Kanuma Tomohiro, Yamamoto Takuya, Kobiyama Kouji, Moriishi Eiko, Masuta Yuji, Kusakabe Takato, Ozasa Koji, Kuroda Etsushi, Jounai Nao, Ishii Ken J
Laboratory of Adjuvant Innovation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
Laboratory of Vaccine Science, World Premier International Immunology Frontier Research Center, Osaka 565-0871, Japan.
J Immunol. 2017 Jun 15;198(12):4707-4715. doi: 10.4049/jimmunol.1600731. Epub 2017 May 15.
DNA vaccines are attractive immunogens for priming humoral and cellular immune responses to the encoded Ag. However, their ability to induce Ag-specific CD8 T cell responses requires improvement. Among the strategies for improving DNA vaccine immunogenicity are booster vaccinations, alternate vaccine formulations, electroporation, and genetic adjuvants, but few, such as extracellular vesicles (EVs), target natural Ag delivery systems. By focusing on CD63, a tetraspanin protein expressed on various cellular membranes, including EVs, we examined whether a DNA vaccine encoding an Ag fused to CD63 delivered into EVs would improve vaccine immunogenicity. In vitro transfection with plasmid DNA encoding an OVA Ag fused to CD63 (pCD63-OVA) produced OVA-carrying EVs. Immunizations with the purified OVA-carrying EVs primed naive mice to induce OVA-specific CD4 and CD8 T cells, whereas immunization with EVs purified from cells transfected with control plasmids encoding OVA protein alone or a calnexin-OVA fusion protein delivered into the endoplasmic reticulum failed to do so. Vaccinating mice with pCD63-OVA induced potent Ag-specific T cell responses, particularly those from CD8 T cells. CD63 delivery into EVs led to better CD8 T cell responses than calnexin delivery into the endoplasmic reticulum. When we used a mouse tumor implantation model to evaluate pCD63-OVA as a therapeutic vaccine, the EV-delivered DNA vaccination significantly inhibited tumor growth compared with the control DNA vaccinations. These results indicate that EV Ag delivery via DNA vaccination offers a new strategy for eliciting strong CD8 T cell responses to the encoded Ag, making it a potentially useful cancer vaccine.
DNA疫苗是引发针对编码抗原的体液免疫和细胞免疫反应的有吸引力的免疫原。然而，它们诱导抗原特异性CD8 T细胞反应的能力有待提高。改善DNA疫苗免疫原性的策略包括加强免疫、替代疫苗配方、电穿孔和基因佐剂，但很少有策略，如细胞外囊泡（EVs），针对天然抗原递送系统。通过关注CD63（一种在包括EVs在内的各种细胞膜上表达的四跨膜蛋白），我们研究了编码与CD63融合的抗原的DNA疫苗递送至EVs中是否会提高疫苗免疫原性。用编码与CD63融合的OVA抗原的质粒DNA（pCD63-OVA）进行体外转染产生了携带OVA的EVs。用纯化的携带OVA的EVs免疫未接触过抗原的小鼠可诱导OVA特异性CD4和CD8 T细胞，而用仅编码OVA蛋白的对照质粒或递送至内质网的钙连蛋白-OVA融合蛋白转染的细胞纯化的EVs免疫则不能诱导。用pCD63-OVA给小鼠接种疫苗可诱导强烈的抗原特异性T细胞反应，尤其是来自CD8 T细胞的反应。与将钙连蛋白递送至内质网相比，将CD63递送至EVs可导致更好的CD8 T细胞反应。当我们使用小鼠肿瘤植入模型评估pCD63-OVA作为治疗性疫苗时，与对照DNA疫苗接种相比，通过EV递送的DNA疫苗接种显著抑制了肿瘤生长。这些结果表明，通过DNA疫苗接种进行EV抗原递送为引发针对编码抗原的强烈CD8 T细胞反应提供了一种新策略，使其成为一种潜在有用的癌症疫苗。
