title: EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice.
abstract: Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major (EGFP) or L. major (EGFP-LUC)) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wild-types that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study.
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.|Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
authors.forename: Samira
authors.initials: S
authors.lastname: Seif
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.|Department of Genetics Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Fereshteh
authors.initials: F
authors.lastname: Kazemi
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Elham
authors.initials: E
authors.lastname: Gholami
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Negar
authors.initials: N
authors.lastname: Seyed
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Yasaman
authors.initials: Y
authors.lastname: Taslimi
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Sima
authors.initials: S
authors.lastname: Habibzadeh
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Bahareh
authors.initials: B
authors.lastname: Azarian
authors.affiliation: Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
authors.forename: Shahram
authors.initials: S
authors.lastname: Jamshidi
authors.affiliation: Department of Genetics Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Hashemi
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Sima
authors.initials: S
authors.lastname: Rafati
authors.affiliation: Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran. Tahereh_t@yahoo.com.
authors.forename: Tahereh
authors.initials: T
authors.lastname: Taheri
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology*; D018320:Leishmania major / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000276:immunology* / Q000473:pathology; D008156:Luciferases / Q000235:genetics / Q000276:immunology; D008807:Mice, Inbred BALB C; D059208:Parasite Load; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases
keywords: EGFP; In vivo infectivity; Leishmania major; Luciferase; Reporter gene