Optimization of HIV glycoproteins as vaccine candidates

 描述（由申请人提供）：开发针对HIV的预防性疫苗在阻止病毒传播的全球运动中至关重要。大多数新的候选疫苗是基于（或包含）包膜糖蛋白（Env）的版本，所需的广泛中和抗体的目标。然而，即使在成功发现和初步测试疫苗候选物之后，仍存在与生产产率、纯化策略和体外稳定性相关的障碍，这可能阻碍其作为治疗化合物的开发。在这项提案中，我们的目标是优化疫苗开发管道，以加快将有希望的线索推向临床的过程。在一所研究型大学（波多黎各大学）、一家生物技术初创企业（CDI实验室）和当地生物制药合作伙伴（安进制造公司、礼来公司）的咨询投入之间的合作中，我们将应用最先进的分析工具来优化三聚体gp 145的生产，这是一种有前途的新候选疫苗，由亨利M.杰克逊军事科学基金会和NIH疫苗研究计划。我们的小组和其他人已经表明，在CHO细胞中表达的三聚体gp 145的产率始终较低（3-4 mg/L），并且尚未开发出信息分析方法来测量试剂质量或预测其功效。因此，我们建议开发一套分析工具来快速评估聚糖组成、聚集、结合活性和蛋白质降解（目标#1）。然后，我们将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gp 145的低产量是由于其对生产宿主CHO-K1的毒性（目标#4）。所提出的工作是我们小组中涉及这种特定候选疫苗的努力的继续。我们的团队已经开发了一种MALDI-ToF质谱法，用于测量三聚体gp 145的聚糖组成，并且我们已经使用所得信息来评估修改培养基组成对聚糖分布的影响。我们已经开发了使用动态光散射检测蛋白质聚集体和使用生物传感器测定定量蛋白质结合的方法。我们还首次表明，外源性添加三聚体gp 145到CHO-K1细胞中，大大损害了它们在培养中的活力，从而表明在疫苗开发的早期阶段最好解决潜在的毒性问题。所有拟定的gp 145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至生产设施。总之，本提案中开发的实验工具将有助于对三聚体gp 145的生产做出更好的决策，可能会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.