Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system

Abstract

Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV's have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5' leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

Keywords: protein expression; translation enhancing element; vaccine development; vaccinia virus.

Fig. 1.

VACV-driven protein expression. (a) Luciferase reporter plasmids containing the slp were transfected into HeLa cells which were subsequently infected with either the Copenhagen (wt) or NYVAC strains of VACV. Luciferase protein expression was measured via luciferase assay, and results made relative to those obtained from the wt-infected cells. (b) HeLa cells were transfected with luciferase reporter plasmids containing either the slp or hTEE-658 upstream of the coding region. Cells were then infected with either wt or NYVAC virus. Luciferase expression was determined by luciferase assay and results made relative to those obtained from cells containing the plasmid with slp, for the individual viruses. (c) Luciferase assay results from cells transfected with either an slp- or hTEE-658-containing plasmid, which were then infected with wt or NYVAC virus, respectively. Expression levels were made relative to those from the slp-plasmid transfected, wt-infected cells. (d) mRNA levels for cells transfected with either the slp- or hTEE-658-containing plasmids, and infected with either the wt or NYVAC virus, was determined via quantitative realtime PCR. Levels were made relative to those derived from cells transfected with the slp-containing plasmid in each pairing. All experiments were conducted in triplicate at minimum. Error reported as standard deviation.

Fig. 2.

Effect of 5’ leader length on translation levels. (a) Diagram of reporter constructs containing either the slp or hTEE-658 upstream of a leader sequence of varying length, followed by the coding region for the firefly luciferase protein. Luciferase levels were determined by transfect-infect assay using reporters that contain either no inserted leader sequence (none) or a designed sequence consisting of 30 nucleotides (nts) (b, c), 60 nts (d, e) or 90 nts (f, g). Assays were conducted in HeLa cells using the Copenhagen strain of VACV, and in each case results were made relative to those when no leader sequence was present. All experiments were conducted in triplicate at minimum. Error is reported as standard deviation.

Fig. 3.

Combinatorial effect of translation enhancing elements. Luciferase production levels when reporter plasmids containing either hTEE-658 alone or in combination with a known translation enhancing element (TEE) were used in a transfect-infect assay with either the Copenhagen (a) or NYVAC (b) strain of VACV. Assays were conducted in HeLa cells, with luciferase assay results made relative to those when only hTEE-658 was present upstream of the luciferase coding region. All experiments were conducted in triplicate at minimum, and error is reported as standard deviation.

Fig. 4.

hTEE-658 driven expression of HIV-1 gp120. (a) Representative Western blot analysis of lysates from cells transfected with a plasmid containing either hTEE-658 alone or in combination with hTEE-675 or hTEE-884, and infected with the NYVAC strain of VACV. hTEE-658, or the indicated combination, was positioned within the plasmid immediately upstream of the coding region for the gp120 protein of HIV-1. Results of triplicate experiments are shown. (b) The normalized fluorescence of each band was determined, and then averaged for each sample. The averages were made relative to those when only hTEE-658 was present within the plasmid. (c) Representative Revert total protein staining of samples used for Western blot analysis. (d) Fluorescence of each Revert stained lane was determined, and then averaged for each sample. The averages were made relative to the sample with the highest average fluorescence. In all analyses, error is reported as standard deviation.

Fig. 5.

Recombinant virus containing hTEE-658. (a) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. (b) Luciferase expression following infection of BSC40 cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.