激发巨噬细胞的抗复发性HSK新型DNA疫苗研制及免疫机制研究

Recurrent herpes simplex keratitis (HSK) is a blind-caused eye disease mainly caused by herpes simplex virus type 1 (HSV-1) latent, recurrent infection, which is very difficult to be treated so far. The therapeutic vaccine which can strengthen ocular local anti-viral active immunity may be a potential way. Previously, we developed the DNA vaccines of targeting dendritic cell (DC) or gDogC which can enhance the viral antigen presentation or the complement effects, however, these vaccines are far from fully controlling the recurrent HSK. The possible reason may be that they could not induce local ocular effective anti-viral cellular immune reaction. Based on the inactive viral killing of CD8+ T cells due to the interfering with the MHC-I presentation of viral antigen by HSV-1, we intend to strengthen the killing activity of recurrent virus by macrophages. Therefore, we are going to construct a macrophage targeted DNA vaccine, in which, the gene of HSV-1 antigen gD will be fused into of the gene of the specific monoclonal antibody of the specific receptor-sialoadhesin expressed on macrophage surface, and further combined with the DC targeted and gDogC vaccines forming the triple DNA vaccine, which is packaged by the nanoparticle chitosan and topically inoculated into the eyes of mice. It may trigger effective local ocular immune killing activity to the virus by macrophages as to successfully controlling the recurrent HSK. This new type therapeutic vaccine has never been reported so far, and the success of it and the demonstration of its mechanism may provide a new thought and therapy strategy in treating the recurrent HSK.

复发性单纯疱疹病毒性角膜炎(HSK)是主要由单疱病毒I型(HSV-1)潜伏、复发感染引起的难治型可致盲眼病。加强眼局部主动免疫病毒杀伤的治疗性疫苗是潜在途径。我们前期研制了包括增强对病毒抗原提呈的靶向树突状细胞（DC）及激活补体效应的gD.gC DNA疫苗，但它们远不足以完全抑制或控制疾病的发生与发展。究其可能原因，它们尚不能诱导眼局部产生有效抗病毒细胞免疫反应。鉴于HSV-1干扰了主要组织相容性复合体分子I类介导的自身抗原提呈而不能激活CD8+T细胞对其杀伤，我们拟增强巨噬细胞（Mø）对复发感染病毒的杀伤。依据唾液酸黏附素单抗与Mø特异结合的特性，拟构建靶向Mø的DNA疫苗；由此进一步联合靶向DC及gD.gC的三联DNA疫苗经纳米壳聚糖包裹滴眼接种疾病小鼠后，可能激发眼局部Mø对病毒的有效杀伤以控制复发性HSK。该新型治疗性疫苗的成功研制及机制研究将为复发性HSK提供新的防治思维与策略。

单纯疱疹病毒性角膜炎（HSK）是主要由单疱病毒1型（HSV-1）感染引起的难治性可致盲眼病。加强眼局部主动免疫病毒杀伤的疫苗或药物是潜在途径。我们前期研制了包括增强对病毒抗原提呈的靶向树突状细胞（DC）及激活补体效应的gD.gC DNA疫苗，但它们远不足以完全抑制或控制疾病的发生与发展。我们发现HSK小鼠角膜、结膜和眼引流淋巴结（下颌下淋巴结）中巨噬细胞显著增多，其中眼表主要以M1型巨噬细胞为主，而引流淋巴结主要以CD169+巨噬细胞为主，针对这两类细胞我们进行了疫苗及药物的研发。我们构建了神经节苷脂GM1（良好靶向CD169的生物来源配体）修饰的包裹HSV-1 gD蛋白的脂质体疫苗，验证了其靶向性和生物安全性后，发现它抑制了HSV-1在眼表的复制及在三叉神经节中的潜伏，促进CD169+巨噬细胞向M1极化，并激活T细胞促进适应性免疫。同时，我们也筛选并鉴定了PKHB1多肽，体内动物实验表明，PKHB1多肽抑制HSV-1复制并降低HSK疾病的严重程度，增加了M1巨噬细胞的数量和下颌下淋巴结中CD8+T细胞的数量。体外实验进一步研究发现，细胞免疫原性死亡的发生可能是PKHB1作用的机制。这些研究明确了激活巨噬细胞是防治HSK的干预靶点，神经节苷脂修饰的包裹HSV-1 gD蛋白的脂质体疫苗和PKHB1药物可能是防治HSK潜在的治疗途径，为HSK的防治提供新靶点和新方向。