Introduction

The use of nucleic acid vaccines is being investigated worldwide as a viable option for the prevention of infectious diseases (1,2,3,4,; reviewed in 5,6). A major advantage of nucleic acid vaccines results from endogenous expression of the encoded protein(s) that may result in either direct processing of the protein in an antigen presenting cell or in cross presentation (7). Thus, expressed proteins are processed and presented through major histocompatibility complex (MHC) class I and class II pathways and stimulate CD4+ and CD8+ T-lymphocytes (1,8,9,10). DNA vaccines, first introduced in 1988 (11), are able to generate good immunity in small animals, particularly in common laboratory animals, but with a few exceptions have not been equally effective in non-human primates and humans (12). Furthermore, the risk of integration of the introduced DNA resulting in insertional mutagenesis is a recognized, although perhaps only theoretical, risk (13).

An alternative is to use RNA vaccines that retain the advantages of DNA vaccines without the disadvantages, including the aforementioned insertional mutagenesis and the possibility of inducing autoimmunity. RNA-based vaccines can be delivered in a number of different formulations (14). In particular, RNA replicons derived from positive-sense RNA viruses have considerable potential as vaccine delivery vehicles (15) and may be delivered as DNA, naked RNA, or RNA that is packaged into virus-like particles. We have previously used Kunjin virus replicon RNA to generate cytotoxic T cell (CTL) responses in mice (16), and this replicon shows promise as a vaccine delivery vehicle (17,18). RNA vaccines have the potential to induce strong cell mediated immunity (CMI) including CTL as well as antibody responses that would be advantageous in combating viral infections (19,20).

A general assumption regarding RNA vaccines, however, is that they are unstable and expensive. A recent review (14) presented a convincing argument that since it is possible to synthesize large amounts of RNA in vitro from a DNA template and because the bulk of the costs of a vaccine are related to regulatory affairs, this assumption is incorrect. Furthermore, it is axiomatic that RNA is only unstable in the presence of RNases and consequently it should be possible to synthesize and store RNA for a considerable period if contact with nucleases can be avoided.

Trehalose is a diglucose molecule that accumulates in certain organisms that naturally survive dehydration (21) and has demonstrated a protective effect when freezing mammalian cells (22). The addition of trehalose to reverse transcription PCR (RT-PCR) or ligase reactions can result in thermo-stabilization of enzymes and resolve secondary structures of RNA resulting in increased yield of long cDNA transcripts (23).

Consequently, the aim of this study was to develop a protocol for the synthesis and storage of the RNA replicon which was devoid of protein, DNA, and nucleases, and to examine whether the addition of trehalose increased RNA stability as a first step toward the more general introduction of self-replicating RNA vaccines.

Materials and Methods

In Vitro Transcription of RNA

The Kunjin replicon vector plasmid encoding either the hepatitis C virus (HCV) NS3 or HCV core proteins (24) was linearized using the restriction enzyme XhoI according to the manufacturer's recommendations (Promega, Sydney, Australia), and the DNA purified using High Pure PCR purification kit (Roche, West End, Australia). RNA was transcribed in vitro in a 20-µL reaction mix using the SP6 mMessage mMachine kit containing a 7-mG cap analog (Ambion, Thebarton, Australia) with the addition of 1 mM guanosine-5′-triphosphate (GTP), as recommended by the manufacturer. After different DNase treatments (described in the Results section), the RNA was purified using RNeasy kit spin purification columns (Qiagen, Doncaster, Australia), eluted from the column with nuclease-free water, and adjusted to a final concentration of 50 µg/mL in nuclease-free water (Promega). The quality and integrity of the RNA was assessed by gel electrophoresis in a 1% agarose TAE gel and visualized under UV light using 1 µg/mL ethidium bromide.

DNase Treatment

The efficiency of removal of the DNA template from the RNA preparation was compared using three sources of DNase: a) DNase I supplied with the mMessage mMachine in vitro transcription kit (Ambion), b) TURBO-DNase (Ambion) and c) the DNase-set for use with the spin purification columns (QIAGEN). These were each used according to the manufacturer's instructions individually or in combination as described in Results. The integrity of the treated RNA was assessed on a 1% agarose Tris-acetate-EDTA (TAE) gel and visualized under UV light using 1 µg/mL ethidium bromide.