FUSION PROTEIN AS IMMUNOGENS AGAINST HIV INFECTION

控制艾滋病的一个可取的方法是研制一种疫苗 可以赋予人类长期免疫力 免疫缺陷病毒（HIV），同时很容易 在全球范围内管理。 我们已经鉴定出两种高度抗原性的 牛痘病毒蛋白的14 K和39 K，并表明，当我们 将14 K与HIV-1 env或gag共价连接，表达融合蛋白 在感染HIV-牛痘病毒重组体的细胞中， 观察到以下结果：1）融合蛋白定位于 细胞表面以寡聚体的形式，2）不被切割或 3）env糖基化较差，4）当 将重组病毒接种于小鼠体内， 抗HIV env和gag的特异性抗体。 这些的安全性 HIV重组痘苗病毒在 免疫抑制小鼠 鉴于14 K融合的独特性， 蛋白，我们认为HIV-牛痘融合蛋白可能提供 增强和改变B和T细胞应答的有效手段 对抗艾滋病病毒感染。 本提案的目的是确定 是否由HIV-牛痘融合蛋白引起的免疫应答 对艾滋病病毒更有效， 天然蛋白质。 我们将使用小鼠模型，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， 灵长类动物，最终在人类身上。 拟议项目将 包括以下主要目标： 1. 构建HIV-痘苗重组病毒（野生型和 减毒的）表达融合蛋白，所述融合蛋白含有 融合到融合子中的牛痘14 K或39 K蛋白的全长序列 HIV-1 env和gag的全序列或部分序列及研究 它们在培养细胞中的表达模式。 2. 描述体液免疫反应的程度 在用HIV-牛痘重组病毒致敏的小鼠中引发 并用活的重组病毒、纯化的 非融合或纯化的融合蛋白，并定义 通过特异性抗血清抑制HIV细胞致病性。 3. 为了表征细胞免疫应答的程度， 不同的小鼠 单倍型免疫，如目的2所述。 如果融合蛋白能引发更有效和持续的体液免疫， 和细胞免疫反应的艾滋病毒抗原比非融合 蛋白质，那么这种新的方法可能会产生一种有用的疫苗 用于血清阴性（作为预防性 测量）或血清阳性人类受试者（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).