Investigation of a special neutralizing epitope of HEV E2s

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

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 . 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 .
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, Pep-3D-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 482 -Thr 484 , Ser 487 -Gly 490 , Ser 527 -Pro 540 , Tyr 559 -Asn 560 , Asn 562 -Gln 568 , Asn 573 and Ser 582 -Thr 586 , 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 482 -Thr 484 , Ser 487 -Gly 490 , Ser 527 -Pro 540 , Tyr 559 -Asn 560 , Asn 562 -Gln 568 , Asn 573 and Ser 582 -Thr 586 ) 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 , 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 regions (Ser 527 -Thr 535 , Tyr 559 -Asn 560 , and Tyr 584 -Thr 586 ) predicted by the three programs based on mimotopes, there are four additional binding regions (Leu 521 , Arg 524 -Pro 525 , Leu 570 , Ala 602 -Pro 604 ) on the first subunit and one region (Thr 552 -Lys 554 ) on the second subunit from the prediction by ZDOCK (Fig. 1C and 1D). All the predicted interaction regions are located on the groove or near-groove of the E2s dimer structure.
To verify the prediction results functionally, all predicted sites on E2s with solvent accessibility were individually mutated to Ala and the mutants were expressed in E. coli. The wild type E2s and its mutants were subjected to nonreducing SDS-PAGE and Western blotting against 8H3. All samples except Thr564Ala and Ser582Ala were resolved mainly as dimers ( Fig. 2A). In previous studies, we have demonstrated that the dimerization of E2s is essential for HEV-host interactions and antibody neutralization, and Thr 564 and Ser 582 can stabilize the dimer (Li et al., 2009;Li et al., 2005). Therefore, the Thr564Ala and Ser582Ala The epitope residues shown in purple correspond to their interactions to 8H3 by hydrogen bonds. The epitope residues shown in brown are located on the second subunit (in orange) of E2s. (D) The major clusters of 8H3 epitope on E2s are shown in ball and stick model and colored in deep purple. The epitope residues labeled with underline are located on the second subunit of E2s. The 8H3 Fab is shown as surface representation, H-chain is shown in light blue, and L-chain is green. (E) Depicts the electrostatic potential surface of the epitope on the E2s (red, negative; blue, positive; and gray, neutral) with the key residues for interaction from 8H3 Fab represented as sticks. The figure was prepared using the program PyMOL (Delano, 2002). mutants lose the binding ability to 8H3 due to the collapse of dimeric form of E2s. The Western blotting results showed that the wild type dimeric E2s and 15 mutants, including the ones with mutated sites (Thr 489 , Ser 527 , Gln 531 , Gln 568 and Pro 604 ) responsible for binding to 8H3 by hydrogen-bond as the prediction results of ZDOCK, were reactive with 8H3 ( Fig. 2A). Since Ile529Ala and Asn560Ala mutants in dimeric form abolished the 8H3 reactivity, Ile 529 and Asn 560 contribute to the binding of 8H3 with E2s ( Fig. 2A).
We have shown previously that mAb 8C11 enhances the binding of 8H3 to E2s . If 8C11 can't enhance the binding of 8H3 to the E2s mutants, the E2s mutants can be assumed of losing the binding capacity to 8H3 and the epitope of 8H3 on E2s was destroyed by the site mutations. In order to investigate the enhancement of 8C11 on the binding of 8H3 to the Ile529Ala or Asn560Ala mutants, CLIA (chemiluminescent immunoassay) was carried out using microplates coated with purified E2s mutants and pre-incubated with buffer containing 8C11, then reacted with HRP labeled 8H3. Binding of the labeled antibodies was measured in RLU (relative light unit). The results showed that the Ile529Ala and Asn560Ala mutants retained the binding activity to 8C11, but they can't bind to 8H3 with the presence of 8C11 (Fig. 2B), suggesting that the epitope of 8C11 was retained, but the epitope of 8H3 was damaged in mutants Ile529Ala and Asn560Ala. From the structure of E2s (PDB: 3GGQ or 3RKD), we found that the amino acid residues around Ile 529 in 6 Å included Gln 530 , Thr 528 , Gln 531 , Ser 527 , Val 538 and Gln 568 , and residues around Asn 560 in 6 Å included Thr 489 , Ser 582 , Asp 567 , Ala 565 and Thr 564 (Fig. 2C). The mutations of Asn560Ala and Ile529Ala may have resulted in conformational changes of the 8H3 epitope, especially those amino acid residues located around Asn 560 and Ile 529 in 6 Å. These results collectively indicated that Ile 529 and Asn 560 were the key sites to maintain the epitope of 8H3 and confirmed the prediction results that 8H3 targets the groove region of E2s.  E2  T489A  G490A  R524A  P525A  S527A  T528A  I529A  Q530A  Q531A  P540A  T552A  T553A  K554A  N560A  T564A  S566A  Q568A  S582A  P604A   I529A  Mutational studies on the predicted binding-region of 8H3. All residues from the predicted binding-region were mutated to Ala. The wild type E2s and its mutants were subjected to non-reducing SDS-PAGE and Western blotting with 8H3.
[+] denotes dimerization or reactivity with 8H3, [−] denotes loss of dimerization or reactivity with 8H3. (B) Effects of 8C11 on the binding of 8H3 with E2s mutants. (C) Residues around the two key amino acid residues in dark blue Ile 529 and Asn 560 in 6 Å are circled in light green and light blue, respectively. The sites in red and purple are the predicted epitope of 8H3 and the residues with interactions to 8H3 by hydrogen bonds are shown in purple.