The ultrasonically treated nanoliposomes containing PCV2 DNA vaccine expressing gC1qR binding site mutant Cap is efficient in mice

FIGURE 1. Construction of the plasmids and preparation of the porcine circovirus type 2 (PCV2) DNA vaccines. (A) Shematic structure of pVAX-Cap, pVAX-Capmut, pVAX-A-Cap-W, pVAX-A-Capmut-W, pVAX-A-spCap-W, pVAX-A-spCapmut-W, and the plasmids expressing CXCL12, CCL22, and CCL25. Human cytomegalovirus (CMV) represents the promoter of the vectors, sp represents the IL-2 secretory signal peptides. (B) The pVAX-Cap, pVAX-Capmut, pVAX-A-Cap-W, pVAX-A-Capmut-W, pVAX-A-spCap-W, and pVAX-A-spCapmut-W plasmids were transfected into 293T cells with equal amount for 48 h, then the mRNA levels of Cap were measured by qPCR. (C) The pVAX-Cap, pVAX-Capmut, pVAX-A-Cap-W, pVAX-A-Capmut-W, pVAX-A-spCap-W, and pVAX-A-spCapmut-W plasmids were transfected into 293T cells with equal amount for 48 h, the expression levels of Cap in the transfected cells and in the supernatants of these cells were examined by western blot. (D) The polyethylenimine (PEI) containing plasmids were treated with ultrasonic for 2, 4, 6, 8, 10, and 12 min, respectively. The particle sizes of the treated mixtures were analyzed. (E,F) The ultrasonic PEI containing pVAX-Cap and 1/10 pEGFP-N1 and the non-ultrasonic PEI containing pVAX-Cap and…

FIGURE 2. The gC1qR binding site mutation Cap induces higher humoral immune response than the wild-type Cap in mice. (A) Vaccination schedule and blood collection, spleen collection, lung collection, and challenge times. (B) The serum samples of the vaccinated mice were assayed for porcine circovirus type 2 (PCV2) Cap-specific antibody levels by enzyme-linked immunosorbent assay (ELISA). (C) PCV2 were incubated with the serum samples of the vaccinated mice, and then infected to PK-15 cells for 72 h. The PCV2 levels were detected, and the PCV2 neutralizing antibody levels were calculated. The same small letters (a∼n) represent no significant difference among the groups, and different small letters represent significant differences among the groups in (B,C).

FIGURE 3. The gC1qR binding site mutation Cap induces higher cellular immune response than the wild-type Cap in mice. At 28 days post-vaccination, half of the mice in each group were euthanized, and then the spleen lymphocytes were isolated for further assays. (A) The spleen lymphocytes of mice in each group were plated into 96-well plates, and stimulated by the prokaryotic expressed Cap, ConA as positive control, or medium as negative control for 68 h. Then the proliferation level of spleen lymphocytes in mice was detected by 3- (4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay. (B,C) The IL-4 (B) and IFN-γ (C) levels in the supernatant of these lymphocytes stimulated by the prokaryotic expressed Cap were detected by enzyme-linked immunosorbent assay (ELISA). The same small letters (a∼e) represent no significant difference among the groups, and different small letters represent significant differences among the groups in (A–C).

FIGURE 4. The gC1qR binding site mutation Cap is more efficient in protecting mice from porcine circovirus type 2 (PCV2) infection and the viral infection-induced lung lesions. (A) The PCV2 copy numbers in the serum of the different groups mice were measured by qPCR every 7 days after challenge. (B) At 56 days post-vaccination, all groups mice were euthanized, the lungs were made into paraffin tissue sections, stained with HE, and the pathological changes of the lung tissues were observed with a microscope. (C) The PCV2 copy numbers in the lungs of the different groups mice were measured by qPCR at 56 days post-vaccination. Bar = 20 μm. ND means not detected, and the same small letters (a∼j) represent no significant difference among the groups, and different small letters represent significant differences among the groups in (A).