Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus (ZikV) and the four serotypes of dengue virus (DenV1-4) must elicit broadly neutralizing antibodies (bnAbs) to prevent the risk of enhancement of infection by non-neutralizing antibodies. We recently discovered new orthoflavivirus-directed bnAbs, including F25.S02, which neutralizes DenV1-4 and ZikV with comparable or superior potency to the previously characterized E dimer epitope (EDE) bnAbs. Mutagenesis studies of viral envelope proteins showed that the epitope specificity of F25.S02 is distinct from EDE1 bnAbs. Here, we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F25.S02 at the molecular level. We obtained a ∼4.2 Å cryoEM structure of F25.S02 Fab bound to a stabilized DenV3 soluble E protein dimer and a 2.3 Å crystal structure of F25.S02 Fab bound to ZikV soluble E protein dimer. Like previously described EDE1 bnAbs, the structural epitope of F25.S02 is at the E dimer interface, encompassing predominantly conserved regions in domain II, including the fusion loop. However, unlike EDE1 bnAbs, F25.S02 binding is almost entirely dependent on the heavy chain and is shifted slightly away from the dimer symmetry axis. Our findings emphasize the importance of this cross-neutralizing site of vulnerability for DenV and ZikV that can facilitate rational design of vaccines and therapeutics.

为了预防由非中和抗体引发的感染增强风险，针对共同流行的蚊媒正黄病毒（如寨卡病毒(ZikV) 和四种血清型登革热病毒(DenV1-4)）的安全有效的疫苗必须诱导广谱中和抗体(bnAbs)。我们最近发现了一些新的正黄病毒导向bnAbs，包括F25.S02，它可以与E二聚体表位(EDE) bnAbs相比，以相等或更优的效力中和DenV1-4 和ZikV。对病毒包膜蛋白进行突变研究后发现，F25.S02 的表位特异性区别于EDE1 bnAbs。在这里，我们使用冷冻电子显微镜(cryoEM)和X射线晶体学来了解F25.S02 在分子水平上的交叉中和机制。我们得到了一个与稳定化的DenV3可溶性E蛋白二聚体结合的F25.S02 Fab结构（分辨率为约4.2 Å），以及一个与ZikV可溶性E蛋白二聚体结合的F25.S02 Fab结构（分辨率为2.3 Å）。如同之前描述的EDE1 bnAbs，F25.S02 的结构性表位位于E二聚体界面，在II域中包含主要保守区域，包括融合环。然而，与EDE1 bnAbs不同的是，F25.S02 结合几乎完全依赖于重链，并且稍微偏离了二聚体对称轴。我们的研究结果强调了DenV 和ZikV的这一交叉中和脆弱位点的重要性，这可以促进疫苗和治疗药物的理性设计。