Preliminary Studies For Constructing HCMV Multi-epitope DNA Vaccine

Aim: The rate of hCMV infection is very high. HCMV generally persistents latent in thebody, however, active infection occurs when the immunity of organism descends, and thencauses severe diseases. So far there are not any effective therapies. It is necessary to developvaccines which can restore the immunity of organism. Traditional prophylactic vaccines can notinduce effective immune responses in the infected individuals, it is imperative to develop noveltherapeutic vaccines to sufficiently induce CD8⁺T cell immunity so as to clear up the hCMVlatent in the organism. Epitope is the minimal functional unit to induce immune response, if thenumber of hCMV specific T cells can be augmented by peptide epitope vaccines, the hCMVlatent in the organism can be eliminated. Because of restriction of TCR recognition, one singlepeptide epitope can not be recognized by a majority of population. To develop a multi-epitopevaccine which can be used in most population, we combined six epitopes restricted by highfrequency HLAs to construct multi-epitope DNA eukaryotic expressing plasmids, thentransfected B cells in the PBMCs of hCMV positive and HLA-A*0201-restricted individual withthe multi-epitope DNA recombinant plasmid and analyzed the activation of hCMV specific Tcells. In this study we provided primary data for the further development of hCMV vaccines andexperimental models for the digital and visual recognition of T cell.Methods: The DNA sequence of hCMV_6epitopes, which include HLA-A2, All andA24-restricted epitopes was synthesized through overlap PCR, and inserted into the vectorplasmid pVAX1 to construct eukaryotic expressing plasmid. For the studies of evaluatinghCMV_6epitopes expression of transfected cells and the activation of T cells, pEGFP-hCMV_6epitopesand pVAX1-hCMV_6epitopes-GFP were constructed by inserting hCMV_6epitopes and the reportinggene GFP DNA sequence into vector plasmids pEGFP-N1 and pVAXl-hCMV_6epitopesrespectively. Three plasmids mentioned above were identified by PCR identifications,dual-enzyme digest, gel electrophoresis and sequencing. Hela cells were transfected respectivelywith plasmids pEGFP-hCMV_6epitopes and pVAX1-hCMV_6epitopes-GFP by lipofectamine to test theexpression of protein encoded by target sequence in eukaryotic cells. The B cells in theindividual of hCMV positive and HLA-A*0201-restricted were transfected with plasmidpEGFP-hCMV_6epitopes by Nucleofetor TM device, the transfection rate in the B cells and the activation rate of hCMV specific T cells were analyzed by FCS.Results: The multi-epitope DNA sequence hCMV_6epitopes had been combined successfully.pVAX1-hCMV_6epitopes, pVAX1-hCMV_6epitopes-GFP and pEGFP-hCMV_6epitopes were constructedcorrectly, hCMV_6epitopes DNA and GFP DNA respectively inserted into plasmid vectors correctly.Recombinant plasmids pVAX1-hCMV_6epitopes and pEGFP-hCMV_6epitopes were able to expresstarget proteins in Hela cells, showing green fluorescent. When the B cells in the PBMCs ofhCMV positive and HLA-A*0201-restricted individual were transfected with recombinantplasmid pEGFP-hCMV_6epitopes, tranfecting rate was only 0.97%. But they were able to inducerecognitions and activations of hCMV specific CTLs, with CD69 expression rate reaching11.48%. While in the control group, the tranfection rate of pEGFP-N1 in B cells was 37.44%,and the activation rate of hCMV specific CTL was only 2.01%. It showed differencesignificantly between the two groups.Conclusions: hCMV_6epitopes DNA was synthesized successfully. Recombinant plasmidspVAX1-hCMV_6epitopes, pVAX1-hCMV_6epitopes-GFP and pEGFP-hCMV_6epitopes were constructedsuccessfully and the target proteins could be expressed in the Hala cells. The B cells in thePBMCs of hCMV positive and HLA-A*0201-restricted individual transfected with plasmidpEGFP-hCMV_6epitopes could correctly express, process and present hCMV specific antigenepitopes, and could induce the activations of hCMV specific T cells.