Production of glycosylphosphatidylinositol-anchored proteins for vaccines and directed binding of immunoliposomes to specific cell types

Abstract Background: Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. Furthermore, the introduction of a universal immunoglobulin-binding domain can make liposomes targetable to virtually any desired receptor for which antibodies exist. Methods: We developed a system for the production of recombinant proteins with GPI anchors and histidine tags and Strep-tags for simplified purification from cells. This system was applied to i) the green fluorescent protein (GFP) as a reporter, ii) the promising Plasmodium falciparum vaccine antigen PfRH5 and iii) a doubled immunoglobulin Fc-binding domain termed ZZ from protein A of Staphylococcus aureus. As the GPI-attachment domain, the C-terminus of murine CD14 was used. After the recovery of these three recombinant proteins from Chinese hamster ovary (CHO) cells and association with liposomes, their vaccine potential and ability to target the CD4 receptor on lymphocytes in ex vivo conditions were tested. Results: Upon immunization in mice, the PfRH5-GPI-loaded liposomes generated antibody titers of 103 to 104, and showed a 45% inhibitory effect on in vitro growth at an IgG concentration of 600 µg/mL in P. falciparum cultures. Using GPI-anchored ZZ to couple anti-CD4 antibodies to liposomes, we created immunoliposomes with a binding efficiency of 75% to CD4+ cells in splenocytes and minimal off-target binding. Conclusions: Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes. Importantly, the CHO-cell and GPI-tagged produced PfRH5 elicited invasion-blocking antibodies qualitatively comparable to other approaches.

摘要 背景：脂质体（liposomes）是递送药物或抗原的高价值载体。糖基磷脂酰肌醇（glycosylphosphatidylinositol，GPI）锚定蛋白与脂质体结合，可通过提升抗原的表面浓度，潜在增强疫苗抗原的免疫原性。此外，引入通用免疫球蛋白结合结构域，可使脂质体靶向结合现有抗体所针对的任意目标受体。

方法：本研究构建了一套可制备带有GPI锚、组氨酸标签（histidine tag）及Strep标签（Strep-tag）的重组蛋白的系统，实现从细胞中便捷纯化目标蛋白。该系统被应用于三类研究对象：其一为作为报告蛋白的绿色荧光蛋白（green fluorescent protein，GFP）；其二为极具开发前景的恶性疟原虫（Plasmodium falciparum）疫苗抗原PfRH5；其三为源自金黄色葡萄球菌（Staphylococcus aureus）A蛋白的双重免疫球蛋白Fc结合结构域ZZ。本研究选用小鼠CD14的C端作为GPI锚定结构域。从中国仓鼠卵巢（Chinese hamster ovary，CHO）细胞中回收上述三种重组蛋白并使其与脂质体结合后，我们测试了它们的疫苗研发潜力，以及在离体条件下靶向淋巴细胞表面CD4受体的能力。

结果：对小鼠进行免疫接种后，负载PfRH5-GPI的脂质体诱导产生的抗体效价可达10³至10⁴；在恶性疟原虫体外培养体系中，当IgG浓度为600 μg/mL时，其对疟原虫生长的抑制率可达45%。利用GPI锚定的ZZ将抗CD4抗体偶联至脂质体后，我们制备的免疫脂质体对脾细胞中CD4⁺细胞的结合效率达75%，且脱靶结合水平极低。

结论：通过GPI锚定结构域，蛋白可高效结合至脂质体以形成蛋白脂质体（proteoliposome）颗粒，该颗粒可应用于多种场景，包括疫苗制备以及脂质体的抗体介导靶向。值得注意的是，经CHO细胞生产的带GPI标签的PfRH5，其诱导的侵袭阻断抗体在性质上可与其他方法制备的同类产品相媲美。