Development of a Malaria DNA Vaccine with Enchanced Immunogenicity

描述（由申请人提供）：尽管DNA疫苗提供了许多优势，但它们无法在非人类灵长类动物和人类中引发持续的保护性免疫反应，这令人失望。基于来自肿瘤模型系统的大量证据和对候选约氏疟原虫疫苗的初步研究，本提案假设，通过在未成熟树突状细胞（iDC）上表达疟疾蛋白抗原作为与受体趋化因子配体连接的融合蛋白，可以开发出一种有效的基于dna的疟疾疫苗。该疫苗模型与相关方法的不同之处在于，它将适当的抗原呈递细胞吸引到接种部位，并通过将感兴趣的疫苗表位与趋化因子融合，确保抗原的有效吸收进行处理。这一假设将在约尔疟原虫感染和免疫原性的小鼠模型中得到验证。具体来说，我们将1。评估一种候选多表位DNA疫苗在BALB/c小鼠体内引起的体外反应，该疫苗通过体内电穿孔给药，针对约氏疟原虫的红细胞前阶段。待评估的候选疫苗将由表达巨噬细胞炎症蛋白31 （MIP- 31）的质粒组成，该质粒融合到(a) BALB/c小鼠中SYVPSAEQI免疫优势的P. yoelii i类限制性T细胞表位(b) BALB/c小鼠中（QGPGAP）4免疫优势的P. yoelii b细胞表位和(c) BALB/c小鼠中YNRNIVNRLLGDALNGKPEEKA免疫优势的ii类限制性T细胞表位。将创建缺乏MIP-31成分的类似DNA结构，并将建立几个额外的阳性和阴性对照免疫组。免疫结果将通过干扰素γ (IFN-3) Elispot测定、ELISA和免疫荧光测定抗体浓度以及流式细胞术评估中枢记忆T细胞的发育来测定。2. 评估该候选疫苗在小鼠疟疾攻毒模型中的保护效果。将利用实时聚合酶链式反应（real-time PCR）对小鼠肝脏样本中的疟疾基因表达以及免疫小鼠与非免疫小鼠的血液寄生虫血症水平进行定量分析。公共卫生相关性：该项目旨在研制疟疾疫苗。如果这种方法取得成功，这种疫苗可以挽救每年因这种感染而死亡的100多万人。

DESCRIPTION (provided by applicant): Although DNA vaccines offer many advantages, their inability to elicit sustained protective immune responses in non-human primates and humans has been disappointing. Based on substantial evidence derived from tumor model systems and preliminary studies with a candidate Plasmodium yoelii vaccine, this proposal hypothesizes that an effective DNA-based vaccine against malaria can be developed by expressing malarial protein antigens as fusion proteins linked to chemokine ligands for receptors on immature dendritic cells (iDC). This vaccine model differs from related approaches in that it attracts the appropriate antigen-presenting cell to the inoculation site and by fusing the vaccine epitopes of interest to the chemokine, ensures the efficient uptake of the antigen for processing. This hypothesis will be tested in a murine model of Plasmodium yoelii infection and immunogenicity. Specifically, we will 1. Evaluate ex vivo the responses elicited in BALB/c mice by a candidate multi-epitope DNA vaccine administered by in vivo electroporation that targets the pre-erythrocytic stages of P. yoelii. The candidate vaccine to be evaluated will consist of a plasmid that expresses macrophage inflammatory protein 31 (MIP- 31) fused to (a) The SYVPSAEQI immunodominant P. yoelii Class I-restricted T cell epitope in BALB/c mice (b) The (QGPGAP)4 immunodominant P. yoelii B cell epitope in BALB/c mice and (c) The YNRNIVNRLLGDALNGKPEEKA immunodominant Class II-restricted T cell epitope in BALB/c mice. A similar DNA construct will be created that lacks the MIP-31 component and several additional positive and negative control immunization groups will also be established. Outcomes of immunization will be measured by interferon gamma (IFN-3) Elispot assays, antibody concentrations measured in ELISA and immunofluorescence assays, and by flow cytometry evaluation the development of central memory T cells. 2. Evaluate the protective efficacy of this candidate vaccine in mouse malaria challenge models. Outcomes will be followed using real-time PCR to quantitate malaria gene expression in liver samples from the mice and levels of blood parasitemia attained in immune compared to nonimmune mice. PUBLIC HEALTH RELEVANCE: This project is designed to develop a vaccine for malaria. Should this approach be successful, this vaccine could save the more than 1 million deaths that occur annually as a result of this infection.