PACKAGING OF VEE-REPLICON VECTORS WITH RECOMBINANT POXVIRUSES

Abstract

A full-length infectious cDNA clone of the genome of Venezuelan equine encephalomyelitis virus (VEE) has been modified to form an expression vector by Pushko et al. (1997a; 1997b). The vaccine vector is composed of a self-replicating RNA, termed a "VEE-replicon". In the replicon system, the structural genes encoding the viral capsid (C) and glycoproteins (GP) are deleted from the viral genome and replaced with a heterologous gene of interest (Pushko et al. 1997a; Pushko et al. 2001; Lee et al. 2001). In order to package the VEE replicon into virus replicon particles (VRPs), the capsid (C) and the glycoproteins (GP) are supplied in trans by co-transfection of RNA transcribed from the replicon and structural protein helper plasmids (Pushko et al.1997a; Pushko et al.1997b). Recombination between these RNA's can occur at high frequency in alphavirus-infected cells, making it theoretically possible that recombination between the replicon and helper RNA's might generate replication-competent VEE virus (Weiss and Schlesinger 1991; Kenneth 1997; Schlesinger 1999). The generation of replication-competent virus has been observed in other alphavirus replicon expression systems (Weiss and Schlesinger 1991; Rice et al.1987). Recombination events between the components of these expression systems usually requires that the template RNA's contain the viral 5' and 3'- terminal cisacting sequences, therefore an alternative method of structural protein expression is desirable. The purpose of this study was to determine if the packaging of VEE replicons could be carried out using recombinant vaccinia (VACC) helper viruses expressing the VEE virus structural proteins in lieu of the C and GP RNA helpers (Kinney et al.1988). For these studies, the replicon contains the GFP protein gene or for the influenza virus hemagglutinin (HA) glycoprotein, which is responsible for the hemagglutination and attachment of influenza virus to the host cell (Taylor et at. 1991). Several recombinant vaccinia viruses were used as helpers to package the VEE-replicon. The recombinant vaccinia virus that generated the highest VRP titers was VACC TRD-1A that contains a truncated cDNA corresponding the 26S mRNA of wild type VEE subtype IA/B. This vaccinia virus helper produced approximately 10⁶ infectious units (IU) of VRPs per ml, 24 hours post-infection (h.p.i.) in BHK cells. Plaque assays of the VRP supernatant on VERO-E6 cells showed plaque formation. Due to the potential generation of replication-competent VEE virus by recombination, assays were designed to identify the virus generating plaques on VERO-E6 cells. The VRP supernatants were inoculated on CHO cells, which are non-permissive for vaccinia virus but fully permissive for VEE. No plaques were observed on CHO cells indicating that no replication-competent VEE virus was generated when vaccinia virus helpers were used to package the VEE replicon. Further experiments showed that the virus produced from these cells was vaccinia virus. Attempts were made to purify VEE-VRPs from vaccinia virus using various Low Protein Binding Durapore (LPBD) filters as well as packaging in CHO cells. These methods were unsuccessful indicating that other methods for production of VRPs will need to be examined.