Research and Development Center for Mucosal Vaccines, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
HanaVax Inc., Tokyo 103-0012, Japan.
Mol Pharm. 2021 Apr 5;18(4):1582-1592. doi: 10.1021/acs.molpharmaceut.0c01003. Epub 2021 Feb 23.
We previously developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan. Here, we generated three pneumococcal surface protein A (PspA) fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel and mixed the three resulting monovalent formulations into a trivalent nanogel-PspA formulation. First, to characterize the nanogel-PspA formulations, we used native polyacrylamide gel electrophoresis (PAGE) to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, we adopted two methods-a densitometric method based on lithium dodecyl sulfate (LDS)-PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)-to determine the PspA content in the nanogel formulations. Third, treatment of nanogel-PspA formulations by adding methyl-β-cyclodextrin released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. However, when nanogel-PspA formulations were heat-treated at 80 °C for 16 h, CD spectroscopy showed that each PspA was released in a denatured form. Fourth, we confirmed that the nanogel-PspA formulations were internalized into nasal mucosa effectively and that each PspA was gradually released from the nanogel in epithelial cells in mice. Fifth, LDS-PAGE densitometry and ELISA both indicated that the amount of trivalent PspA was dramatically decreased in the heat-treated nanogel compared with that before heating. When mice were immunized nasally using the heat-treated formulation, the immunologic activity of each PspA was dramatically reduced compared with that of the untreated formulation; in both cases, the immunologic activity correlated well with the content of each PspA as determined by LDS-PAGE densitometry and ELISA. Finally, we confirmed that the trivalent nanogel-PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent PspA nasal vaccine formulation.
我们之前开发了一种安全有效的鼻腔疫苗输送系统，该系统使用源自阳离子化胆甾醇普鲁兰的自组装纳米水凝胶（纳米凝胶）。在这里，我们生成了三种肺炎球菌表面蛋白 A（PspA）融合抗原作为通用肺炎球菌鼻腔疫苗，然后将每个 PspA 封装到纳米凝胶中，并将三种单价制剂混合到三聚体纳米凝胶-PspA 制剂中。首先，为了表征纳米凝胶-PspA 制剂，我们使用天然聚丙烯酰胺凝胶电泳（PAGE）来确定每个纳米凝胶分子中封装的 PspA 分子的平均数量。其次，我们采用了两种方法-基于十二烷基硫酸钠（LDS）-PAGE 的密度测定法和涉及夹心酶联免疫吸附测定（ELISA）的生物学方法-来确定纳米凝胶制剂中的 PspA 含量。第三，通过添加甲基-β-环糊精处理纳米凝胶-PspA 制剂，以其天然形式释放出每种 PspA，这通过圆二色性（CD）光谱得到证实。但是，当纳米凝胶-PspA 制剂在 80°C 下热处理 16 小时时，CD 光谱显示每种 PspA 均以变性形式释放。第四，我们证实纳米凝胶-PspA 制剂有效地被内化到鼻黏膜中，并且每种 PspA 逐渐从纳米凝胶中在小鼠的上皮细胞中释放。第五，LDS-PAGE 密度测定和 ELISA 均表明，与加热前相比，热处理的纳米凝胶中三价 PspA 的量大大减少。当使用经热处理的制剂经鼻免疫小鼠时，与未经处理的制剂相比，每种 PspA 的免疫活性大大降低；在这两种情况下，免疫活性与通过 LDS-PAGE 密度测定和 ELISA 确定的每种 PspA 的含量密切相关。最后，我们证实三聚体纳米凝胶-PspA 制剂在免疫小鼠中诱导了等效滴度的 PspA 特异性血清 IgG 和黏膜 IgA Ab。这些结果表明，我们开发的规格方法有效地表征了我们基于纳米凝胶的三聚体 PspA 鼻腔疫苗制剂。
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