Structural Requirements for Broadly Protecting Antibodies to Influenza A & B

描述(申请人提供)：流感仍然是一个主要的医学问题，是对人类健康的持续威胁。自20世纪40年代第一次接种以来，流感疫苗已经为全球提供了针对流感感染的季节性保护。然而，由于流感倾向于通过一种称为“抗原漂移”的过程不断改变其血凝素(HA)蛋白，以及通过“抗原转移”来改变大流行毒株，目前的疫苗需要 近年度重新制定和准确预测即将到来的季节的循环毒株。新发现的流感血凝素(HA)干区表位是罕见的广谱中和抗体(BNAbs)的靶点，这让人们再次乐观地认为，开发一种能够诱导这些类型的bNAbs的“通用”流感疫苗是可能的。然而，最近生产这种疫苗的尝试只显示出有限的成功。我们现在从2009年H1N1大流行期间对B细胞反应的分子研究中认识到，在诱导bNAbs的保护水平方面的限制不是由于固有的抗原性缺乏，而是由于对HA球状的免疫显性抗体反应，这是反复的年度记忆B细胞对季节性流感疫苗的扩张的结果，在数量和资源上超过了干细胞定向记忆B细胞。我们的初步数据指出了一种选择或诱导bNAbs的有效方法，包括合理设计HA上的糖基化位点，通过对头部表位的“糖链屏蔽”和对茎表位的揭开，将Abs“重定向”到茎区域。在目标1中，我们是否会建议使用我们的约500亿成员的人-抗体噬菌体展示文库来研究葡聚糖屏蔽和去掩蔽作为一种策略来筛选广泛反应的茎结合抗体(BrAbbs)的广泛适用性。回收的BrAbs将进行病毒学和免疫清除特性(如ADCC、CDC)测试。在目标2中，我们将通过FACS对有流感经验的捐赠者的记忆B细胞/浆母细胞进行分选，来研究HA-葡聚糖变异体是否能够加强干细胞定向BR/bNAbs的分离。我们将通过比较从Aim 1/2wt HA与糖变异体筛选中回收的BR/bNAbs的种系基因使用、体细胞突变、相对数量和多样性来缩小糖变异体的范围。在目标3中，我们将进行结构研究，以绘制最有希望的bR/bNAbs的表位。这里的基本原理是，理解表位结构/位置以及种系与体细胞氨基酸替换的贡献将在原子水平上提供重要的见解，以了解什么类型(S) 我们希望通过接种疫苗来诱导保护性的、茎定向的BrAbs。在目标4中，我们将通过在人源化和Balb/c小鼠身上进行体内疫苗研究来测试我们的领先HA-葡聚糖变异体(S)通过优先刺激先前存在的干细胞定向的人类记忆B细胞的扩增或体内启动幼小鼠B细胞以分泌保护性的BR/bNAb抗体来诱导BR/bNab应答的能力。这些多聚糖变异体应为设计基于HA的重组通用流感疫苗提供“先导抗原”。

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.