EVALUATION OF NEW CARRIERS AND ADJUVANTS FOR HIV-1 VACCINES.

(1)项目目标：-鉴定一种能够增加HIV-1亚基免疫原性的载体，并能够在已有免疫缺陷的患者中召回抗hiv - B记忆细胞。-确定一种载体，该载体可以增加感染个体的TH1/TH2比率，并有利于细胞反应的产生，包括细胞毒性细胞（CTL）和趋化因子的产生。(2)实验方法：将革兰氏阴性流产布鲁氏菌（Ba）及其细胞壁脂多糖（Ba- LPS）作为灭活HIV-1病毒粒子、gp120 （SF2）糖蛋白或HIV-1 (MN) env的v3环衍生肽的载体进行测试。用不同的偶联物免疫不同程度T细胞缺乏症小鼠。四只恒河猴也接种了Ba-V3结合物疫苗。-体液免疫反应和细胞毒性免疫反应均被测量，包括全身和粘膜抗体反应。在合胞体抑制试验中测定生物学相关抗体。在体外研究中，通过聚合酶链反应（PCR）和生物检测，评估了人类T细胞和来自正常以及HIV-1感染患者的洗脱单核细胞对Ba和Ba- lps的淋巴因子产生的反应。- pcr引物用于测试Ba、Ba- lps和Ba/DNA诱导人PBL趋化因子的能力。(3)主要发现：- Ba与含有b细胞表位和CTL表位（N3v3）的肽结合，产生能够杀死hiv感染目标的中和抗体和细胞毒性T细胞。用Ba-N3V3偶联物免疫后，CD4 - depletion小鼠仍能产生抗hiv中和抗体和CTL。-从安全角度来看，Ba或其脂多糖对动物的毒性比大肠杆菌衍生的脂多糖小得多。通过诱导淋巴因子il - 2和ifn - γ，发现- Ba和Ba- lps可直接激活纯化的人cd4阳性TH1细胞，并在较小程度上激活cd8阳性细胞。来自HIV-1感染者的PBL也有应答性。Ba和Ba- lps也能激活人单核细胞分泌IL12。恒河猴在血清和粘膜表面产生高滴度的hiv -1中和抗体。

(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. - From a safety point of view, Ba or its LPS are much less toxic to animals than E. Coli derived LPS. - 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. - 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.