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

描述(申请人提供)：在没有保护措施的平民或军事人群中再次出现天花(或衍生)可能会导致大规模伤亡、恐慌和功能崩溃。因此，一种安全有效的痘病毒疫苗的可获得性可能会显著缓解其中一些担忧。在痘病毒编码的蛋白中，有四种膜蛋白(A33R、B5R、L1R和A27L)(称为4-POX疫苗)可产生保护性中和抗体反应，而宿主范围蛋白2(HRP2，又称018L)已被证明能在人类白细胞抗原A2转基因小鼠中产生保护性T细胞介导的免疫应答。针对这些关键天花抗原的DNA疫苗是控制天花感染的一种潜在可行的方法。这种DNA疫苗策略的一个局限性是编码抗原的DNA自然免疫原性低，导致疫苗效力有限。基因枪皮内注射DNA疫苗是一种将DNA疫苗导入体内专业APC的有效方法。本申请的重点是基于针对APC的策略来研究增强效力的不同方法。通过选择五种依赖于不同的抗原提呈机制的免疫增强方法，希望能产生新的相加或协同效应来产生有效的免疫。这些方法包括：1)优化4-POX抗原的密码子以增强抗原的表达；2)将痘病毒抗原与钙网蛋白(CRT)连接以加强抗原的处理和呈递；3)与编码产肠毒素的不耐热肠毒素(LT)的DNA共同接种以增强免疫原性；4)与表达GM-CSF的质粒共同接种以吸引APC和佐剂DNA疫苗；以及5)添加编码已知的细胞免疫靶蛋白(即痘病毒HRP2蛋白)的DNA疫苗。因此，在目前的应用中，我们将测试密码子优化的4-POX DNA疫苗和/或包含GM-CSF或LT DNA和/或通过基因枪连接到CRT的HRP2 DNA皮内递送是否能够增强抗原特异性体液和T细胞介导的免疫反应和体内抗活体痘病毒感染的抗病毒效果。拟议研究的成功实施将产生一种可能适合临床开发的改进的痘病毒DNA疫苗。

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.