Project 1: Immunogens and Manufacturing

项目一：免疫原及制造

• 批准号: 8478317
• 负责人: HARRIET L ROBINSON
• 金额: $ 185.9万
• 依托单位: GEOVAX, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8478317
• 关键词: 3' Untranslated Regions; AIDS/HIV problem; Address; Adjuvant; Animals; Antibodies; Antibody Formation; Antigens; Avidity; B-Lymphocytes; Binding; Bioreactors; Birds; Calmodulin; Cell Line; Cells; Chick Embryo; Clinical Research; Collaborations; Country; Developed Countries; Development; Ducks; Fibroblasts; Frameshift Mutation; France; GMP lots; Generations; Genes; Goals; Growth; HIV; HIV Vaccine Trials Network; HIV vaccine; Human; Individual; Interruption; Laboratories; Length; Life; Macaca mulatta; Mammalian Cell; Membrane; Methods; MicroRNAs; Modeling; Mosses; National Institute of Allergy and Infectious Disease; Pharmaceutical Preparations; Phase; Point Mutation; Positioning Attribute; Preparation; Process; Production; Recombinants; Running; SIV; Seeds; Silent Mutation; Stem cells; TNFSF5 gene; Testing; Time; Toxic effect; Treatment Protocols; Vaccination; Vaccine Design; Vaccine Production; Vaccines; Virus; Virus Diseases; Virus-like particle; Work; cell bank; continuous cell line; design; drug resistant virus; gp160; improved; manufacturing process; meetings; mutant; prevent; programs; research clinical testing; response; safety testing; stability testing; stem; vector

Our goal is to design and produce a clade C HIV MVA vaccine that will elicit antibodies capable of preventing HIV acquisition. Our clade B HIV VLP-expressing MVA-vectored vaccine has been tested clinically and shown good ability to Induce Env-specific antibodies. The clade B HIV vaccine, however, is not designed to express full length Env that we have found to prime the most protective antibody responses using the SIV rhesus macaque model (see Program Overview). Two technical approaches are proposed to increase the induction of protective Ab responses; the development of a MVA-vectored HIV vaccine encoding full-length, clade 0 gp160 and the use of MVA-expressed CD40L as a B-cell adjuvant. Specifically,we will 1) Construct a clade C VLP MVA vaccine expressing full-lengfh gp160 Env to optimize the protective avidity and neutralizing activity of elicited Env-specific Ab, 2) Insert target sequences for EB66 stem cellspecific miRNAs into 3' untranslated regions of vaccine Env inserts to suppress insert expression and enhance titers and vector stability during manufacture in the EB66 cell line and 3) Construct CD40Lexpressing MVA to serve as a strong B cell adjuvant for the titer and avidity of Env-specific Ab. Production of MVA vaccines is a challenge because of the growth of these vaccines in chick embryo fibroblasts that require sourcing and producing large batches of primary cells. Because of this, GeoVax has explored the use of continuous cell lines for growth of MVA and has found the EB66 duck stem cell-derived line developed by Vivalis LLC. Nantes France to be a promising option. GeoVax has produced dedicated Master and Working EB66 cell banks. The goals for this project include 1) Qualification of media and production processes for EB66 cells as the MVA manufacturing cell substrate focusing on "end-of-production" testing and the generation of Master seed stocks, 2) Production of a "GMP-like" pilot/feasibility lot of MVA-vectored clade C vaccine and the MVA encoding CD40L to validate the manufacturing process and to produce material for animal safety testing and 3) Production of a GMP lot of each vaccine to be used in Phase 1 clinical testing by the HVTN.

我们的目标是设计和生产一种进化枝C HIV MVA疫苗， 预防艾滋病毒感染。我们的进化枝B HIV VLP表达MVA载体疫苗已经过测试 在临床上显示出良好的诱导Env特异性抗体的能力。然而，进化枝B HIV疫苗并不是设计来表达全长Env的，我们已经发现使用SIV恒河猴模型引发最具保护性的抗体应答（参见程序概述）。提出了两种技术方法来增加保护性Ab应答的诱导;开发编码全长、进化枝0 gp 160的MVA载体HIV疫苗和使用MVA表达的CD 40 L作为B细胞佐剂。具体地，我们将1）构建表达全-Env gp 160 Env的进化枝C VLP MVA疫苗以优化引发的Env特异性Ab的保护性亲合力和中和活性，2）将EB 66干细胞特异性miRNA的靶序列插入疫苗Env插入物的3'非翻译区中，以在EB 66细胞系中制备期间抑制插入物表达并增强滴度和载体稳定性，以及构建表达MVA的CD 40 L，作为Env特异性Ab的效价和亲合力的强B细胞佐剂。 MVA疫苗的生产是一个挑战，因为这些疫苗在鸡胚中生长 成纤维细胞需要采购和生产大批量的原代细胞。因此，GeoVax探索了使用连续细胞系生长MVA，并发现了Vivalis LLC开发的EB 66鸭干细胞衍生系。法国南特是一个很有前途的选择。GeoVax生产了专用的主细胞库和工作EB 66细胞库。本项目的目标包括：1）确认EB 66细胞作为MVA生产细胞基质的培养基和生产工艺，重点是“生产结束”检测和主种子储备液的生成，2）生产MVA的“GMP样”中试/可行性批次-载体化进化枝C疫苗和编码CD 40 L的MVA，以验证生产工艺并生产用于动物安全性试验的材料，以及3）HVTN生产用于1期临床试验的每种疫苗的GMP批次。