Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of i
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RESEARCH ARTICLE
Open Access
Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine Ya‑Yi Chen1,2†, Wei‑Chen Yang3†, Yu‑Kang Chang4,5, Chi‑Young Wang6, Wei‑Ru Huang3,10, Jyun‑Yi Li3,10, Kuo‑Pin Chuang7, Hung‑Yi Wu8, Ching‑Dong Chang8, Brent L. Nielsen9 and Hung‑Jen Liu3,10,11,12,13*
Abstract To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as con‑ firmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and C D4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections. Keywords: PCV2, Cap protein, baculovirus surface display vectors, 4Cap(d41) vaccine, virus neutralization test, cellular immune response, CD4+ T cells, IFN-γ
*Correspondence: [email protected] † Ya-Yi Chen and Wei-Chen Yang contributed equally to this work 3 Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan Full list of author information is available at the end of the article
Introduction Porcine circoviruses (PCVs) are very small non‐enveloped animal viruses with circular single‐stranded DNA genomes of approximately 2 kb, which belong to the genus Circovirus in the family Circoviridae [1]. In pigs, four circovirus species (PCV1, PCV2, PCV3, and PCV4) within the genus Circovirus have been identified [1–3]. PCV1 is non-pathogenic and exists in contaminated pig kidney cell lines (porcine kidney
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