CRISPR/Cas9 approach to generate an auxotrophic BCG strain for unmarked expression of LTAK63 adjuvant: a tuberculosis vaccine candidate
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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 (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.
Moraes L, Trentini MM, Fousteris D, Eto SF, Chudzinski-Tavassi AM, Leite LCC, et al. CRISPR/Cas9 approach to generate an auxotrophic BCG strain for unmarked expression of LTAK63 adjuvant: a tuberculosis vaccine candidate. Front. Immunol. 2022 Mar;13:867195. doi:10.3389/fimmu.2022.867195.
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