Project 2. cGMP manufacture of HIV-1 Env trimer sortase A-conjugated nanoparticles

项目2. HIV-1 Env三聚体分选酶A结合纳米颗粒的cGMP生产

• 批准号: 10369070
• 负责人: Jason Dickens
• 金额: $ 331.11万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-17 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10369070
• 关键词: 2019-nCoV; Address; Adopted; Affinity; Antibody Response; Antigens; Binding; Binding Sites; Cell Line; Chimeric Proteins; Clinical Trials; Complex; Consumption; Cyclic GMP; Development; Enzymes; Evaluation; Ferritin; Funding; Future; Generations; Genes; Goals; Grant; HIV Vaccine Trials Network; HIV-1; HIV-1 vaccine; Human; Immune response; Immune system; Immunology; Infection; Link; Methods; Molecular Conformation; Nature; Pharmaceutical Preparations; Phase I Clinical Trials; Process; Production; Protein Engineering; Proteins; Recombinants; Regimen; Research; Safety; Series; Specificity; Staphylococcus aureus; Surface; System; Testing; Time; Toxicology; Translating; Vaccination; Vaccine Design; Vaccines; Viral Vaccines; Work; cGMP production; cost; design; expectation; experimental study; human pathogen; immunogenicity; innovation; iterative design; misfolded protein; nanoparticle; nanoparticle drug; neutralizing antibody; pathogen; phase I trial; polypeptide; preservation; programs; research clinical testing; response; safety and feasibility; sortase; stability testing; vaccine candidate; vaccine development

ABSTRACT-PROJECT 2 A key goal of HIV-1 vaccine development is to induce long-lasting protective broadly neutralizing antibody (bnAb) responses that can inhibit HIV-1 infection. However, such protective immune responses have been difficult to induce in the setting of vaccination. One strategy to induce these protective responses is to present to the immune system the target for neutralizing antibodies, HIV-1 envelope (Env) trimers in their native form. Multimerizing Env trimers on nanoparticles (NPs) enhances their immunogenicity, thus a goal for immunogen design is to present multiple copies of well-folded Env trimers. Env is a metastable protein that adopts multiple conformations. The primary obstacle facing Env trimer NP vaccines is that Env fusion nanoparticles, recombinantly expressed as a single polypeptide chain, can adopt a mixture of well-folded and misfolded conformations. The misfolded Env trimers may induce non-neutralizing antibody responses that change Env conformation upon binding or compete with bnAb precursors for binding to HIV-1 Env. For HIV-1 Env trimer NPs to become a rapid and widely-used platform for HIV-1 vaccine design, a nanoparticle platform that easily incorporates well-folded Env trimers without the need for costly, year-long protein engineering experiments is needed. To overcome the hurdles of HIV-1 Env nanoparticle vaccines, we have innovated a rapid system for generating well-folded HIV-1 Env trimer nanoparticles using a sortase A conjugation method. The HIV-1 Env trimers and ferritin are produced separately and to a high purity, allowing for elimination of misfolded protein. By mixing HIV-1 Env trimer, ferritin and sortase A enzyme, the HIV-1 Env trimer is covalently linked to the ferritin nanoparticle. The resulting sortase A-conjugated nanoparticles (scNPs) display the Env trimer in the desired conformation. The overall goals of Project 2 are to translate sortase A-conjugated nanoparticles into a cGMP-capable process for generation of viral vaccines against numerous pathogens and to manufacture two HIV-1 bnAb-targeting immunogens for evaluation in a Phase I trial conducted by the HIV Vaccine Trials Network (HVTN). To accomplish this goal, we will first determine optimal expression system for ferritin and sortase A and establish target product profiles for platform production (Aim 1). Then, we will develop, manufacture, and quality release recombinant sortase A and ferritin platform molecules for the conjugation of Env stabilized trimer to ferritin nanoparticles (Aim 2). Next, we will cGMP produce and quality release >1 g of two different stabilized Env trimer scNP drug substances and drug products for toxicology studies and clinical testing in a Phase I clinical trial (Aim 3). The first Env trimer scNP, CH505 TF Env trimer scNP, will be used as the first boosting immunogen in a CH505 Env NP sequential vaccine targeting CD4 binding site bnAbs. Finally, we will produce an additional sequential Env trimer scNP to select for further maturation of the same CD4 binding site bnAb lineage (Aim 3). The impact of translating this platform to cGMP manufacturing is that it will enable rapid production and quicker testing in clinical trials of high-quality HIV-1 Env trimer NP vaccines.

项目2摘要 HIV-1疫苗开发的一个关键目标是诱导持久的保护性广泛中和抗体 （bnAb）反应，可以抑制HIV-1感染。然而，这种保护性免疫反应已经被 在接种疫苗的情况下很难诱导。诱导这些保护性反应的一种策略是 中和抗体的靶标HIV-1包膜（Env）三聚体以其天然形式进入免疫系统。 在纳米颗粒（NPs）上多聚化Env三聚体增强了它们的免疫原性，因此免疫原性研究的目标是 设计是呈现良好折叠的Env三聚体的多个拷贝。Env是一种亚稳态蛋白， 构象Env三聚体NP疫苗面临的主要障碍是Env融合纳米颗粒， 重组表达为单条多肽链，可以采用良好折叠和错误折叠的混合物， 构象错误折叠的Env三聚体可诱导非中和抗体应答， 构象或与bnAb前体竞争结合HIV-1 Env。对于HIV-1 Env三聚体 NPs成为HIV-1疫苗设计的快速和广泛使用的平台， 融合了折叠良好的Env三聚体，而不需要昂贵的、长达一年的蛋白质工程实验， needed.为了克服HIV-1 Env纳米颗粒疫苗的障碍，我们创新了一种快速系统， 使用分选酶A缀合方法产生良好折叠的HIV-1 Env三聚体纳米颗粒。HIV-1 Env 三聚体和铁蛋白分别产生，并达到高纯度，从而消除错误折叠的蛋白质。 通过混合HIV-1 Env三聚体、铁蛋白和分选酶A酶，HIV-1 Env三聚体共价连接至铁蛋白。 铁蛋白纳米颗粒。所得的分选酶A缀合的纳米颗粒（scNP）显示Env三聚体， 理想的构造。项目2的总体目标是将分选酶A缀合的纳米颗粒转化为 用于产生针对多种病原体的病毒疫苗的具有cGMP能力的方法， HIV-1 bnAb靶向免疫原在HIV疫苗试验进行的I期试验中进行评估 网络（HVTN）。为了实现这一目标，我们将首先确定铁蛋白的最佳表达系统， 分选酶A并建立用于平台生产的目标产物谱（目标1）。然后，我们将开发， 生产和质量放行重组分选酶A和铁蛋白平台分子，用于缀合 Env稳定的三聚体与铁蛋白纳米颗粒（目的2）。接下来，我们将cGMP生产和质量放行>1 g的 两种不同的稳定化Env三聚体scNP药物物质和药物产品，用于毒理学研究和临床 在I期临床试验中进行测试（目标3）。第一个Env三聚体scNP，CH 505 TF Env三聚体scNP，将用作 靶向CD 4结合位点bnAb的CH 505 Env NP序贯疫苗中的第一个加强免疫原。最后， 我们将产生另外的连续Env三聚体scNP以选择相同的CD 4进一步成熟 结合位点bnAb谱系（Aim 3）。将该平台转化为cGMP生产的影响是， 使高质量HIV-1 Env三聚体NP疫苗的临床试验能够快速生产和更快检测。