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 I.

doi:10.3389/fimmu.2022.867195

Cited by 7 publications

(3 citation statements)

References 28 publications

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“…Image analysis software (ImageJ, National Institutes of Health, USA) was used to determine the pulmonary area affected. Briefly, five images at 40 x magnification per lung lobule, totaling 25 images per group, were randomly selected, and analyzed for the qualitative evaluation of the cell infiltrate and intra-alveolar regions ( 17 – 19 ). To measure the areas of interest, the images were transformed into 8-bit and treated with threshold and percentage of the measured area.…”

Section: Methodsmentioning

confidence: 99%

Recombinant BCG expressing the LTAK63 adjuvant improves a short-term chemotherapy schedule in the control of tuberculosis in mice

Trentini

Kanno²,

Rodríguez³

et al. 2022

Front. Immunol.

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Tuberculosis (TB) is one of the deadliest infectious diseases around the world. Prevention is based on the prophylactic use of BCG vaccine, effective in infants but as protection wanes with time, adults are less protected. Additionally, chemotherapy requires the use of many antibiotics for several months to be effective. Immunotherapeutic approaches can activate the immune system, intending to assist chemotherapy of TB patients, improving its effectiveness, and reducing treatment time. In this work, the recombinant BCG expressing LTAK63 (rBCG-LTAK63) was evaluated for its immunotherapeutic potential against TB. Bacillary load, immune response, and lung inflammation were evaluated in mice infected with Mycobacterium tuberculosis (Mtb) and treated either with BCG or rBCG-LTAK63 using different routes of administration. Mice infected with Mtb and treated intranasally or intravenously with rBCG-LTAK63 showed a reduced bacillary load and lung inflammatory area when compared to the group treated with BCG. In the spleen, rBCG-LTAK63 administered intravenously induced a higher inflammatory response of CD4+ T cells. On the other hand, in the lungs there was an increased presence of CD4+IL-10+ and regulatory T cells. When combined with a short-term chemotherapy regimen, rBCG-LTAK63 administered subcutaneously or intravenously decreases the Mtb bacillary load, increases the anti-inflammatory response, and reduces tissue inflammation. These findings highlight the potential of rBCG-LTAK63 in assisting chemotherapy against Mtb.

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Section: Methodsmentioning

confidence: 99%

Recombinant BCG expressing the LTAK63 adjuvant improves a short-term chemotherapy schedule in the control of tuberculosis in mice

Trentini

Kanno²,

Rodríguez³

et al. 2022

Front. Immunol.

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“…A recombinant BCG expressing a genetically detoxified subunit A of heat-labile toxin from Escherichia coli , proved to induce improved protection against M. tuberculosis in mice, including a hypervirulent Beijing strain [ 91 ]. CRISPR/Cas9 has also been used to remove the antibiotic resistance marker for recombinant construction, so the unmarked auxotrophic BCG strain could be used as a vaccine in humans [ 92 ].…”

Section: Crispr In the Study Of The Immune Response Against ...mentioning

confidence: 99%

The future of CRISPR in Mycobacterium tuberculosis infection

et al. 2023

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Clustered Regularly Interspaced Short Palindromic repeats (CRISPR)-Cas systems rapidly raised from a bacterial genetic curiosity to the most popular tool for genetic modifications which revolutionized the study of microbial physiology. Due to the highly conserved nature of the CRISPR locus in Mycobacterium tuberculosis, the etiological agent of one of the deadliest infectious diseases globally, initially, little attention was paid to its CRISPR locus, other than as a phylogenetic marker. Recent research shows that M. tuberculosis has a partially functional Type III CRISPR, which provides a defense mechanism against foreign genetic elements mediated by the ancillary RNAse Csm6. With the advent of CRISPR-Cas based gene edition technologies, our possibilities to explore the biology of M. tuberculosis and its interaction with the host immune system are boosted. CRISPR-based diagnostic methods can lower the detection threshold to femtomolar levels, which could contribute to the diagnosis of the still elusive paucibacillary and extrapulmonary tuberculosis cases. In addition, one-pot and point-of-care tests are under development, and future challenges are discussed. We present in this literature review the potential and actual impact of CRISPR-Cas research on human tuberculosis understanding and management. Altogether, the CRISPR-revolution will revitalize the fight against tuberculosis with more research and technological developments.

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“…In order to move forward towards clinical applications, we have recently constructed an unmarked strain of rBCG expressing LTAK63 through auxotrophic complementation. The BCG auxotroph, obtained by the CRISPR/cas9 approach, was transformed with a complementation vector expressing the antigen without an antibiotic resistance marker [15]. This strain will be suitable for clinical studies.…”

Section: Introductionmentioning

confidence: 99%

Recombinant BCG-LTAK63 Vaccine Candidate for Tuberculosis Induces an Inflammatory Profile in Human Macrophages

et al. 2022

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Tuberculosis (TB) is one of the top 10 leading causes of death worldwide. The recombinant BCG strain expressing the genetically detoxified A subunit of the thermolabile toxin from Escherichia coli (LTAK63) adjuvant (rBCG-LTAK63) has previously been shown to confer superior protection and immunogenicity compared to BCG in a murine TB infection model. To further investigate the immunological mechanisms induced by rBCG-LTAK63, we evaluated the immune responses induced by rBCG-LTAK63, BCG, and Mycobacterium tuberculosis (Mtb) H37Rv strains in experimental infections of primary human M1 and M2 macrophages at the transcriptomic and cytokine secretion levels. The rBCG-LTAK63-infected M1 macrophages more profoundly upregulated interferon-inducible genes such as IFIT3, OAS3, and antimicrobial gene CXCL9 compared to BCG, and induced higher levels of inflammatory cytokines such as IL-12(p70), TNF-β, and IL-15. The rBCG-LTAK63-infected M2 macrophages more extensively upregulated transcripts of inflammation-related genes, TAP1, GBP1, SLAMF7, TNIP1, and IL6, and induced higher levels of cytokines related to inflammation and tissue repair, MCP-3 and EGF, as compared to BCG. Thus, our data revealed an important signature of immune responses induced in human macrophages by rBCG-LTAK63 associated with increased inflammation, activation, and tissue repair, which may be correlated with a protective immune response against TB.

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

Cited by 7 publications

References 28 publications

Recombinant BCG expressing the LTAK63 adjuvant improves a short-term chemotherapy schedule in the control of tuberculosis in mice

Recombinant BCG expressing the LTAK63 adjuvant improves a short-term chemotherapy schedule in the control of tuberculosis in mice

The future of CRISPR in Mycobacterium tuberculosis infection

Recombinant BCG-LTAK63 Vaccine Candidate for Tuberculosis Induces an Inflammatory Profile in Human Macrophages

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