Second Generation Single-Round Infectious Particle (SRIP)-Producing Flavivirus Vaccine

The genus Flavivirus includes the important human arthropod-borne viral pathogens dengue virus (DENV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). Vaccine research targeting these pathogenic flaviviruses has been driven by the need for inexpensive, efficacious, safe, and stable preparations applicable for use in the developing world. One promising vaccine strategy is to utilise flavivirus genomes which have large internal deletions within the capsid (C) gene, thus preventing packaging of the genome and formation of infectious particles. These dC vaccines lead to secretion of large amounts of immunogenic prM/E particles from self-replicating RNA but cannot form a spreading infection. Our laboratory has previously developed a DNA-based vaccine that trans-complements the deleted C within the same plasmid DNA and produces single-round infectious particles (SRIPs) in vivo, leading to an amplifying single round of infection in the vaccinee (1). The first generation SRIP-producing DNA, though protective against lethal WNV challenge, generated low amounts of both SRIPs and prM/E particles. In this project we aimed at increasing production of prM/E particles and SRIPs by generating smaller internal C deletions via the removal of specific alpha helices, by optimizing trans-C expression, and by investigating the benefits of envelope (E) protein glycosylation. New C-deletion constructs were investigated and assessment of E protein secretion and infectious assays demonstrated that removing alpha helices 1 and 2 led to secretion of relatively large amounts of prM/E particles while retaining the non-infectious phenotype. Trans-complementation of C-deleted Kunjin cDNA by addition of different forms of C protein under control of different promoters was investigated in regards to efficiency of SRIP production. The stronger EF1α promoter encoding a membrane-targeted extended form of C was demonstrated to lead to the formation of the highest titres of SRIPs. Additionally, SRIP titres further improved via the incorporation of a glycosylation motif in the E gene. Intradermal immunisation of mice with SRIP-producing vaccines led to recovery of infectious particles and induction of an immune response consistent with protection from virulent virus. As this optimised vaccine yields ≈ 2 log greater titres of SRIPs than the 1st generation vaccine, it appears to be a very promising candidate for further characterisation. 1. Chang DC, Liu WJ, Anraku I, Clark DC, Pollitt CC, Suhrbier A, Hall RA, Khromykh AA. 2008. Single-round infectious particles enhance immunogenicity of a DNA vaccine against West Nile virus. Nat Biotechnol 26:571-577