Kunjin virus replicon vaccine vectors induce protective CD8+ T-cell immunity

Abstract

The ability of self-replicating RNA (replicon) vaccine vectors derived from the Australian flavivirus Kunjin (KUN) to induce protective alphabeta CD8+ T-cell responses was examined. KUN replicons encoding a model immunogen were delivered by three different vaccine modalities: (i) as naked RNA transcribed in vitro, (ii) as plasmid DNA constructed to allow in vivo transcription of replicon RNA by cellular RNA polymerase II (DNA based), and (iii) as replicon RNA encapsidated into virus-like particles. A single immunization with any of these KUN replicon vaccines induced CD8+ T-cell responses at levels comparable to those induced by recombinant vaccinia virus encoding the same immunogen. Immunization with only 0.1 microg of DNA-based KUN replicons elicited CD8+ T-cell responses similar to those seen after immunization with 100 microg of a conventional DNA vaccine. Naked RNA immunization with KUN replicons also protected mice against challenges with recombinant vaccinia virus and B16 tumor cells. These results demonstrate the value of KUN replicon vectors for inducing protective antiviral and anticancer CD8+ T-cell responses.

FIG. 1.

Schematic representation of the replicon gene expression and delivery systems based on the KUN replicon (A) and of the KUN replicon constructs encoding Mpt (B). (A) KUN replicon RNA can be transcribed in vitro from plasmid DNA incorporating the bacteriophage SP6 promoter and delivered as naked RNA via transfection or injection (25, 52). It can also be synthesized in vivo by cellular RNA polymerase II from transfected or injected plasmid DNA incorporating the cytomegalovirus (CMV) promoter (53). In addition, KUN replicon RNA can be first packaged into VLPs by using a packaging system described previously (27) and then delivered by infection (52). Regardless of the delivery mode, once in the cytoplasm, KUN replicon RNA initiates self-replication, leading to production of multiple RNA copies, translation of which results in enhanced production of encoded HG products. CAP represents the cap structure added to the 5′ terminus of replicon RNA molecules either synthetically during in vitro transcription or naturally during in vivo transcription or KUN RNA replication, to ensure efficient initiation of translation. (B) KUN replicon constructs contain sequences required for KUN RNA replication, i.e., 5′ and 3′ UTRs; sequences coding for the first 20 amino acids of the KUN C protein (C20) and the last 22 amino acids of the KUN E protein (E22); and the entire nonstructural region coding for the KUN nonstructural proteins NS1 (shown as NS1) and NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (shown as NS2-NS5). In addition, constructs contain either SP6 or CMV promoters upstream of the KUN 5′ UTR to drive in vitro or in vivo RNA transcription, respectively, and the antigenomic sequence of the hepatitis delta virus ribozyme (HDVr) and the polyadenylation signal from simian virus 40 (pA) inserted downstream of the 3′ UTR to ensure production of KUN replicon RNA molecules with precise 3′ termini for efficient initiation of replication. To allow cytoplasmic release of Mpt peptide from the KUN C20 and E22 peptides, the constructs contain two copies of the 2A autoprotease of the foot-and-mouth disease virus (FMDV2A), one upstream and another downstream of the Mpt sequence. Pro and Leu variants contain amino acid Pro or Leu at position 250 in the KUN NS1 gene product, respectively.

FIG. 2.

Evidence of replication of KUN replicon constructs encoding Mpt in transfected BHK21 cells. (A) IF analysis of BHK21 cells transfected with DNA-based or RNA-based KUN replicons encoding Mpt. Cells on coverslips were transfected with KUN replicon-polytope DNAs (DNALeuMpt and DNAProMpt) or RNAs (RNALeuMpt and RNAProMpt) and assayed for expression of KUN NS3 proteins by IF at 48 h after transfection. (B) Northern blot of total RNA from BHK21 cells transfected with KUN replicon-polytope DNAs and RNAs. Transfection of cells, RNA isolation, and Northern blotting were performed as described in Materials and Methods. The left panel shows results of Northern blotting with a KUN-specific probe representing the KUN 3′ UTR, and the right panel shows results of Northern blotting of the same membrane rehybridized with the probe representing the Mpt sequence. Samples from DNA-transfected cells contain 6 μg of total RNA, and samples from RNA-transfected cells contain 12 μg of total RNA. pKUNβRep2(dGDD) lines show RNA samples isolated from BHK cells transfected with pKUNβRep2(dGDD) DNA producing nonreplicating KUN replicon RNA encoding β-galactosidase (53). The position of this RNA in the left panel is indicated by the asterisk. Control RNA represents 10 ng of in vitro-transcribed RNALeuMpt RNA.

FIG. 3.

Cytotoxic T-lymphocyte responses specific for YPHFMPTML (YPH), RPQASGVYM (RPQ), TYQRTRALV (TYQ), and SYIPSAEKI (SYI) epitopes induced by immunization with various KUN replicon-based vectors encoding Mpt immunogen. (A) ELISPOT analysis of BALB/c (n = 4 per group) mouse splenocytes following immunization with the indicated vaccines. (B) ⁵¹Cr release assay of restimulated splenocyte populations from BALB/c mice (n = 3 per group) immunized with VLPProMpt. A standard 6-h Cr release assay was performed using labeled target cells sensitized with (filled squares) or not sensitized with (empty squares) the indicated peptides. SE, standard error.

FIG. 4.

Cytotoxic T-lymphocyte responses specific for SYIPSAEKI induced after immunization with different doses of DNA-based KUN replicons encoding the Mpt. DNALeuMpt and DNAProMpt were serially diluted in PBS, and the indicated doses were injected into quadriceps muscles of BALB/c mice (n = 4 per group). Mice were sacrificed 2 weeks later, and cytotoxic T-lymphocyte responses were measured by the ELISPOT assay. SE, standard error.

FIG. 5.

Recombinant vaccinia virus and B16-OVA tumor challenge following RNALeuMpt vaccination. (A) Recombinant vaccinia virus challenge. BALB/c mice (n = 6 per group) were vaccinated once with RNALeuMpt or RNALeuControl RNAs and then challenged with rVVMpt. Vaccinia virus titers in ovaries were measured at day 4 after infection. (B) B16-OVA challenge. BALB/c mice (n = 6 per group) were immunized twice with either RNALeuControl or RNALeuMpt RNAs or once with recombinant vaccinia virus encoding Mpt (rVVMpt) and were then challenged with B16-OVA cells. The top graph shows the average tumor area for 12 tumor sites for each of the three groups of mice. The tumor growth lines stop on the date when the first tumor in a group reached the size of 15 by 15 mm, requiring the animal's euthanatization. The bottom graph represents Kaplan-Meier survival curves of the three groups of mice. Mice were euthanatized when the tumor size reached 15 by 15 mm. SE, standard error.