Howland Shanshan W, Tsuji Takemasa, Gnjatic Sacha, Ritter Gerd, Old Lloyd J, Wittrup Karl Dane
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
J Immunother. 2008 Sep;31(7):607-19. doi: 10.1097/CJI.0b013e318181c87f.
Saccharomyces cerevisiae stimulates dendritic cells (DCs) and represents a promising candidate for cancer vaccine development. Effective cross-presentation of antigen delivered to DCs is necessary for successful induction of cellular immunity. Here, we present a yeast-based vaccine approach that is independent of yeast's ability to express the chosen antigen, which is instead produced separately and conjugated to the yeast cell wall. The conjugation method is site-specific (based on the SNAP-tag) and designed to facilitate antigen release in the DC phagosome and subsequent translocation for cross-presentation. We demonstrate that nonsite-specific chemical conjugation of the same protein hinders cross-presentation. Phagosomal antigen release was further expedited through the insertion of the invariant chain ectodomain as a linker, which is rapidly cleaved by Cathepsin S. The dose of delivered antigen was increased in several ways: by using yeast strains with higher surface amine densities, by using yeast hulls (cell wall fragments) instead of whole cells, and by conjugating multiple layers of antigen. The novel multilayer conjugation scheme takes advantage of Sfp phosphopantetheinyl transferase and remains site-specific; it enables the antigen dose to grow linearly with the number of layers. We show that whole yeast cells coated with 1 layer of the cancer-testis antigen NY-ESO-1 and yeast hulls bearing 3 layers were able to cross-prime naive CD8 T cells in vitro, with the latter resulting in higher frequencies of antigen-specific cells after 10 days. This cross-presentation-efficient antigen conjugation scheme is not limited to yeast and can readily be applied toward the development of other particulate vaccines.
酿酒酵母可刺激树突状细胞（DCs），是癌症疫苗开发的一个有潜力的候选者。有效交叉呈递递送至DCs的抗原对于成功诱导细胞免疫是必要的。在此，我们提出一种基于酵母的疫苗方法，该方法独立于酵母表达所选抗原的能力，所选抗原而是单独产生并与酵母细胞壁偶联。偶联方法是位点特异性的（基于SNAP标签），旨在促进抗原在DC吞噬体中的释放以及随后的转运以进行交叉呈递。我们证明相同蛋白质的非位点特异性化学偶联会阻碍交叉呈递。通过插入恒定链胞外结构域作为连接子进一步加速吞噬体抗原释放，该连接子可被组织蛋白酶S快速切割。通过几种方式增加递送抗原的剂量：使用具有更高表面胺密度的酵母菌株、使用酵母壳（细胞壁片段）而非完整细胞以及偶联多层抗原。新颖的多层偶联方案利用了Sfp磷酸泛酰巯基乙胺基转移酶且保持位点特异性；它使抗原剂量随层数呈线性增加。我们表明，包被1层癌胚抗原NY-ESO-1的完整酵母细胞和带有3层抗原的酵母壳能够在体外交叉启动初始CD8 T细胞，10天后后者产生更高频率的抗原特异性细胞。这种具有高效交叉呈递能力的抗原偶联方案不限于酵母，可容易地应用于其他颗粒疫苗的开发。
