SARS-CoV-2 vaccines based on RBDs with engineered glycosylation sites

我们正在开发针对SARS-CoV-2的疫苗抗原，将抗体反应集中在病毒Spike (S)蛋白受体结合域（RBD）的中和表位上。来自SARS-CoV-2变体的增强抗原将特别受益于限于RBD，因为全长S蛋白中存在大量保守但非中和的表位。然而，由于RBD上的疏水性斑块在天然S蛋白中形成亚基间界面，仅RBD疫苗面临聚集和表达不良的技术限制。我们通过在RBD中加入N链糖基化位点来克服这些限制。这些聚糖还有助于将免疫反应从RBD的脱靶面转移到有效中和抗体反应的靶标上。我们将进一步扩展这一策略，将抗体反应集中在RBD中在SARS-CoV-2变体中保守的中和表位上。RBD抗原将作为脂质纳米颗粒(LNP)-mRNA疫苗进行测试。在该项目的一期研究中，我们将增强RBD抗原作为LNP-mRNA疫苗的固有免疫原性。在第二阶段，我们将在此平台上比较基于变体衍生的RBD和变体衍生的全长S的增强剂，并优化RBD抗原，将抗体反应集中在SARS-CoV-2变体中保守的中和表位上。该项目产生的抗原将利用四个水平的免疫聚焦来引发或增强识别RBD中保守表位的抗体反应，并中和抗原不同的SARS-CoV-2变体。

We are developing vaccine antigens for SARS-CoV-2 that focus the antibody response onto neutralizing epitopes in the receptor binding domain (RBD) of the viral Spike (S) protein. Booster antigens derived from variants of SARS-CoV-2 would especially benefit from being limited to the RBD, due to the preponderance of conserved but non-neutralizing epitopes in the full-length S protein. However, RBD-only vaccines face the technical limitations of aggregation and poor expression, due to hydrophobic patches on the RBD that form the inter-subunit interfaces in the native S protein. We have overcome these limitations by engineering N- linked glycosylation sites into the RBD. These glycans also help to focus the immune response away from off-target faces of the RBD, and onto the targets for potent neutralizing antibody responses. We will extend this strategy further, to focus the antibody response onto neutralizing epitopes in the RBD that are conserved among variants of SARS-CoV-2. The RBD antigens will be tested as lipid nanoparticle (LNP)-mRNA vaccines. In Phase I of this project, we will enhance the intrinsic immunogenicity of RBD antigens delivered as LNP-mRNA vaccines. In Phase II, we will build upon this platform to compare boosters based on variant- derived RBD versus variant-derived full-length S, and to optimize RBD antigens for focusing antibody responses onto neutralizing epitopes that are conserved among variants of SARS-CoV-2. The antigens generated by this project will exploit four levels of immunofocusing to elicit or boost antibody responses that recognize conserved epitopes in the RBD and neutralize antigenically-distinct variants of SARS-CoV-2.