A novel method to incorporate bioactive cytokines as adjuvants on the surface of virus particles.
A novel method to incorporate bioactive cytokines as adjuvants on the surface of virus particles.
复制标题
一种将生物活性细胞因子作为佐剂掺入病毒颗粒表面的新方法。
DOI：
10.1089/jir.2008.0017
复制
发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Sundick,RoysS
中科院分区：
文献类型：
--
作者：
Yang,Yufang;Leggat,David;Herbert,Andrew;Roberts,PaulC;Sundick,RoysS
细胞因子已被广泛用作疫苗中的佐剂。然而，实际的考虑限制了它们的使用;从抗原扩散，半衰期短和额外的生产成本。为了解决这些问题，我们开发了一种技术，有效地生产灭活的，全病毒流感疫苗携带膜结合的细胞因子。为了提供“原理证明”，我们选择了鸡白细胞介素-2（IL-2）和鸡粒细胞-巨噬细胞集落刺激因子。产生融合构建体，其中它们的编码区分别与神经氨酸酶和血凝素基因的流感病毒跨膜编码结构域连接。这些融合构建体用于建立稳定的Madin-Darby犬肾细胞系，组成型表达膜结合细胞因子。细胞表面表达通过免疫荧光和精氨酸特异性生物测定来验证。从感染的携带细胞中收获的流感病毒被纯化、灭活，并通过免疫荧光、蛋白质印迹和生物测定证实包括膜结合的细胞因子。使用几种标准病毒灭活方案保留细胞因子生物活性。两种含精氨酸的流感疫苗目前正在体内进行免疫原性测试。初步实验表明，与给予常规疫苗的鸡相比，注射携带IL-2的流感病毒的鸡具有升高的抗病毒抗体水平。总之，该技术提供了一种利用细胞因子和其他免疫刺激分子作为病毒疫苗佐剂的新方法。
Cytokines have been used extensively as adjuvants in vaccines. However, practical considerations limit their use; diffusion from antigen, short half-lives and additional production costs. To address these problems we have developed a technology that efficiently produces inactivated, whole-virus influenza vaccine bearing membrane-bound cytokines. To provide “proof of principle,” we chose chicken interleukin-2 (IL-2) and chicken granulocyte–macrophage colony-stimulating factor. Fusion constructs were generated in which their coding regions were linked to the influenza virus transmembrane encoding domains of the neuraminidase and hemagglutinin genes, respectively. These fusion constructs were used to establish stable Madin–Darby Canine Kidney cell lines, constitutively expressing membrane-bound cytokine. Cell surface expression was verified by immunofluorescence and cytokine-specific bioassays. Influenza virus harvested from infected cytokine-bearing cells was purified, inactivated, and confirmed to include membrane-bound cytokine by immunofluorescence, Western blotting and bioassay. Cytokine bioactivity was preserved using several standard virus inactivation protocols. Both cytokine-bearing influenza vaccines are now being tested for immunogenicityin vivo. Initial experiments indicate that chickens injected with IL-2-bearing influenza have elevated antiviral antibody levels, compared to chickens given conventional vaccine. In conclusion, this technology offers a novel method to utilize cytokines and other immunostimulatory molecules as adjuvants for viral vaccines.