Evaluation of new carriers and adjuvants for HIV-1 vaccines.

(1)项目目标：- 鉴定一种载体，该载体将增加HIV-1亚单位的免疫原性，并且将能够在具有预先存在的免疫缺陷的患者中回忆起抗HIV B记忆细胞。- 为了鉴定一种载体，该载体将增加感染个体的TH 1/TH 2比率，并将有利于产生细胞应答，包括细胞增殖细胞（CTL）和β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）和来源于其细胞壁的LPS（Ba-LPS）作为灭活的HIV-1病毒体、gp 120（SF 2）糖蛋白或来源于HIV-1（MN）env的V3环的肽的载体进行测试。不同的缀合物用于免疫具有不同程度的T细胞缺陷的小鼠。 还用Ba-V3缀合物接种了4只恒河猴。- 测量体液和细胞免疫应答，包括系统和粘膜抗体应答。在合胞体抑制测定中测量生物学相关抗体。 --通过PCR和生物学测定，评估了正常和HIV-1感染患者的人T细胞和淘析的单核细胞对Ba和Ba-LPS应答的淋巴因子产生。 - 设计PCR引物以测试Ba、Ba-LPS和Ba/DNA从人PBL中引发趋化因子的能力（3）主要发现：- Ba与含有B细胞表位和CTL表位的肽（N3 v3）缀合，产生能够杀死HIV感染的靶的中和抗体和细胞毒性T细胞。 CD 4耗尽的小鼠在用Ba-N3 V3缀合物免疫后保留了它们产生抗HIV中和Ab和CTL的能力。- 从安全性的角度来看，Ba或其LPS对动物的毒性远小于E。大肠杆菌衍生的LPS。 - 发现Ba和Ba-LPS直接激活纯化的人CD 4阳性TH 1细胞，并且在较小程度上激活CD 8阳性细胞，如通过诱导淋巴因子IL 2和IFN-γ所判断的。 来自HIV-1感染者的PBL也有反应。 Ba和Ba-LPS也可以激活人单核细胞分泌IL 12。- 恒河猴的血清和粘膜表面产生高滴度的HIV-1中和抗体。- 发现灭活的Ba以及Ba衍生的DNA和LPS诱导人PBL中RANTES、MIP-1a和MIP-1b的mRNA（20小时内），以及这些趋化因子从CD 8+和CD 4+细胞和单核细胞的分泌。这些趋化因子将增加接种疫苗的个体的抗病毒能力。β趋化因子可以通过结合和/或下调CCR 5 HIV- 1辅助受体来阻断和预防R5病毒的感染。

(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.