Structure-Based Design of Porcine Circovirus Type 2 Chimeric VLPs (cVLPs) Displays Foreign Peptides on the Capsid Surface

Abstract

Although porcine circovirus-like particles can function as a vector to carry foreign peptides into host cells, displaying foreign peptides on the surface of virus-like particles (VLPs) remains challenging. In this study, a plateau, consisting of the middle portion of Loop CD (MP-Lcd) from two neighboring subunits of PCV2 capsid protein (Cap), was identified as an ideal site to insert various foreign peptides or epitopes and display them on the surface of PCV2 VLPs. One of the goals of this work is to determine if the surface pattern of this plateau can be altered without compromising the neutralizing activity against PCV2 infections. Therefore, biological roles of MP-Lcd regarding VLPs assembly, cell entry, and antigenicity were investigated to determine whether this was a universal site for insertion of foreign functional peptides. Three-dimensional (3D) structure simulations and mutation assays revealed MP-Lcd was dispensable for PCV2 Cap assembly into VLPs and their entry into host cells. Notably, substitution of MP-Lcd with a foreign peptide, caused surface pattern changes around two-fold axes of PCV2 VLPs based on 3D structure simulation, but was not detrimental to VLPs assembly and cell entry. Moreover, this substitution had no adverse effect on eliciting neutralizing antibodies (NAbs) against PCV2 infection in pigs. In conclusion, MP-Lcd of the PCV2 Cap was a promising site to accommodate and display foreign epitopes or functional peptides on the surface of PCV2 VLPs. Furthermore, chimeric VLPs (cVLPs) would have potential as bivalent or multivalent vaccines and carriers to deliver functional peptides to target cells.

The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation

“…In addition, PCV2 VLPs have also been extensively used to study the structure of the PCV2 capsid (4, 5), PCV2 binding receptors on cell surface (27), PCV2 entry and infection (28,29), as well as the mechanism of the PCV2 capsid assembly (30). In addition, PCV2 VLPs have been used as a vehicle to display various foreign epitopes or functional peptides in vivo (31)(32)(33)(34). Understanding the mechanism of PCV2 VLP assembly will also greatly advance applications of the VLPs as a novel multivalent vaccine and a vector for gene delivery.…”

“…The eight ␤-strands are connected by seven loops (loops BC, CD, DE, EF, FG, GH, and HI), which contribute to the PCV2 surface features (except loop FG) and stabilize the capsid via extensive interactions with neighboring loops (35). Loop CD is exposed on the capsid surface and frequently exploited as a site to display foreign epitopes on the surface for multivalent vaccine design (33,34). In loop DE, a unique cysteine was reported to play a critical role in the assembly of PCV2 VLPs since the point mutant (C108S) failed to self-assemble into VLPs in vitro (30).…”

Conformational Dynamics of Nonenveloped Circovirus Capsid to the Host Cell Receptor

“…The crystal structure of the PCV2 virus-like particle (VLP) revealed that the capsid protein comprises two β-sheets, each containing four antiparallel β-strands, which were labeled as B to I ( Khayat et al., 2011 ). The residues between the β-strands form eight loops, among which the CD and C-terminal loops are exposed on the VLP exterior and can accommodate the insertion of short foreign peptides ( Liu et al., 2016 ; Wang et al., 2016 , 2018 ). The capsid protein was also reported to have the neutralizing and non-neutralizing epitope sites and the putative canonical HS-binding motif 98 IRKVKV 103 ( Mahe et al., 2000 ; Misinzo et al., 2006 ; Shang et al., 2009 ).…”

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate

“…More recently, experimental vaccines based on the chimeric viruses PCV1 and PCV2 were formulated in chimeric porcine circovirus-Cap proteins, where small Cap loops were replaced with other epitopes of interest. These chimeric proteins were expressed in Escherichia coli (E. coli), preserving the structure of the VLP [30][31][32], and this approach was demonstrated as an alternative against PCV2 infection.…”