DNA and RNA-based vaccines: principles, progress and prospects

Abstract

DNA vaccines were introduced less than a decade ago but have already been applied to a wide range of infectious and malignant diseases. Here we review the current understanding of the mechanisms underlying the activities of these new vaccines. We focus on recent strategies designed to enhance their function including the use of immunostimulatory (CpG) sequences, dendritic cells (DC), co-stimulatory molecules and cytokine- and chemokine-adjuvants. Although genetic vaccines have been significantly improved, they may not be sufficiently immunogenic for the therapeutic vaccination of patients with infectious diseases or cancer in clinical trials. One promising approach aimed at dramatically increasing the immunogenicity of genetic vaccines involves making them 'self-replicating'. This can be accomplished by using a gene encoding RNA replicase, a polyprotein derived from alphaviruses, such as Sindbis virus. Replicase-containing RNA vectors are significantly more immunogenic than conventional plasmids, immunizing mice at doses as low as 0.1 microg of nucleic acid injected once intramuscularly. Cells transfected with 'self-replicating' vectors briefly produce large amounts of antigen before undergoing apoptotic death. This death is a likely result of requisite double-stranded (ds) RNA intermediates, which also have been shown to super-activate DC. Thus, the enhanced immunogenicity of 'self-replicating' genetic vaccines may be a result of the production of pro-inflammatory dsRNA, which mimics an RNA-virus infection of host cells.

Fig. 1. Transfection of host cells with plasmid DNA

Plasmid (◯) is actively or passively taken up by host cells. Antigen (ν) produced by transfected myocytes can be taken up by bone marrow (BM)-derived APCs. Alternatively, BM-APC can be transfected directly. Antigen-bearing APC then can process and present peptides complexed with MHC-molecules to the immune system after migrating to lymphoid tissue.

Fig. 2. ‘Super-activation’ of APC after vaccination with genetic vaccines

APC are initially stimulated in the injected tissue by either needle or gene-gun delivery of plasmid. Immunostimulatory sequences (ISS) stimulate host APCs, T cells and NK cells. APC interact with T cells through both TCR/MHC and CD40/CD40L, triggering the upregulation of B7 expression on the APC. ISS also polyclonally stimulate B-cells and induce the release of IL-6 and IL-12, further promoting a Th1 response.

Fig. 3. Self-replicating genetic vaccines

The first product of the self-replicating RNA is a four-subunit-replicase which uses the (+) strand RNA as a template to make (−) strand RNA and more copies of full length (+) strand ‘genomic’ RNA and (+) strand ‘subgenomic’ mRNA for the encoded antigen. Due to the high number of RNA-copies, the main product of the transfected cells becomes the encoded antigen. The host cell eventually undergoes apoptosis.

Fig. 4. Potential factors contributing to the high immunogenicity of self-replicating genetic vaccines

(Starting in the upper centre and moving clockwise): Accumulation of antigen in the transfected cell can result in highly efficient MHC-I-loading. A number of ‘danger signals’ may be generated such as interferon production and interferon release from infected cells resulting from the presence of dsRNA. Interferon may also be produced by bystander cells in response to dsRNA released from dead and lysed transfected cells. Heat shock proteins (HSP) have also been shown to be produced in response to the presence of dsRNA in the cells. Ingestion of antigen-loaded apoptotic cells by APCs can also result in the elicitation of powerful immune responses. Finally, the local release of large amounts of antigen at the site of injection by transfected cells may be fed into resident APC.

Fig. 5. dsRNA might be a central element in the immunogenicity of replicase-based genetic vaccines

dsRNA is produced in cells infected with RNA-viruses, but is also expected to be an intermediate in the replicase-mediated duplication of mRNA after delivery of ‘self-replicating’ genetic vaccines. dsRNA is a potent inducer of interferon, which in turn induces the expression of PKR and 2-5A synthetase. These enzymes are activated by dsRNA and mediate at least some of the cellular effects associated with viral infection, including apoptotic cell death, aimed at preventing viral spread. The ‘danger signals’ resulting from the activation of these two enzymes could account for the increased immunogenicity of replicase-based genetic vaccines, providing an adjuvant-effect.