Expression of FMD virus-like particles in yeast Hansenula polymorpha and immunogenicity of combine with CpG and aluminum adjuvant

Figures

• 

  Fig. 1. The target protein was further purified by three-step chromatography. (A) Anion exchange chromatography (Q Sepharose High Performance; GE Healthcare). (B) A hydrophobic interaction chromatograph (Butyl Sepharose 4FastFlow; GE Healthcare). (C) Based on molecular chromatography (Superdex 200 pg; GE Healthcare). The shaded part in the figure is the purified VLPs collected by chromatography. VLP, virus-like particle.

• 

  Fig. 2. Identification of virus-like particles after ultracentrifugation. (A) The protein curve of collected samples determined by A280. The sodium dodecyl sulfate polyacrylamide gel electrophoresis and WB detection results of samples near the peak. WB analyses of recombinant proteins using rabbit type O foot-and-mouth disease virus antibody (lane M is the protein Marker; lanes 1, 2, and 3 are the three sample tubes at the peak, respectively). (B) The purity of the peak sample (lane 2 sample) measured by high-performance liquid chromatography. (C) The peak sample (lane 2 sample) with particles around 25 nm observed by electron microscopy. WB, Western blot.

• 

  Fig. 3. Liquid phase blocking ELISA of FMD type O antibody. BALB/c mice were immunized every 2 wk for a total of four times. Blood samples were collected 2 wk after immunization for antibody detection. The last blood samples were collected 4 wk after the last immunization for antibody detection by FMD type O antibody liquid phase blocking ELISA. The antibody titers of each group were transformed logarithmically, and the bars represent mean ± standard error of the mean. I: PBS represents the immune PBS solution, II: FMD inactivated vaccine represents the immune inactivated vaccine group, III: sephadexVLPs (10 μg) represents the immune group containing 10 μg VLPs, IV: sephadexVLPs (5 μg) represents the immunized group containing 5 μg VLPs. Titer = 0.6 means the minimum dilution assay in which no positive signal was detected. ELISA, enzyme-linked immunosorbent assay; FMD, foot-and-mouth disease; PBS, phosphate buffered saline; VLP, virus-like particle. ap < 0.05; bp < 0.001.

• 

  Fig. 4. ELISA results of mouse serum IgG, IgG, IgG2a, and IgG1/IgG2a. BALB/c mice were immunized every 2 wk for a total of four times. Blood samples were collected 2 wk after immunization for antibody detection, and the last blood samples were collected 4 wk after the last immunization for antibody detection by ELISA. The antibody titers of each group were transformed logarithmically, and the bars represent mean ± standard error of the mean. (A-C) The production of serum IgG, IgG1, and IgG2a after immunization. (D) The IgG1/IgG2a ratio after multiple immunizations. I: PBS represents the immune PBS solution; II: FMD inactivated vaccine represents the immune inactivated vaccine group; III: sephadexVLPs (10 μg) represents the immune group containing 10 μg VLPs; IV: sephadexVLPs (5 μg) represents the immunized group containing 5 μg VLPs. Titer = 1 means the minimum dilution assay in which no positive signal was detected. ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; FMD, foot-and-mouth disease; PBS, phosphate buffered saline; VLP, virus-like particle.

• 

  Fig. 5. Detection of the IFN-γ secretion level and positive conversion rate in splenocytes by ELISpot. After the mice were sacrificed, splenocytes were taken and stimulated with peptide pools. The number of IFN-γ-producing T cells was assessed by ELISpot. The Bars are presented as the mean ± standard error of the mean. (A) The representative spot plot. (B) The corresponding statistical results. PBS represents the immune PBS solution, FMD inactivated vaccine represents the immune inactivated vaccine group, sephadexVLPs (10 μg) represents the immune group containing 10 μg VLPs, sephadexVLPs (5 μg) represents the immunized group containing 5 μg VLPs. IFN, interferon; con, control; ELISpot, enzyme-linked immunospot assay; IgG, immunoglobulin G; FMD, foot-and-mouth disease; PBS, phosphate buffered saline; VLP, virus-like particle.

• 

  Fig. 6. Flow cytometry results of mouse splenocytes B cells. Fluorescence-activated cell sorting were conducted 28 d after the last injection to analyze the B cells, GC B, MBC, and plasma B cells, respectively. (A) The representative B cell flow cytometry and the statistical results of the B cell data. (B) The representative GC B flow cytometry and the statistical results of the GC B data. (C) The representative MBC flow cytometry and the statistical results of the MBC data. (D) The representative plasma B cells flow cytometry and the statistical results of plasma B cells data. The data are presented as the mean ± standard error of the mean. I: PBS represents the immune PBS solution, II: FMD inactivated vaccine represents the immune inactivated vaccine group, III: sephadexVLPs (10 μg) represents the immune group containing 10 μg VLPs, IV: sephadexVLPs (5 μg) represents the immunized group containing 5 μg VLPs. GC B, Germinal center B cell; MBC, memory B cell; FMD, foot-and-mouth disease; PBS, phosphate buffered saline; VLP, virus-like particle. ap < 0.05; bp < 0.01; cp < 0.001.

• 

  Fig. 7. Flow cytometry results of T cells in mouse splenocytes. T cells, CD4+ T cells, and CD8+ T cells were labeled with CD3-AF700, CD4-BV510, and CD8a-FITC (eBioscience), respectively. (A) The flow data of T cells analyzed by FlowJo with TSNE dimension reduction. (B) The number of CD4+ T cells, CD8+ T cells, and the ratio of CD4+/CD8+. The data are presented as the mean ± standard error of the mean. I: PBS represents the immune PBS solution, II: FMD inactivated vaccine represents the immune inactivated vaccine group, III: sephadexVLPs (10 μg) represents the immune group containing 10 μg VLPs, IV: sephadexVLPs (5 μg) represents the immunized group containing 5 μg VLPs. FMD, foot-and-mouth disease; PBS, phosphate buffered saline; VLP, virus-like particle. ap < 0.05; bp < 0.01; cp < 0.001.

• 

  Fig. 8. Results of mouse splenocyte-specific cytokine assay by flow cytometric. Fluorescence-activated cell sorting were conducted 28 d after the last injection to analyze the FMD-specific CD4+ cells producing IL-2 (A), IL-4 (B), TNF-α (C), and IL-17A(D), including the representative cells flow cytometry and the statistical results, respectively. The data are presented as the mean ± standard error of the mean. I: PBS represents the immune PBS solution; II: FMD inactivated vaccine represents the immune inactivated vaccine group; III: sephadexVLPs (10 μg) represents the immune group containing 10 μg VLPs; IV: sephadexVLPs (5 μg) represents the immunized group containing 5 μg VLPs. FMD, foot-and-mouth disease; PBS, phosphate buffered saline; VLP, virus-like particle. ap < 0.05; bp < 0.01; cp < 0.001.