Investigation of a special neutralizing epitope of HEV E2s

Abstract

After 14-years of development, the first prophylactic vaccine against the Hepatitis E virus (HEV) has been marketed since 2012 (Wu et al., 2012). However, the neutralizing epitopes of HEV are not completely defined. E2s, a protruding homodimer domain of HEV capsid protein, is responsible for interacting with host cells to initiate infection (Li et al., 2009; Li et al., 2005). It was shown that two monoclonal antibodies (mAb), 8C11 and 8H3, could neutralize the infectivity of HEV in rhesus, and the two mAbs bind to different neutralizing conformational epitopes on E2s (Zhang et al., 2005). The epitope for 8C11 was reported based on the crystal structure of HEV protruding domain E2s in complex with 8C11 Fab (Tang et al., 2011). In a HEV antibody panel consisting of 30 mAbs, including 8C11, none of them can cross block the binding of 8H3 to E2s. These findings suggest that 8H3 recognizes a special epitope which is different from the epitope for 8C11 and other mAbs. X-ray crystallography is not a viable option to identify the 8H3 epitope because 8H3 binds to the E2s with a low affinity (Zhang et al., 2005). Conformational epitopes, which often escape identification by linear peptide screening, can be identified and characterized from studies with mimotopes (Cardoso et al., 2009). Most mimotopes obtained from phage displayed peptide libraries can be employed to facilitate the identification of novel peptide sequences that mimic binding sites for mAbs (Mayrose et al., 2007). We panned three phagedisplayed peptide libraries (ph.D.-C7C and ph.D.-12 displaying peptide on the pIII protein, and lib C10C displaying peptide on the pVIII protein) to select 8H3 mimotopes (Table S1). After three rounds of panning, phage clones were tested for binding specificity to 8H3. Mimotopes which reacted to 8H3 without cross-reacting with three HEV related antibodies (8C11, 12G8, and 8G12) and two HEV non-related antibodies (13D4 against AIV and 42B6 against HBV) were considered positive. Finally, 21 mimotopes to 8H3 were obtained for further analysis (Table S2). The 21 mimotopes to the 8H3 mAb were processed individually by three efficacious prediction programs, Pep3D-Search program, Pepsurf and EpiSearch for 8H3 epitope mapping (Huang et al., 2008; Mayrose et al., 2007; Negi and Braun, 2009). The E2s structure of the HEV (PDB: 3GGQ) was used as a template for epitope prediction (Li et al., 2009). The mimotope sequences listed in Table S2 were used to deduce the best cluster by default parameters. The prediction results from the three programs were shown in Table S3. Overlapping regions of the predicted clusters from the three programs, composed of Gln-Thr, SerGly, Ser-Pro, Tyr-Asn, Asn-Gln, Asn and Ser-Thr, were considered parts of the 8H3 epitope. These overlapping regions (shown in rose red in Fig. 1A) are located on the groove of E2s, and they are independent from the epitope of 8C11. The prediction results based on the mimotope sequences provide general information on the binding region on the antigen, but it does not provide the details on antigen-antibody contacts, for example, the amino acids involving in hydrogen-bonding contacts and the binding sites on the antibody. The rigid-body protein-protein docking program ZDOCK was then used to map the antigen-antibody contact sites. Fast Fourier Transform (FFT) algorithm was applied to perform a global docking to search for potential binding positions of two component proteins (Pierce et al., 2014). Since validity of the ZDOCK analysis is affected by the accuracy of the search algorithm as well as the proteinprotein complex to be predicted, some of the top-scoring predictions resulted from the soft scoring function of the program could be false positives (Wiehe et al., 2008). Combining the results from epitope prediction softwares based on mimotope and ZDOCK may lead to a more reliable result. The overlapping regions (Gln-Thr, SerGly, Ser-Pro, Tyr-Asn, Asn-Gln, Asn and Ser-Thr) predicted from the three programs were further investigated by ZDOCK. The 3-dimensional model of mAb 8H3 was generated by a homology modeling protocol. Given the facts that the epitope of antibody 8H3 is different from that of 8C11, and the binding of 8H3 to E2s can be enhanced by 8C11 (Zhang et al., 2005), the structure of 8C11 Fab-E2s complex (PDB: 3RKD) was used as the antigen for the ZDOCK program to search for the best combination model. As a result, the surface on antigen E2s for binding to antibody 8H3 were shown in red (Fig. 1B). The regions of E2s binding to 8H3 were further analyzed and shown in dark shade (Fig. 1C). In addition to the same three

DOI: 10.1007/s13238-014-0115-3

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Cite this paper

@inproceedings{You2014InvestigationOA, title={Investigation of a special neutralizing epitope of HEV E2s}, author={Min Kyoung You and Lu Xin and Yi Yang and Xiao Zhang and Yingwei Chen and Hai Yu and Shaowei Li and Jun Zhang and Zhiqiang An and Wenxin Luo and Ningshao Xia}, booktitle={Protein & Cell}, year={2014} }