Dev. of Optimized/Adjuvanted Smallpox DNA Vaccine (Gene Gun)

描述（由申请人提供）：天花（或衍生物）在未受保护的平民或军人中重新出现可能会导致大规模伤亡、恐慌和功能崩溃。因此，安全有效的痘病毒疫苗的出现可能会显着缓解其中一些担忧。在痘病毒编码蛋白中，四种膜蛋白（A33R、B5R、L1R 和 A27L）（称为 4 痘疫苗）已被证明可产生保护性中和抗体反应，而宿主范围蛋白 2（HRP2，也称为 018L）已被证明可在 HLA-A2 转基因小鼠中产生针对痘病毒攻击的保护性 T 细胞介导的免疫反应。针对这些关键天花抗原的 DNA 疫苗代表了一种控制天花感染的潜在可行方法。这种 DNA 疫苗接种策略的一个限制是 DNA 编码抗原的天然低免疫原性，导致疫苗效力有限。通过基因枪皮内注射 DNA 疫苗是将 DNA 疫苗递送到专业 APC 体内的有效方法。本申请的重点是研究基于针对 APC 的策略来增强效力的不同方法。通过选择五种依赖于改善抗原呈递的不同机制的免疫增强方法，希望能够引发新的相加或协同效应，从而产生有效的免疫力。这些方法包括：1) 4-pox 抗原的密码子优化以增强抗原表达； 2) 痘病毒抗原与钙网蛋白 (CRT) 连接以增强抗原加工和呈递； 3)与来自产肠毒素大肠杆菌的编码不耐热肠毒素(LT)的DNA共同施用以增强免疫原性； 4) 与表达GM-CSF的质粒共同施用以吸引APC和佐剂DNA疫苗； 5) 添加编码已记录的细胞介导免疫靶蛋白（即痘病毒 HRP2 蛋白）的 DNA 疫苗。因此，在当前的应用中，我们将测试通过包含GM-CSF或LT DNA和/或通过基因枪与CRT连接佐剂的密码子优化的4痘DNA疫苗和/或HRP2 DNA的皮内递送是否会导致增强的抗原特异性体液和T细胞介导的免疫反应以及针对体内活痘病毒感染的抗病毒作用。拟议研究的成功实施将产生一种改进的痘病毒 DNA 疫苗，可能适合临床开发。

DESCRIPTION (provided by applicant): The reemergence of smallpox (or derivative) in an unprotected civilian or military population would likely cause mass casualties, panic, and functional breakdown. Thus, the availability of a safe effective poxvirus vaccine may significantly alleviate some of these concerns. Among the poxvirus-encoded proteins, four membrane proteins (A33R, B5R, L1R and A27L) (called 4-pox vaccine) have been shown to generate protective neutralizing antibody responses and host range protein 2 (HRP2, also called 018L) has been shown to generate protective T cell-mediated immune response against poxvirus challenge in HLA-A2 transgenic mice. DNA vaccines targeting these key smallpox antigens represent a potentially plausible approach for the control of smallpox infections. A limitation of this DNA vaccination strategy is the naturally low immunogenicity of DNA encoding antigen, resulting in limited vaccine potency. Intradermal administration of DNA vaccines via gene gun represents an efficient method for delivery of DNA vaccines into professional APCs in vivo. The focus of the present application is to investigate different methods of enhancing potency based upon strategies that target the APCs. By selecting five methods of immune enhancement that rely on different mechanisms of improving antigen presentation, it is hoped that novel additive or synergistic effects will be elicited to generate effective immunity. These methods include: 1) codon optimization of the 4-pox antigens to enhance antigen expression; 2) linkage of poxvirus antigen to calreticulin (CRT) to enhance antigen processing and presentation; 3) co-administration with DNA encoding heat labile enterotoxin (LT) from enterotoxigenic E. coli to enhance immunogenicity; 4) co-administration with a plasmid expressing GM-CSF to attract APCs and adjuvant DNA vaccines; and 5) addition of a DNA vaccine encoding a documented target protein of cell-mediated immunity (i.e. poxvirus HRP2 protein). Thus, in the current application, we will test if intradermal delivery of a codon-optimized 4-pox DNA vaccines and/or HRP2 DNA adjuvanted by inclusion of GM-CSF or LT DNA and/or linkage to CRT via gene gun would lead to enhanced antigen-specific humoral and T cell-mediated immune responses and antiviral effects against live poxvirus infections in vivo. The successful implementation of the proposed studies will result in an improved poxvirus DNA vaccine potentially suitable for clinical development.