Table 1.
Primer Sequence of EgAgB8/1 and Eg-01883.
Fig 1.
Schematic illustration of the experimental process.
Schematic illustration of the experimental workflow. The parasite Echinococcus granulosus was isolated from infected dogs, and genomic DNA was subsequently extracted.Recombinant proteins rEgAgB8/1 and rEg-01883 were expressed, purified, and evaluated for immunogenicity through protein microarray analysis using sera from patients with cystic echinococcosis. The antigenicity of rEgAgB8/1 and Trx was further assessed in a mouse immunization model. Flow cytometry was performed to analyze the proportions of antibody-secreting cells (plasmablasts and plasma cells), memory B cells, germinal center (GC) B cells, and T follicular helper (Tfh) cells in the spleens of mice immunized with rEgAgB8/1, Trx, or PBS. Lymphocyte proliferation in response to rEgAgB8/1 and Trx immunization was measured by CCK-8 assay, while serum anti-rEgAgB8/1 antibody levels were analyzed using protein microarrays. Additionally, the frequencies of IFN-γ- and IL-10-producing T cells in mouse.
Fig 2.
Expression, purification and serological evaluation of rEgAgB8/1 and rEg-01883.
(A) Schematic diagram of protein structure of Echinococcus granulosus protein (EgAgB8/1 and Eg-01883) amino acid (aa) sequences. (B) Polymerase chain reaction (PCR, left) amplification for Eg-01883 (lane 1) and EgAgB8/1 (lane 2). Coomassie blue-stained SDS-PAGE gel (middle) of eluted, purified and concentrated rEg-01883 (lane 1) and rEgAgB8/1 (lane 2). Western blot analysis (right) of rEg-01883 (lane 1) and rEgAgB8/1 (lane 2) using an anti-HIS rabbit monoclonal antibody. (C) Antibody levels of rEgAgB8/1 and rEg-01883 protein in sera from healthy individuals and patients with cystic echinococcosis. The data are shown as the mean ± SD. P values were calculated using Student’s t‑test. Significant differences between groups are denoted on the graph: *** P < 0.001, nonsignificant (ns), significant differences are indicated (P > 0.05).
Fig 3.
Humoral immune response induced by rEgAgB8/1 and Trx in mice.
(A) Antibody titers of sera obtained from rEgAgB8/1 and Trx or PBS-immunized mice. Blood samples were collected on days 14, 28, and 42 of post-immunization (left). Sera from immunized mice was arrayed in duplicate in a series of two-fold dilutions (from 1: 250 to 1: 32,000) (right). (B) Western blot confirming the presence of rEgAgB8/1 in Echinococcus granulosus lysate using antibodies from the sera of mice immunized with rEgAgB8/1 (lane 1), Trx (lane 2) or PBS (lane 3). (C) The percentage comparison of Memory B cells detected by FACS in the spleen of rEgAgB8/1, Trx and PBS-immunized mice. (D) The percentage comparison of PB (Plasmablasts) and PC (Plasma cells) detected by FACS in the spleen of rEgAgB8/1, Trx and PBS-immunized mice. (E) The percentage comparison of GC Tfh and GC B cells detected by FACS in the spleen of rEgAgB8/1, Trx and PBS-immunized mice. The data are shown as the mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Significant differences between groups are denoted on the graph: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4.
IFN-γ/IL-10-producing T cell responses in rEgAgB8/1-immunized mice.
(A) Flow cytometry gating strategy for IFN-γ/IL-10-producing T cells upon rEgAgB8/1 antigen. (B) Lymphoproliferative activity of splenocytes isolated from experimental groups and PBS group, concanavalin A (Con A) worked as a positive control. Measurement of IFN-γ-producing (C) and IL-10-producing (D) CD4+ and CD8+ T cell levels in mice by flow cytometry. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Significant differences between groups are denoted on the graph: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5.
Frequency of memory T cells in response to the stimulation of rEgAgB8/1 antigen.
(A) The representative gating strategy to identify T naïve cells (Tnaïve, CD44int/low CD62L+), T central memory cells (TCM, CD44high CD62L+), T effector memory cells (TEM, CD44high CD62L−) and T terminally differentiated cells (TEMRA, CD44int/low CD62L−). Percentage comparison of Tnaïve (B), TCM (C), TEM (D) and TEMRA (E) in CD4+ and CD8+ T cells after stimulating the splenocytes of rEgAgB8/1, Trx and PBS-immunized mice by rEgAgB8/1 in vitro. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Significant differences between groups are denoted on the graph: *P < 0.05, **P < 0.01, ***P < 0.001.
Table 2.
Physicochemical parameters and antigenicity prediction of rEgAgB8/1 and rEg-01883 proteins.