Synthetic glycolipids to investigate the interactome of viral glycan-binding proteins

基于RNA的大流行性病毒，如SARS-CoV 2和甲型流感病毒株，与不同的突变负担有关，以逃避适应性免疫系统组分的攻击。它们通常快速进化的性质使这些病毒对疫苗开发具有挑战性：流感疫苗必须每年更新，中和抗血清效力受损的新SARS-CoV 2毒株正在出现。这些病毒由人类宿主细胞产生，在Asn-Xaa-Ser/Thr序列子上高度糖基化（例如SARS-Cov 2 Spike的22）。糖基化位点的添加或去除比肽序列的抗原漂移慢得多，其中一些聚糖对于病毒进入是必需的。鉴于病毒糖蛋白的大部分表面被聚糖覆盖，糖肽可能是抗体识别的重要靶标。例如，通过冷冻电子显微镜偶然发现了一种识别冠状病毒（包括SARS-CoV 2）上保守糖肽表位的中和抗体。表位占据了300 μ 2的大表面，但关键依赖于聚糖的存在。然而，该领域目前缺乏诊断技术来分析针对定义的糖肽的免疫应答，这仅仅是因为这些探针比非糖基化肽更不容易合成和纯化。同时，评估来自感染或接种个体的血清中和感染的能力依赖于繁琐的低通量细胞培养实验。我们假设抗病毒糖肽抗体在中和中起着至关重要的作用。该项目将利用合成糖肽的潜力开发靶向诊断方法，以SARS-CoV 2为案例研究，分析患者血清对呼吸道病毒的中和能力。我们将开发一种方法，将光开关脂质标签附加到合成聚糖上。光电开关将被设计成能够通过聚糖微阵列分析进行结合实验。这一物理科学创新将使我们能够覆盖一个大的结构空间，因为可以探测聚糖的不同方向。因此，我们的数据将用于诊断设备和优化疫苗抗原的生成，这些疫苗抗原应该比纯肽表位对抗原漂移更具抗性。

Pandemic RNA-based viruses such as SARS-CoV2 and influenza A strains are associated with a varying mutational burden to evade attack by components of the adaptive immune system. Their often rapidly evolving nature has made these viruses challenging targets for vaccine development: influenza vaccines have to be updated annually, and new SARS-CoV2 strains with impaired efficacy of neutralizing antisera are emerging. Being produced by human host cells, these viruses are highly glycosylated on Asn-Xaa-Ser/Thr sequons (e.g. 22 for SARS-Cov2 Spike). Addition or ablation of glycosylation sites is considerably slower than the antigenic drift of peptide sequences, with some glycans being essential for virus entry. Given that a large part of the surface of viral glycoproteins is covered by glycans, glycopeptides are likely to be important targets for antibody recognition. For instance, a neutralizing antibody recognizing a conserved glycopeptide epitope on coronaviruses including SARS-CoV2 was found serendipidously by cryo-electron microscopy. The epitope occupies a large 300 Å2 surface but crucially relies on the presence of the glycan. However, the field is currently lacking diagnostic technologies to profile immune responses against defined glycopeptides simply because these probes are much less straightforward to synthesise and immobilize than non-glycosylated peptides. At the same time, assessing the capability of sera from infected or vaccinated individuals to neutralise infection relies on cumbersome, low-throughput cell culture experiments. We hypothesise that antibodies against viral glycopeptides play a crucial role for neutralisation. This project will leverage the potential of synthetic glycopeptides to develop targeted diagnostics that profile the neutralising capacity of patient sera against respiratory viruses, with SARS-CoV2 as a case study. We will develop a method to append photo-switchable lipid tags to synthetic glycans. Photoswitches will be designed to enable binding experiments by glycan microarray analysis. This physical sciences innovation will allow us to cover a large structural space, as different orientations of glycans can be probed. Thereby, our data will feed into the generation of diagnostic devices and optimized vaccine antigens that should be much more resistant towards antigenic drift than pure peptide epitopes.