GENETIC ENGINEERING AND EXPRESSION ANALYSIS

这项提议的目的是发展重组人的使用 痘苗病毒作为活病毒载体用于运送疫苗 提供全面和长期的保护，防止多个 HIV-1毒株。痘病毒生物学在本实验室的研究 为我们对病毒的理解做出了具体贡献 增长和复制已直接应用于 重组牛痘疫苗的制备。已经制定了一项计划， 正在衍生病毒的减毒形式而不影响其 作为疫苗的有效性，以及不含药物和诱变剂的选择 为了快速选择重组子，已经开发了一种方案。 在单个病毒中多种抗原的调节表达具有 已经实现，并且新的概念在同源的应用中 重组正在进行中。用于开发一种 有效的艾滋病疫苗，重组牛痘病毒 将构建表达HIV-1和SIV的多种抗原 并用于研究对这些病原体的免疫反应。 这些重组体将包括那些表达基因产物的重组子。 单价、二价和三价的env、Gag、Poll和SOR 构象。一种多价重组病毒代表了 可能的候选疫苗，因为它提供了最多的 来自HIV-1的抗原，并有可能诱导最广泛的免疫 回应。单价和双价重组牛痘疫苗将 帮助描绘单个抗原对 免疫反应，以及抗原之间可能的协同作用。未来 一代代的重组子将基于 体液和细胞介导性研究结果的同化 用第一代重组体免疫，并进行了研究 特定表位和突变基因的免疫原性。为 例如，编码单个多肽产品的基因 Env、Gag和Pol将被分离和操纵以用于表达， 和包膜中的免疫显性非中和结构域 糖蛋白将利用定点突变被删除。 包膜中观察到的菌株多样性问题 糖蛋白也将被提及。所有研究加在一起将会 最终导致制备可供使用的候选疫苗 在人体I期临床试验中。

The objective of this proposal is to develop the use of recombinant vaccinia virus as a live viral vector for the delivery of a vaccine which yields total and long-term protection against multiple strains of HIV-1. Studies of poxvirus biology in this laboratory have made specific contributions to our understanding of virus growth and replication which have had direct application in the preparation of vaccinia recombinants. A program is in place which is deriving attenuated forms of the virus without affecting its efficacy as a vaccine, and a drug- and mutagen-free selection scheme has been developed for the rapid selection of recombinants. The modulated expression of multiple antigens in a single virus has been achieved, and novel concepts in the application of homologous recombination are being pursued. For the development of an effective vaccine against AIDS, recombinant vaccinia virus which express numerous antigens from HIV-1 and SIV will be constructed and used in studies of the immune response to these pathogens. These recombinants will include those expressing the gene products of env, gag, pol, and sor, in monovalent, divalent, and trivalent conformations. A multivalent recombinant virus represents the most likely candidate vaccine as it presents the largest number of antigens from HIV-1 and will potentially elicit the broadest immune response. The monovalent and divalent recombinant vaccinia will aid in delineating the contribution of individual antigens to the immune response, and possible synergy between antigens. Future generations of recombinants will be constructed based on the assimilation of results from studies of humoral and cell-mediated immunity using the first generation of recombinants, with studies of the immunogenicity of specific epitopes and mutated genes. For example, the genes encoding the individual polypeptide products of env, gag, and pol will be isolated and manipulated for expression, and immunodominant, non-neutralizing domains in the envelope glycoprotein will be deleted utilizing site-directed mutagenesis. The issue of the strain diversity observed in the envelope glycoprotein will also be addressed. All studies combined will ultimately lead to the preparation of candidate vaccines for use in human Phase I clinical trials.