Engineered Dengue EDIIIs as Broad Immunogens

作为广泛免疫原的工程登革热 EDIII

• 批准号: 9224550
• 负责人: Jonathan R. Lai
• 金额: $ 20.88万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9224550
• 关键词: ART protein; Affinity; Amino Acids; Antibodies; Antibody Response; Antibody-Dependent Enhancement; Antigens; Area; Attenuated; Attenuated Live Virus Vaccine; Bacteriophages; Binding; Biochemical; Brazil; Cell Culture Techniques; Cells; Cessation of life; Characteristics; Communicable Diseases; Country; Culicidae; Dengue; Dengue Vaccine; Dengue Virus; Development; Dimerization; Disease; Engineering; Epitopes; Evaluation; Exhibits; Fever; Flavivirus; GTP-Binding Protein alpha Subunits, Gs; Geometry; Glycoproteins; Goals; Head; Hospitalization; Human; Immune response; Immunity; Immunization; Immunize; Immunocompetent; Immunodominant Epitopes; In Vitro; Individual; Infection; Lead; Libraries; Masks; Membrane Fusion; Methods; Mexico; Modeling; Molecular Conformation; Mus; Mutation; Phage Display; Philippines; Population; Primary Infection; Production; Property; Protein Engineering; Recombinants; Safety; Serotyping; Serum; Shock; Structure; Subunit Vaccines; Syndrome; Tail; Testing; Vaccines; Variant; Vertebral column; Viral; Virus; Work; Yellow fever virus; base; biophysical properties; cross reactivity; design; dimer; experimental study; immunogenicity; improved; in vivo; innovation; lumazine; neutralizing antibody; next generation; nonhuman primate; novel; particle; phase III trial; premature; prevent; receptor; receptor binding; response; screening; secondary infection; vaccine candidate; vaccine development; vector vaccine; virus envelope

SUMMARY Dengue virus is a mosquito-transmitted flavivirus that causes hundreds of millions of human infections world- wide each year. There are four serotypes of Dengue (DENV1-4) that co-circulate in hyperendemic regions. Primary infection by a single serotype results in febrile illness and subsequent lifelong immunity to that serotype. Secondary infections by heterotypic serotypes, however, can lead to severe shock syndrome and death. Severe Dengue disease is thought to be caused by antibodies that are elicited during primary infection that can bind multiple serotypes but not neutralize them, and facilitate entry and infection in Fcγ receptor positive cells thus causing "antibody-dependent enhancement" (ADE) of infection. While a live-attenuated four-component chimeric vaccine was recently implemented in three countries, this vaccine did not protect naïve individuals against symptomatic infection. There is therefore significant rationale to explore alternative platforms such as subunit vaccines (immunogens) as either next-generation primary vaccines or as boosting agents to improve existing live attenuated virus vaccines. Furthermore, identification and development of DENV immunogens that elicit broadly neutralizing antibody (bNAb) responses is highly desirable. DENV E glycoprotein domain III (EDIII) is a small (~100 residue) β-sandwich domain that is an attractive candidate for immunogen design, since many bNAbs target this region. However, immunodominant regions of this domain lie outside of critical neutralization epitopes, which has hampered its advancement as a subunit vaccine. We have developed a phage display platform for EDIII immunogen engineering and used it to produce "resurfaced" EDIIIs (rsDIIIs) in which non-productive epitopes are masked by mutation. Our structure-based design strategy involves production and screening of EDIII-based phage libraries in which residues that are not involved in interactions with model bNAbs are varied with restricted amino acid diversity. This approach has yielded a panel of rsDIIIs that exhibit reactivity toward a prototypic bNAb, thus maintaining the conformational integrity of the broadly susceptible epitope, but not toward type-specific or non-neutralizing antibodies. We propose to perform a full in vitro characterization of this immunogen panel for biochemical properties such as folding stability and binding affinity. Monovalent as well as bivalent and polyvalent immunogen presentation formats also will be explored. The most promising rsDIII immunogen candidates will be tested in mice for immunogenicity and ability to confer protective immunity in a serum transfer challenge experiment. The overarching goal of this work is to use innovative protein engineering approaches to overcome the traditional barriers with EDIII-based immunogens. This work will provide proof-of-concept in mice for novel subunit vaccine candidates against DENV, and possibly other flaviviruses of global concern.

概括 登革热病毒是一种由蚊子传播的黄病毒，导致全世界数亿人感染—— 每年有四种登革热血清型 (DENV1-4) 在高流行地区共同流行。 单一血清型的初次感染会导致发热性疾病，并随后对该血清型产生终生免疫力 然而，异型血清型的继发感染可导致严重的休克综合征和 严重登革热病被认为是由初次感染期间产生的抗体引起的。 可以结合多种血清型但不中和它们，并促进 Fcγ 受体的进入和感染 阳性细胞因此引起“抗体依赖性增强”（ADE）感染，同时是减毒活病毒。 四成分嵌合疫苗最近在三个国家实施，这种疫苗没有保护 因此，探索替代方案具有重要的理由。 亚单位疫苗（免疫原）等平台作为下一代初级疫苗或加强疫苗 此外，还鉴定和开发了改进现有减毒活病毒疫苗的药物。 引起广泛中和抗体（bNAb）反应的 DENV 免疫原是非常理想的。 糖蛋白结构域 III (EDIII) 是一个小的（约 100 个残基）β-夹心结构域，是一个有吸引力的候选结构域 免疫原设计，因为许多 bNAb 靶向该区域，然而，该区域的免疫显性区域。 位于关键中和表位之外，这阻碍了其作为亚单位疫苗的发展。 开发了用于 EDIII 免疫原工程的噬菌体展示平台，并用其生产 “重新出现”的 EDIII（rsDIII），其中非生产性表位被我们基于结构的突变所掩盖。 设计策略涉及基于 EDIII 的噬菌体文库的产生和筛选，其中残基 不参与与模型 bNAb 相互作用的氨基酸多样性有限。 已经产生了一组 rsDIII，它们对原型 bNAb 表现出反应性，从而保持了 广泛敏感表位的构象完整性，但不针对类型特异性或非中和性 我们建议对该免疫原组进行完整的体外生化表征。 性质，例如折叠稳定性和结合亲和力。 还将探索最有前途的 rsDIII 免疫原候选物。 在小鼠中测试免疫原性和在血清转移挑战中赋予保护性免疫的能力 这项工作的总体目标是使用创新的蛋白质工程方法来进行实验。 这项工作将提供基于 EDIII 的免疫原的概念验证。 小鼠用于针对 DENV 以及可能的其他全球关注的黄病毒的新型亚单位候选疫苗。