Recombinant Marek’s disease virus expressing VP1 and VP2 proteins provides robust immune protection against chicken infectious anemia virus

Figure 1. Strategy for the construction and generation of recombinant virus (rMDV). (A–C) Strategy for the construction of recombinant virus. (D) Identification of purification of recombinant virus. Cells from isolated plaque were lysed, and then the recombinant virus was isolated through five rounds of plaque isolation by FACS and identified via PCR using primers located at the flanking region of the insertion site. The bands (700 bp) amplified from passaged recombinant virus-infected cells indicate the presence of the parental virus. None of the amplified bands indicating the pure recombinant strain were obtained by clone screening to the 5th generation. (E,F) PCR analysis of viral genome isolated from infected CEF cells using specific primers to identify the VP1 and VP2 gene. VP1: 1,200 bp; VP2: 600 bp. DNA Marker: 2,000 bp. (G) Confirmation of rMDV expressing the VP1 and VP2 proteins by indirect immunofluorescence assay. CEF cells were infected with the recombinant virus at 100 PFU. Then, the CEF cells were harvested at 72 h.p.i. and expression of the genes was determined by IFA. The plasmids pEGFP-N1, pCAGGS-VP1 and pCDNA3.1-VP2 were transfected into DF-1 cells used as positive…

Figure 2. Characterization of the recombinant MDV virus (rMDV). (A) Comparison of the replication kinetics of the recombinant virus. Data are presented as the means ± standard deviations (S.D.) from three independent experiments. Statistical significance was set at p < 0.05. (B) VP1 and VP2 genes from rMDV passaged 20 times in CEFs were amplified by PCR. M: 2,000 DNA Marker. (C) VP1 and VP2 expression from rMDV passaged 20 times in CEFs with indirect immunofluorescence assay. The plasmids pCAGGS-VP1 and pCDNA3.1-VP2 were transfected into DF-1 cells used as positive controls, respectively. Scale bar, 125 μm, respectively.

Figure 3. Replication and exogenous protein expression of rMDV in vivo. (A) rMDV replication kinetics in the thymus after vaccination according to RT-PCR analysis in vivo. Data are presented as the means ± standard deviations (S.D.) from three independent experiments. Statistical significance was set at p < 0.05. (B) In vivo expression analysis of the viral VP1 protein in the thymus, liver, and spleen by western blot at 14 and 42 days post-vaccination. CIAV HeN/193001 was detected as the positive control. rMS∆Meq was detected as the negative control. The VP1 protein of CIAV was located at approximately 55 KDa.

Figure 4. CIAV specific serum antibody response in the experimental chicken. Serum samples were collected weekly and detected using a commercial CIAV Antibody Test Kit for competitive ELISA. (A) Detection the S/N value of CIAV antibody in immunized animals during 42 days post-vaccination. (B) Detection of CIAV antibodies titers against CIAV in immunized chickens during 42 days post-vaccination. The presence of antibody was determined as positive for samples with a (S/N) ratio ≤ 0.6 or antibody titers ≥2,050 for each sample. Data are presented as the means ± standard deviations (S.D.) from five birds in each group. Statistical significance was set at p < 0.05.

Figure 5. Assessment of cell-mediated immune (CMI) response in chicken after vaccination. Lymphocyte proliferation response in chicken at 3, 4, and 5 weeks after vaccination using (A) ConA- and (B) LPS-activated PBMCs. The PBMCs from the experimental birds (n = 5) were stimulated, and antigen-specific lymphocyte proliferation was expressed as the stimulation index (mean ± SE). Different superscripted capital letters indicate the time effect (p < 0.0001), while small superscripted letters indicate the treatment effect (p < 0.0001) in the vaccinated groups. (C) Flow cytometric analysis for CD4+ and CD8+ T-cell subsets in the blank group and vaccination group. (D) Percentage of CD8+ T cells in PBMCs at 3, 4, and 5 weeks after vaccination in birds in different groups. Bars (mean ± SE) indicate the representative data of a single experiment, and data with different small superscripted letters indicate the treatment effect (p < 0.0001). (E) Detection of IFN- γ antibody titers of immunized animals by ELISA at 3 and 4 weeks after vaccination. Data are presented as the means ± standard deviations (S.D.) from five birds in each group. Statistical significance was set at p < 0.0002.

Figure 6. Protective efficacy of the recombinant vaccines against CIAV challenge. (A) Hematocrit value (Hct) after CIAV challenge. Hct < 27 indicates anemia. (B) Incidence rate of thymic atrophy evaluated by the thymus index scores for chickens after CIAV challenge. A score < 0.8 means thymic atrophy. (C) Determination of viral load in thymus at 12 days post-challenge. Data are presented as the means ± standard deviations (S.D.) from seven birds in of the vaccinated group and five birds in the blank control group and CIAV challenge group. Statistical significance was set at p < 0.05. (D) Representative images of the microscopic appearance of the thymus in chicks challenged with JL17P10. Chicks were vaccinated with rMDV (vaccinated chicks) or PBS (unvaccinated chicks) and then challenged intramuscularly with 1.5 mL of JL17P10 (5.5 × 105 copy/mL). The thymuses were examined macroscopically at 7 days post-challenge.