Generation of Zika virus-specific recombinant antibodies

寨卡病毒特异性重组抗体的产生

• 批准号: 9226804
• 负责人: MICHAEL D GUNN
• 金额: $ 23.85万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-12 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226804
• 关键词: Advanced Development; Affinity; Antibodies; Antibody Formation; Antigens; Aspergillus fumigatus; B-Lymphocytes; Bacteria; Bacteriophages; Binding; Biological Assay; Blood; Brazil; Cells; Characteristics; Child; Clinical; Culicidae; Dengue; Dengue Virus; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Disease Outbreaks; Emerging Communicable Diseases; Engineering; Enzyme-Linked Immunosorbent Assay; Epidemiologic Monitoring; Flavivirus; Funding; Generations; Hand; Harvest; Healthcare; Human; Hybridomas; Immune; Individual; Infection; Laboratories; Lead; Length; Libraries; Microcephaly; Mothers; Mus; Pathogen detection; Patients; Phage Display; Procedures; Process; Proteins; Public Health; RNA; Reagent; Recombinant Antibody; Recombinants; Research Personnel; Salmonella paratyphi; Salmonella typhi; Sampling; Sensitivity and Specificity; Serum; Staging; Symptoms; System; Techniques; Technology; Testing; Time; Validation; Viral Antigens; Viral Proteins; Virus; Virus Diseases; West Nile virus; Yellow Fever; Zika Virus; base; cost effective; cross reactivity; diagnostic assay; experience; global health; improved; individual patient; murine antibody; novel; pathogen; prototype; research clinical testing; screening

Zika Virus (ZIVR) is a very rapidly emerging infectious disease that has been associated with microcephaly in the children of infected mothers in an ongoing outbreak in Brazil. ZIVR is similar to other flavaviruses, such as Dengue, Yellow Fever, and West Nile, which are transmitted to humans by mosquitos. Controlling ZIVR from a public health perspective and appropriately managing individual patients will require that the diagnosis of ZIVR infection can be made rapidly and accurately. Because the symptoms of ZIVR infection are similar to those of other infections, the diagnosis cannot be made on clinical grounds alone and a diagnostic assay is required. Unfortunately, no routine and accurate assay for the diagnosis of ZIVR infection is currently available. Existing assays are either limited to specialized laboratories or give false positive results in patients that have been infected with other flaviviruses. We propose to significantly advance the development of a ZIVR diagnostic assay by generating antibodies that specifically recognize ZIVR, but not other flavaviruses, and using these to construct a prototype ZIVR antigen ELISA assay. Virus-specific antibodies are the key reagents required for the most widely used types of diagnostic assays, which detect the presence of viral proteins in the blood. Such assays have the advantage of yielding rapid results and being applicable to almost any health care setting. However, to be accurate, such assays must use antibodies that are specific for a single virus. Here, we will use single-chain antibody phage display technology to generate antibodies specific to the NS1 protein produced by ZIVR. We have chosen NS1 because it has proven to be an excellent diagnostic target for other viral infections such as Dengue. The specific procedures that will be used include immunizing mice against ZIVR NS1, harvesting the antibody-producing B cells from these mice, and using them to make a library of single-chain antibodies that are expressed on the tips of a bacteriophage (viruses that infect bacteria). This phage library will then be screened for antibody clones that bind to ZIVR, but not to related viruses using standard phage display procedures. Once antibody phage clones with the appropriate binding characteristics are identified, they will be converted into full-length antibodies suitable for use in a diagnostic assay. At all stages of the process, candidate antibodies will be tested to validate their sensitivity and specificity for ZIVR NS1. The successful completion of this project will result in the availability of validated high affinity highly specific anti-ZIVR NS1 Abs that can be used to construct sensitive and specific ZIVR diagnostic assays. We will also develop a ZIVR NS1 ELISA suitable for clinical testing in the course of these studies. This will markedly improve our ability to accurately diagnose ZIVR infection, thus significantly improving our capacity to track, control, and appropriately treat this disease.

寨卡病毒（ZIVR）是一种非常迅速出现的传染病，与小头畸形症有关， 在巴西的一次持续爆发中，受感染母亲的孩子。ZIVR类似于其他黄病毒，如 登革热、黄热病和西尼罗河病毒，这些病毒通过蚊子传播给人类。控制ZIVR从一个 从公共卫生角度和适当管理个体患者的角度来看， 可以快速准确地进行感染。因为ZIVR感染的症状与 对于其他感染，不能仅根据临床理由进行诊断，需要进行诊断测定。 不幸的是，目前还没有用于诊断ZIVR感染的常规和准确的测定法。现有 检测或者局限于专门的实验室，或者在已经进行过检测的患者中给出假阳性结果。 感染其他黄病毒。我们建议大大推进ZIVR诊断的发展， 通过产生特异性识别ZIVR但不识别其他黄病毒的抗体进行检测，并使用这些抗体 构建原型ZIVR抗原ELISA测定。病毒特异性抗体是检测病毒感染所需的关键试剂。 最广泛使用的诊断检测类型，用于检测血液中病毒蛋白的存在。等 测定具有产生快速结果和适用于几乎任何卫生保健环境的优点。 然而，为了准确起见，这种测定必须使用对单一病毒特异的抗体。在这里，我们将 利用单链抗体噬菌体展示技术产生针对NS 1蛋白的特异性抗体 由ZIVR制作。我们之所以选择NS1，是因为它已被证明是一个很好的诊断目标， 病毒感染，如登革热。将使用的具体程序包括免疫小鼠， ZIVR NS1，从这些小鼠中收获产生抗体的B细胞，并使用它们来制备抗体库。 在噬菌体（感染细菌的病毒）顶端表达的单链抗体。这 然后，利用噬菌体文库筛选与ZIVR结合但不与相关病毒结合的抗体克隆， 标准噬菌体展示程序。一旦抗体噬菌体克隆具有适当的结合特性 被鉴定，它们将被转化为适用于诊断测定的全长抗体。根本 在该过程的各个阶段，将测试候选抗体以验证其对ZIVR的灵敏度和特异性。 NS 1。该项目的成功完成将导致高度验证的高亲和力的可用性 特异性抗ZIVR NS1 Ab，其可用于构建灵敏和特异的ZIVR诊断测定。我们 在这些研究过程中，ZIVR还将开发适用于临床检测的ZIVR NS1 ELISA。这将 显著提高我们准确诊断ZIVR感染的能力，从而显著提高我们的能力， 跟踪、控制和适当治疗这种疾病。