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

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN IMMUNOLOGY; v. 13, p. 10-pg., 2022-03-30.

Resumo

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)

• Processo FAPESP: 17/24832-6 - Desenvolvimento de vacinas baseadas em BCG recombinante: Tuberculose, Pertussis, Pneumococo e Schistosoma
• Beneficiário: Luciana Cezar de Cerqueira Leite
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 17/17218-0 - Implantação da plataforma de CRISPR-Cas9 em micobactéria: investigação do sistema ESX-1 na imunogenicidade de BCG
• Beneficiário: Luana Moraes
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 19/06454-0 - Avaliação de BCG expressando o adjuvante LTAK63 em um modelo de camundongo humanizado como vacina terapêutica para Tuberculose
• Beneficiário: Monalisa Martins Trentini
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado