Recombinant Technology
High level expression of a human rabies virus-neutralizing monoclonal antibody by a rhabdovirus-based vector

https://doi.org/10.1016/S0022-1759(01)00353-2Get rights and content

Abstract

Humans exposed to rabies virus must be promptly treated by passive immunization with anti-rabies antibody and active immunization with rabies vaccine. Currently, antibody prepared from pooled human serum or from immunized horses is utilized. However, neither of these reagents are readily available, entirely safe, or consistent in their biological activity. An ideal reagent would consist of a panel of human monoclonal antibodies. Such antibodies are now available, their only drawback being the cost of production. Using recombinant technology, we constructed a rabies virus-based vector which expresses high levels (∼60 pg/cell) of rabies virus-neutralizing human monoclonal antibody. The vector is a modified vaccine strain of rabies virus in which the rabies virus glycoprotein has been replaced with a chimeric vesicular stomatitis virus glycoprotein, and both heavy and light chain genes encoding a human monoclonal antibody have been inserted. This recombinant virus can infect a variety of mammalian cell lines and is non-cytolytic, allowing the use of cell culture technology routinely employed to produce rabies vaccines.

Introduction

Human rabies is a worldwide public health problem. Nearly 500,000 people annually receive rabies post-exposure prophylaxis (Steele, 1988), which includes the use of anti-rabies virus immunoglobulin together with the administration of rabies vaccine (Wilde et al., 1989). Equine anti-rabies immunoglobulin (ERIG) and human anti-rabies immunoglobulin (HRIG), which are currently used for rabies post-exposure prophylaxis, have either been associated with severe adverse effects or are, as in the case of HRIG, extremely expensive. There are also safety concerns for HRIG because it is prepared from pooled human sera and, therefore, could be potentially contaminated with human pathogens.

As a first step toward the production of safer reagents, human monoclonal antibodies (hMAbs) to rabies virus have been produced Ueki et al., 1990, Champion et al., 2000. Several of these hMAbs neutralize a broad spectrum of rabies virus strains and were able to protect hamsters against a lethal rabies virus infection when administered after infection (Dietzschold et al., 1990). Although these findings indicate the usefulness of hMAbs for the rabies post-exposure treatment of humans, the low expression levels (∼1 mg/l) of antibody using mouse–human heterohybrid cells currently preclude the cost-effective production of hMAbs.

An alternative technique for the production of hMAbs involves the insertion of the nucleotide sequences encoding for the immunoglobulin (Ig) heavy (H) and light (L) chains of these hMAbs into suitable expression vectors and expressing the inserted protein-coding sequences in appropriate cells, preferably eukaryotic cells. Thus, far, Ig L and H chains of several MAbs have been cloned into different expression vectors, which have enabled the expression of functional antibodies in a variety of cells, including lymphoid and non-lymphoid mammalian cells (Ovens and Young, 1994), insect cells (Liang et al., 1997) and plants (Whitelam et al., 1994). Some of these expression systems, in particular mouse myeloma cells, are able to produce high levels of antibodies. Here we describe a novel rhabdovirus-based expression vector for Ig H and L chain genes which expresses high levels of a rabies virus-neutralizing hMAb in mammalian cells.

Section snippets

cDNA cloning of human IgG H and L chains from JA-3.3A5 hybridoma cells

Total RNA was isolated from JA-3.3A5 hybridoma cells (Champion et al., 2000) using RNAzol B (Biotech Labs., Houston, TX). Reverse transcriptase (RT) reactions were performed at 42°C for 1 h with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and oligo(dT) primer. A portion of the RT product was subjected to polymerase chain reaction (PCR) amplification using chain-specific primers: IgG-HF1 (5′-ACCATGGAGTTTGGGCTGAG-3′; start codon of H chain underlined; (GenBank

Antibody expression in tissue cultures infected with SPBN-L, SPBN-H, and SPBN-H+L

Immunofluorescence analysis using FITC-conjugated antibodies specific for human kappa chains or human IgG1 revealed expression of the genes encoding IgH and IgL chains in BSR cells infected with SPBN-H and SPBN-L, respectively. BSR cells infected with SPBN-H+L expressed both Ig chains (data not shown).

To determine whether functional antibodies are expressed by the chimeric rhabdovirus SPBN vector, NA cells, BSR cells, CHO cells, and Sp2/0 cells were infected with SPBN-H+L or double-infected

Discussion

The ready availability of safer, economical, and effective products is needed for the post-exposure prophylaxis of human rabies. Evidence indicates that the currently used HRIG or ERIG can be replaced by a cocktail of rabies virus-neutralizing hMAbs which provide the advantages of high specific protective activity, consistency in biological activity, and lack of infection risk and adverse effects such as serum sickness.

Hybridoma technology for production of hMAbs is available and several clones

Acknowledgements

We tank Suchita Santosh Hodawadekar for excellent technical help. This work was supported by Public Health Service Grant AI45097.

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