Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess

Abstract

Background: Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.

Methodology/principal findings: A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.

Conclusions/significance: Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

Fig 1. Reactivity of sera from hamsters immunized with C-Igl and sham-immunized hamsters to C-Igl, rIgl1, and rIgl2 in ELISA (A), Western immunoblot (B) and dot blot analysis (C).

(A) Mean value of ELISA results from serial dilutions of sera from C-Igl-immunized hamsters reacted with C-Igl (●), rIgl-1 (■), or rIgl-2 (▲) and sera from sham-immunized hamsters reacted with C-Igl (▽), rIgl-1 (◇), or rIgl-2 (○). Error bars represent the standard errors of the means calculated from each individuals from three independent experiments.*P < 0.001. (B) Western immunoblot analysis of the reactivity of pooled sera from immunized hamsters to E. histolytica trophozoites. Crude antigen from the HM-1: IMSS strain was subjected to SDS-PAGE under reduced conditions. The strip in lane 1 was stained with CBB. Strips were treated with 1:400 diluted sera from C-Igl-immunized hamsters (lane 2) and sham-immunized hamsters (lane 3). Biostep Prestained Protein Marker (Tanon, China) was used and numbers to the left indicate the molecular masses of the marker. (C) Various concentrations (0.01, 0.1, and 1 μg) of rIgl-1 and rIgl-2 were spotted on nitrocellulose membranes. One strip was stained with Coomassie brilliant blue (CBB). Other strips were treated with different dilutions of pooled sera. HRP-conjugated goat antibody to hamster IgG was used as a secondary antibody.

Fig 2. Effects of sera from hamsters vaccinated with the rIgl-1 (A) and C-Igl (B) on the adherence of CHO cells to E. histolytica. Trophozoites were pretreated with 1:1,000, 1:100, 1:10 diluted pooled sera from immunized hamsters and were then incubated with CHO cells. The number of trophozoites with at least three adherent CHO cells was scored by examining at least 300 trophozoites. Adherence was expressed as the percentage of adherence seen in phosphate-buffered saline -treated controls. Error bars represent the standard deviations of the means calculated from three independent replicates. *P < 0.001; **P < 0.001. (C) Complement-mediated lysis of E. histolytica by the sera from hamsters vaccinated with C-Igl.

Trophozoites were incubated with different dilutions of serum from C-Igl immunized or sham-immunized hamsters and different dilutions of guinea pigs’ complement or heat-inactivated hamster serum (without complement). EGTA, which inhibits the classical complement cascade, served as additional controls for specificity. Cell lysis was measured 60 min after incubation. Heat-inactivated serum was used as a control. Error bars represent the standard deviations of the means calculated from four independent replicates. *P < 0.05; **P < 0.001 (vs. sham-immune control).

Fig 3. One-step RT-PCR amplification of cytokine genes in spleen cells from hamsters immunized with C-Igl (A) and quantitative real-time PCR assays of cytokine response induced by C-Igl in spleen cells from hamsters immunized with C-Igl (B-F).

(A) The RT-PCR products were analyzed by electrophoresis. IL-4, IL-10, IFN-γ, TNF-α, IL-6, and β-actin data were included. (B-F) The expression level of cytokine genes of spleen cells from immunized hamsters was represented by the 2^-ΔΔCt of target cytokine gene relative to the β-actin gene and normalized with the corresponding values of sham-immunized PBS-stimulated group. White bars represent sham-immunized group data and gray bars represent C-Igl immunized group data. The Y axes were linear coordinates and numbers corresponded to the fold increase over value 1.0 given to the sham-immunized PBS stimulated control group. Error bars represent the standard errors of the means calculated from three independent replicates. Abscissa means different stimulates. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4. Quantitative real-time PCR assays of PD1 (A), PD1-L1 (B) and PD1-L2 (C) genes induced by C-Igl in spleen cells from hamsters immunized with C-Igl.

The expression level of cytokine genes of spleen cells from immunized hamsters was represented by the 2^-ΔΔCt of target gene relative to the β-actin gene and normalized with the corresponding values of sham-immunized PBS-stimulated group. White bars represent sham-immunized group data and gray bars represent C-Igl immunized group data. The Y axes were linear coordinates and numbers corresponded to the fold increase over value 1.0 given to the sham-immunized PBS stimulated control group. Error bars represent the standard errors of the means calculated from three independent replicates. Abscissa means different stimulates. *P < 0.05.