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.
Recombinant Kunjin replicon virus-like particle (VLP), vaccinia virus (rVV) and DNA vaccines were tested in a large series of prime-boost vaccinations using interferon (IFN)c ELISPOT assays that reflected effector (E), effector memory (EM) and central memory (CM) responses. All vaccine constructs encoded the murine polytope immunogen and responses to four CD8 T-cell epitopes (TYQRTRALV, SYIPSAEKI, YPHFMPTNL and RPQASGVYM) were measured. VLP/rVV out performed (by 14-to 20-fold) DNA/rVV for induction of CM responses, whereas EM responses were only marginally increased. DNA/VLP induced more EM, but not CM responses, than VLP alone, illustrating that DNA priming is not universally beneficial. rVV/VLP gave comparable results to VLP/rVV combinations, although the former induced approximately threefold more E responses, illustrating the utility of poxvirus priming in this setting. Although higher doses of VLP and rVV increased responses after single immunizations, such dose increases provided only marginal benefit in heterologous prime-boost settings. Triple combinations also provided no benefit over two vaccinations. DNA vaccination was associated with broad CM, but not EM responses, and the breadth of EM and E responses was significantly improved by increasing viral vector dose. VLP/rVV, rather than DNA priming, induced T cells with consistently high IFNc secretion profiles across all ELISPOT measures. Vector-specific CD8 T-cell responses generally correlated well with immunogen-specific responses, although, as expected, single use of each vector reduced the relative levels of vectorspecific responses. These experiments illustrate the utility of replicons in heterologous prime-boost vaccinations, and illustrate the diversity of data that can be obtained from ELISPOT analyses. Keywords: ELISPOT; prime-boost; replicon; vaccinia Heterologous prime-boost vaccination, where the same immunogen is delivered sequentially by different delivery modalities, has been widely adopted as a strategy for infectious diseases and cancer. 1-4 Heterologous prime-boost vaccination tend to induce substantially higher levels of CD8 T-cell responses than those obtained by repeated vaccination using a single modality (homologous prime-boost vaccination). 5,6 Priming with naked DNA followed by boosting with a viral vector (often a poxvirus-based modality) has been extensively studied 3 and many human immunodeficiency virus trials are testing, or have tested, this strategy. 6,7 DNA priming is believed to convey a number of advantages such as (i) effective generation of central memory (CM) CD8 T cells that can be efficiently boosted by viral vectors to produce a large pool of memory cells, [8][9][10] (ii) induction of CD8 T cells with enhanced interferon (IFN)g secretion profiles 11,12 and (iii) induction of broad CD8 T-cell responses directed evenly at multiple epitopes. 7,13,14 Unfortunately, DNA vaccination has not emerged to be particularly immunogenic in humans, 15,16 with multiple injections of high doses of DNA usually required to obtain ...
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