The Development of Methods for the Production of New Molecular Vaccines and Appropriate RNA Fragments to Counteract Unwanted Genes: A Pilot Study.

The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations.

Upregulation of Natural Killer Cells Proliferation by Cytokine Stimulation.

Natural killer (NK) cells can discriminate between normal and cancer cells and are known to directly recognize and kill malignant cells or induce apoptosis. Thus, activation of NK cells is considered as a promising strategy for cancer treatment. However, clinical application has been somewhat limited because of difficulties in the preparation of sufficient number of highly cytotoxic/activated NK cells in vitro. We used cytokine stimulation to provide a suitable environment (activating receptor-ligand interactions) for the expansion of NK cells. This method potently expanded NK cells, and the final product was composed of highly proliferating NK cells. The expanded NK cells showed significant upregulation of various activation receptors such as CD69 and NKG2D. The latter is a particularly important receptor for triggering NK cell responses toward tumor cells.