Engineering a new vaccine platform for heterologous antigen delivery in live-attenuated Mycobacterium tuberculosis

Broset, Esther ; Calvet Seral, Juan (Universidad de Zaragoza) ; Arnal, Carmen ; Uranga, Santiago (Universidad de Zaragoza) ; Kanno, Alex I. ; Leite, Luciana C.C. ; Martín, Carlos (Universidad de Zaragoza) ; Gonzalo-Asensio, Jesús (Universidad de Zaragoza)

Resumen: Live 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.

Idioma: Inglés

DOI: 10.1016/j.csbj.2021.07.035

Año: 2021

Publicado en: Computational and Structural Biotechnology Journal 19 (2021), 4273-4283
ISSN: 2001-0370

Factor impacto JCR: 6.155 (2021)

Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 70 / 297 = 0.236 (2021) - Q1 - T1

Factor impacto CITESCORE: 6.0 - Computer Science (Q1) - Biochemistry, Genetics and Molecular Biology (Q2)

Factor impacto SCIMAGO: 1.538 - Biochemistry (Q1) - Biophysics (Q1) - Structural Biology (Q1) - Genetics (Q1) - Computer Science Applications (Q1)

Financiación: info:eu-repo/grantAgreement/ES/MCIU/PID2019-104690RB-I00
Financiación: info:eu-repo/grantAgreement/ES/MCIU/RTI2018- 097625-B-I00
Financiación: info:eu-repo/grantAgreement/ES/MINECO/BES-2012-052937
Financiación: info:eu-repo/grantAgreement/ES/MINECO/BFU2015-72190-EXP

Tipo y forma: Artículo (Versión definitiva)

Área (Departamento): Área Microbiología (Dpto. Microb.Ped.Radio.Sal.Pú.)
Área (Departamento): Proy. investigación HQA (Dpto. Microb.Ped.Radio.Sal.Pú.)