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Download article 		Title: Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis
Author: Carvalho, Gabriela Guimarães; Calarga, Aline Parolin; Zorgi, Nahiara Esteves; Astudillo-trujillo, Carlos A.; Gontijo, Marco Túlio Pardini; Brocchi, Marcelo; Giorgio, Selma; Kabuki, Dirce Yorika
Year: 2022
Is part of: INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, v. 376, p. 109745 -
DOI: https://doi.org/10.1016/j.ijfoodmicro.2022.109745

Citation: Carvalho, Gabriela Guimarães; Calarga, Aline Parolin; Zorgi, Nahiara Esteves; Astudillo-trujillo, Carlos A.; Gontijo, Marco Túlio Pardini; Brocchi, Marcelo; Giorgio, Selma; Kabuki, Dirce Yorika; Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis. INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, v.376, p. 109745-, 2022

Abstract: Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3' UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 x 10(6) L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4(+), TCD8(+) and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-gamma, TNF-alpha and IL-6. In parallel groups of mice, a challenge consisting on 1 x 10(6) virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.



Funding: This work was supported by The Brazilian National Council for Research and Development (CNPq) - Grants No. 405018/2013-4 and202471/2011-0. Sao Paulo State Research Foundation (FAPESP) Grants No. 2015/23767-0;2016/11539-6;2018/23302-6; and2019/06322-6. 		Financed by: