Kunjin replicon-based simian immunodeficiency virus gag vaccines

Abstract

An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines encoded the wild type gag gene, an RNA-optimised gag gene, a codon-optimised gag gene and a modified gag-pol gene construct. The vaccines behaved quite differently for induction of effector memory and central memory responses, for mediation of protection, and with respect to insert stability, with the SIV gag-pol vaccine providing the optimal performance. These results illustrate that for an RNA-based vector the RNA sequence of the antigen can have profound and unforeseen consequences on vaccine behaviour.

Fig. 1

Schematic representation of RNA Kunjin replicon constructs encoding the SIV gag and gag-pol antigens. The Kunjin replicon RNA contained the SP6 RNA promoter, the 5′ and 3′ untranslated regions (UTR), the first 20 amino acids of the Kunjin core protein (C20), ubiquitin (Ub) to allow N-terminal cleavage by ubiquitin hydrolase, the SIV gag or gag-pol genes, the 2A autoprotease sequence of the foot and mouse disease virus (FMDV2A) to allow C-terminal cleavage, the last 22 amino acids of the Kunjin envelope protein (E22), the non-structural proteins (NS1-NS5) that are responsible for RNA replication, the anti-genomic sequence of the hepatitis delta virus ribozyme (HDVr) and the polyadenylation signal from simian virus 40 (pA) . The wild type vaccine construct (WT) contains the full-length native SIV mac239 gag gene. The DX and OPT constructs contain RNA- and codon-optimised versions of this gene, respectively, with the positions of the codon changes schematically represented by vertical lines below the gene box. DX sequence was provided by Dr. Felber and 6% of nucleotides have been changed. The OPT sequence is available from the AIDS Research and Reference Reagent Program web site (catalogue #9422) and 25% of nucleotides have been changed. The Gag-pol construct contains wild-type matrix and capsid from gag and in-frame reverse transcriptase from pol. The gag-pol construct contained a second FMDV2A site in place of the Ub sequence at the N-terminus of gag.

Fig. 2

Analysis of SIV gag expression from the four different Kunjin SIVgag constructs. (A) Analysis of SIV Gag protein expression after infection of Vero cells with Kunjin VLPs. Cells were lysed after 48 h incubation and total cell lysates analysed by western blot using anti-Gag antibody (55-2F12). (B) As for A except that BHK cells were transfected with RNA for each of the vaccine constructs. (C) Comparison of SIV Gag and HIV Gag protein expression after infection of Vero cells as above with WT and HIV-1 VLPs using the AG3.0 antibody. (D) Comparison of the reactivity of AG3.0 in western analysis with serial dilutions of purified recombinant SIV Gag and recombinant HIV Gag proteins.

Fig. 3

T cells responses induced after immunisation with Kunjin-SIVmac239 gag VLP vaccines. (A) Measurement of Gag-specific responses by ex vivo ELISPOT assays. BALB/c mice (n = 3 or 4 per group) were immunised twice with 10⁶ IU of each of the indicated Kunjin VLP vaccines. There were two control groups, one received 10⁶ IU of a control VLP encoding an irrelevant antigen (control VLP) and the other received no vaccination (control naïve). Ten weeks after the second immunisation, the mice were sacrificed and the splenocytes were assayed for Gag-specific T cell responses by ex vivo IFNγ ELISPOT using six pools of overlapping peptides spanning either the SIV Gag protein for SIV gag vaccinated animals, or the HIV-1 Gag protein for HIV-1 gag vaccinated animals. Error bars illustrate the variation in responses to each pool. (B) Measurement of Gag-specific responses by cultured IFNγ ELISPOT assays. The splenocytes from the animals described in (A) were also cultured for 6 days with the pooled SIV or HIV Gag peptides, prior to the ELISPOT assay. (C) Measurement of NS3-specific responses by ex vivo IFNγ ELISPOT assays. The splenocytes from the animals described in (A) were also used in an ex vivo ELISPOT assays using the NS3 CD8 T cell peptide epitope, GYISTRVEL. (D) Measurement of NS3-specific responses by cultured IFNγ ELISPOT assays. The splenocytes from the animals described in (A) were cultured for 6 days with GYISTRVEL peptide prior to an ELISPOT assay using the same peptide.

Fig. 4

Analysis of insert deletions in the Kunjin SIV gag vaccines. (A) Non-denaturing agarose gel and ethidium bromide staining of in vitro transcribed RNA for the indicated constructs prior to transfection of packaging cells and VLP manufacture. Note the double-stranded DNA markers do not accurately illustrate the size of the transcribed single-stranded RNA species, which are about 10.5 kb. (B) Vero cells were infected with WT (lane 2), DX (lane 3), empty VLPs (lane 4) or nothing (lane 5) and RT-PCR performed on extracted RNA using primers flanking the multiple cloning site region into which the gag genes were inserted for the former two constructs. Lane 1 shows the markers and lane 6 no template control. (C) Sequencing of the ≈180 bp fragment from DX transfected cells. The insert deletion is schematically represented with the sequence either side of the insert deletion site given beneath.

Fig. 5

Challenge of Kunjin SIV gag VLP vaccinated mice with A20 cells expressing SIV mac239 Gag. (A) Western blot of A20-EGFP-SIVgag and parental A20 cells with anti-SIV Gag antibody (55-2F12) and anti-GFP antibody. 15 μg of total protein was loaded in each well. (B) Kaplan Meier plot of survival. Groups of mice (n = 4–6) were immunised twice with the indicated VLP vaccines and were then challenged ≈9 months later with A20-EGFP-SIVgag cells. Animals were euthanased when tumours reached 100 mm². (C) Mean growth curves for the tumours from the same experiment. When an animal was euthansed a value of 100 mm² was included for that animal in the mean for all subsequent time points.