CD40 LIGAND AS ADJUVANT FOR RAISING PROTECTIVE ANTI-HIV AB BY DNA/MVA VACCINES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本项目旨在利用猕猴/猴免疫缺陷病毒(SIV)模型评估CD40L增强DNA/MVA疫苗诱导的人类免疫缺陷病毒(HIV)特异性体液和细胞免疫的佐剂效应。 以SIV239疫苗DNA为参照，克隆了猕猴CD40L基因，并在顺式(在同一质粒上)和反式(在另一粒上)中进行了表达。在顺式中，蛋白质以膜结合的形式表达，并被整合到SIV病毒样颗粒中。在转化子中，该蛋白以可溶性分泌形式表达。流式细胞仪检测细胞内CD40L蛋白的表达，Western blotting检测细胞外蛋白的表达。 表达的蛋白对SIV感染猕猴的DC具有很高的刺激活性。这些激活的DC上调CD80，并产生肿瘤坏死因子-α和IL-12。此外，这些蛋白还具有刺激猕猴B细胞的活性。正在进行的实验正在测试这些结构在猕猴身上的佐剂作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project seeks to evaluate the adjuvant effects of CD40L to enhance the human immunodeficiency virus (HIV)-specific humoral and cellular immunity elicited by a DNA/MVA vaccine using a macaque/simian immunodeficiency virus (SIV) model. The macaque CD40L was cloned and expressed in cis (on the same plasmid) and in trans (on a separate plasmid) with respect to the SIV 239 vaccine DNA. In the cis form, the protein was expressed as a membrane bound form that is incorporated into SIV virus-like particles. In the trans form, the protein was expressed as a soluble secreted form. Intracellular expression of the CD40L protein was confirmed by flow cytometry and extracellular protein was confirmed by Western blotting. The expressed proteins were highly active in stimulating DC from SIV-infected macaques. These activated DC upregulated CD80 and produced TNF-a and IL-12. In addition, these proteins were active in stimulating macaque B cells. On going experiments are testing the adjuvant effects of these constructs in macaques.