Two classes of protective antibodies against Pseudorabies virus variant glycoprotein B: Implications for vaccine design

Pseudorabies virus (PRV) belongs to the Herpesviridae family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.

Introduction complement [28]. Further biochemical studies mapped the epitopes of these antibodies to be within three main regions of gB, residues 59-126, 216-279, and 540-734, respectively [29]. Most antibodies target epitopes within residues 540-734 [29]. In order to develop better therapeutics and vaccines, more efforts should be made to characterize the structural and immunogenic properties of gBs of these emerging highly pathogenic PRV variants.
In this work, we immunized mice with soluble PRV gB to generate gB specific mAbs and identified a total of 15 neutralizing antibodies that effectively block PRV entry with either complement dependent or independent mechanisms. To locate their epitopes, we also determined the crystal structure of PRV gB and verified the binding footprints by mutagenesis. These findings would enormously advance our understanding of PRV gB immunogenicity and provide important guidance for antiviral drug design and vaccine development.

Identification of PRV neutralizing antibodies targeting the envelope protein gB
We expressed the ectodomain of PRV gB with the Bac-to-Bac expression system. The purified soluble gB protein was then applied to immunize mice. A total of 43 gB-specific mAbs were originally selected by indirect ELISAs from hundreds of hybridoma cells. Virus entry inhibition assay was then conducted to assess the neutralizing activities of these mAbs. The tests were performed with addition of exogenous rabbit complement or without in parallel to identify the potential complement dependent neutralizing activities (See details in Materials and Methods). Fifteen neutralizing mAbs in total were identified, which effectively blocked PRV entry into pig kidney cells (PK-15) ( Table 1). Among them, fourteen mAbs blocked the virus entry only in the presence of complement, indicating they exerted the neutralizing activity by complement effect. The remaining one, 1H1 mAb, however directly neutralized the virus without addition of complement (Table 1). Thus, 1H1 mAb might block PRV entry by interfering with either the receptor binding or membrane fusion processes. Broad-spectrum neutralizing activity of 1H1 mAb against different PRV strains As the only complement-independent neutralizing antibody, we further analyzed the neutralizing efficacies of 1H1 mAb against eight different PRV strains, including vaccine strains (Bartha and HB98), classical virulent strains (RA and SU), and the current emerging variant strains (HN1201, 188-5, 072-1, and BH1). Interestingly, the 1H1 mAb displayed a broad-spectrum neutralizing activity against all PRV strains tested though with varied efficacies (Fig 1) [29], this region is therefore a suitable immunogen to elicit complement-dependent neutralizing antibodies against PRV infection. In contrast, the 1H1 mAb only reacted with gB but not gB-D_IV (Table 1;    To further characterize these neutralizing antibodies, we sequenced the variable regions of all these mAbs. Three pairs of mAbs were found to share the same genes for the variable regions (6D2/9B10, 3E1/7B11, and 1H9/9A10) ( Table 2). Thus, we actually obtained 12 unique antibodies, among which 11 of them exert complement-dependent neutralizing activities.
Crystal structures of PRB-gB, PRV-gB-D_IV In order to further characterize the immunogenic properties of PRV gB and identify the epitopes of these neutralizing antibodies, we determined the crystal structures of PRV gB and gB-D_IV at 3.1 and 2.7 Å resolution, respectively (Fig 2). The overall structures of PRV-gB and HSV gB are highly similar, with a sequence identity of 52% (S2 Fig). The two structures could be well superimposed with an overall rmsd of 0.962 Å (Fig 2C). Similar to other class III viral fusion proteins, PRV gB exists as homotrimers and each protomer could be divided into five domains. Each domain could be superimposed better with their counterpart in HSV gB than the entire gB molecule, as slight domain movement was observed for domain IV relative to the other portion of gB molecule ( Fig 2C). In the middle of each gB protomer, a potential furin cleavage site was identified and thus the protein would intend to be processed into two fragments during expression in the cell (Fig 2A). Consistent with this feature, the soluble gB displayed three-band SDS-PAGE profile though eluted as a monodispersed peak in size-exclusion chromatography (S3B Fig). Similar cleavage processing was also observed in HSV gB [15], while it is unclear whether the cleavage is required for the fusogenic activity of gB. The trimeric gB is mainly stabilized by the central helix bundle formed by domain III in the membrane-distal portion. Domain IV wraps around the top of the central helix bundle to form a crown in the bottle-shaped gB trimer ( Fig 2B). The trimerization interfaces are highly stable, such that the truncated gB-D_IV could also assemble into trimers (S3C Fig; Fig 2D). The structure of gB-D_IV trimer could be ideally superimposed with the corresponding portion in the context of the entire gB ectodomain, with a rmsd of 0.598 Å (Fig 2E). The highly stable structure of gB-D_IV further supported the conclusion from our ELISA based assays that these complement-dependent neutralizing antibodies target the crown region of PRV gB and very probably within domain IV.

