Enhancing DNA vaccines using modified Bacterial Toxin A1 Subunits as adjuvants

描述（由申请方提供）：霍乱毒素（CT）和相关的不耐热肠毒素（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（CTA 1）和LT（LTA 1）的酶活性A1结构域已被证明特别有希望作为遗传佐剂，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加强和剂量节省效应。在我们的I期研究中，我们鉴定了CTA 1和LTA 1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活性。我们鉴定了LTA 1的突变体，其在小鼠中诱导的抗HIV和抗SIV细胞应答比由“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。在该II期应用中，我们建议筛选额外的突变体并选择前导A1亚基佐剂以将其佐剂性与IL-12 pDNA和电穿孔的佐剂性进行比较。使用原型SIV DNA疫苗，我们将比较小鼠和猕猴的T细胞反应的幅度和多功能性。我们将遵循猕猴研究与同源SIV的挑战，以确定是否有任何定量和定性差异，在免疫应答中观察到的保护。我们提出通过以下具体目的实现这些目标：目的1：鉴定与电穿孔和IL-12 pDNA相比诱导对SIV疫苗抗原相当或上级免疫应答的前导A1亚单位佐剂;目的2：表征在猕猴模型中由前导A1亚单位佐剂与IL-12 pDNA和电穿孔相比佐剂化的抗SIV免疫应答。确定施用由前导A1亚基佐剂化的SIV pDNA疫苗是否提供对同源SIVmac 251攻击的保护，其上级由IL-12 pDNA和电穿孔提供的保护。如果在同源激发模型中证明先导A1亚基基因佐剂上级IL-12 pDNA，则将在异源激发模型中进一步评估该佐剂。2 公共卫生相关性：本项目的目的是开发一种先进的DNA疫苗佐剂，其基于具有增强的酶活性和佐剂性的经修饰的霍乱毒素或不耐热肠毒素的A1亚单位。需要这样的佐剂来增强HIV DNA疫苗接种在人类中的临床效用。

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.