FUSION PROTEIN AS IMMUNOGENS AGAINST HIV INFECTION

控制艾滋病的一个可取的方法是开发一种疫苗 这可能会授予对人类的长期豁免权 同时容易感染免疫缺陷病毒(HIV) 在全球范围内实施。我们已经鉴定出两个抗原性很强的 痘苗病毒14K和39K的蛋白质，并表明，当我们 将14K与HIV-1 env或Gag共价连接并表达融合 HIV-痘苗病毒重组体感染细胞中的蛋白质 观察如下：1)融合蛋白定位于 低聚物形式的细胞表面，2)没有裂解或 从细胞中释放出来，3)env糖基化不良，4)当 重组病毒在小鼠体内接种有诱导作用 抗HIV env和Gag的特异性抗体。这些产品的安全性 对HIVVacinia病毒重组体进一步检测 免疫抑制的小鼠。鉴于14K聚变的独特性 蛋白质，我们认为HIV-痘苗融合蛋白可能提供 增强和修饰B细胞和T细胞反应的有效手段 对抗艾滋病毒感染。这项提案的目标是确定 人类免疫缺陷病毒-牛痘病毒融合蛋白诱导的免疫应答 更有效地对抗艾滋病毒，比由 天然蛋白质。我们将使用鼠标模型，因为它将 提供了更广泛的数据，否则我们无法 实际上是从灵长类动物模型中获得的。长期目标 这项研究的目的是开发一种安全的疫苗，可以在 灵长类动物，最终在人类身上。拟议的项目将 包括以下主要目标： 1.构建HIV-痘苗病毒重组病毒(野生型和 弱化)表达的融合蛋白包含 痘苗病毒14K或39K蛋白融合蛋白全长序列 HIV-1包膜蛋白和Gag基因全序列或部分序列及其研究 它们在培养细胞中的表达方式。 2.确定体液免疫应答的程度 在HIV-Vacinia重组病毒免疫的小鼠中诱导 并用纯化的活重组病毒加强免疫 未融合或纯化的融合蛋白，并确定 特异性抗血清对HIV细胞致病性的抑制作用 3.确定细胞免疫反应的程度。 不同性别的小鼠 单倍型免疫，如目标2所述。 如果融合蛋白诱导出更有效和更持久的体液 而对HIV抗原的细胞免疫应答要比未融合的 蛋白质，那么这种新的方法可能会生产出一种有用的疫苗 在任何一种血清阴性的情况下使用抗艾滋病毒(作为预防措施 测量)或血清阳性的人体受试者(作为治疗剂)。

A desirable approach to control AIDS would be to develop a vaccine that could confer long-term immunity against the human immunodeficiency virus (HIV) and at the same time be easily administered worldwide. We have identified two highly antigenic vaccinia virus proteins of 14K and 39K and shown that, when we covalently link 14K to HIV-1 env or gag and expressed the fusion proteins in cells infected with HIV-vaccinia virus recombinants we observe the following: 1) the fusion proteins are localized on the cell surface in the form of oligomers, 2) are not cleaved or released from the cells, 3) env is poorly glycosylated and 4) when the recombinant viruses are inoculated in mice there is induction of specific antibodies against HIV env and gag. The safety of these HIVvaccinia virus recombinants was further assesed in immunosuppressed mice. In light of the uniqueness of the 14K-fusion protein, we suggest that HIV-vaccinia fusion proteins may provide an effective means of enhancing and modifying B and T-cell responses against HIV infection. The goal of this proposal is to determine whether the immune response elicited by HIV-vaccinia fusion proteins is more effective against HIV than the immune response elicited by the native proteins. We will use the mouse model because it will provide a wider range of data that otherwise we could not practically obtain from a primate model. The long-term objective of this study is to develop a safe vaccine that can be tested in primates and eventually in humans. The proposed project will consist of the following main objectives: 1. To construct HIV-vaccinia recombinant viruses (wild type and attenuated) expressing fusion proteins containing the full-length sequence of vaccinia 14K or 39K proteins fused to complete or partial sequence of HIV-1 env and gag and study their mode of expression in cultured cells. 2. To characterize the extent of the humoral immune response elicited in mice primed with HIV-vaccinia recombinant viruses and boosted with either the live recombinant virus, purified non-fused or purified fused proteins and to define the mode of inhibition of HIV cytopathogenicity by the specific antisera. 3. To characterize the extent of the cellular immune response in mice of different haplotype immunized as described in objective 2. If the fusion proteins elicit a more effective and sustained humoral and cellular immune response to HIV antigens than the non-fused proteins, then this novel approach may produce a useful vaccine against HIV to be used in either seronegative (as a preventive measure) or seropositive human subjects (as therapeutic agent).