Pokholenko I. Construction of model DNA-vaccine and ways to enhance its immunogenic properties

The thesis is devoted to developing approaches to enhance the immunogenicity of recombinant plasmid constructs containing the chimeric gene for the E2 glycoprotein of classical swine fever virus by optimizing the composition of model marker DNA vaccine. A series of recombinant plasmids was created, containing chimeric genes based on the SacI-EcoRI fragment of the E2 fragment of classical swine fever virus, placed under the transcriptional control of the promoter/enhancer of early/immediate genes of human cytomegalovirus in the eukaryotic cassette, and located between inverted terminal repeats of human adeno-associated virus-2 (ITR AAV-2). In a transient expression system, it was demonstrated that the introduction of ITR AAV-2 sequences into the recombinant vector leads to an increase in the accumulation of chimeric antigen in transfected cells of the HEK293 line. The amount of EGFP/E2 in the cells transfected with plasmid pTR-EGFP/E2 was ~113 ± 21 ng per 105 viable cells per 72 h, while transfecting cells with plasmid pEGFP/E2 was ~15 ± 3 ng per 105 viable cells per 72 h. It should be noted that the observed difference in the expression levels of the protein was not due to the decrease in metabolic activity of cells or more significant cytotoxicity of the pEGFP/E2 and polyethyleneimine complexes. We demonstrated that the introduction of ITR AAV-2 into the plasmid vector did not significantly affect the duration of expression and persistence of the transgene in HEK293 cells in the absence of selective pressure. It was found that the recombinant plasmid constructs pTR-BKneo- and pBS-BK can induce the production of antibodies specific to the E2 fragment CSFV in mice. For the first time, it has been demonstrated that the introduction of ITR AAV-2 sequences into the vector construct of a model marker DNA vaccine against CSF leads to an increase in both the intensity and duration of the humoral immune response to immunization. The use of heterologous booster vaccination could significantly increase the immunogenicity of candidate vaccines, reduce their reactogenicity, and the likelihood of the development of an immune response to the vector. Our data indicate that booster immunization with a recombinant fragment of the E2 protein of CSFV performed after double immunizations with the plasmid vector pTR-BKneo- leads to a significant increase in the synthesis of antibodies specific to the target antigen, compared with three injections of pTR-BKneo-. One of the promising strategies to enhance the immune response to DNA vaccination, which has been considered recently, is the introduction of so-called "gene adjuvants" - recombinant vectors that carry genes encoding cytokines, chemokines, or co-stimulatory molecules. In this work, we created recombinant constructs pmIL12-TR and pmIL2-TR, which contain mouse genes interleukin-12 and interleukin-2, respectively, under the transcriptional control of the promoter/enhancer of early/immediate human cytomegalovirus genes in the expression cassette located between ITR AAV-2. We also created their derivatives pTR-mIL2 / EGFP and pTR-mIL2 / EGFP, which contain the chimeric genes of IL2 / EGFP and IL12 / EGFP proteins. In vitro study of the functional activity of the generated vectors showed that chimeric proteins are expressed from the generated recombinant constructs in HEK293 cells and are partially secreted from the cells into the conditioned medium. The combined administration of pTR-BKneo- and genes of murine interleukin-2 and chimeric interleukin-12 in the created recombinant constructs enhances the humoral immune response to E2 CSFV. Moreover, the enhancement effect is the most pronounced in the group of animals that received 10 μg of pmIL12-TR and pmIL2-TR in combination with pTR-BKneo-.