Fusion Expression and Immune Effect of PCV2 Cap Protein Tandem Multiantigen Epitopes with CD154/GM-CSF

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.

Humoral and Cellular Immune Responses Induced by Bivalent DNA Vaccines Expressing Fusion Capsid Proteins of Porcine Circovirus Genotypes 2a and 2b

“…Thus, enhanced immune induction by PCV2b-2a-GI may be supported by these attributes of GM-CSF and IL-4. In agreement with our results, an immunization with the recombinant PCV2 capsid protein fused with GM-CSF increased PCV2-specific antibody production and enhanced the transcription levels of cytokines IL-2, IFN-γ, IL-4, and IL-10 in a mouse model [45]. Likewise, an immunization with a HSV-1 recombinant virus expressing IL-4 induced a humoral immune response of greater magnitude than immunization with the parental control virus [24].…”

Surface Display of porcine circovirus type 2 antigen protein cap on the spores of bacillus subtilis 168: An effective mucosal vaccine candidate

“…The coat protein of spores is considered an ideal platform for antigen delivery because of its excellent mucosal immune adjuvant properties and strong resistance to harsh physical and chemical environments. Cap protein-based PCV2 subunit vaccines have been reported in many studies ( 39 – 41 ). In addition, the 47 amino acids at the N-terminal of Cap protein were NLS (nuclear localization signal) peptides, which did not contain major antigenic epitopes ( 42 ).…”

Matrine Targets Intestinal Lactobacillus acidophilus to Inhibit Porcine Circovirus Type 2 Infection in Mice

“…PCV2 capsid (cap) protein is a viral nucleocapsid protein encoded by open reading frame 2 (ORF2) and contains specific antigenic determinants [3]. Cap is an important immunogenic protein of PCV2, which is involved in the virus replication and viral infection and transmission [4,5]. Pigs have a strong susceptibility to PCV2, which can lead to symptoms such as dyspnea and abortion [6], pathological changes such as liver injury [7], interstitial pneumonia [8], and changes in intestinal flora [9].…”