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.

总结 登革热病毒是一种蚊子传播的黄病毒，在全世界造成数亿人感染- 每一年的宽。有四种血清型登革热（DENV 1 -4）在高流行地区共同传播。 由单一血清型引起的原发性感染导致发热性疾病和随后的终身免疫 血清型然而，异型血清型的继发感染可导致严重的休克综合征， 死亡严重的登革热被认为是由初次感染时产生的抗体引起的 可结合多种血清型但不能中和它们，并促进Fcγ受体的进入和感染 阳性细胞，从而引起感染的“抗体依赖性增强”（ADE）。而减毒活疫苗 四组分嵌合疫苗最近在三个国家实施，这种疫苗不能保护 对有症状的感染者的天真的个人。因此，有充分的理由探索替代方案， 平台，如亚单位疫苗（免疫原）作为下一代初级疫苗或加强疫苗 改进现有的减毒活病毒疫苗。此外，确定和发展 引发广泛中和抗体（bNAb）应答的DENV免疫原是高度期望的。DENV E 糖蛋白结构域III（EDIII）是一个小的（~100个残基）β-夹心结构域，是一个有吸引力的候选者， 免疫原设计，因为许多bNAb靶向该区域。然而，该结构域的免疫显性区域 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。我们 已经开发了用于EDIII免疫原工程的噬菌体展示平台，并将其用于生产 “表面重构”EDIII（rsDIII），其中非生产性表位被突变掩蔽。我们基于结构的 设计策略涉及基于EDIII的噬菌体文库的产生和筛选， 不参与与模型bNAb的相互作用的氨基酸多样性受限制。这种方法 已经产生了一组rsDIII，其表现出对原型bNAb的反应性，从而维持了对原型bNAb的反应性。 广泛易感表位的构象完整性，但不针对类型特异性或非中和性 抗体的我们建议对该免疫原组进行全面的体外表征，以进行生化分析。 例如折叠稳定性和结合亲和力。单价以及二价和多价 还将探索免疫原呈递形式。最有希望的rsDIII免疫原候选物将 在小鼠中测试免疫原性和在血清转移攻击中赋予保护性免疫的能力 实验这项工作的首要目标是使用创新的蛋白质工程方法， 用基于EDIII的免疫原克服传统障碍。这项工作将提供概念验证， 小鼠用于针对DENV和可能的全球关注的其他黄病毒的新型亚单位疫苗候选物。