There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)–specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo–electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 “locks” two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.

Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed. Dengue virus (DENV) is a mosquito-borne pathogen and the causative agent of dengue fever, dengue hemorrhagic fever (DHF), and the life-threatening dengue shock syndrome (DSS). Currently, DENV infects about 50 to 100 million people per year, resulting in 250,000 to 500,000 cases of DHF or DSS, making it a major health, social, and economic problem (1). This virus belongs to the family Flaviviridae, which also includes other major human pathogens, such as yellow fever virus, West Nile virus (WNV), tick-borne encephalitis virus, Japanese encephalitis virus, etc. The four DENV serotypes share high genetic homology, varying in amino acid sequence by about 25 to 40%. Genotypes within a serotype are even more conserved, containing about only 3% variation in amino acid sequence (2, 3).Envelope (E) protein is the major antigenic structure on the surface of DENV (4). Crystal structures of the ectodomain of the E protein showed that it consists of three domains (I, II, and III) and that E proteins likely exist as dimers in solution (5-7). Domain III participates in receptor binding, while domain II facilitates virus fusion via the interaction of its fusion loop at the tip of the domain with the endosomal membrane during virus entry into the cell (4). Cryo-electron microscopy (cryo-EM) structures (8, 9) of the mature DENV showed that the surface of the virus is made from 180 copies of E and 180 copies of membrane (M) proteins that are arranged in an icosahedral manner. There are three individual E proteins in each of the 60 asymmetric units (molecules A, B and C; also shown in Fig. 5B) (8, 9). Each of these E proteins is located close to one of the 2-, 3-, or 5-fold vertices, thereby having a different local chemical environment. The E proteins are organized as 90 head-to-tail homodimers. Three of these homodimers lie parallel to each other, forming a raft, and together with the other 29 rafts, they form a herringbone pattern on the surface of the virus.The previously published cryo-EM structures were done with DENV grown in mosquito cell lines incubated at 28 to 30°C for several days and then kept at 4°C during virus purification pr...

Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection.Subject Categories Microbiology, Virology & Host Pathogen Interaction; Immunology

SUMMARY Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.