Optimization of HIV glycoproteins as vaccine candidates

 描述(由申请人提供)：艾滋病毒预防性疫苗的开发在全球遏制病毒传播的努力中至关重要。大多数新的候选疫苗是基于(或包含)包膜糖蛋白(Env)的版本，包膜糖蛋白是所需的广泛中和抗体的靶标。然而，即使在成功发现和初步测试候选疫苗后，仍存在与产量、纯化策略和体外稳定性相关的障碍，这些障碍可能会阻碍其作为治疗化合物的开发。在这项建议中，我们旨在优化疫苗开发流水线，以加快获得有希望的临床线索的进程。在一所研究型大学(波多黎各大学)、一家生物技术初创公司(CDI实验室)和当地生物制药合作伙伴(安进制造、礼来公司)的咨询意见的合作下，我们将应用最先进的分析工具来优化三聚体gp145的生产，三聚体gp145是亨利·M·杰克逊军事科学基金会和NIH疫苗研究计划合作发现的一种有前途的新候选疫苗。我们和其他人已经证明，在CHO细胞中表达的三聚体gp145的产量一直很低(3-4 mg/L)，而且还没有开发出信息丰富的分析方法来测量试剂质量或预测其有效性。因此，我们建议开发一套分析工具来定量评估多糖的组成、聚集、结合活性和蛋白质降解(目标1)。然后，我们将把这些分析工具应用于克隆选择过程(目标2)和蛋白质表达条件的优化(目标3)。最后，我们解决了一个长期存在的说法，即三聚体gp145的低产率是由于它对生产宿主CHO-K1的毒性(目标#4)。拟议的工作是我们小组涉及这一特定候选疫苗的继续努力。我们团队已经开发了一种MALDI-ToF质谱学方法来测量三聚体gp145的糖链组成，并利用得到的信息来评估改变介质组成对糖链分布的影响。我们开发了使用动态光散射检测蛋白质聚集体的方法，以及使用生物传感器实验对蛋白质结合进行量化的方法。我们还首次表明，在CHO-K1细胞中外源添加三聚体gp145，极大地损害了它们在培养中的活力，从而提出了潜在的毒性问题，最好在疫苗开发的早期阶段加以解决。所有拟议的gp145分析将使用适当的合格方法进行，以促进所有方法最终转移到制造设施中。综上所述，本提案中开发的实验工具将有助于对三聚体gp145的生产做出更好的决策，潜在地提高临床试验过程中的产量和质量。

 DESCRIPTION (provided by applicant): The development of prophylactic vaccines against HIV is of paramount importance in the global drive to halt the spread of the virus. Most new vaccine candidates are based on (or contain) versions of the envelope glycoprotein (Env), the target of the desired broadly neutralizing antibodies. However, even after the successful discovery and initial testing of a vaccine candidate, there are hurdles associated with production yield, purification strategy and in vitro stability that may hinder its development as a therapeuti compound. In this proposal, we aim to optimize the vaccine development pipeline in order to speed up the process of getting promising leads to the clinic. In a partnership between a research university (University of Puerto Rico), a biotechnology start-up (CDI laboratories) and the advisory input of local biopharmaceutical partners (Amgen Manufacturing, Eli Lilly), we will apply state-of-the-art analytical tools to optimize the production of trimeric gp145, a promising new vaccine candidate discovered and partnered between the Henry M. Jackson Foundation for Military Science and the NIH Vaccine Research Program. It has been shown by our group and by others that the yields of trimeric gp145 expressed in CHO cells, are consistently low (3-4 mg/L) and that informative analytical methods have not been developed to measure reagent quality or to predict its efficacy. Thus, we propose to develop a suite of analytical tools to quicly assess glycan composition, aggregation, binding activity and protein degradation (Aim #1). We will then implement these analytical tools to the process of clone selection (Aim #2) and to the optimization of protein expression conditions (Aim #3). Finally, we address a long-standing claim that the low yields of trimeric gp145 are due to its toxicity toward the production host, CHO-K1 (Aim #4). The proposed work is a continuation of efforts in our group involving this particular vaccine candidate. Our group has already developed a MALDI-ToF mass spectrometry method for the measurement of glycan composition of trimeric gp145 and we have used the resulting information to evaluate the effect of modifying the media composition on glycan distribution. We have developed methods for the detection of protein aggregates using dynamic light scattering and for the quantification of protein binding using a biosensor assay. For the first time, we have also shown that exogenously added trimeric gp145 to CHO-K1 cells, dramatically compromise their viability in culture, thus suggesting potential toxicity issues that are best addressed during the early stages of vaccine development. All proposed analyses of gp145 will be performed using appropriately qualified methods to facilitate eventual transfer of all methods into manufacturing facilities. In summary, the experimental tools developed in this proposal will help to make better decisions on the production of trimeric gp145, potentially boosting the production yields and quality en route to clinical trials.