Project 1: Immunogens and Manufacturing

我们的目标是设计和生产一种C级HIV MVA疫苗，它将诱导出能够 预防艾滋病病毒感染。我们的B分支HIV VLP表达MVA载体疫苗已经测试 临床上表现出良好的诱导Env特异性抗体的能力。然而，B分支HIV疫苗并不是为表达全长Env而设计的，而我们已经发现，这种全长Env可以利用SIV恒河猴模型启动最具保护性的抗体反应(参见程序概述)。人们提出了两种技术方法来增加保护性抗体反应的诱导：开发编码全长、分支0 gp160的MVA载体HIV疫苗，以及使用MVA表达的CD40L作为B细胞佐剂。具体地说，我们将1)构建表达全长gp160 Env的CVLP MVA疫苗，以优化诱导的Env特异性抗体的保护亲和力和中和活性；2)将EB66干细胞特异性miRNAs的靶序列插入疫苗Env插入物的3‘非翻译区，以抑制插入表达，提高EB66细胞系生产过程中的滴度和载体稳定性；3)构建表达CD40LMVA的MVA，作为对Env特异性抗体效价和亲和力的强大B细胞佐剂。 MVA疫苗的生产是一个挑战，因为这些疫苗是在鸡胚胎中生长的 需要大量采购和生产原代细胞的成纤维细胞。正因为如此，GeoVax探索了使用连续细胞系来生长MVA，并找到了由Vivalis LLC开发的EB66鸭干细胞衍生系。法国南特是一个很有希望的选择。GeoVax生产了专门的主EB66细胞库和工作中的EB66细胞库。该项目的目标包括1)鉴定EB66细胞作为MVA制造细胞底物的培养基和生产工艺，重点是“生产结束”测试和生成主种子种群；2)生产“类似GMP”的MVA载体分支C疫苗和编码CD40L的MVA，以验证制造工艺并生产动物安全试验材料；3)生产每种疫苗的GMP批次，供HVTN进行第一阶段临床试验。

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.