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

描述（由申请人提供）：天花（或衍生品）在未受保护的平民或军队人口中重新出现可能会造成大规模伤亡，恐慌和功能崩溃。因此，安全有效的痘病毒疫苗的可用性可能会大大减轻这些担忧。在痘病毒编码的蛋白中，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，宿主范围蛋白2 （HRP2，也称为018L）已被证明可在HLA-A2转基因小鼠中产生保护性T细胞介导的免疫应答，以抵抗痘病毒的攻击。针对这些关键天花抗原的DNA疫苗代表了控制天花感染的一种可能可行的方法。这种DNA疫苗接种策略的一个局限性是编码抗原的DNA的天然低免疫原性，导致疫苗效力有限。通过基因枪皮内给药DNA疫苗是一种有效的将DNA疫苗输送到体内专业APCs的方法。本申请的重点是研究基于针对apc的策略提高效力的不同方法。通过选择五种依赖于不同抗原呈递机制的免疫增强方法，希望能激发出新的加性或协同效应，从而产生有效的免疫。这些方法包括：1)优化4痘抗原密码子，提高抗原表达；2)痘病毒抗原与钙网蛋白（CRT）连锁，增强抗原加工和递呈；3)与产肠毒素大肠杆菌编码热不稳定肠毒素（LT）的DNA共给药，增强免疫原性；4)与表达GM-CSF的质粒共给药，以吸引apc和佐剂DNA疫苗；5)添加一种DNA疫苗，编码一种记录的细胞介导免疫的靶蛋白（即痘病毒HRP2蛋白）。因此，在目前的应用中，我们将测试皮内递送密码子优化的4痘DNA疫苗和/或HRP2 DNA佐剂，包括GM-CSF或LT DNA和/或通过基因枪连接到CRT，是否会导致抗原特异性体液和T细胞介导的免疫反应增强，以及体内对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.