Function And Immunogenicity Analysis Of Mycobacterium Tuberculosis Main Secretory Proteins

Tuberculosis?TB?is a highly contagious disease of human beings and bovines caused by Mycobacterium tuberculosis?Mtb?and Mycobacterium bovis?M.bovis?,respectively.Both species share almost 99%genomic identity and can infect each other's host with less severity of disease than infecting their own host.This observation leads to the development of Bacillus Calmette–Guérin?BCG?vaccine?a modified form of M.bovis?for human TB.Currently,BCG is the only available vaccine for TB,offers no booster and efficacy in adults.The promising adjunct or BCG complement remains major challenge to overcome TB menace.Considering the fact,a subunit vaccine was designed from the proteins of three secretory systems in Mtb:twin arginine translocation?TAT?,Esx and Sec by using immunoinformatics approach.Immunogenic epitopes of the secretory proteins were predicted and used for vaccine scheming.Vaccine was then evaluated in silico by applying various online tools.These tools declared the vaccine stable,non-allergic,antigenic?immunogenic?with binding affinity for toll like receptor 2?TLR2?.For the intracellular expression of vaccine protein,amino acid sequence was reverse translated to nucleotide sequence and cloned into adeno associated virus vector?p AAV?.The virus vectors without and with vaccine protein were packaged into AAV-Dj/8 bicistronic expression system.Both viruses were in vitro characterized for DNA and RNA copy number estimation by q PCR and q RT-PCR respectively.The copy numbers for both viruses were in acceptable range i.e.10⁹/m L-10¹¹/m L.Both viruses were made ready to use for pre-clinical studies.For further characterization of subunit vaccine,the function analysis of 18 proteins whose epitopes participated in the subunit vaccine design were predicted for host DNA binding.6 showed positive prediction and 2 of them:Esx T?Rv3444c?and Esx U?Rv3445c?were selected for analysis.Both belongs to the Esx-4 cluster?functionally unknown?and present throughout mycobacteriaceae family.Both proteins were nuclear localized,alone and together,detected by immunofluorescent assay.Similarly,both were physically interacting identified by co-immunoprecipitation.For determining interaction of both proteins with host genome,lentivirus induced THP1 stable cell lines,THP1-OE?expressing both genes?and THP1-GFP?control?were generated and used for histone modification,RNA sequencing,cytokines analyses and bacilli viability assay.Tri-methylation and acetylation at lysine 27 position of histone H3 were performed.Tri-methylation showed no significant feature,whereas,acetylation provided several enriched regions and transcriptional factors?TF?in both cell lines.Only THP1-OE cell line enriched regions were involved in regulating host immunity for the progress of Mtb infection.The RNA-seq data provided differentially expressed genes where majority of upregulated genes were belonged to nucleic acid binding class.RNA-seq data also demonstrated that immunological profile of THP1-OE cell line was suppressed compared to THP1-GFP.This was supported by cytokines analysis data?pro-inflammatory cytokines fold expression reduced?and Mtb viability assay?viability enhanced?in THP1-OE cell line.Functional study of both proteins in Mtb was further executed by generating combined knock down?KD?strain via CRISPRi,technique.RNA-seq was again performed by infecting THP1 cells with WT/KD strains.It revealed that highly down regulated genes were belonged to the nucleic acid binding class.Here,the immunological profile of KD infected THP1 cells was improved compared to WT infected ones.This was confirmed by the cytokines analysis data?pro-inflammatory cytokines fold expression raised?and Mtb viability assay?viability reduced?in KD infected THP1 cells.In vivo?mouse model?study also proved that the pro-inflammatory cytokines level raised?lungs?and Mtb viability decreased?lung and spleen?in KD infected mice compared to WT infected one's.However,histopathological micrographs of lung and spleen samples did not show significant pathological effects in KD infected mice than WT infected ones.Overall this study has comprehensively described the subunit vaccine design for Mtb using immunoinformatics approach and functionally analyzed two vaccine participatory proteins.Both proteins in combination have strong interaction with host genome in regulating host immune status and Mtb survival.