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

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

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.867195

DOI=10.3389/fimmu.2022.867195

ISSN=1664-3224

ABSTRACT=<p>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 <italic>Escherichia coli</italic>, induces improved protection against <italic>Mycobacterium tuberculosis</italic> (<italic>Mtb</italic>) 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 <italic>lysA</italic> gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying <italic>lysA</italic> and <italic>ltak63</italic> 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 <italic>Mtb</italic> 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.</p>