+Treg)in mouse splenocytes in each treatment group was significantly lower than that in the normal group(P<0.0001),that in Vaccae vaccine group,ag85 ab DNA group and W541 DNA group were significantly higher than those in the saline group and p VAX-1 vector group(P<0.0001 or P<0.01 or P<0.05).The IFN-γ levels in the culture supernatant of mouse spleen lymphocytes in the ag85 ab DNA group and W541 DNA group were significantly higher than those in the normal control group,saline group,p VAX-1 vector group,and Vaccae vaccine group(P<0.0001 or P<0.01 or P<0.05).The IL-2 level in the culture supernatant of spleen lymphocytes in the ag85 ab DNA group was significantly higher than those in the p VAX-1 vector group,Vaccae vaccine group,and the normal control group(P < 0.0001 or P < 0.01 or P <0.05),and that in W541 DNA group and the saline group were also significantly higher than that in the normal control group(P < 0.0001 or P< 0.05).Compared with the saline group and p VAX-1 vector group,the IL-4 levels in the culture supernatant of spleen lymphocytes in the three vaccine groups decreased slightly,but there was no statistically significant difference among them.Compared with the p VAX-1 vector group and normal control group,the IL-6 levels in the culture supernatant of splenic lymphocytes in the three vaccine groups were slightly higher,but there was no also statistically significant difference among them(P>0.05).The number of lung viable bacteria in the 4-week MTB infection group was 7.78Log10,indicating that the mouse MTB infection model was successfully established.The number of pulmonary viable bacteria in the12-week chemotherapy group decreased to 0 CFU,indicating that chemotherapy was effective,and inhibited the active proliferation of MTB in the mouse lungs.With the prolongation of MTB infection time,the number of lung viable bacteria in the 16-week infection group and 29-week infection group were 6.90Log10 and 7.33Log10,respectively,which decreased slightly,suggesting that BALB/c mice had a certain natural resistance to MTB.After themouse LTBI modelwas treated with hormonefor 3 weeks and stopped hormone injection for 3 weeks,the lung CFUs values of the normal saline group and vector group increased from 0after chemotherapy to 4.07Log10 and 5.20Log10 respectively,suggesting that the latent MTB after chemotherapyhad an endogenous resurgence,which proved that the mouse LTBI activation model was successfully established.The mouse LTBI model was prophylactically treated with DNA vaccine,and then induced latent MTB endogenous activation with hormone,to evaluate the effect of the vaccine on preventing latent MTB activation.Compared with the saline group and p VAX-1 vector group,the lung and liver colony count in the three vaccine treatment groups reduced at different degrees.The number of the liver colonyin the ag85 ab DNA group and W541 DNA group was 0 CFU,suggesting that Vaccae vaccine group,ag85 ab DNA group,and W541 DNA group had certain immunosuppressive and scavenging effects on latent MTB,could reduce the number of viable bacteria in mice and inhibit the latent MTB reburning.Groups of mice lung tissue pathology The pathological results of lung tissue showed that the range of lung lesion range in the W541 DNA group,Vaccae vaccine group,and ag85 ab DNA of mice were less than the saline groupand p VAX-1vector group.In conclusion,MTB epitope-tandem protein W541 has multiple potential B-cell and T cell epitopes,of which T cell epitope is dominant,with good immunogenicity and very low homology with humans.W541 DNA vaccine can significantly enhance the specific cellular immune function of mice,mainly stimulate Th1-type immune response,preventive treatment of mouse LTBI model can reduce the number of viable bacteria in lung and liver tissue,significantly reduce the lesion area of lung tissue,and alleviate the pathological changes.Its therapeutic effect is similar to that of the existing commercial LTBI prophylactic preparation,Vaccae vaccine,whichprovides a good foundation for the development of new LTBI vaccines. |
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