Inhibition of HIV by Novel Chimeric Receptors

新型嵌合受体对 HIV 的抑制

• 批准号: 6721231
• 负责人: Jonathan S Allan
• 金额: $ 25.35万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6721231
• 关键词: HIV envelope protein gp41; antiAIDS agent; antiviral agents; biological signal transduction; chimeric proteins; cytotoxic T lymphocyte; flow cytometry; helper T lymphocyte; human immunodeficiency virus 1; human tissue; northern blottings; polymerase chain reaction; receptor; receptor binding; western blottings

DESCRIPTION (provided by applicant): To develop an effective T cell-based immunotherapy against HIV-1, one confronts three complicating factors: 1) an extreme sequence diversity among HIV-1 strains; 2) polymorphism of HLA among human population; and 3) the very cells required to mount effective immune responses are infected by viruses. To overcome these factors, we have fused a single chain Fv (scFv) of human monoclonal antibody to transmembrane and signal transduction domains of two subunits of type I interferon receptors IFNAR1 and IFNAR2L. The high affinity antibody recognizes a conserved epitope in HIV-1 gp41. We hypothesized that by expressing chimeric receptors on the surface of T cells, they will recognize gp41 on the surface of viral particles or HIV-1-infected cells and trigger the JAK/STAT/IRF signal transduction pathway. As a result, transduced CD4 T cells will become resistant to and CD8 T cells will mount a vigorous response against HIV-1. In the preliminary studies, the genes encoding chimeric receptors and EGFP control were transduced into chronically HIV-1-infected ACH-2 cells and properly expressed. Expression of chimeric receptors does not alter cell growth and CD4 and CXCR4 expression. However, HIV-1 release and spread are substantially inhibited in cells transduced with both scFv-IFNAR1 (SR1) and scFv-IFNAR2L (SR2L) or with SR2L alone. HIV-1 spread, but not release, in cells transduced with SR1 alone is also inhibited to a lesser degree. In this proposal, we will first characterize the mechanisms of HIV-1 inhibition of chimeric receptors in chronically HW-1-infected cells. We will determine if chimeric receptors can bind to gp41 on the surface of viral particles and trigger the JAK/STAT/IRF signal transduction pathway and if so, whether the signal transduction pathway triggered by chimeric receptors is a prerequisite for their inhibition of HIV-1. Second, we will study anti-HIV-1 activity of chimeric receptors in primary human CD4 and CD8 T cells. We will transduce the genes encoding chimeric receptors and EGFP into primary human CD4 and CD8 T cells. The transduced CD4 T cells will be challenged with primary HW-1 isolates to test if expression of chimeric receptors renders them resistant to acute HW- 1 infection. If so, we will determine at what step(s) of the viral cycle inhibition takes place. We will test if expression of chimeric receptors in transduced CD8 Tcells will enable them to effectively recognize and kill or inhibit viral replication in H1V-1-infected cells through cytolytic and non-cytolytic means. If successful, this strategy will have broad applications in immunotherapy and immune reconstitution against HIV-1.

描述（由申请人提供）：为了开发针对HIV-1的有效的基于T细胞的免疫疗法，人们面临三个复杂的因素：1）HIV-1毒株之间的极端序列多样性; 2）人类群体中HLA的多态性;和3）启动有效免疫应答所需的细胞被病毒感染。为了克服这些因素，我们融合了单链抗体（scFv）的人单克隆抗体的跨膜和信号转导结构域的两个亚基的I型干扰素受体IFNAR 1和IFNAR 2L。高亲和力抗体识别HIV-1 gp 41中的保守表位。我们假设，通过在T细胞表面表达嵌合受体，它们将识别病毒颗粒或HIV-1感染细胞表面的gp 41，并触发JAK/STAT/IRF信号转导通路。因此，转导的CD 4 T细胞将对HIV-1产生抗性，而CD 8 T细胞将对HIV-1产生强烈的应答。在初步研究中，编码嵌合受体和EGFP对照的基因被转导到慢性HIV-1感染的ACH-2细胞中并适当表达。嵌合受体的表达不改变细胞生长以及CD 4和CXCR 4表达。然而，在用scFv-IFNAR 1（SR 1）和scFv-IFNAR 2L（SR 2L）两者或单独用SR 2L转导的细胞中，HIV-1释放和扩散基本上被抑制。HIV-1的传播，但不释放，在细胞中单独转导SR 1也被抑制到较低的程度。在这个提议中，我们将首先描述HIV-1抑制嵌合受体在慢性HW-1感染细胞中的机制。我们将确定嵌合受体是否可以与病毒颗粒表面的gp 41结合并触发JAK/STAT/IRF信号转导途径，如果是这样，嵌合受体触发的信号转导途径是否是其抑制HIV-1的先决条件。其次，我们将研究嵌合受体在原代人CD 4和CD 8 T细胞中的抗HIV-1活性。我们将编码嵌合受体和EGFP的基因转染到原代人CD 4和CD 8 T细胞中。转导的CD 4 T细胞将用原代HW-1分离株攻击，以测试嵌合受体的表达是否使其对急性HW- 1感染具有抗性。如果是，我们将确定病毒周期抑制发生在哪个步骤。我们将测试转导的CD 8 T细胞中嵌合受体的表达是否能够使它们通过溶细胞和非溶细胞手段有效地识别和杀死或抑制H1 V-1感染细胞中的病毒复制。如果成功，这种策略将在针对HIV-1的免疫治疗和免疫重建中具有广泛的应用。