Structural Requirements for Broadly Protecting Antibodies to Influenza A & B

广泛保护甲型流感抗体的结构要求

• 批准号: 8918922
• 负责人: Wayne A. Marasco
• 金额: $ 64.54万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-11 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8918922
• 关键词: Affinity; Amino Acid Substitution; Antibodies; Antibody Formation; Antigens; B-Lymphocytes; Binding; Cells; Centers for Disease Control and Prevention (U.S.); Complement; Crystallography; Data; Development; Electron Microscopy; Engineering; Epitope Mapping; Epitopes; Frequencies; Genes; Genetic; Goals; Head; Health; Hemagglutinin; Human; Immune; Immunity; Immunologic Surveillance; Immunology procedure; In Vitro; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Influenza Hemagglutinin; Influenza vaccination; Lead; Libraries; Location; Masks; Medical; Medical Research; Memory; Memory B-Lymphocyte; Methods; Modification; Molecular; Morbidity - disease rate; Mus; Nature; Pattern; Phage Display; Plasmablast; Polysaccharides; Process; Property; Proteins; Recombinants; Recovery; Relative (related person); Resources; Role; Seasons; Site; Somatic Mutation; Sorting - Cell Movement; Structure; Surface; Testing; Vaccination; Vaccines; Variant; Viral; Virus; Work; X-Ray Crystallography; anti-influenza; antibody-dependent cell cytotoxicity; base; biosecurity; design; experience; glycosylation; immune clearance; in vivo; influenza virus strain; influenza virus vaccine; influenzavirus; insight; member; mortality; mouse model; neutralizing antibody; novel; optimism; pandemic disease; response; seasonal influenza; stem; structural biology; success

DESCRIPTION (provided by applicant): Influenza remains a major medical problem and is a constant threat to human health. Since their first administration in the 1940s, influenza vaccines have provided global seasonal protection against influenza infections. However, because of influenza's propensity to continuously change its hemagglutinin (HA) protein by a process known as "antigenic drift", and for pandemic strains by "antigenic shift", current vaccines require near-annual reformulation and accurate prediction of circulating strains for the upcoming season. The newly discovered epitopes in influenza's hemagglutinin (HA) stem region to which rare broadly neutralizing antibodies (BnAbs) are directed has led to renewed optimism that the development of a "universal' influenza vaccine that elicits these types of BnAbs will be possible. However, recent attempts to produce such a vaccine have shown only limited success. We now recognize from molecular studies of B cell responses during the 2009 H1N1 pandemic in which a high frequency of BnAbs was seen, that the limitation in eliciting protective levels of BnAbs is not due to an inherent lack of antigenicity, but rather to an immunodominant Ab response to the HA globular, a result of repeated annual memory B cell expansion to seasonal influenza vaccines which outcompete stem-directed memory B cells in numbers and for resources. Our preliminary data point to an effective approach to selecting or eliciting BnAbs, involving the rational engineering of glycosylation sites on HA that "redirect" Abs to the stem region via "glycan shielding" of head epitopes and unmasking of stem epitopes. In aim 1, will we propose to investigate the broad applicability of glycan shielding and deshielding as a strategy to select broadly reactive stem-binding Abs (BrAbs) using our circa 50 billion member human-Ab phage display library. The recovered BrAbs will be tested for virologic and immune clearance properties (e.g ADCC, CDC). In aim 2, we will investigate whether HA-glycan variants can enhance the isolation of stem- directed Br/BnAbs by FACS sorting of memory B cells/plasmablasts from influenza-experienced donors. We will narrow the glycan variants by comparing Ab germline gene useage, somatic mutations, relative numbers and diversity of Br/BnAbs recovered from aim 1/2 wt HA vs glycan variant screens. In aim 3, we will perform structural studies to map the epitopes of the most promising Br/BnAbs. The rationale here is that under- standing the epitope structure/location and the contribution of germline vs somatic amino acid substitutions will provide important insights, at the atomic level, into what type(s) of protective, stem-directed BrAbs we want to elicit by vaccination. In aim 4, we will test our lead HA-glycan variant(s) through in vivo vaccine studies in humanized & Balb/c mice, for their ability to induce Br/BnAb responses that are protective against heterosubtypic virus challenge, by preferentially stimulating the expansion of pre-existing stem-directed human memory B cells or priming of naive mouse B cells in vivo to secrete protective Br/BnAbs. These glycan variants should provide "lead antigens" for the design of recombinant HA-based universal influenza vaccines.

描述（由申请人提供）：流感仍然是一个主要的医学问题，并且对人类健康构成持续威胁。自 20 世纪 40 年代首次接种以来，流感疫苗已为全球提供针对流感感染的季节性保护。然而，由于流感倾向于通过称为“抗原漂移”的过程不断改变其血凝素（HA）蛋白，并且对于大流行毒株来说，通过“抗原漂移”，当前的疫苗需要 几乎每年都会重新配制并准确预测即将到来的季节的流行菌株。罕见的广泛中和抗体 (BnAb) 所针对的流感血凝素 (HA) 干区中新发现的表位让人们重新乐观起来，认为开发出引发这些类型 BnAb 的“通用”流感疫苗将是可能的。然而，最近生产此类疫苗的尝试仅取得了有限的成功。我们现在从 B 细胞反应的分子研究中认识到 在 2009 年 H1N1 大流行期间，BnAb 出现频率很高，BnAb 引起保护性水平的限制并不是由于固有的抗原性缺乏，而是由于对 HA 球状蛋白的免疫显性抗体反应，这是记忆 B 细胞每年重复扩增到季节性流感疫苗的结果，在数量和资源上超过了干定向记忆 B 细胞。我们的初步数据 指出了选择或引发 BnAb 的有效方法，包括对 HA 上的糖基化位点进行合理的改造，通过头表位的“聚糖屏蔽”和茎表位的暴露将抗体“重定向”至茎区域。在目标 1 中，我们是否建议研究聚糖屏蔽和去屏蔽的广泛适用性，作为使用我们的方法选择广泛反应性茎结合抗体 (BrAbs) 的策略 大约 500 亿成员的人类抗体噬菌体展示库。回收的 BrAb 将进行病毒学和免疫清除特性测试（例如 ADCC、CDC）。在目标 2 中，我们将研究 HA-聚糖变体是否可以通过对来自流感经历过的供体的记忆 B 细胞/浆母细胞进行 FACS 分选来增强干定向 Br/BnAb 的分离。我们将通过比较缩小聚糖变体范围 Ab 种系基因使用、体细胞突变、从 Target 1/2 wt HA 与聚糖变体筛选中回收的 Br/BnAb 的相对数量和多样性。在目标 3 中，我们将进行结构研究来绘制最有希望的 Br/BnAb 的表位图。这里的基本原理是，了解表位结构/位置以及种系与体细胞氨基酸取代的贡献将提供重要的见解，在 原子级别，分为什么类型 我们希望通过疫苗接种来引发保护性、干导向的 BrAb。在目标 4 中，我们将通过人源化小鼠和 Balb/c 小鼠的体内疫苗研究来测试我们的主要 HA-聚糖变体，通过优先刺激预先存在的干定向人类记忆 B 细胞的扩增或体内启动幼稚小鼠 B 细胞分泌保护性 Br/BnAb，诱导针对异亚型病毒攻击的 Br/BnAb 反应的能力。这些聚糖变体应该为设计基于重组 HA 的通用流感疫苗提供“先导抗原”。