Integrase Defective Lentiviral Vectors for Antibody Delivery against Influenza

 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，我们往往仍然无法迅速保护非免疫人群。被动免疫疗法是一种快速而成功的保护方法，但单克隆抗体（mAb）价格昂贵，需要高剂量重复使用才能有效。因此，这种方法不适合大规模使用。使用病毒载体递送mAb最近已被提出作为mAb直接注射的有吸引力的替代方案。为此，已经使用了多种病毒载体 然而，载体免疫和安全性问题阻碍了这一策略的发展。 本提案的主要目的是解决使用整合酶缺陷型慢病毒载体（IDLV）递送mAb的可行性。IDLV具有优于其他病毒载体的几个优点，包括不存在预先存在的抗载体免疫以及非整合和非复制的安全特征。IDLV也维持在非分裂细胞中，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表达抗传染病mAb的可行性，我们将设计IDLV以表达抗甲型流感病毒（IAV）血凝素（HA）的mAb，并将测试其抗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的。此外，被动免疫和超免疫血清已成功地用于预防和治疗IAV。在我们的初步数据中，我们表明IDLV可以在体外转导细胞中表达抗HA mAb，并且我们定量了水平。基于对保护所需的mAb量的估计，我们假设IDLV将产生足够水平的mAb以保护小鼠免受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，我们将测试将mAb和保守流感抗原的遗传递送组合以保护免受IAV的有效性。我们将通过以下目标来检验我们的假设：1。测量小鼠血清或肺中由IDLV产生的mAb的水平和持久性，并确定用于攻击实验的最佳剂量，2.评估表达抗HA mAb的IDLV保护免于致死性IAV攻击的能力，和3.评价表达抗HA mAb的IDLV和表达NP的IDLV在给药后不同时间共同给药保护免受IAV的能力。 如果成功的话，我们的研究将是第一个显示IDL作为遗传递送mAb的工具的有效性的研究，这些mAb可以防止感染性病原体。此外，单克隆抗体沿着保护性抗原的表达是疾病预防中的新应用，其可对预防IAV和其它传染病具有广泛影响。鉴于非整合的安全性特征，该载体的成功开发将代表基因递送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.