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

基本信息

批准号：
10541868
负责人：
Jason Dickens
金额：
$ 372.47万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10541868
关键词：
2019-nCoV Acceleration Adopted Affinity Antibodies 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 Reproducibility 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 manufacture 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

项目摘要

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.

抽象项目2 HIV-1疫苗开发的关键目标是诱导持久的保护性广泛中和抗体（BNAB）可以抑制HIV-1感染的反应。但是，这种保护性免疫反应已经在疫苗接种的情况下很难诱导。诱导这些保护性反应的一种策略是到免疫系统中和抗体的靶标，HIV-1包膜（ENV）的天然形式。纳米颗粒（NP）上的多层次ENV三聚体增强其免疫原性，因此是免疫原的目标设计是呈现多个折叠式ENV三聚机的副本。 env是一种亚稳态的蛋白质，采用多种构象。 Env Trimer NP疫苗面临的主要障碍是Env Fusion纳米颗粒，重组表示为单个多肽链，可以采用折叠式和折叠倍数的混合物构象。错误折叠的ENV三聚体可能会引起不中和抗体反应，以改变env 结合或与BNAB前体竞争后，与HIV-1 Env结合。对于HIV-1 ENV TRIMER NP成为HIV-1疫苗设计的快速且广泛使用的平台，这是一个纳米颗粒平台合并充分折叠的Env三聚体，而无需进行长期，长达一年的蛋白质工程实验是需要。为了克服HIV-1 ENV纳米颗粒疫苗的障碍，我们已经创新了一个快速的系统使用分类酶一种共轭方法生成折叠良好的HIV-1 env-Trimer纳米颗粒。 HIV-1环境三聚体和铁蛋白分别产生，并具有很高的纯度，从而消除了错误折叠的蛋白质。通过混合HIV-1 env三聚体，铁蛋白和排序酶A酶，HIV-1 env-Trimer共价链接到铁蛋白纳米颗粒。所得的排序酶A结合的纳米颗粒（SCNP）在所需的构象。项目2的总体目标是将排序酶A结合的纳米颗粒转化为具有CGMP能力的过程，用于生成许多病原体的病毒疫苗，并制造两种 HIV-1 BNAB靶向免疫剂在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类A并建立用于平台生产的目标产品配置文件（AIM 1）。然后，我们将发展，制造和质量释放重组分子酶A和铁蛋白平台分子，用于结合 ENV稳定了铁蛋白纳米颗粒（AIM 2）。接下来，我们将CGMP生产和质量释放> 1 g 两种不同的稳定的Env Trimer SCNP药物和用于毒理学研究和临床的药物在I期临床试验中进行测试（AIM 3）。第一个ENV Trimer SCNP CH505 TF ENV TRIMER SCNP将被用作 CH505 ENV NP NP顺序疫苗靶向CD4结合位点BNABS中的第一个增强免疫原。最后，我们将产生一个额外的顺序Env -Trimer SCNP，以选择相同CD4的进一步成熟结合位点BNAB谱系（AIM 3）。将该平台转换为CGMP制造的影响是在高质量的HIV-1 Env Trimer NP疫苗的临床试验中，可以快速生产和更快的测试。