Abstract
Porcine circovirus associated diseases (PCVAD) is a contagious disease of swine caused by porcine circovirus type 2 (PCV2). The capsid protein (Cap) is the sole structural protein and the main antigen of PCV2. Cap is the principal immunogenic protein and induces humoral and cellular immunity. CD154 and GM-CSF are immune adjuvants that enhance responses to vaccines. However, whether these two cellular molecules could produce an enhanced effect in PCV2 vaccines still needs to be further studied. The results of PCR and restriction enzyme showed that the recombinant lentiviral plasmids pCDH-TB-Cap, pCDH-TB-Cap-CD154 and pCDH-TB-Cap were successfully constructed. Western blot and IFA showed that the three fusion proteins TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF were stably expressed in CHO-K1 cells. Indirect ELISA assay showed that mice immunized with TB-Cap-CD154 and TB-Cap-GM-CSF fusion proteins produced higher PCV2-specific antibodies than mice immunized with the TB-Cap and a commercial vaccine (p < 0.0001). Moreover, lymphocyte proliferation and flow cytometry showed that the cellular immune response of each immune group was significantly enhanced (p < 0.0001). After PCV2 challenge, the results revealed that the viral loads in serum, lung and kidney of all vaccinated groups were significantly lower than the PBS group (p < 0.0001). The transcription levels of IL-2, IFN-gamma, IL-4 and IL-10 cytokines in the TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF groups were significantly higher than those in the PBS and recombinant vaccine groups (p < 0.0001). These results indicated that CD154 and GM-CSF could enhance the ability of TB-Cap protein to induce the body to produce PCV2 specific antibodies and increase the transcription level of cytokines. Thus, CD154 and GM-CSF molecules were a powerful immunoadjuvant for PCV2 subunit vaccines. The novel TB-Cap-CD154 and TB-Cap-GM-CSF subunit vaccine has the potential to be used for the prevention and control of PCVAD.
Highlights
Porcine circovirus (PCV) is the smallest known animal virus, belonging to the genus Circovirus in the Circoviridae family and can be divided into three serotypes, Porcine circovirus type 1 (PCV1), Porcine circovirus type 2 (PCV2) and Porcine circovirus type 3 (PCV3) [1]
PCV2 infection in pigs causes a series of Porcine circovirus associated diseases (PCVAD), such as postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis nephropathy syndrome (PDNS) and swine reproductive disorder syndrome (SRDS) and other varieties of clinical syndromes, which brings great economic losses to the global pig industry [4,5]
The PCV2 commercial vaccine selected in this experiment is a subunit vaccine with Cap as the antigen, which can indirectly serve as a control group without the addition of the dominant epitope of TB cells
Summary
Porcine circovirus (PCV) is the smallest known animal virus, belonging to the genus Circovirus in the Circoviridae family and can be divided into three serotypes, Porcine circovirus type 1 (PCV1), Porcine circovirus type 2 (PCV2) and Porcine circovirus type 3 (PCV3) [1]. The development of enhancing the immunogenicity of the PCV2 subunit vaccine is important. The Cap protein is the major immunogenic structural protein of PCV2 and is responsible for neutralizing antibodies and inducing humoral and cellular immunity [9,10,11]. Studies have shown that cytokines and multiple epitopes can enhance the immune effect of vaccines [12,13]. Experiments conducted by Yunyan Wu in our laboratory confirmed that the combination of the dominant epitopes of T and B cells with Cap can enhance the immunogenicity of the vaccine. The PCV2 commercial vaccine selected in this experiment is a subunit vaccine with Cap as the antigen, which can indirectly serve as a control group without the addition of the dominant epitope of TB cells. The recombinant fusion proteins of TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF were expressed in mammalian expression systems and the recombinant fusion proteins were used to further evaluate its vaccine potential in terms of immunogenicity and protection against PCV2 challenge in BALB/c mice
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