Sungsuwan Suttipun, Wu Xuanjun, Huang Xuefei
Michigan State University, East Lansing, MI, United States.
Michigan State University, East Lansing, MI, United States.
Methods Enzymol. 2017;597:359-376. doi: 10.1016/bs.mie.2017.06.030. Epub 2017 Jul 26.
Tumor-associated carbohydrate antigens (TACAs) are attractive targets for anticancer vaccine development. Due to the low immunogenicity of TACAs, a powerful carrier system is needed to boost immune responses. Virus-like particles (VLPs) are an exciting platform for delivering TACAs to the immune system. The high symmetry of VLPs enables the display of TACAs in an organized manner, which in turn can potently activate antibody secreting B cells, eliciting high titers of antiglycan IgG antibodies. In this chapter, the protocol for conjugating a prototypical TACA, the Tn antigen to a VLP, bacteriophage Qβ, is presented. On an average around 370 copies of Tn can be attached to each Qβ capsid. Immunization of mice with Qβ-Tn conjugate leads to over two orders of magnitude higher IgG antibodies compared to control mice receiving Qβ only without the Tn antigen. Antibodies induced by Qβ-Tn recognize Tn-expressing tumor cells strongly and protect mice from tumor-induced death. The techniques for evaluating antibody titers by enzyme-linked immunosorbent assay, antibody binding to tumor cells by flow cytometry, and the protection efficacy of the vaccine in a therapeutic model of tumor are discussed in this chapter.
肿瘤相关碳水化合物抗原（TACAs）是抗癌疫苗研发中颇具吸引力的靶点。由于TACAs的免疫原性较低，因此需要强大的载体系统来增强免疫反应。病毒样颗粒（VLPs）是将TACAs递送至免疫系统的一个令人兴奋的平台。VLPs的高度对称性使得TACAs能够以有序的方式展示，进而能够有效地激活分泌抗体的B细胞，引发高滴度的抗聚糖IgG抗体。在本章中，介绍了将一种典型的TACA，即Tn抗原与一种VLPs，噬菌体Qβ进行偶联的方法。平均每个Qβ衣壳可连接约370个Tn拷贝。与仅接受不含Tn抗原的Qβ的对照小鼠相比，用Qβ-Tn偶联物免疫小鼠可使IgG抗体水平高出两个数量级以上。Qβ-Tn诱导产生的抗体能强烈识别表达Tn的肿瘤细胞，并保护小鼠免于肿瘤诱导的死亡。本章讨论了通过酶联免疫吸附测定评估抗体滴度、通过流式细胞术检测抗体与肿瘤细胞的结合以及在肿瘤治疗模型中评估疫苗保护效果的技术。
