Conventional vaccination/immunisation
Historically, protocols for vaccination or immunisation against proteins have required the availability of purified immunogens. This commonly involved time-consuming and inefficient procedures to purify the immunogen from cell or tissue extracts. Alternatively, selected peptide sequences have been synthesised as immunogens. Extensive purification can damage labile proteins, and synthetic peptides are non-glycosylated, both of which compromise the authenticity of the immunogen. With the advent of recombinant DNA technology in the late seventies, it became possible to clone the protein-coding sequences of genes into expression vectors, and to express the proteins either in bacteria or in eukaryotic cell culture systems. Recent progress in the sequencing of entire genomes from a wide range of species means that the coding sequences of literally hundreds of thousands of proteins have been identified, and these are therefore amenable to expression using routine recombinant DNA and protein technologies. However, although the strategies to clone and express the gene of interest are fairly straightforward, considerable time and effort is usually required to devise protocols to purify the recombinant-derived protein away from the bacterial or host cell proteins.
DNA vaccination/immunisation
DNA vaccination offers a way to avoid the time and cost involved in extracting and purifying recombinant-derived proteins for vaccination/immunisation purposes, while at the same time ensuring that immunogens are properly folded and post-translationally modified. With this technology, protein-coding sequences are cloned into eukaryotic (usually mammalian) expression vectors, which are introduced into host cells using chemical or electrical transfection procedures. Animals are vaccinated by the subcutaneous or intra-muscular implantation of stably transfected cells, and immunisation occurs in response to the expression of the gene of interest using the host cell's own transcriptional and translational machinery.
Disadvantages of current DNA vaccination/immunisation
Current DNA vaccination technologies suffer from several disadvantages. Although the immunogens appear to be correctly expressed and to undergo appropriate post-translational modification, the overall amount of protein produced is low as a result of inefficient expression and post translational gene silencing. Additionally, the foreign proteins accumulate in the cell cytoplasm where they are detected by the cell's “quality control apparatus”. As a result, they are earmarked for destruction by the covalent attachment of one or more ubiquitin molecules and then cleaved into small peptides of 7-13 amino acids in the proteosome, the cells major proteolytic organelle. These cleavage products become bound to MHC class I proteins which become displayed on the cell surface, triggering cell-mediated immune response mechanisms. The production of antibody (i.e. the humoral immune response) is therefore a minor event with current DNA vaccination technologies.
Indeed, with current DNA vaccination technologies, antibody production occurs is essentially a by-product, the result of cell lysis releasing un-degraded protein into the extra-cellular environment, where it induces an HLA II response. A further disadvantage is that the animal is constantly exposed to the immunogen, in contrast to typical immunisation schedules where the immunogen is administered at intervals to enhance the humoral response. Continuous exposure to immunogen is usually detrimental to optimal antibody production as it could lead to the situation where antibody class is never switched from IgM to the more useful IgG molecule. To optimise antibody production by DNA vaccination, it would be advantageous if the MHC class I aspects were inhibited and HLA II mediated responses accentuated. This could be achieved by over-expressing intact protein that is the actively secreted from the cell or displayed in its native state on the cell surface. Therefore, it is desirable to:
|
·
|
Maximise target protein production
|
|
·
|
Minimise proteosomal degradation
|
|
·
|
Direct target proteins to the secretory pathway
|
|
·
|
Activate MHC class II response Control target protein expression (click “More” for next page)
|
