Integrase Defective Lentiviral Vectors for Antibody Delivery against Influenza

 描述（由申请人提供）：全球化、耐药性和生物恐怖主义都加剧了传染病的威胁，但尽管医学取得了进步，我们通常仍然无法快速保护非免疫人群。被动免疫疗法是一种快速且成功的保护方法，但单克隆抗体 (mAb) 价格昂贵，并且需要高重复剂量才能有效。因此，这种方式不适合大规模使用。最近提出使用病毒载体递送mAbs作为直接注射mAbs的有吸引力的替代方案。为此，使用了多种病毒载体 表达单克隆抗体；然而，病媒免疫和安全问题阻碍了这一战略的制定。 该提案的主要目标是解决使用整合酶缺陷型慢病毒载体（IDLV）递送单克隆抗体的可行性。 IDLV 与其他病毒载体相比具有多种优势，包括不存在预先存在的抗载体免疫力以及非整合和非复制的安全特性。 IDLV 也在非分裂细胞中维持，并且可以在体内表达稳定水平的功能蛋白数月。 为了证明使用 IDLV 表达可预防传染病的单克隆抗体的可行性，我们将设计 IDLV 以表达针对甲型流感病毒 (IAV) 血凝素 (HA) 的单克隆抗体，并测试其预防 IAV 感染的能力。使用 IAV 作为模型的优点是 HA 特异性抗体介导的保护机制是明确定义的。此外，被动免疫和超免疫血清已成功用于预防和治疗IAV。在我们的初步数据中，我们表明 IDLV 可以在体外的转导细胞中表达抗 HA mAb，并且我们对其水平进行了量化。根据对保护所需的单克隆抗体数量的估计，我们假设 IDLV 将产生足够水平的单克隆抗体来保护小鼠免受 IAV 攻击。此外，由于我们表明表达流感 NP 的 IDLV 可以引发针对 IAV 的保护性免疫，因此我们将测试结合 mAb 和保守流感抗原的基因递送来预防 IAV 的有效性。我们将通过以下目标检验我们的假设：1. 测量小鼠血清或肺部中 IDLV 产生的 mAb 的水平和持久性，并确定攻击实验中使用的最佳剂量，2. 评估表达抗 HA mAb 的 IDLV 抵御致命 IAV 攻击的能力，以及 3. 评估给药后不同时间共同施用表达抗 HA mAb 的 IDLV 和表达 NP 的 IAV 防护能力。 如果成功，我们的研究将首次证明 IDL 作为单克隆抗体基因传递工具的有效性，以防止传染源。此外，单克隆抗体与保护性抗原的表达是疾病预防中的一种新应用，可能对 IAV 和其他传染病的预防产生广泛影响。鉴于非整合的安全特性，该载体的成功开发将代表基因传递和疫苗开发的重要一步。

 DESCRIPTION (provided by applicant): Globalization, drug resistance, and bioterrorism all contribute to the growing threat from infectious diseases, but despite medical advances, we are often still unable to rapidly protect non-immune populations. Passive immunotherapy is a fast and successful method of protection, but monoclonal antibodies (mAbs) are expensive and require high, repeated doses to be effective. Therefore, this approach is not suitable for large-scale use. The delivery of mAbs using a viral vector has been recently proposed as an attractive alternative to the direct injection of mAbs. To this end, a variety of viral vectors have been used to express mAbs; however, vector immunity and safety issues have hampered the development of this strategy. The main of objective of this proposal is to address the feasibility of using integrase-defective lentiviral vectors (IDLV) to deliver mAbs. IDLV have several advantages over other viral vectors, including the absence of pre-existing anti-vector immunity and the safety features of non-integration and non-replication. IDLV are also maintained in non-dividing cells, and can express steady levels of functional proteins in vivo for months. As proof of the feasibility of using IDLV to express mAbs that protect against infectious disease, we will engineer IDLV to express mAbs against the influenza A virus (IAV) hemagglutinin (HA), and will test their ability to protect against IAV infection. An advantage of using IAV as a model is that HA-specific antibody- mediated mechanisms of protection are well-defined. Furthermore, both passive immunization and hyperimmune sera have been successfully used to prevent and treat IAV. In our preliminary data, we show that IDLV can express anti-HA mAbs in vitro in transduced cells, and we quantify the levels. Based on an estimate of the amount of mAbs needed for protection, we hypothesize that IDLV will produce an adequate level of mAbs to protect against IAV challenge in mice. In addition, since we showed that IDLV expressing influenza NP could elicit protective immunity against IAV, we will test the effectiveness of combining the genetic delivery of a mAb and a conserved influenza antigen to protect against IAV. We will test our hypothesis through the following aims: 1. measure the levels and persistence of mAbs produced from IDLV in the serum or lungs of mice and determine the optimal dose for use in challenge experiments, 2. assess the ability of IDLV expressing anti-HA mAbs to protect against lethal IAV challenge, and 3. evaluate the ability of co-administration of IDLV expressing anti-HA mAbs and IDLV expressing NP to protect from IAV at different times after administration. If successful, our study would be the first to show the effectiveness of IDL as a tool for genetic delivery of mAbs that protect against infectious agents. Additionally, expression of mAbs along with a protective antigen is a novel application in disease prevention that could have a broad impact on the prevention of IAV and other infectious diseases. Given the safety feature of non-integration, the successful development of this vector would represent a significant step in gene delivery and vaccine development.