Integrase-defective lentiviral based influenza vaccines

描述（由申请人提供）：疫苗开发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在人细胞中活跃的启动子区域的转录控制下构成的。它们是细胞免疫反应的良好诱导剂，但它们需要大量的DNA和三次给药才能有效。慢病毒载体（IC-LV）是细胞介导和体液免疫反应的强大诱导剂，然而它们携带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）在整合酶基因中有突变，不整合到基因组中，也不复制，但在没有整合的情况下，它们在细胞核中作为外遗体环状DNA （E-DNA）维持。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，由ID-LV表达的蛋白被免疫系统识别。此外，在体内单次肌肉注射后，表达GFP的ID-LV引发了对GFP的持久免疫反应，这表明单次给药可能足以诱导保护性反应。作为ID-LV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IC-LV所获得的应答。使用流感病毒有几个好处。首先，细胞介导免疫可以保护机体免受多种毒株的病毒攻击。其次，在动物模型中，DNA疫苗已被证明在诱导保护性细胞介导免疫方面是有效的。在目的1中，我们将比较ID-LV、IC-LV和表达流感NP的DNA疫苗免疫引起的体液和细胞免疫反应。在目标2中，我们将测试ID-LV保护动物免受亚致死或致死挑战的能力，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从ID-LV表达的蛋白质在体内保护免受病毒攻击的功效，并比较非整合和整合载体与传统DNA疫苗之间的保护功效和免疫参数。这些研究将确定ID-LV作为递送载体的一般效用。如果成功，考虑到非整合的安全性，这种方法将代表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被免疫系统识别，并且在体内单次肌内注射后可以引发持久的免疫反应。在本研究中，作为ID-LV作为疫苗有效性的概念验证，我们将比较表达流感病毒NP蛋白的ID-LV与传统DNA疫苗和整合酶胜任型ID-LV诱导的应答。流感小鼠模型提供了第一次机会来测试从ID-LV表达的蛋白质在体内保护免受病毒攻击的功效，并比较ID-LV和IC-LV与传统DNA疫苗之间的保护功效和免疫参数。这些研究将确定ID-LV作为递送载体的效用。如果成功，考虑到非整合的安全性，这种方法将代表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.