Implantation of the CRISPR-Cas9 platform in mycobacteria: investigation of ESX-1 system in the immunogenicity of BCG

• Grant number: 17/17218-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: January 01, 2018
• End date: November 30, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Luciana Cezar de Cerqueira Leite
• Grantee: Luana Moraes
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated scholarship(s): 21/13879-7 - Multiplex CRISPR/Cas9 gene editing - generation of unmarked knockout BCG compatible with DIVA skin test, BE.EP.DD

Abstract

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis that persists as one of the major public health problems. The only vaccine to date against tuberculosis is Calmette-Guérrin Bacillus (BCG), a live attenuated vaccine of the pathogenic strain Mycobacterium bovis. Molecular analyzes demonstrate a deletion of large non-genomic fragments of BCG strains and, in particular, a deletion of a 9.5 kb fragment called RD1, present in the pathogenic strain and absent in all BCG subcepts as the main cause in the attenuation of virulence. RD1 contains part of ESX-1, a system for exporting virulence factors related to pathogenicity, virulence and immunogenicity. Individually, several antigens contained in RD1 are immunogenic and, despite demonstrating vaccine potential, restoration of the export system through reintroduction of whole RD1 into BCG demonstrates more promising results. We hypothesize that it is a reconstruction of RD1 in its genomic loci. Possibility of restoration of the antigen export system, raise immunogenicity and, potentially, induce better protection against M. tuberculosis. In this project, we suggest a reintroduction of RD1 into its genomic loci through the new CRISPR-Cas9 genetic editing technique. There are some studies of the use of technicians in mycobacteria, but only for gene silencing. It is necessary to establish this methodology; therefore, we propose using the GFP reporter system as a model. A construction of mycobacterial plasmids for an expression of Cas9 and sgRNA is carried out aiming at a construction and subsequent genetic characterization of the mutant strains for the knock-in of RD1 in BCG. A functional characterization of the ESX-1 system in BCG in vitro and immunological characterization of rBCG::RD1 in animal models will be performed. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

KANNO, I, ALEX; BARBOSA, MAYRA M. F.; MORAES, LUANA; LEITE, LUCIANA C. C.. SARS-CoV-2 vaccine development and how Brazil is contributing. GENETICS AND MOLECULAR BIOLOGY, v. 44, n. 1, 1, 2021. (17/17218-0, 17/24832-6, 20/07547-9)

ALEX I. KANNO; MAYRA M.F. BARBOSA; LUANA MORAES; LUCIANA C.C. LEITE. SARS-CoV-2 vaccine development and how Brazil is contributing. GENETICS AND MOLECULAR BIOLOGY, v. 44, n. 1, 2021-04-02. (17/17218-0, 20/07547-9, 17/24832-6)

MORAES, LUANA; TRENTINI, MONALISA MARTINS; FOUSTERIS, DIMITRIOS; ETO, SILAS FERNANDES; CHUDZINSKI-TAVASSI, ANA MARISA; DE CERQUEIRA LEITE, LUCIANA CEZAR; KANNO, ALEX ISSAMU. CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate. FRONTIERS IN IMMUNOLOGY, v. 13, p. 10-pg., 2022-03-30. (17/24832-6, 17/17218-0, 19/06454-0)