Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania

Moreira, Paulo Otávio Lourenço; Nogueira, Paula Monalisa; Monte-Neto, Rubens L.

doi:10.3390/microorganisms11041043

Cited by 6 publications

(6 citation statements)

References 163 publications

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“…Live parasites have the advantage of the better efficacy to induce long-term protective immune responses as compared to killed parasite vaccines; however, the obstacle to using a live vaccine is the potential risk of disease development which might be overcome by engineering a second generation live-attenuated parasite which can confer protection without associated pathology (25) (26). Therefore, it is believed that generation of attenuated parasites through targeted disruption of genes coding for virulence or optimal growth factors is effective approaches for vaccine development against leishmaniasis (27).…”

Section: Discussionmentioning

confidence: 99%

“…So far, several genes of the Leishmania parasite such as centrin, gp63, CPs, P27 and other genes have been used as possible target for production of attenuated strains (7,8). The discovery of CRISPR/Cas based gene editing allowed genetic manipulation of parasites and/or vectors for disease control and selection of safer mutant live-attenuated Leishmania parasites to investigate as vaccine candidate (9,10).…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence inLeishmania majorparasites

Darzi,

Khamesipour,

Bahrami

et al. 2024

Preprint

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The CRISPR/Cas9 system has emerged as a powerful tool for precise genome editing, allowing for the deletion of genes, generation of point mutations, and addition of tags to endogenous genes. We employed an efficient CRISPR/Cas9 technique in Leishmania major to assess its efficiency in editing a kDNA-associated gene, universal minicircle sequence binding protein (UMSBP), which is involved in mitochondrial respiration and kinetoplast division. We generated UMSBP C-tagged and UMSBP single knockout L. major (LmUMSBP+/−) parasites using the CRISPR/Cas9 toolkit. C-tagged parasite were confirmed by PCR, flow cytometry and Western blot analyses. Gene expression of mitochondrial redox regulating enzymes, tryparedoxin peroxidase (TXNPx) and trypanothione synthetase (TryS), were analysed by real-time RT-PCR. Growth rate of promastigotes in culture and infectivity rate in macrophages were analysed in vitro. Mice were immunized by LmUMSBP+/− mutant strain and lesion size and parasite burden were measured upon challenge with live wild type (WT) L. major. Cytokines were titrated on supernatant of lymph nodes cell culture by sandwich ELISA. Complete UMSBP deletion (LmUMSBP-/- null mutant) impaired promastigote survival, suggesting its essential role in parasite fitness. Despite this, we were able to produce attenuated LmUMSBP+/- parasites, which showed significant reduced growth in culture (P<0.05), increase in apoptosis (P<0.05) and downregulation of TXNPx and TryS gene expressions during growth of promastigotes compared to WT L. major. LmUMSBP+/- mutant strains did not cause ulcerative lesions in susceptible BALB/c mouse model. Furthermore, immunization of mice with LmUMSBP+/- parasites elicited a Th1 immune response with significantly high IFN-γ and low IL4 production in cell culture (P<0.001) associated with partial protection against L. major WT challenge, as evidenced by reduced parasite burden and lesion development in BALB/c mice. Our findings demonstrate the potential of CRISPR/Cas9-edited LmUMSBP+/- parasites as live attenuated vaccine candidate against Leishmania infection.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence inLeishmania majorparasites

Darzi,

Khamesipour,

Bahrami

et al. 2024

Preprint

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“…CRISPR/Cas9-mediated gene targeting and editing facilitates deletion of essential genes (either single or multigene families) to observe the functional and phenotypic effects on living cells in a time-dependent manner ( Zhang and Matlashewski, 2015 ; Adaui et al, 2020 ). The fast development of this technology will certainly accelerate the production of knockout mutants in Leishmania instead of homologous recombination-based gene replacement making more live attenuated parasites available in near future ( Singh et al, 2022 ; Moreira et al, 2023 ).…”

Section: Live Attenuated Parasite Vaccines Most Likely Can Replace Le...mentioning

confidence: 99%

Live attenuated-nonpathogenic Leishmania and DNA structures as promising vaccine platforms against leishmaniasis: innovations can make waves

Seyed,

Taheri,

Rafati

2024

Front. Microbiol.

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Leishmaniasis is a vector-borne disease caused by the protozoan parasite of Leishmania genus and is a complex disease affecting mostly tropical regions of the world. Unfortunately, despite the extensive effort made, there is no vaccine available for human use. Undoubtedly, a comprehensive understanding of the host-vector-parasite interaction is substantial for developing an effective prophylactic vaccine. Recently the role of sandfly saliva on disease progression has been uncovered which can make a substantial contribution in vaccine design. In this review we try to focus on the strategies that most probably meet the prerequisites of vaccine development (based on the current understandings) including live attenuated/non-pathogenic and subunit DNA vaccines. Innovative approaches such as reverse genetics, CRISP/R-Cas9 and antibiotic-free selection are now available to promisingly compensate for intrinsic drawbacks associated with these platforms. Our main goal is to call more attention toward the prerequisites of effective vaccine development while controlling the disease outspread is a substantial need.

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“…On the other hand, disease development is mainly driven by the production of non-protective Th2-associated cytokines IL-4, IL-10, IL-13, and TGF-β [ 5 , 6 ]. Durable protective immunity acquired following leishmanization led to the development of several live attenuated Leishmania strains as prophylactic vaccines [ 7 ]. The possibility of using genetically modified Leishmania spp.…”

Section: Introductionmentioning

confidence: 99%

Immunization with centrin-Deficient Leishmania braziliensis Does Not Protect against Homologous Challenge

Avendaño-Rangel,

Agra-Duarte,

Borba

et al. 2024

Vaccines

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Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin-deficient Leishmania braziliensis (LbCen−/−) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen−/− failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen−/− was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major, L. donovani, and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin-deficient L. donovani (LdonCen−/−) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen−/− to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.

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Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania

Cited by 6 publications

References 163 publications

CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence inLeishmania majorparasites

CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence inLeishmania majorparasites

Live attenuated-nonpathogenic Leishmania and DNA structures as promising vaccine platforms against leishmaniasis: innovations can make waves

Immunization with centrin-Deficient Leishmania braziliensis Does Not Protect against Homologous Challenge

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