BACKGROUND Proprotein convertase subtilisin/kexin 9 (PCSK9) is an important regulator of low-density lipoprotein receptor (LDLR) and plasma levels of LDL cholesterol (LDL-C). PCSK9 inhibition is an efficient therapeutic approach for the treatment of dyslipidemia. We tested the therapeutic effect of a PCSK9 vaccine on dyslipidemia and atherosclerosis. METHODS Lipid film hydration method was used to prepare negatively charged nanoliposomes as a vaccine delivery system. An immunogenic peptide called immunogenic fused PCSK9-tetanus (IFPT) was incorporated on the surface of nanoliposomes using DSPE-PEG-maleimide lipid (L-IFPT) and adsorbed to Alhydrogel® (L-IFPTA+). The prepared vaccine formulation (L-IFPTA+) and empty liposomes (negative control) were inoculated four times with bi-weekly intervals in C57BL/6 mice on the background of a severe atherogenic diet and poloxamer 407 (thrice weekly) injection. Antibody titers were evaluated 2 weeks after each vaccination and at the end of the study in vaccinated mice. Effects of anti-PCSK9 vaccination on plasma concentrations of PCSK9 and its interaction with LDLR were determined using ELISA. To evaluate the inflammatory response, interferon-gamma (IFN-γ)- and interleukin (IL)-10-producing splenic cells were assayed using ELISpot analysis. RESULTS L-IFPTA+ vaccine induced a high IgG antibody response against PCSK9 peptide in the vaccinated hypercholesterolemic mice. L-IFPTA+-induced antibodies specifically targeted PCSK9 and decreased its plasma consecration by up to 58.5% (- 164.7 ± 9.6 ng/mL, p = 0.0001) compared with the control. PCSK9-LDLR binding assay showed that generated antibodies could inhibit PCSK9-LDLR interaction. The L-IFPTA+ vaccine reduced total cholesterol, LDL-C, and VLDL-C by up to 44.7%, 51.7%, and 19.2%, respectively, after the fourth vaccination booster, compared with the control group at week 8. Long-term studies of vaccinated hypercholesterolemic mice revealed that the L-IFPTA+ vaccine was able to induce a long-lasting humoral immune response against PCSK9 peptide, which was paralleled by a significant decrease of LDL-C by up to 42% over 16 weeks post-prime immunization compared to control. Splenocytes isolated from the vaccinated group showed increased IL-10-producing cells and decreased IFN-γ-producing cells when compared with control and naive mice, suggesting the immune safety of the vaccine. CONCLUSIONS L-IFPTA+ vaccine could generate long-lasting, functional, and safe PCSK9-specific antibodies in C57BL/6 mice with severe atherosclerosis, which was accompanied by long-term therapeutic effect against hypercholesterolemia and atherosclerosis.

中文翻译：

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纳米脂质体 PCSK9 疫苗在动脉粥样硬化小鼠模型中的治疗作用。

背景前蛋白转化酶枯草杆菌蛋白酶 9 (PCSK9) 是低密度脂蛋白受体 (LDLR) 和 LDL 胆固醇 (LDL-C) 血浆水平的重要调节剂。PCSK9 抑制是治疗血脂异常的有效治疗方法。我们测试了 PCSK9 疫苗对血脂异常和动脉粥样硬化的治疗效果。方法采用脂质膜水化法制备带负电荷的纳米脂质体作为疫苗递送系统。使用 DSPE-PEG-马来酰亚胺脂质 (L-IFPT) 将一种称为免疫原性融合 PCSK9-破伤风 (IFPT) 的免疫原性肽掺入纳米脂质体表面，并吸附至 Alhydrogel® (L-IFPTA+)。在严重致动脉粥样硬化饮食和泊洛沙姆 407（每周三次）注射的背景下，将制备的疫苗制剂 (L-IFPTA+) 和空脂质体（阴性对照）接种四次，每两周一次。在每次接种疫苗后 2 周和接种小鼠的研究结束时评估抗体滴度。使用 ELISA 确定了抗 PCSK9 疫苗接种对 PCSK9 血浆浓度的影响及其与 LD​​LR 的相互作用。为了评估炎症反应，使用 ELISpot 分析对产生干扰素-γ (IFN-γ) 和白细胞介素 (IL)-10 的脾细胞进行了测定。结果 L-IFPTA+ 疫苗在接种疫苗的高胆固醇血症小鼠中诱导了针对 PCSK9 肽的高 IgG 抗体反应。与对照相比，L-IFPTA+ 诱导的抗体特异性靶向 PCSK9 并降低其血浆浓度高达 58.5%（- 164.7 ± 9.6 ng/mL，p = 0.0001）。PCSK9-LDLR 结合测定显示产生的抗体可以抑制 PCSK9-LDLR 相互作用。与第 8 周的对照组相比，在第四次加强免疫后，L-IFPTA+ 疫苗的总胆固醇、LDL-C 和 VLDL-C 分别降低了 44.7%、51.7% 和 19.2%。长期对接种疫苗的高胆固醇血症小鼠的研究表明，L-IFPTA+ 疫苗能够诱导针对 PCSK9 肽的持久体液免疫反应，同时在初次免疫后 16 周内 LDL-C 显着降低高达 42%与对照相比。与对照组和幼稚小鼠相比，从接种组分离出的脾细胞显示产生 IL-10 的细胞增多，产生 IFN-γ 的细胞减少，表明疫苗的免疫安全性。结论 L-IFPTA+疫苗可在严重动脉粥样硬化的C57BL/6小鼠体内产生长效、功能性、安全的PCSK9特异性抗体，并伴有对高胆固醇血症和动脉粥样硬化的长期治疗作用。