Zika virus (ZIKV) is a flavivirus that is structurally highly similar to the related viruses, dengue virus (DENV), West Nile virus, and yellow fever virus. ZIKV causes an acute infection that often results in mild symptoms but that can cause severe disease in rare instances. Following infection, individuals mount an adaptive immune response, composed of antibodies (Abs) that target the envelope (E) glycoprotein of ZIKV, which covers the surface of the virus. Groups have studied monoclonal antibodies and polyclonal immune sera isolated from individuals who recovered from natural ZIKV infections. Some of these antibodies bind to domain III of E (EDIII), but the functional importance of these antibodies is unknown. In this study, we aimed to determine if EDIII is a major target of the potent serum neutralizing antibodies present in people after ZIKV infection. By generating a chimeric virus containing ZIKV EDIII in a DENV4 virus backbone, our data show a minor role of EDIII-targeting antibodies in human polyclonal neutralization. These results reveal that while monoclonal antibody (MAb) studies are informative in identifying individual antibody epitopes, they can overestimate the importance of epitopes contained within EDIII as targets of serum neutralizing antibodies. Additionally, these results argue that the major target of human ZIKV neutralizing antibodies resides elsewhere in E; however, further studies are needed to assess the epitope specificity of the neutralizing response at the population level. Identification of the major epitopes on the envelope of ZIKV recognized by serum neutralizing antibodies is critical for understanding protective immunity following natural infection and for guiding the design and evaluation of vaccines.
KEYWORDS: Zika virus, chimeric virus, epitope, neutralizing antibodies
Zika virus (ZIKV) was isolated in Uganda in 1947 and introduced into Latin America where it caused an epidemic with millions of infections. ZIKV is genetically and antigenically similar to related flaviviruses such as dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (1, 2). Decades of research into the immune response that occurs following DENV infection revealed that neutralizing antibodies (Abs) targeting the envelope protein are a critical component of protective immunity (1). Despite their protective role, antibodies are also implicated in enhancing disease in secondary infections. Because of the high degree of homology between DENV and ZIKV, there is extensive antibody cross-reactivity (both neutralizing and enhancing) (3). However, there is growing evidence that in people, prior DENV infection partially protects against subsequent ZIKV infection (4, 5). It is critical to fully define the human immune response to ZIKV natural infection to better evaluate next-generation vaccine design (1, 6).
Following ZIKV infection, individuals mount an IgG response that is predominantly directed against the envelope glycoprotein (E) (1). Multiple groups have sought to identify the epitopes targeted by human monoclonal antibodies (MAbs) against ZIKV, as they can be informative of the polyclonal antibody repertoire (3, 7,–11). While MAbs have been identified that target all regions of E (domains I, II, and III), the majority of antibodies described target EDIII (3, 7,–11). Additionally, multiple groups have estimated that a large fraction of polyclonal immune sera and the B-cell repertoire also target EDIII, concluding that this is therefore the primary target of ZIKV antibodies (7, 9, 11, 12). In contrast, following DENV or WNV infection, only a small fraction of antibodies target EDIII, and those that do contribute very little to total polyclonal neutralization (1, 13). Importantly, there have not been any comprehensive studies directly comparing the roles of EDIII antibodies against DENV, WNV, and ZIKV. People infected with ZIKV develop high levels of ZIKV-specific serum neutralizing antibodies, but it is unknown if EDIII is a major target of these antibodies. Using reverse genetics, we sought to develop a tool to track ZIKV EDIII-specific antibodies and to estimate their contribution to ZIKV neutralization.
Across the E ectodomain, ZIKV has high degrees of homology with DENV1 to DENV4 in EDI and EDII, which contain highly conserved regions (e.g., fusion loop) (Fig. 1A and andB)B) (3, 12). EDIII is the least conserved, containing highly variable regions (Fig. 1A and andB)B) (3, 12). To map ZIKV EDIII-targeting antibodies, we generated a chimeric recombinant DENV4 virus containing EDIII from ZIKV (rDENV4/ZIKV-EDIII) (Fig. 1C). The chimeric virus encodes 52 ZIKV amino acids that differ from DENV4, including the addition of three (Fig. 1D). These amino acids span EDIII and include surface-exposed as well as internally facing and cryptic residues (Fig. 1E).
ZIKV E homology and recombinant virus design. (A) (Top) ZIKV E protein sequence homology with DENV1 to DENV4, graphed as the percentage of DENV residues that match ZIKV residues (e.g., a ZIKV residue matching two DENV serotypes = 50% conserved), color-coded by domains (with EDI, EDII, and EDIII color-coded as red, yellow, and blue, respectively). The numbers at the top of the graph correspond to amino acid position. (Bottom) The heat map displays the same ZIKV homology as displayed in the graph (black = 100% conserved, white = 0% conserved). (B) ZIKV protein dimer (PDB 5IZ7) with bottom monomer color-coded by domains and top monomer color-coded by homology to DENV as shown in panel A. (C) Design of rDENV4/ZIKV-EDIII chimeric virus. (D) EDIII amino acid alignment of DENV, ZIKV, and chimeric rDENV4/ZIKV-EDIII. Amino acids missing in DENV4 are highlighted in pink. (E) DENV protein dimer (PDB 1OAN) showing altered residues (highlighted in cyan).
rDENV4/ZIKV-EDIII reached a lower titer compared to both DENV4 and ZIKV (Fig. 2A) and had smaller foci morphology relative to the parental DENV4 strain (Fig. 2B). It is possible that chimerization, in addition to attenuating the virus, altered another aspect of virus biology, such as maturation. To confirm ablation of the DENV4 EDIII epitope and transplantation of ZIKV EDIII, the viruses were evaluated for their ability to be neutralized by EDIII MAbs. rDENV4/ZIKV-EDIII was not neutralized by DENV4 EDIII-specific MAb D4-E75, whereas it was potently neutralized by three different ZIKV EDIII-specific MAbs (ZKA64, ZKA190, and ZKC6), with comparable 50% focus reduction neutralization titers (FRNT50) (Fig. 2C and andD)D) (3, 14). To ensure that distal, non-EDIII epitopes were not disrupted and that their presentation was not altered, we measured neutralization by DENV4 and ZIKV EDI/II hinge antibodies D4-131 and Z3L1 (10, 15). rDENV4/ZIKV-EDIII maintained neutralization by D4-131 and did not gain neutralization to Z3L1 (Fig. 2C and andD),D), confirming that distal epitopes were not disrupted, nor was nonspecific ZIKV neutralization gained.