CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate

Author(s)

Moraes, Luana ; Trentini, Monalisa Martins ; Fousteris, Dimitrios ; Eto, Silas Fernandes ; Chudzinski-Tavassi, Ana Marisa ; de Cerqueira Leite, Luciana Cezar ; Kanno, Alex Issamu

• Document type: Journal article
• Source: FRONTIERS IN IMMUNOLOGY; v. 13, p. 10-pg., 2022-03-30.

Abstract

Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCG Delta-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCG Delta-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCG Delta-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field. (AU)

• FAPESP's process: 17/24832-6 - Development of vaccines based on recombinant BCG: Tuberculosis, Pertussis, Pneumococcus and Schistosoma
• Grantee: Luciana Cezar de Cerqueira Leite
• Support Opportunities: Research Projects - Thematic Grants
• 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)
• FAPESP's process: 19/06454-0 - Evaluation of BCG expressing adjuvant LTAK63 in a humanized mouse model as a therapeutic vaccine for Tuberculosis
• Grantee: Monalisa Martins Trentini
• Support Opportunities: Scholarships in Brazil - Post-Doctoral