Evaluation of new carriers/adjuvants for HIV-1 vaccines

摘要：(1)项目目标： -确定一种载体，该载体将增加HIV-1亚单位的免疫原性，并能够在先前存在免疫缺陷的患者中召回抗HIV B记忆细胞。 -确定一种载体，它可以增加感染者的TH1/TH2比率，并有助于产生包括细胞毒细胞(CTL)和β趋化因子在内的细胞反应。 (2)实验方法： -革兰氏阴性流产布鲁氏菌(BA)及其细胞壁来源的内毒素(BA-LPS)作为灭活的HIV-1病毒粒子、gp120(SF2)糖蛋白或来自HIV-1(MN)env的V3环的多肽的携带者。用不同的结合物免疫不同程度T细胞缺陷的小鼠。4只恒河猴也接种了巴-V3结合物。 -体液和细胞毒性免疫反应，包括系统抗体和粘膜抗体反应。在合胞体抑制试验中检测生物学相关抗体。 --用人T细胞和正常人和HIV-1感染患者的淋巴因子的体外研究，用聚合酶链式反应和生物学方法评估了它们对巴和巴-内毒素的反应。 设计了一对聚合酶链式反应(PCR)引物，用来检测BA、BA-LPS和BA/DNA从人PBL中诱导趋化因子的能力 (三)主要发现： -BA与含有B细胞表位和CTL表位(N3v3)的多肽结合，产生中和抗体和细胞毒性T细胞，能够杀死HIV感染的目标。在用BA-N3V3结合物免疫后，CD4耗竭的小鼠仍能产生抗HIV中和抗体和CTL。 经IL-2和干扰素-γ诱导，发现-BA和BA-内毒素可直接激活纯化的人CD4+TH1细胞，也可直接激活CD8+细胞。HIV-1感染者的PBL也有反应。BA和BA-LPS还能激活人单核细胞分泌IL-12。 -恒河猴在血清和粘膜表面产生高滴度的HIV-1中和抗体。此外，在BA-gp120免疫后，检测产生IFNgAMA的细胞和产生β-趋化因子的细胞。 灭活的BA、BA衍生的DNA和内毒素可诱导人PBL(20小时内)RANTES、MIP-1a和MIP-1b的mRNA表达，并可诱导CD8+、CD4+细胞和单核细胞分泌这些趋化因子。这些趋化因子将增加接种疫苗的个体的抗病毒毫里程。β趋化因子可以通过结合和/或下调CCR5 HIV-1辅助受体来阻断和预防R5病毒的感染。

Summary: (1) Goals of Project: - To identify a carrier which will increase the immunogenicity of the HIV-1 subunits, and will be able to recall anti-HIV B memory cells in patients with pre- existing immune deficiency. - To identify a carrier which would augment the TH1/TH2 ratio of infected individuals and will favor generation of cellular responses including cytotxic cells (CTL) and beta chemokines production. (2) Experimental approaches: - The gram negative Brucella abortus (Ba), and LPS derived from its cell wall (Ba- LPS), were tested as carriers for either inactivated HIV-1 virions, gp120 (SF2) glycoprotein, or peptide derived from the V3-loop of HIV-1 (MN) env. The different conjugates were used to immunize mice with different degrees of T cell deficiency. Four Rhesus macaques were also vaccinated with a Ba-V3 conjugate. -Both humoral and cytotxic immune responses were measured including sytemic and mucosal antibody responses. Biologically relevant antibodies were measured in syncytia inhibition assays. --In vitro studies with human T cells and elutriated monocytes from normal as well as HIV-1 infected patients were assessed for their lymphokine production in response to Ba and Ba-LPS by PCR and biological assays. -PCR primers were designed to test the ability of Ba ,Ba-LPS,and Ba/DNA, to elicit chemokines from human PBL (3) Major findings: - Ba conjugated to a peptide containing B-cell epitope and CTL epitope (N3v3), generated both neutralizing antibodies and cytotoxic T cells capable of killing HIV-infected targets. CD4 - depleted mice retained their ability to generate anti-HIV neutralizing Ab and CTL after immunization with the Ba-N3V3 conjugate. - Ba and Ba-LPS were found to directly activate purified human CD4-positive TH1 cells, and to a lesser degree, CD8-positive cells, as judged by induction of the lymphokines IL2 and IFN-gamma. PBL from HIV-1 infected individuals were also responsive. Ba and Ba-LPS can also activate IL12 secretion by human monocytes. - Rhesus macacques produced high titer HIV-1-neutralizing antibodies in the serum and mucosal surfaces.In addition,IFNgama producing cells and beta-chemokine producing cells were measured after Ba-gp120 immunization. - Inactivated Ba , as well as Ba derived DNA and LPS were found to induce mRNA for RANTES, MIP-1a, and MIP-1b in human PBL (in 20 hr) , and secretion of these chemokines from CD8+ and CD4+ cells and from monocytes. These chemokines will add to the anti-viral millieu in vaccinated individuals. The beta chemokines can block and prevent infection of R5 viruses by binding and/or downregulation of the CCR5 HIV-1 coreceptor.