Enhancing DNA vaccines using modified Bacterial Toxin A1 Subunits as adjuvants

描述（由申请人提供）：霍乱毒素（CT）和相关的热不稳定肠毒素（LT）是具有细胞靶向B结构域和酶活性A结构域的AB毒素。CT （CTA1）和LT （LTA1）具有酶活性的A1结构域作为遗传佐剂已显示出特别的前景，可以增强DNA疫苗在小型和大型动物中的免疫原性，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I期研究中，我们发现CTA1和LTA1突变体在体外具有增强的酶活性，在体内具有增强的佐剂性。我们发现LTA1突变体在小鼠中诱导的抗hiv和抗siv细胞应答比“金标准”佐剂IL-12质粒DNA （pDNA）或体内电穿孔诱导的应答高近2倍。在这个II期申请中，我们建议筛选额外的突变体，并选择一个先导A1亚基佐剂，以比较其与IL-12 pDNA和电穿孔的佐剂的佐剂性。使用SIV DNA疫苗原型，我们将比较小鼠和猕猴中T细胞反应的大小和多功能性。我们将继续对猕猴进行同源SIV攻击研究，以确定在免疫反应中观察到的任何定量和定性差异是否与保护有关。我们建议通过以下具体目标来实现这些目标：目标1：与电穿孔和IL-12 pDNA相比，鉴定一种先导A1亚基佐剂，可诱导对SIV疫苗抗原的相当或更好的免疫反应；目的2：在猕猴模型中表征由A1亚基佐剂对IL-12 pDNA和电穿孔佐剂的抗SIV免疫应答。目的3：确定由A1亚基佐剂的SIV pDNA疫苗是否比IL-12 pDNA和电穿孔佐剂更能提供对同源SIVmac251攻击的保护。如果A1亚基先导遗传佐剂在同源攻击模型中优于IL-12 pDNA，该佐剂将在异源攻击模型中进一步评估。2

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.