The unique glycosylation modification of 1H1 mAb
As the only complement-independent neutralizing antibody identified, the 1H1 mAb recognize PRV gB in regions different from all the other 14 mAbs. To further characterize the antigen recognition properties of this mAb, we solved the crystal structure of 1H1 Fab at a resolution of 2.5 Å (Fig 3A). In this structure, we observed an unusual N-linked glycosylation modification at residue N103 of the HCDR3 loop (S5 Fig). To testify whether the glycans participate in antigen recognition of 1H1 mAb, we introduced site mutations to disrupt the "NXS" motif to eliminate the glycosylation modification ( Fig 3B). The mutants were then subjected to bio-layer interferometry (BLI) assay to test their binding affinities to PRV gB. Compared with the wild type 1H1 mAb, the affinities of both the NL and SL mutants decreased almost 1000 times and the binding kinetics also displayed significant differences. The intact 1H1 mAb binds PRV gB with a quite slow kinetics but seems not to dissociate, while the two mutants are much faster in both binding and dissociation processes (Fig 3C-3E). These observations demonstrated that the glycans in the HCDR3 loop play a key but not indispensible role for antigen recognition of 1H1 mAb.

Identification of the 1H1 binding epitope
To precisely identify the epitope of 1H1 mAb, we made great efforts to determine the structure of PRV gB-1H1_Fab complex. Unfortunately, we failed to obtain high quality diffractive crystals of the complex. As an alternative approach, we conducted 3-dimensional (3D) reconstruction by negative stain electron microscopy (EM) method. A 35 Å resolution EM map of PRV gB in complex with 1H1 Fab was obtained (Fig 4; S6 Fig). In this complex, there are three copies of 1H1 Fab binding to the three protomers in the bottle-shaped gB trimer, which follows the rule of 3-fold symmetry (Fig 4A-4C). The atomic structures of PRV gB and 1H1 Fab were perfectly fitted into the density. With the 3-fold symmetry and the hinged structure of 1H1 Fab, we could correctly identify the orientations of heavy chain and light chain (Fig 4A-4C). Thus, a pseudo-atomic model of PRV gB in complex with 1H1 Fab was built so that the interaction details could be inferred.
According to the structure, the epitope of 1H1 was unambiguously mapped to the bottom of domain I in PRV gB (Fig 4A and 4C). The binding of 1H1 Fab to PRV gB is likely mediated by the HCDR3, LCD1 and LCDR3 loops, among which the HCDR3 probably interacts with the 220-strand region and the LCDR1 and LCDR3 mainly recognize the 210-helix (Fig 4D and  4E). We then analyzed all the residues within 5 Å distance in the binding interface to identify the key residues governing the interactions. Among them, four residues in the 210-helix (Q206, D210, R214 and R215) and two in the 220-strand (K221 and E223) seemed to contribute the most interactions, which form a two-portion discrete footprint on the surface of PRV gB (Fig 4D and 4E). Though only one glycan residue was observed in the density map of 1H1 Fab (Fig 3A), there is probably a long glycan chain attached to N103 in the HCDR3 loop as the glycosylation modification in eukaryotic cells often involves multiple glycan residues. Therefore, the space between the 210-helix and 220-strand could possibly accommodate the glycan chain and the 220-strand is probably involved in the interactions with glycans (Fig 4D and 4E). As glycans play a minor role in the interaction as shown by previous biochemical studies, the main binding footprint would thus fall into the 210-helix region. To further verify the location of 1H1 epitope, we performed mutagenesis on the four candidate residues in the 210-helix to test their effects on the interactions.
The 293T cells were transiently transfected with plasmids encoding full-length wild type or mutant PRV gB and the 1H1 mAb was applied to stain the transfected cells. The binding was visualized and quantified by flow cytometry (Fig 5). As expected, a PRV gB mutant with all the four residues replaced by alanine (Mut4) completely abolished the binding (Fig 5F). Interestingly, Q206A and D210A single mutations displayed no obvious effect on the binding, and mutant R215A only slightly reduced the binding affinity (Fig 5B, 5C and 5E). The R214A single mutation, however, significantly impaired the reactivity of PRV gB to 1H1 Fab (Fig 5D), with the same effect as the quadruple mutant (Mut4). To exclude the possibility that gB mutants failed to be displayed on cell surface due to misfolding, we also stained the cells transfected with Identification of gB specific Pseudorabies virus neutralizing antibodies gB mutants encoding plasmids by 5G12 mAb which targets the crown region of gB. As shown by the flow cytometry-based assays, all five mutants could be detected on the surface of transfected cells with similar expression levels as the wild type gB (S7 Fig), which demonstrated that the inability of 1H1 mAb to bind gB mutants expressing cells solely results from the substitution of key interacting residues within the epitope. Collectively, these observations implied that the main epitope of 1H1 neutralizing antibody is probably located in the 210-helix region of PRV gB, among which the residue R214 plays a critical role in the interactions.

