GENETIC ENGINEERING AND EXPRESSION ANALYSIS

本提案的目的是开发重组 牛痘病毒作为用于递送疫苗的活病毒载体 它提供全面和长期的保护， HIV-1病毒株。 本实验室痘病毒生物学研究 对我们理解病毒有特殊贡献 增长和复制，直接应用于 牛痘重组体的制备。 一项计划已经到位， 是在不影响病毒 作为疫苗的有效性，以及无药物和诱变剂的选择 已开发出用于重组体的快速选择的方案。 单一病毒中多种抗原的调节表达， 已经实现，并在同源的应用新的概念， 重组正在进行中。 对于发展建立一套 一种抗艾滋病有效疫苗， 将构建表达多种HIV-1和SIV抗原的 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体 env、gag、pol和sor的单价、二价和三价 构象 多价重组病毒代表了 可能的候选疫苗，因为它提供了最大数量的 HIV-1的抗原，并可能引发最广泛的免疫 反应 单价和二价重组牛痘将 有助于描述单个抗原对免疫反应的贡献 免疫应答和抗原之间可能的协同作用。 未来 将基于以下构建几代重组体： 体液和细胞介导的研究结果的同化 使用第一代重组体的免疫， 特异性表位和突变基因的免疫原性。 为 例如，编码以下的单个多肽产物的基因： 将分离env、gag和pol并操作用于表达， 和包膜中的免疫显性非中和结构域 糖蛋白将利用定点诱变而缺失。 信封中观察到的菌株多样性问题 糖蛋白也将被处理。 所有研究将 最终导致候选疫苗的制备 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.