Increased expression of capsid protein in trans enhances production of single-round infectious particles by West Nile virus DNA vaccine candidate.

West Nile virus (WNV; genus Flavivirus, family Flaviviridae) is an emerging pathogenic arbovirus responsible for outbreaks of encephalitis around the world. Whilst no vaccines are currently available to prevent WNV infection of humans, the use of cDNA copies of flavivirus RNA genomes with large internal deletions within the capsid (C) appears promising. C-deleted vaccines are able to replicate and secrete large amounts of non-infectious immunogenic subviral particles (SVPs) from transfected cells. We have previously generated a WNV DNA vaccine candidate pKUNdC/C where C-deleted WNV cDNA was placed under the control of one copy of the cytomegalovirus (CMV) promoter and the C gene was placed under the control of a second copy of the CMV promoter in the same plasmid DNA. This DNA was shown to generate single-round infectious particles (SRIPs) capable of delivering self-replicating C-deleted RNA producing SVPs to surrounding cells, thus enhancing the vaccine potential. However, the amounts of both SRIPs and SVPs produced from pKUNdC/C DNA were relatively low. In this investigation, we aimed at increasing SRIP production by optimizing trans-C expression via incorporating different forms of C and the use of a more powerful promoter. The construct containing an elongation factor EF1α promoter encoding an extended form of C was demonstrated to produce the highest titres of SRIPs and was immunogenic in mice. Additionally, SRIP and SVP titres were further improved via incorporation of a glycosylation motif in the envelope protein. The optimized DNA yielded ~100-fold greater titres of SRIPs than the original construct, thus providing a promising candidate for further vaccine evaluation.