Discussion
Outbreaks of newly emerging highly pathogenic PRV variants in Chinese pig farms have caused serious public concerns [5,[8][9][10]. Poor protective efficacy of marketed vaccines and lack of effective therapeutic drugs further raised the threat that the epidemics might cross the border to affect surrounding countries. Better understanding the structures and antigenic properties of PRV proteins is therefore urgently required for developing effective vaccines and therapeutic drugs.
In common with other members in the Herpesviridae family, PRV harbors a pool of glycoproteins embedded in its envelope to form a huge machinery for virus entry, among which gB is the main fusogen responsible for inducing membrane fusion [2,17,27]. Previous studies have indicated gB as an effective immunogen to elicit complement-dependent neutralizing antibodies against PRV infections [26,28,29]. Our studies further supported the conclusion and identified other antigenic sites to elicit direct neutralizing antibodies as well. Combining our findings with previous reports, we can conclude that domain IV is the immunodominant region of PRV gB. Based on the gB structure we present in this study, domain IV is located on the apexes of the gB trimer to form a "crown", making it fully accessible for both antibody and potential receptor recognition. This domain is quite conceivable to become a hotspot for antibody targeting and thus a suitable candidate to develop subunit vaccines.
It has been established that the entry of herpesviruses involves multiple viral glycoproteins and possibly multiple receptors as well, and gB was also shown to play a role in receptor binding [17]. However, all the antibodies targeting domain IV of gB reported to date do not directly block PRV entry but dependent on the complement effect instead, as shown by cell-based assays in vitro [26,28,29]. This phenomenon implies that domain IV is probably not a receptor binding site for PRV gB, and that it might retain the same fold before and after membrane fusion with only domain rearrangement in the process of gB conformational changes to mediate membrane fusion. These findings would thus provide important clues to understand the entry mechanisms of PRV and other herpesviruses.
Besides, we also identified a direct neutralizing antibody that effectively blocked the entry of PRV in the absence of complement, 1H1, which targets the domain I of gB. This is the first complement-independent neutralizing epitope of PRV gB reported to date to our knowledge. The 1H1 mAb binds gB at the bottom of domain I, which is very close to the fusion loops. Although the atomic interaction details cannot be elucidated by the low-resolution EM structure, the binding region of 1H1 mAb can be definitely determined, which is also confirmed by the mutagenesis work. Three copies of Fab fragment surround the peripheral of fusion loops, making it probably unable to reach the membrane of host cells. In the context of full-length IgG, the other Fab arm might render extra steric hindrance, which further blocks the interactions between gB and cell membrane. This hypothesis is strongly supported by the observation that gB fusion loops directly interact with lipid bilayer captured by cryo-EM imaging [30]. In analogy to other class III viral fusion proteins, e.g. the glycoprotein of vesicular stomatitis virus (VSV GP), the domain I of gB might adopt similar fold before and after membrane fusion [31,32]. Therefore, the 1H1 mAb could probably recognize gB in different conformations, including pre-fusion, post-fusion and the intermediates in between, which is possibly the reason contributing to its high neutralizing efficacy. In addition, these observations also indicate that domain I of gB could possibly serve as an ideal immunogen to elicit direct neutralizing antibodies against herpesviruses.
In summary, we combined both immunological and structural approaches to systematically characterize the envelope protein gB of an emerging highly pathogenic PRV variant. We identified two classes of neutralizing antibodies that effectively block PRV infection in vitro, which utilize different mechanisms with complement dependence or without respectively. These two classes of antibodies recognize gB with epitopes in two separate domains and thus indicate these domains as potential subunit vaccines to prevent PRV infections. These findings would intensify our understanding of the immunogenic properties of PRV glycoproteins and provide important guidance for antiviral drug design and vaccine development.

