Nanocapsule-mediated topical delivery of VLP-generating Dengue DNA vaccine

描述(申请人提供)：该项目的最终目标是开发一种安全有效的登革热病毒(DENV)局部疫苗。据估计，DENV每年导致3,000万至5,000万例致人衰弱的发烧病例，导致全世界20,000多人死亡。与目前临床试验中的DENV候选疫苗相比，基于DNA的疫苗具有巨大的潜力，因为它们可以更快地产生平衡的免疫反应，生产成本更低，并且具有更好的温度稳定性。然而，DNA疫苗历来存在免疫原性低的问题。我们提出的疫苗是通过三种关键方式克服这一成功障碍的。首先，美国疾病控制和预防中心(CDC)疫苗结合了一些进展，将体液免疫从非保护性和潜在致病抗体的产生重新定向，以增加有效的中和和保护性抗体的产生。其次，疫苗通过GeneSegues(GSI)局部注射的亚50纳米(S50)胶囊直接传递到皮肤中的免疫效应细胞网络。第三，s50微囊化疫苗利用高效、大小敏感的脂筏摄取途径，直接进入免疫效应细胞的细胞核，解决了DNA传递的主要障碍。在这项第一阶段的研究中，我们建议开发一种专注于2型DENV(DENV-2)的局部DENV DNA疫苗，有四个特定的目标。首先，我们将在体内试验s50 DENV-2疫苗注射研究的基础上，在小鼠模型中确定最佳局部注射部位参数。其次，对于选定的投放和应用部位，我们将通过比较早期效应细胞转染率和抗原表达与随后的中和抗体应答和持久性，从机械上评估不同的佐剂和剂量参数。第三，使用AIMS 1和AIMS 2中确定的两个最佳S50候选疫苗，我们将通过检查DENV-2特异性免疫记忆的建立和持久性来表征细胞介导的免疫反应。第四，在另一项单独的研究中，我们将在一组半固体载体(乳液和/或凝胶)中复合s50 DENV-2 DNA疫苗，以获得最大和统一的剂量输送，并在具有最佳复合候选的小鼠身上进行原理验证研究，关键目标是通过电穿孔和肌肉注射获得与裸露DNA相同或更好的保护性中和抗体效价。未来的工作将扩大和过渡到剩下的三个登革热血清型(DENV-1、-3和-4)，并确定在两到三个月内诱导四价平衡、保护性和持久免疫的最佳配方。将通过使用DENV疫苗和疾病AG129小鼠模型来评估疫苗的有效性和安全性，包括致死挑战和亚致死抗体依赖的增强感染研究。我们还将进行制造规模扩大和其他必要的任务，以推进IND提交和人体临床试验。

DESCRIPTION (provided by applicant): The ultimate goal of this project is to develop a safe and effective topical vaccine against the dengue virus (DENV). DENV causes an estimated 30 to 50 million cases of debilitating fever leading to over 20,000 deaths worldwide every year. DNA-based vaccines have great potential against DENV because they can more quickly generate a balanced immune response, are less expensive to produce, and have greater temperature stability than current DENV vaccine candidates in clinical trial. However, DNA vaccines have historically suffered from low immunogenicity. The vaccine that we propose is engineered to overcome this barrier-to- success in three key ways. First, the US Centers for Disease Control and Prevention (CDC) vaccine incorporates advances that redirect humoral immunity away from the production of non-protective and potentially pathogenic antibodies to increase the production of potently neutralizing and protective antibodies. Second, the vaccine is delivered directly to the network of immune effector cells in the skin via GeneSegues' (GSI's) topically administered sub-50 nanometer (s50) capsules. Third, the s50 encapsulated vaccine exploits the efficient, size-sensitive lipid raft uptake pathway to traffic directly to the nucleusof immune effector cells, addressing a major hurdle to DNA delivery. In this Phase 1 study, we propose to develop a topical DENV DNA vaccine by focusing on serotype-2 (DENV-2), with four specific aims. First, we will build upon pilot in vivo s50 DENV-2 vaccine delivery studies to determine optimal topical delivery site parameters in a mouse model. Second, for the selected delivery and application site, we will mechanistically assess differential adjuvants and dosing parameters, by comparing early percent effector cell transfection and antigen expression with subsequent neutralizing antibody response and persistence. Third, using the two best s50 vaccine candidates identified in Aims 1 and 2, we will characterize cell-mediated immune responses by examining the establishment and persistence of DENV-2 specific immunological memory. Fourth, in a separate study arm, we will compound the s50 DENV-2 DNA vaccine in a panel of semisolid vehicles (lotions and/or gels) to obtain maximum and uniform dose delivery, and execute a proof-of-principle study in mice with the best compounded candidate, with a key goal of achieving equivalent or superior protective neutralizing antibody titers vs. naked DNA delivered via electroporation and via intramuscular injection. Future work will expand and transition to the three remaining dengue serotypes (DENV-1, -3 and -4) and determine the optimum formulation to elicit tetravalent balanced, protective, and long lasting immunity within two to three months. Vaccine efficacy and safety will be assessed by using the DENV vaccine and disease AG129 mouse model, including lethal challenge and sublethal antibody-dependent enhancement of infection studies. We will also conduct manufacturing scale-up and other tasks necessary to progress to IND submission and human clinical trials.