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EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

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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.

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Acknowledgments

We would like to acknowledge Dr. Behrouz Vaziri (Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran) for providing us with imaging facility and Shahram Alizadeh (Department of Immunotherapy and Vaccine Research in Leishmania, Pasteur Institute of Iran, Tehran, Iran) for his technical assistance.

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    Authors and Affiliations

    1. Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran

      Samira Seif, Fereshteh Kazemi, Elham Gholami, Negar Seyed, Yasaman Taslimi, Sima Habibzadeh, Sima Rafati & Tahereh Taheri

    2. Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

      Samira Seif & Shahram Jamshidi

    3. Department of Genetics Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran

      Fereshteh Kazemi & Mehrdad Hashemi

    4. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

      Bahareh Azarian

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    1. Samira Seif
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    Correspondence to Tahereh Taheri.

    Ethics declarations

    All applicable institutional guidelines for the care and use of animals were followed. All mouse experiments including maintenance, feeding, handling program, and euthanasia were approved by the Institutional Animal Care and Research Advisory Committee of Pasteur Institute of Iran, based on the Specific National Ethical Guidelines for Biomedical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education of Iran. All mice were kept in plastic cages with free access to tap water and standard rodent pellets in an air-conditioned room under a constant 12:12 h light–dark cycle at room temperature. This article does not contain any studies with human participants performed by any of the authors.

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    This project was fulfilled by a grant from Pasteur Institute of Iran (grant number 653).

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    All authors of the manuscript declare that they have no conflict of interests.

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    Samira Seif and Fereshteh Kazemi contributed equally to this work.

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    Seif, S., Kazemi, F., Gholami, E. et al. EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice. Appl Microbiol Biotechnol 100, 3923–3934 (2016). https://doi.org/10.1007/s00253-015-7201-1

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