Integrated System for Purification and Assembly of PCV Cap Nano Vaccine Based on Targeting Peptide Ligand

Fangyu Wang,^1,* Junfang Hao,^1,2,* Ning Li,^3,* Guangxu Xing,¹ Man Hu,¹ Gaiping Zhang<sup>1,4

1</sup>Key Laboratory for Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, Henan 450002, People’s Republic of China; ²College of Biology and Food, Shangqiu Normal University, Shangqiu, Henan 476000, People’s Republic of China; ³College of Food Science and Technology, Henan Agricultural University, Zhengzhou, Henan 450000, People’s Republic of China; ⁴College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450000, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Gaiping Zhang
Key Laboratory for Animal Immunology, Henan Academy of Agricultural Sciences, 116# Huayuan Road, Zhengzhou, Henan Province 450002, People’s Republic of China
Email zhanggaiping2003@163.com

Purpose: The vaccine design has shifted from attenuated or inactivated whole pathogen vaccines to more pure and defined subunit vaccines. The purification of antigen proteins, especially the precise display of antigen regions, has become a key step affecting the effectiveness of subunit vaccines.
Materials and Methods: This work presents the application of molecular docking for a peptide ligand designed for PCV2 Cap purification and assembly in one step. Based on the PCV2 Cap protein affinity peptide (L11-DYWWQSWE), the amino terminal of PCV2 Cap was covalently coupled with the polylactic acid–glycolic acid copolymer (PLGA) carboxyl terminal through the EDC/NHS method.
Results: The PLGA had an average diameter of 106 nm. The average diameter increased to 122 nm after the PCV2 Cap protein conjugation, and the Zeta potential shifted from − 13.7 mV to − 9.6 mV, indicating that the PCV2 Cap protein stably binds to the PLGA. Compared with the free PCV2 Cap protein group, the neutralizing antibody titer was significantly increased on the 14th day after the PLGA-Cap immunization (P < 0.05). The neutralizing antibody level was extremely significant on the 28th day (P < 0.001). The CCK-8 analysis showed that PLGA-Cap had an obvious cytotoxic effect on RAW264.7 cells at the PLGA nanoparticle concentration up to 200 μg/mL but had no obvious cytotoxic effect on DC2.4 cells. Compared with the Cap protein group, the antigen-presenting cells had a stronger antigen uptake capacity and a higher fluorescence in the PLGA-Cap group. The immune effect showed that the level of the neutralizing antibody produced by this structure is much better than that of purified protein and helps improve the immune system response.
Conclusion: This technology provides a potential new perspective for the rapid enrichment of the antigen protein with the affinity peptide ligand.

Keywords: PCV2 Cap, peptide ligand, antigen display, protein assembly, nano vaccine, neutralizing antibody