Immunogenicity evaluation of inactivated virus and purified proteins of porcine circovirus type 2 in mice

Abstract

Background: Vaccination is considered as an effective and economical way to against PCV2 infection. However, some of commercial available vaccines are based on inactivated viruses, while the others are based on purified protein of PCV2. In the present study, we aimed to compare the immunogenicity of inactivated virus and purified proteins of porcine circovirus type 2 in mice.

Results: The results showed that positive antiserum titers were significantly increased after second, third and fourth immunization using inactivated PCV2 or purified proteins as coating antigen. Moreover, the inactivated PCV2 induced significantly higher levels of PCV2-specific antibodies than that of PCV2 subunit proteins. After PCV2 wild strain challenged, the average daily gain was comparable with that of mice in the mock group, and the sera from both inactivated PCV2-immunized animals and subunit protein Cap+ORF3 + Rep immunized animals had significantly higher neutralizing antibody titers than that of the PBS group. As expected, the neutralizing antibody in the inactivated PCV2 group was significantly higher than that of the subunit protein group. These results indicated that positive antiserum induced by the inactivated PCV2 had a better reactivity and specificity than that of the positive antiserum induced by the purified proteins.

Conclusions: The results in the present study demonstrated inactivated PCV2 is more effective than PCV2 subunit proteins in stimulating immune response to against PCV2 infection.

Keywords: Immunogenicity; Inactivated; Mice; Porcine circovirus type 2 (PCV2); Subunit; Vaccine.

Fig. 1

Serum antibody level after different immunizations in mice. Inactivated PCV2 and subunit protein Cap+ORF3 + Rep were used as antigens to raise antiserum, respectively. After each immunization, the antisera were collected as described in the Materials and methods section. ELISA was performed using PCV2 as coating antigen. (a) Second immune; (b) Third immune; (c) Fourth immune

Fig. 2

Serum antibody level after fourth immunization using purified proteins (Cap + Rep, 1:1) as antigen. Inactivated PCV2 and subunit protein Cap+ORF3 + Rep were used as antigens to raise antiserum, respectively. After fourth immunization, the antisera were collected and analyzed

Fig. 3

Average daily gain. Thirty female BABL/C mice, 5 weeks old, were randomly divided into three groups and immunized subcutaneously with 200 μL of the purified proteins or inactivated virus. Two weeks following fourth immunization, all the mice were infected with 200 μL of PCV2 at 7 log10 TCID₅₀/mL or 100 μL of PBS by intramuscular injection, respectively. All mice were observed three times daily for changes in physical appearance and deaths (if any) for up to 14 days post virus exposure. Generally, mice were weighed every other day to determine the average weight gain of the group

Fig. 4

ELISA results of different groups used as coating antigen. a Inactivated PCV2 as coating antigen; b Cap protein as coating antigen; c Rep protein as coating antigen

Fig. 5

Neutralizing assay of the immunized sera. Serum samples were heat inactivated at 56 °C for 30 min, followed by serial dilution in 2-fold increments in Dulbecco’s modified Eagle medium (DMEM) supplemented with 2% of fetal bovine serum (FBS), and mixed 1:1 with 100 TCID₅₀ of PCV2 strain CC1 for 2 h at 37 °C. Thereafter, 100 μL of serum-virus mixture was added to 50% confluency of PK-15 cells and incubated for 1 h at 37 °C. After incubation, the serum-virus mixture was removed, and the cells were washed with PBS for three times and further incubated in DMEM supplemented with 2% of FBS at 37 °C and 5% CO₂. After incubation for 72 h, an indirect fluorescence assay (IFA) was performed to assess the neutralizing activity of the immunized sera. The results represent the means ± S.D. of three replicates of each group. P-values were calculated using two-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001