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.

Lactococcus lactis expressing sand fly PpSP15 salivary protein confers long-term protection against Leishmania major in BALB/c mice

“…Although there was no significant difference between G1 (L. lactis-PpSP15-EGFP cwa ) and G2 (L. lactis-EGFP cwa ) in both ST and LT experiments, the mean±SD in G1 was lower than in G2. However, immunogenicity of EGFP is a controversial subject [50][51][52]. However, foodpad swelling in mice by itself is not a valid indicator of the potential efficacy of vaccination or the infectivity rate [53].…”

The outcome of arginase activity inhibition in BALB/c mice hosting Leishmania tropica

“…The regional draining popliteal lymph nodes (dLNs) were dissected from each individual mice (n = 5) and homogenized for parasite burden calculation, as previously described. 15 Briefly, the homogenates at each dLNs were serially diluted 5-fold (ranging from ~1 1 to ~10 15 ) and dispensed in 96-well micro-titration flat-bottom plates in duplicate. Plates were examined by inverted microscope, and the last dilution with at least one viable promastigote was selected as the end titre.…”

“…At different intervals (1 and 3 mo after the Ltrop inoculation, and 1 and 2 mo after Lmj inoculation), popliteal dLNs of mice (5 mice per group) were extracted and homogenized to be used for parasite burden quantification. The homogenized dLNs were serially diluted 5-fold (ranging from ~1 1 to ~1015 ) and dispensed into 96-well plates in duplicate (A and C). All wells were examined by inverted microscope, and the last dilution with viable promastigote was selected as the final titre for calculation of parasite burden.…”

The mKate fluorescent protein expressed by Leishmania mexicana modifies the parasite immunopathogenicity in BALB/c mice

“…Other reports have identified immunodominant epitopes of EGFP and DsRed proteins, which induce a CTL response in BALB/c and C57BL/6 mice . A further study by Seif et al . showed that despite a lack of evidence of EGFP immunogenicity in Leishmania major infected BALB/c mice, they found enhanced lesion sizes and increased parasite burdens .…”

“…A further study by Seif et al . showed that despite a lack of evidence of EGFP immunogenicity in Leishmania major infected BALB/c mice, they found enhanced lesion sizes and increased parasite burdens . The aforementioned experimental models using reporter proteins call for an analysis of the possible immunogenicity of fluorescent proteins when used in transgenic parasite models for biological studies.…”