Enhancing DNA vaccines using modified Bacterial Toxin A1 Subunits as adjuvants

描述(申请人提供)：霍乱毒素(CT)和相关的不耐热肠毒素(LT)是具有靶向B结构域和酶活性A结构域的细胞的AB毒素。CT(CTA1)和LT(LTA1)的酶活性A1结构域已经显示出作为遗传佐剂的特殊前景，可以增强DNA疫苗在小动物和大动物中的免疫原性，并可能在人类中提供必要的增强和剂量节约效应。在我们的第一阶段工作中，我们鉴定了CTA1和LTA1的突变体，它们在体外具有增强的酶活性，在体内具有增强的佐剂作用。我们鉴定了LTA1的一个突变体，它在小鼠中诱导的抗HIV和抗SIV细胞反应比“金标准”佐剂IL-12质粒DNA(PDNA)或体内电穿孔诱导的细胞反应高近2倍。在这个第二阶段的应用中，我们建议筛选更多的突变体，并选择一个先导A1亚单位佐剂来比较其佐剂与IL-12 pDNA和电穿孔的佐剂。使用SIV DNA疫苗原型，我们将比较小鼠和猕猴T细胞反应的大小和多功能性。我们将在猕猴研究之后进行相应的SIV挑战，以确定在免疫反应中观察到的任何数量和质量的差异是否与保护相关。我们建议通过以下具体目标来实现这些目标：目的1：确定与电穿孔和IL-12 PDNA相比，能够诱导与SIV疫苗抗原相似或更好的免疫应答的Lead A1亚单位佐剂；目标2：在猕猴模型中表征Lead A1亚单位佐剂与IL-12 PDNA和电穿孔相比的抗SIV免疫反应。目标3：确定由Lead A1亚单位佐剂接种的SIV PDNA疫苗是否提供了对同源SIVmac251攻击的保护，其效果优于IL-12 PDNA和电穿孔。如果先导A1亚基基因佐剂在同源挑战模型中被证明优于IL-12 PDNA，则将在异源挑战模型中进一步评估该佐剂。2. 与公共卫生相关：该项目的目标是开发一种先进的DNA疫苗佐剂，该佐剂基于霍乱毒素或不耐热肠毒素的改良A1亚基，具有增强的酶活性和佐剂活性。这种佐剂是提高艾滋病毒DNA疫苗在人类中的临床效用所必需的。

DESCRIPTION (provided by applicant): Cholera toxin (CT) and the related heat-labile enterotoxin (LT) are AB toxins with cell targeting B domains and enzymatically active A domains. The enzymatically active A1 domains of both CT (CTA1) and LT (LTA1) have demonstrated particular promise as genetic adjuvants that can enhance the immunogenicity of DNA vaccines in small and large animals and may provide necessary boosting and dose sparing effects in humans. In our Phase I efforts, we identified mutants of CTA1 and LTA1 with enhanced enzymatic activity in vitro and enhanced adjuvanticity in vivo. We identified a mutant of LTA1 that induced anti-HIV and anti-SIV cellular responses in mice nearly 2-fold higher than those induced by the "gold-standard" adjuvant IL-12 plasmid DNA (pDNA) or in vivo electroporation. In this Phase II application, we propose screen additional mutants and select a lead A1 subunit adjuvant to compare its adjuvanticity to that of IL-12 pDNA and electroporation. Using a prototype SIV DNA vaccine, we will compare the magnitude and polyfunctionality of the T cell response in both mice and macaques. We will follow the macaque studies with a homologous SIV challenge to determine whether any of the quantitative and qualitative differences observed in the immune response are relevant to protection. We propose to achieve these goals through the following specific aims: Aim 1: Identify a lead A1 subunit adjuvant that induces comparable or superior immune responses to SIV vaccine antigens as compared to electroporation and IL-12 pDNA; Aim 2: Characterize the anti-SIV immune responses adjuvanted by the lead A1 subunit adjuvant vs. IL-12 pDNA and electroporation in a macaque model Aim 3: Determine if administration of a SIV pDNA vaccine adjuvanted by the lead A1 subunit provide protection from homologous SIVmac251 challenge that is superior to that provided by IL-12 pDNA and electroporation. If the lead A1 subunit genetic adjuvant proves to be superior to IL-12 pDNA in the homologous challenge model, this adjuvant will be further evaluated in a heterologous challenge model. 2 PUBLIC HEALTH RELEVANCE: The objective of this project is to develop an advanced DNA vaccine adjuvant based on a modified A1 subunit of cholera toxin or heat-labile enterotoxin with enhanced enzymatic activity and adjuvanticity. Such an adjuvant is needed to enhance the clinical utility of HIV DNA vaccination in humans.