Classical swine fever (CSF) is a highly contagious and often fatal disease of swine. It is caused by classical swine fever virus (CSFV), one of the members of the genus Pestivirus of the Flaviviridae family (1). An epidemic often causes huge economic losses, so CSF is one of 16 OIE list A diseases and under strict surveillance (7). The genome of CSFV, a positive-stranded RNA genome, comprises a single long open reading frame (ORF) coding for a poly-protein encompassing all the viral proteins. The structural proteins comprise the nucleocapsid protein C and three envelope glycoproteins, Erns, E1, and E2. Erns and E2 are located at the surface of infected cells (12), induce virus-neutralizing antibodies and mount protective immunity in the natural host (2, 5, 11).
Studies have showed that CSFV envelope gly-coprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge (9). With a panel of 13 MAbs, four antigenic domains, A, B, C and D, have been identified on the E2 gene of CSFV strain Brescia. Domains A, B and C contain epitopes for neutralizing MAbs (13). These antigenic domains have been mapped to the N-terminal half of E2 and are located on two independent structural units in a proposed model of the antigenic structure of E2 (10). Until now, attenuated live vaccines have been the most effective weapons to defend against pathogen infection. However, Genghini et al reported obvious pig chromosome aberrations after vaccination with live vaccine, which serves as a reminder of the potential danger of live vaccines. So it is necessary to find an alterative safer vaccine against CSFV.
Retroviral vectors are believed to be advantageous for single gene transfer, which may allow for the simultaneous achievement of more efficientgene delivery and longer-term transgene expression. The ability of retrovirus vectors delivering a particular gene to target cells has been applied commonly in both experimental and clinical settings. In this report, we have cloned and expressed four major antigen domains of E2 gene of CSFV in eukaryotic cells, and analyzed the immunological activity in rabbits. The antigenicity and immunogenicity of the construct was monitored in rabbits in a first step to evaluate the possibility of developing a subunit vaccine.
In order to demonstrate appropriate expression of the CSFV E2 proteins, infected PK15 cells were detected by IFA, Western blot, ELISA and PCR. The analysis of IFA demonstrated that CSFV E2 protein was detected in PK15 cells infected with artificial retrovirus (Fig. 1A), which form specific fluorescence, while the blank cell control (Fig. 1B) with the same cells did not show any fluorescence emission.
Figure 1. Expression of the E2 gene in PK-15 cells detected by fluorescent antibody. PK15 cells were analyzed for expression of CSFV E2 protein by IFAT. A: The screened positive cells. B: Normal PK15 cell control.
For detecting the activity of expressed E2 protein in vitro, the medium of the infected cells was harvested and analyzed by Western blot and ELISA. As seen in Fig. 2, the picture revealed a major band of 26 kDa in cells infected with"artificial retrovirus", whereas no E2 protein band was found in blank cells. In ELISA assay (as show in Table 1), E2 protein was expressed in PK15-E2 cell culture and culture supernatant according the ELISA kit assessment standard.
Figure 2. Western blot used to detect the activity of CSFV E2 protein in the infected cells. M, Protein marker; 1 and 2, SDS-PAGE detection of screened positive PK15-E2 cell lysate; 3 SDS-PAGE detection of negative PK15 cell lysate; 4 and 5, Western-Blot detection of screened positive PK15 cell lysate; 6, Western-Blot detection of negative PK15 cell lysate.
Table 1. E2 protein of cell culture detected by ELISA
The CSFV E2 gene was amplified from infected PK15-E2 cells, and a 0.5 kb DNA fragment (using E2-f and E2-r as the primers) was visible (Fig. 3). The fragment was sequenced to confirm its stability and reliability throughout the entire process.
Figure 3. Genetic stability of E2 in infected cells. M, DL2000 Marker. 1, PCR identification of infected 1st generation cells; 2, PCR identification of infected 10th generation cells; 3, PCR identification of infected 20th generation cells; 4, PCR identification of infected 30th generation cells; 5, Blank cell control.
In groups A and B, rabbits showed a T-lymphocyte proliferation response (Fig. 4). But none of animals in groups C showed a T-lymphocyte response. Rabbits of group A vaccinated with recombinant antigen alone developed an enhanced response after the second vaccination. Analysis of T-lymphocyte proliferation response with software of biological statistics showed that there was no significant difference between groups A and B, but there was a significant difference between groups A and group C.
The CSFV specific antibody levels in the sera that were measured by a commercial competitive ELISA are presented in Table. 2. Groups A that were inoculated with PK15-E2 protein were seropositive on day 2 weeks; this result was also observed in group B that was vaccinated with the C strain vaccine. No animals seroconverted in the control group (inoculated with 0.9% NaCl).
Table 2. Detection of CSFV specific antibodies in immunized rabbits by blocking ELISA
If rabbits displayed typical fever after neither the first challenge nor the second one, it means that they are not susceptible to the common dose (100 FD min) of C-strain. In this study, the negative control (group C) suffered a typical fever after challenge with C-strain, while the same dose of C-strain was not able to induce typical fever in all rabbits of the test group (group A) or the positive control group (group B) (Table. 3).
Table 3. Challenge results with C-strain.