Refined methods for propagating vesicular stomatitis virus vectors that are defective for G protein expression

Abstract

Propagation-defective vesicular stomatitis virus (VSV) vectors that encode a truncated G protein (VSV-Gstem) or lack the G gene entirely (VSV-ΔG) are attractive vaccine vectors because they are immunogenic, cannot replicate and spread after vaccination, and do not express many of the epitopes that elicit neutralizing anti-VSV immunity. To consider advancing nonpropagating VSV vectors towards clinical assessment, scalable technology that is compliant with human vaccine manufacturing must be developed to produce clinical trial material. Accordingly, two propagation methods were developed for VSV-Gstem and VSV-ΔG vectors encoding HIV gag that have the potential to support large-scale production. One method is based on transient expression of G protein after electroporating plasmid DNA into Vero cells and the second is based on a stable Vero cell line that contains a G gene controlled by a heat shock-inducible transcription unit. Both methods reproducibly supported production of 1×10 7 to 1×10 8 infectious units (I.U.s) of vaccine vector per ml. Results from these studies also showed that optimization of the G gene is necessary for abundant G protein expression from electroporated plasmid DNA or from DNA integrated in the genome of a stable cell line, and that the titers of VSV-Gstem vectors generally exceeded VSV-ΔG.

Complementing Defective Viruses That Express Separate Paramyxovirus Glycoproteins Provide a New Vaccine Vector Approach

“…However, the production of such defective vectors requires complementation by expression of VSV G. Since VSV G expression is toxic to cells (1), large-scale production of such vectors is problematic because it requires transient DNA transfection or use of a cell line in which VSV G expression is tightly regulated (31,36).…”

“…VSV⌬G recombinants expressing NiV G or F copropagate in the absence of VSV G complementation. Although VSV⌬G recombinants expressing foreign proteins can be highly effective vaccines (19,26), their preparation requires complementation with VSV G. This is typically performed in cells transiently transfected with a DNA encoding VSV G or in an inducible cell line expressing VSV G (31,36). To determine whether the VSV⌬G recombinants expressing NiV G or F might be grown as a complementing pair, we initially infected BHK-21 cells separately or simultaneously with VSV⌬G-G(NiV) and VSV⌬G-F(NiV) that had been complemented with VSV G. The coinfected cells showed extensive fusion resulting in syncytia across the entire dish (Fig.…”

“…Although these defective vectors have been just as effective as live-attenuated recombinants in preclinical trials (26), production using complementing cell lines is a major limitation (36). We describe here a new approach for growing such defective recombinants using a complementing pair of VSV⌬G recombinants, each expressing one of the two glycoproteins that are both required for the entry of Nipah virus (NiV), an emerging zoonotic virus in the paramyxovirus family.…”

Neurovirulence and Immunogenicity of Attenuated Recombinant Vesicular Stomatitis Viruses in Nonhuman Primates

“…These vectors were further modified to express HIV-1 strain HXBc2 gag p55 from an extra TU inserted in the first position of the virus genome, generating rVSVN3CT1gag1, rVSVN4CT1gag1, rVSVN4CT9gag1, and rVSVM NCP CT1gag1 (60). Propagation-defective rVSVGstem-gag1 lacking the G gene was generated as described previously (62). The rVSVgag5 vector used in this work as an nonattenuated reference standard during immunogenicity studies was generated by cloning the gag p55 open reading frame into an XhoI/NheI expression cassette located between the virus G and L genes (34).…”

Single-dose replication-defective VSV-based Nipah virus vaccines provide protection from lethal challenge in Syrian hamsters

“…The two-dose HeVsG vaccine formulation requires the action of two adjuvants, Allhydrogel and CpG oligodeoxynucleotide (ODN) 2006 (Bossart et al, 2012) in order to generate a Th1 immune response, since Allhydrogel alone typically induces a Th2 response which is not appropriate against viral infections (Coffman et al, 2010; Steinhagen et al, 2011; Weeratna et al, 2001). The HeVsG vaccine however holds an advantage over the VSV-ΔG vector in that the vaccine antigen is more easily produced on a large scale versus the production of infectious VSV-ΔG particles which require multiple plasmid transfections (Pallister et al, 2011; Witko et al, 2010). The results reported in this study underscore the safety profile and robust protection conferred by a single dose of these replication-deficient single-cycle vectors against NiV.…”