Optimization of HIV glycoproteins as vaccine candidates

作为候选疫苗的 HIV 糖蛋白的优化

基本信息

批准号：
9506694
负责人：
Abel J Baerga-Ortiz
金额：
$ 130.39万
依托单位：
UNIVERSITY OF PUERTO RICO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9506694
关键词：
Academia Address Affect Amino Acids Binding Binding Proteins Biological Biological Assay Biological Products Biosensor Biotechnology Cell Culture Techniques Cell Line Cells Chinese Hamster Ovary Cell Clinic Clinical Clinical Trials Collaborations Data Detection Development Ethnic group Foundations Glycoproteins HIV HIV Envelope Protein gp120 HIV diagnosis HIV vaccine Heterogeneity Immunologics In Vitro Individual Industry Infection Laboratories Lipids MALDI-TOF Mass Spectrometry Measurement Measures Mediating Messenger RNA Methods Military Science Monitor Neurons Pathway interactions Peptide Mapping Perfusion Polysaccharides Positioning Attribute Preventive vaccine Principal Investigator Process Production Proteins Puerto Rico Reagent Reporting Research Role Route Sampling Scheme Site Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Speed System Techniques Testing Time Toxic effect United States United States National Institutes of Health Universities Vaccine Clinical Trial Vaccine Research Vaccines Variant Viral Virus Visit Volatile Fatty Acids Work analytical method analytical tool base cytotoxicity drug development experimental study glycosylation improved light scattering macrophage manufacturing facility mass spectrometer molecular mass neutralizing antibody novel novel vaccines pandemic disease programs protein aggregate protein aggregation protein degradation protein expression public health relevance tool transcriptomics trend vaccine candidate vaccine development

项目摘要

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.

描述（由申请人提供）：针对 HIV 的预防性疫苗的开发对于阻止病毒传播的全球努力至关重要。大多数新候选疫苗都是基于（或包含）包膜糖蛋白 (Env) 的版本。然而，即使在成功发现并初步测试候选疫苗之后，仍存在与生产产量、纯化策略和体外稳定性相关的障碍，这可能会阻碍其作为治疗化合物的发展。根据提案，我们的目标是优化疫苗开发渠道，以加快将有希望的线索推向临床的进程。根据当地生物制药合作伙伴（安进制造公司、礼来公司）的咨询意见，我们将应用最先进的分析工具来优化三聚体 gp145 的生产，这是一种有前途的新候选疫苗，由亨利·M·杰克逊基金会发现并合作军队科学和 NIH 疫苗研究计划。我们的小组和其他人已经表明，CHO 细胞中表达的三聚体 gp145 的产量始终很低（3-4 mg/L），并且尚未开发出信息丰富的分析方法。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）。最后，我们解决了一个长期存在的观点，即三聚体 gp145 的低产量是由于其对生产宿主 CHO- 的毒性。 K1（目标#4）。拟议的工作是我们小组涉及这种特定候选疫苗的努力的延续。我们小组已经开发了一种用于测量三聚体聚糖组成的 MALDI-ToF 质谱方法。 gp145，我们使用所得信息来评估改变培养基成分对聚糖分布的影响，我们首次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。同时，我们还表明，将三聚体 gp145 外源添加到 CHO-K1 细胞中，会极大地损害其在培养物中的活力，因此表明最好在疫苗开发的早期阶段解决潜在的毒性问题。 gp145 将使用合格的方法进行适当的操作，以促进所有方法最终转移到生产设施中。总之，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，从而有可能提高生产产量和质量。进入临床试验的途径。