Abstract
Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.
Highlights
Hantavirus is a genus of the family Bunyaviridae, containing more than 20 different hantavirus species [1]
We have demonstrated that the inserted Puumala virus (PUUV)-Gc segment was exposed on the surface of virus-like particles (VLPs) and elicited formation of Gc-specific antibodies
The His-tagged PUUV Kazan Gc (PUUV-Gc) protein was purified from the insoluble fraction of yeast cell lysate using nickel chelate affinity chromatography (Figure 2A, lane 1)
Summary
Hantavirus is a genus of the family Bunyaviridae, containing more than 20 different hantavirus species [1]. Hantaviruses with pathogenicity for human are carried by rodent reservoirs and are mainly transmitted to humans by inhalation of aerosols derived from excreta of infected rodents [2]. During the previous years a large number of novel shrew-, mole- and bat-borne, hantaviruses of unknown pathogenicity for human has been discovered [3]. The hantavirus virion contains three genome segments S, M and L, that encode the nucleocapsid (N) protein, glycoprotein precursor (GPC). RNA-dependent RNA-polymerase, respectively [4]. GPC is a polyprotein of 1133–1158 amino acid (aa) residues in length [5]. Cotranslational cleavage of GPC at a site C-terminal to a conserved
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