Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen.

Corinna M. Bremer,Dieter Glebe,Indre Kucinskaite-Kodze,Milda Pleckaityte,Aurelija Zvirbliene,Alma Gedvilaite

doi:10.1186/s12896-015-0203-3

Abstract

BackgroundVirus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules.ResultsPolyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface.ConclusionsPolyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections.

Highlights

Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast
Generation in yeast of recombinant proteins harbouring anti-hepatitis B virus surface antigen (HBsAg) molecule To generate pseudotype VLPs with surface-exposed HBsAg-specific antibody molecule, the gene encoding anti-HBsAg was fused with gene encoding modified Hamster polyomavirus (HaPyV) minor capsid protein VP2 and co-expressed together with the gene encoding HaPyV major capsid protein VP1
Two recombinant proteins harbouring HBsAgspecific antibody molecule were produced in yeast S. cerevisiae strain AH22-214p: one carrying the single-chain fragment variable (scFv)-Fc molecule and one carrying the scFv without the Fc part (Table 1, Fig. 1)

Summary

Introduction

Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Hamster polyomavirus (HaPyV) VP1 protein has been exploited for the generation of either chimeric VLPs harbouring foreign epitopes, or pseudotype VLPs when co-expressed with the minor capsid protein VP2 that binds within the central 5-fold cavity of each VP1 pentamer [8,9,10,11]. Based on the structure of polyomavirus capsids [13, 14], it was predicted that the inserted target sequence can be displayed on the surface of pseudotype VLPs. In contrast to chimeric VLPs that do not tolerate long inserts, this approach allowed efficient presentation of large-sized foreign protein sequences as demonstrated for the full-length human tumor-associated antigen Her2 [15, 16], prostatespecific antigen [17], green fluorescent protein [18], cellular marker p16INK4A [19] and the antibody molecule [12]. An approach to display multiple functionally active antibody molecules on the surface of pseudotype VLPs that can be purified by density-gradient centrifugation may provide a promising alternative to other currently available methods for producing recombinant antibodies

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