Integrase-defective lentiviral based influenza vaccines

描述(申请人提供)：疫苗开发中的一个关键问题是找到一种有效的方法来诱导细胞免疫。DNA疫苗是由编码靶抗原的质粒组成的，受人类细胞中活跃的启动子区域的转录控制。它们是细胞免疫反应的良好诱导者，但它们需要高含量的DNA和三次给药才能有效。慢病毒载体(IC-LV)是细胞免疫和体液免疫反应的强大诱导者，但它们携带着与宿主基因组整合相关的内在风险。非整合慢病毒载体(ID-LV)的整合酶基因发生突变，不整合到基因组中，也不复制，但在没有整合的情况下保持为核内的环状DNA(E-DNA)。我们之前已经证明，E-DNA是稳定的，可以表达功能蛋白。在细胞培养中，ID-LV表达的蛋白质被免疫系统识别。此外，表达GFP的ID-LV在体内单次肌肉注射后可诱导对GFP的持久免疫反应，提示单次给药可能足以诱导保护性反应。作为对ID-LV有效性的概念验证，我们将比较表达流感病毒NP蛋白的ID-LV与传统DNA疫苗和IC-LV获得的应答。使用流感病毒有几个好处。首先，细胞介导的免疫提供了针对病毒挑战的多种毒株的保护。其次，DNA疫苗已被证明在动物模型中诱导保护性细胞免疫是有效的。在目标1中，我们将比较表达流感病毒NP的ID-LV、IC-LV和DNA疫苗免疫诱导的体液免疫和细胞免疫应答。在目标2中，我们将测试ID-LV保护动物免受亚致死或致命攻击的能力，并解决交叉保护的问题。流感小鼠模型提供了第一次机会来测试ID-LV表达的蛋白在体内保护病毒攻击的有效性，并比较非整合载体和整合载体与传统DNA疫苗的保护效果和免疫学参数。这些研究将确定ID-LV作为递送媒介的一般用途。如果成功，考虑到非整合的安全特性，这种方法将代表着疫苗开发的重要一步。公共卫生相关性：我们之前已经证明，整合酶缺陷慢病毒载体(ID-LV)表达的蛋白质在细胞培养中可被免疫系统识别，并可在体内单次肌肉注射后引发持久的免疫反应。在这项建议中，作为对ID-LV作为疫苗有效性的概念证明，我们将比较表达流感病毒NP蛋白的ID-LV与传统DNA疫苗和整合酶活性LV所获得的应答。流感小鼠模型提供了第一次机会来测试ID-LV表达的蛋白对体内病毒攻击的保护效果，并比较ID-LV和IC-LV与传统DNA疫苗的保护效果和免疫学参数。这些研究将确定ID-LV作为递送媒介的效用。如果成功，鉴于非整合的安全特性，这种方法将代表着疫苗开发的重要一步。

DESCRIPTION (provided by applicant): A key problem in vaccine development is finding an effective way to induce cellular immunity. DNA vaccines are constituted by a plasmid encoding the target antigen under the transcriptional control of a promoter region active in human cells. They are good inducers of cellular immune responses however they require a high amount of DNA and three administrations to be effective. Lentiviral vectors (IC-LV) are powerful inducers of cell mediated and humoral immune responses, however they carry the intrinsic risk associated with integration into the host genome. Non-integrating lentiviral vectors (ID-LV) have a mutation in the integrase gene, do not integrate into the genome, nor do they replicate, but are maintained as episomal circular DNA in the nucleus (E-DNA) in the absence of integration. We have previously shown that E-DNA is stable and can express functional proteins. Proteins expressed by ID-LV are recognized by the immune system in cell culture. In addition, ID-LV expressing GFP elicited a long-lasting immune response to GFP after a single intramuscular injection in vivo suggesting that a single administration may be sufficient to induce a protective response. As proof-of-concept of the effectiveness of ID-LV, we will compare the responses induced by ID-LV expressing the NP protein of influenza virus with those obtained with conventional DNA vaccines and IC-LV. Use of influenza virus offers several advantages. First, cell-mediated immunity confers protection from multiple strains towards viral challenge. Second, DNA vaccines have been proven to be effective in inducing protective cell-mediated immunity in animal models. In Aim 1 we will compare humoral and cellular immune response, elicited by immunization with ID-LV, IC-LV and DNA vaccine expressing influenza NP. In Aim 2 we will test the ability of ID-LV to protect the animal from sublethal or lethal challenge and address the question of cross-protection. The influenza mouse model provides the first opportunity to test the efficacy of a protein expressed from ID-LV to protect from a viral challenge in vivo and to compare both protection efficacy and immunologic parameters among the non-integrated and integrated vector and a conventional DNA vaccine. These studies will define the general utility of ID-LV as a delivery vector. If successful, this approach would represent a significant step in vaccine development given the safety feature of non integration. PUBLIC HEALTH RELEVANCE: We have previously shown that proteins expressed by integrase-defective lentiviral vectors (ID-LV) are recognized by the immune system in cell culture and can elicit a long-lasting immune response after a single intramuscular injection in vivo. In this proposal, as proof-of-concept of the effectiveness of ID-LV as a vaccine, we will compare the responses induced by ID-LV expressing the NP protein of influenza virus with those obtained with conventional DNA vaccines and integrase-competent-LV. The influenza mouse model provides the first opportunity to test the efficacy of a protein expressed from ID-LV to protect from a viral challenge in vivo and to compare both protection efficacy and immunologic parameters among the ID-LV and IC-LV and a conventional DNA vaccine. These studies will define the utility of ID-LV as a delivery vector. If successful, this approach would represent a significant step in vaccine development given the safety feature of non-integration.