Full text
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| Author(s): |
Luana Moraes
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2024-08-30 |
| Examining board members: |
Luciana Cezar de Cerqueira Leite;
Andrea Borrego;
Simone Guedes Calderano;
Luis Carlos de Souza Ferreira;
Andre Kipnis;
Luiz Cosme Cotta Malaquias
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| Advisor: | Luciana Cezar de Cerqueira Leite; Alex Issamu Kanno |
| Abstract | |
Tuberculosis (TB) is a serious global public health issue and has a significant impact on the livestock economy, particularly in bovine tuberculosis (bTB). In humans, the primary causative agent is Mycobacterium tuberculosis (Mtb), while in cattle, it is M. bovis (Mb). The only currently licensed vaccine against TB is BCG, a live-attenuated vaccine derived from Mb, effective in protecting against severe forms of TB in children and widely used worldwide. However, BCG\'s protection against pulmonar tuberculosis in adults is highly variable, whilst it is not applicable in cattle as it interferes with diagnostic tests required for bTB control. CRISPR/Cas9 is a powerful gene editing tool successfully applied in many organisms and could potentially be used to improve the efficacy of BCG (recombinant BCG- rBCG). We proposed establishing CRISPR/Cas9 in BCG to develop an improved TB vaccine and, compatible with diagnostic tests for use in cattle. To achieve this, mycobacterial vectors containing the CRISPR/Cas9 system were generated. When used for lysA gene deletion, mutations were observed in both M. smegmatis (25%) and BCG (5%). The mutant BCG strain (ΔlysA) was used to express the adjuvant LTAK63 (genetically detoxified subunit A of E. coli thermolabile toxin) thus generating rBCGΔ-LTAK63. Mice immunized with rBCGΔ-LTAK63 showed increased protection against Mtb challenge and improved resolution of the inflammatory process. Application of CRISPR/Cas9 to nocaute mpb70, mpb83, espA, espC, and esxS genes in BCG to enable its compatibility with a diagnostic test to differentiate infected from vaccinated animals (DIVA), successful mutations were achieved in four of the five planned genes: mpb70 (75%), mpb83 (20%), espC (4.3%), and esxS (15.7%). This study demonstrates the establishment and effective application of CRISPR/Cas9 in developing an improved vaccine against tuberculosis, compatible with the diagnostic test in bovines. A single vaccine to control both human TB and bTB would greatly benefit public health as well as the economy. (AU) | |
| FAPESP's process: | 17/17218-0 - Implantation of the CRISPR-Cas9 platform in mycobacteria: investigation of ESX-1 system in the immunogenicity of BCG |
| Grantee: | Luana Moraes |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |