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

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...

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Autores principales: Moraes, Luana, Trentini, Monalisa Martins, Fousteris, Dimitrios, Eto, Silas Fernandes, Chudzinski-Tavassi, Ana Marisa, Leite, Luciana Cezar de Cerqueira, Kanno, Alex Issamu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9005855/
https://www.ncbi.nlm.nih.gov/pubmed/35432328
http://dx.doi.org/10.3389/fimmu.2022.867195
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author Moraes, Luana
Trentini, Monalisa Martins
Fousteris, Dimitrios
Eto, Silas Fernandes
Chudzinski-Tavassi, Ana Marisa
Leite, Luciana Cezar de Cerqueira
Kanno, Alex Issamu
author_facet Moraes, Luana
Trentini, Monalisa Martins
Fousteris, Dimitrios
Eto, Silas Fernandes
Chudzinski-Tavassi, Ana Marisa
Leite, Luciana Cezar de Cerqueira
Kanno, Alex Issamu
author_sort Moraes, Luana
collection PubMed
description 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.
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spelling pubmed-90058552022-04-14 CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate Moraes, Luana Trentini, Monalisa Martins Fousteris, Dimitrios Eto, Silas Fernandes Chudzinski-Tavassi, Ana Marisa Leite, Luciana Cezar de Cerqueira Kanno, Alex Issamu Front Immunol Immunology 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. Frontiers Media S.A. 2022-03-30 /pmc/articles/PMC9005855/ /pubmed/35432328 http://dx.doi.org/10.3389/fimmu.2022.867195 Text en Copyright © 2022 Moraes, Trentini, Fousteris, Eto, Chudzinski-Tavassi, Leite and Kanno https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Moraes, Luana
Trentini, Monalisa Martins
Fousteris, Dimitrios
Eto, Silas Fernandes
Chudzinski-Tavassi, Ana Marisa
Leite, Luciana Cezar de Cerqueira
Kanno, Alex Issamu
CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate
title CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate
title_full CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate
title_fullStr CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate
title_full_unstemmed CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate
title_short CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate
title_sort crispr/cas9 approach to generate an auxotrophic bcg strain for unmarked expression of ltak63 adjuvant: a tuberculosis vaccine candidate
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9005855/
https://www.ncbi.nlm.nih.gov/pubmed/35432328
http://dx.doi.org/10.3389/fimmu.2022.867195
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