000108527 001__ 108527
000108527 005__ 20230519145454.0
000108527 0247_ $$2doi$$a10.1016/j.csbj.2021.07.035
000108527 0248_ $$2sideral$$a125120
000108527 037__ $$aART-2021-125120
000108527 041__ $$aeng
000108527 100__ $$0(orcid)0000-0003-4155-749X$$aBroset, Esther
000108527 245__ $$aEngineering a new vaccine platform for heterologous antigen delivery in live-attenuated Mycobacterium tuberculosis
000108527 260__ $$c2021
000108527 5060_ $$aAccess copy available to the general public$$fUnrestricted
000108527 5203_ $$aLive vaccines are attractive vehicles for antigen delivery as a strategy to immunize against heterologous pathogens. The live vaccine MTBVAC is based on rational attenuation of Mycobacterium tuberculosis with the objective of improving BCG protection against pulmonary tuberculosis. However, the development of recombinant mycobacteria as antigen-presenting microorganisms has been hindered due to their fastidious genetic manipulation. In this study, we used MTBVAC as a genetic platform to deliver diphtheria, tetanus, or pertussis toxoids, which are the immunogenic constituents of the DTP vaccine. When using nonoptimal genetic conditions, the expression of these immunogens was barely detectable. Accordingly, we pursued a rational, step-by-step optimization of the genetic components to achieve the expression and secretion of these toxoids. We explored variants of the L5 mycobacteriophage promoter to ensure balanced antigen expression and plasmid stability. Optimal signal sequences were identified by comparative proteomics of MTBVAC and its parental strain. It was determined that proteins secreted by the Twin Arginine Translocation pathway displayed higher secretion in MTBVAC, and the Ag85A secretion sequence was selected as the best candidate. Because the coding regions of diphtheria, tetanus, and pertussis toxoids significantly differ in G + C content relative to mycobacterial genes, their codon usage was optimized. We also placed a 3xFLAG epitope in frame with the C-terminus of these toxoids to facilitate protein detection. Altogether, these optimizations resulted in the secretion of DTP antigens by MTBVAC, as demonstrated by western blot and MRM-MS. Finally, we examined specific antibody responses in mice vaccinated with recombinant MTBVAC expressing DTP antigens.
000108527 536__ $$9info:eu-repo/grantAgreement/ES/MCIU/PID2019-104690RB-I00$$9info:eu-repo/grantAgreement/ES/MCIU/RTI2018- 097625-B-I00$$9info:eu-repo/grantAgreement/ES/MINECO/BES-2012-052937$$9info:eu-repo/grantAgreement/ES/MINECO/BFU2015-72190-EXP
000108527 540__ $$9info:eu-repo/semantics/openAccess$$aby-nc-nd$$uhttp://creativecommons.org/licenses/by-nc-nd/3.0/es/
000108527 590__ $$a6.155$$b2021
000108527 592__ $$a1.538$$b2021
000108527 594__ $$a6.0$$b2021
000108527 591__ $$aBIOCHEMISTRY & MOLECULAR BIOLOGY$$b70 / 297 = 0.236$$c2021$$dQ1$$eT1
000108527 593__ $$aBiochemistry$$c2021$$dQ1
000108527 593__ $$aBiophysics$$c2021$$dQ1
000108527 593__ $$aStructural Biology$$c2021$$dQ1
000108527 593__ $$aGenetics$$c2021$$dQ1
000108527 593__ $$aComputer Science Applications$$c2021$$dQ1
000108527 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000108527 700__ $$0(orcid)0000-0002-3428-2267$$aCalvet Seral, Juan$$uUniversidad de Zaragoza
000108527 700__ $$0(orcid)0000-0002-5152-1071$$aArnal, Carmen
000108527 700__ $$0(orcid)0000-0001-7866-2803$$aUranga, Santiago$$uUniversidad de Zaragoza
000108527 700__ $$aKanno, Alex I.
000108527 700__ $$aLeite, Luciana C.C.
000108527 700__ $$0(orcid)0000-0003-2993-5478$$aMartín, Carlos$$uUniversidad de Zaragoza
000108527 700__ $$0(orcid)0000-0001-8841-6593$$aGonzalo-Asensio, Jesús$$uUniversidad de Zaragoza
000108527 7102_ $$11011$$2630$$aUniversidad de Zaragoza$$bDpto. Microb.Ped.Radio.Sal.Pú.$$cÁrea Microbiología
000108527 7102_ $$11011$$2X$$aUniversidad de Zaragoza$$bDpto. Microb.Ped.Radio.Sal.Pú.$$cProy. investigación HQA
000108527 773__ $$g19 (2021), 4273-4283$$pComput. struct. biotechnol. j.$$tComputational and Structural Biotechnology Journal$$x2001-0370
000108527 8564_ $$s1918679$$uhttps://zaguan.unizar.es/record/108527/files/texto_completo.pdf$$yVersión publicada
000108527 8564_ $$s2867462$$uhttps://zaguan.unizar.es/record/108527/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000108527 909CO $$ooai:zaguan.unizar.es:108527$$particulos$$pdriver
000108527 951__ $$a2023-05-18-14:48:46
000108527 980__ $$aARTICLE