DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response

Optimization of DNA vaccine delivery improves the potency of the immune response and is crucial to clinical success. Here, we inquired how such optimization impacts the magnitude of the response, its specificity and type. BALB/c mice were DNA-immunized with two model immunogens, HIV-1 protease and reverse transcriptase by intramuscular or intradermal injections with electroporation. DNA immunogens were co-delivered with DNA encoding luciferase. Delivery and expression were monitored by in vivo bioluminescence imaging (BLI). The endpoint immune responses were assessed by IFN-γ/IL-2 FluoroSpot, multiparametric flow cytometry and antibody ELISA. Expression and immunogenicity were compared in relation to the delivery route. Regardless of the route, protease generated mainly IFN-γ, and reverse transcriptase, IL-2 and antibody response. BLI of mice immunized with protease- or reverse transcriptase/reporter plasmid mixtures, demonstrated significant loss of luminescence over time. The rate of decline of luminescence strongly correlated with the magnitude of immunogen-specific response, and depended on the immunogenicity profile and the immunization route. In vitro and in vivo BLI-based assays demonstrated that intradermal delivery strongly improved the immunogenicity of protease, and to a lesser extent, of reverse transcriptase. Immune response polarization and epitope hierarchy were not affected. Thus, by changing delivery/immunogen expression sites, it is possible to modulate the magnitude, but not the type or fine specificity of the induced immune response.

优化DNA疫苗递送方式可提升免疫应答效力，对临床成功至关重要。本研究旨在探究此类递送优化如何影响免疫应答的强度、特异性与类型。实验以BALB/c小鼠为对象，采用肌内注射或皮内注射联合电穿孔的方式，以两种模式免疫原——HIV-1蛋白酶（HIV-1 protease）与逆转录酶（reverse transcriptase）对小鼠进行DNA免疫。免疫原DNA与编码荧光素酶（luciferase）的DNA共递送，通过体内生物发光成像（in vivo bioluminescence imaging, BLI）监测递送过程与蛋白表达水平。采用干扰素γ/白细胞介素2（IFN-γ/IL-2）荧光免疫斑点法（FluoroSpot）、多参数流式细胞术（multiparametric flow cytometry）与抗体酶联免疫吸附试验（antibody ELISA）评估终点免疫应答，并基于递送途径对比免疫原的表达情况与免疫原性。结果显示，无论采用何种递送途径，HIV-1蛋白酶主要诱导干扰素γ应答，而逆转录酶则主要引发白细胞介素2应答与抗体应答。对注射HIV-1蛋白酶/报告质粒混合物或逆转录酶/报告质粒混合物的小鼠进行BLI检测，发现随时间推移生物发光信号显著衰减。该信号的衰减速率与免疫原特异性应答的强度呈显著正相关，且受免疫原性特征与免疫接种途径的影响。基于BLI的体外与体内实验结果表明，皮内递送可显著提升HIV-1蛋白酶的免疫原性，对逆转录酶的免疫原性提升效果则相对较弱。免疫应答极化与表位层级未受递送途径影响。综上，通过改变递送途径或免疫原表达位点，可调节诱导产生的免疫应答强度，但无法改变其类型与精细特异性。