Novel strategy for expression and characterization of rabies virus glycoprotein

Highlights

• A new method for improved expression of viral glycoproteins in mammalian cell system.

• A stable CHO clone with several thousand folds higher RABV G protein expression at 50 mg/L.

• The recombinant RABV G protein displayed ideal level of immunoreactivity and immunogenicity.

• The recombinant RABV G protein is suitable for serology, vaccinology and structural studies.

Abstract

Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few μg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection.

Introduction

Rabies is a fatal zoonosis which menaces humans and animals worldwide since antiquity [1]. The disease is caused by rabies virus (RABV), known for neurotropism, belonging to genus Lyssavirus, family Rhabdoviridae and order Mononegavirales [2]. The RABV is composed of a highly stable and organized complex of genomic RNA and nucleoprotein contained in a lipid envelope derived from the host cell membrane [3]. The genome of RABV consists of a single, negative stranded, non-segmented RNA which is approximately 12 kb in size [4]. It encodes for five viral proteins: nucleoprotein, matrix protein, phosphoprotein, glycoprotein and large protein/RNA-directed RNA polymerase [5,6]. The glycoprotein (G protein) plays a pivotal role in viral attachment to neurons [7,8] and determination of tissue tropism [9], retrograde trans-synaptic spread of RABV in the central nervous system [1] and induction of cellular and humoral immune response required for conferring complete protection from lethal challenge [10,11]. The G protein, a type 1 membrane glycoprotein, consists of 524 amino acids including three domains (Fig. 1A) such as cytoplasmic domain, transmembrane domain and ectodomain exposed on the surface of mature virus particle [7,12]. The G protein is anchored in viral envelope by transmembrane domain made up of 22 amino acids from 460 to 480 residues [4]. The cytoplasmic domain consisting of 44 amino acids extends into the cytoplasm of infected cells where it interacts with the matrix protein to complete the viral assembly. The ectodomain exists as homotrimer spikes with each monomer having 439 amino acid residues [13].

As an important viral protein, the G protein has been evaluated for the development of a subunit rabies vaccine and used as reference antigen for Enzyme linked immunosorbent assay (ELISA)/Single radial immune diffusion (SRID)-based rabies vaccine potency determination. Purified G protein is preferred to whole virus antigen because of its highly defined and homogenous nature devoid of interfering substances. Moreover, it provides better evaluation of specific virus neutralizing antibodies (VNA) in sera of vaccinated subjects [14]. However, the production of native G protein had been shown to be tedium, high cost and long turnaround time. Many attempts have been made to express G protein using a recombinant system. The glycosylated nature of the G protein necessitates a eukaryotic system for expression rather than a prokaryotic system which lacks glycosylation machinery. Thus far, full-length version of G protein has been expressed in eukaryotic systems such as insect cells [15], CHO cells [16], yeast [17] and plant [18] albeit with varying levels of success and expression. In this study, high-level and stable extracellular expression of natively folded recombinant G protein ectodomain in CHO cells was achieved with a yield of 50 mg/L and 95% purity. Most importantly, the recombinant RABV G protein was characterized and found to possess native folding and immunologically relevant antigenic sites including a neutralizing epitope. The immunogenicity of the G protein was demonstrated when the induced antibodies showed broader reactivity with inactivated antigens of at least three fixed RABV strains. The data pertaining to expression and characterization of the recombinant RABV G protein are presented and discussed. Taken together, the purified recombinant RABV G protein has been used to successfully develop an inexpensive rabies serology ELISA kit [19]. It seems to hold promise for the development of a rabies subunit vaccine and elucidation of RABV G protein structure which would help understand the molecular mechanism of RABV pathogenesis.