Ethics statement
The protocol in this study was approved by the Committee on the Ethics of Animal Care and Use of National Research Center for Veterinary Medicine (Permit 20160313088). The study was conducted following the Guide for the Care and Use of Animals in Research of the People's Republic of China.

Cells and viruses
Pig kidney (PK-15) cells (CL33, obtained from ATCC) and African green monkey kidney (Vero) cells (GNO10, obtained from cell resource center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) in a humidified chamber containing 5% CO 2 at 37˚C. PRV strain HN1201 was isolated in 2012 from an affected pig farm in China [5], and was propagated in Vero cells. PRV strains Bartha-K61, HB98, RA, SU, 188-5, 072-1, and BH1 were provided by the National Research Center for Veterinary Medicine, China. To make the current epidemic status and the major molecular changes of PRV clear, a phylogenetic analysis based on all PRV genomes available in the GenBank database was performed using the distance-based neighbor-joining method in MEGA4 software (S8 Fig). The Genbank accession numbers were included in the names of PRV strains.

Expression and purification of PRV-gB and PRV-gB-D_IV soluble proteins
Both proteins (HN1201 strain [5]) were expressed with the Bac-to-Bac baculovirus expression system (Invitrogen). The PRV gB construct covers nearly the full-length ectodomain, including residues A59 to N752, followed by a C-terminal 6×His tag. The PRV-gB-D_IV includes two continuous regions, residues A59 to S148 and N546 to T700, linked by a GGSG polypeptide, and a 6×His tag is fused at the C terminus. The GP67 signal sequence was engineered at the N-terminus of each protein to facilitate secretion during protein production. To produce recombinant proteins, Hi5 cells were infected with high-titer recombinant baculovirus and grown for 48 h. The cell culture supernatant was harvested by centrifugation applied to metal affinity chromatography with a HisTrap HP column (GE Healthcare). The eluted product was further purified by size-exclusion chromatography using a Superdex 200 16/600 pg column (GE Healthcare) equilibrated with a buffer containing 20 mM Tris-HCl and 150 mM NaCl (pH 8.0). The final product reached a purity of~95% as shown by SDS-PAGE.

Generation of gB-specific mAbs
To generate gB-specific mAbs, purified recombinant PRV-gB (100 μg/mouse) was blended with Freund's complete adjuvant and used to immunize 6-week-old female BALB/c mice. Booster immunizations were performed twice with 3-week intervals. Then the spleen cells were isolated and fused with SP2/0 myeloma cells. Hybridoma culture supernatants were screened for reactivity to purified PRV-gB by standard indirect ELISAs. Positive hybridoma clones were subcloned by limited dilution at least three times. The mAbs were initially captured from hybridoma cell culture supernatants by affinity chromatography with a Protein G HP column (GE Healthcare) and further purified by size-exclusion chromatography.

Preparation of 1H1 Fab fragment
The coding sequences for 1H1 variable fragments (VH and VL) were fused to the sequence encoding constant regions of a mouse IgG1 (CH, CL and Fc) to generate chimeric IgG1 expression vector using the backbone of pCAGGS plasmid. Two mutant protein-expressing plasmids (1H1_NL and 1H1_SL) were constructed by site-directed mutagenesis to replace N103 or S105 in HCDR3 with alanine ( Fig 3B). The plasmids were transiently transfected into human embryonic kidney 293T (HEK293T) cells for protein expression. After three to seven days post transfection, the supernatants containing secreted IgG1s were harvested and subjected to HiTrap ProteinG chromatography (GE Healthcare). Target proteins were eluted with 0.1 M glycine (pH 3.0) and further purified by size-exclusion chromatography using a Superdex 200 16/600 pg column (GE Healthcare). Fab fragments were generated by ficin digestion and purified using the pierce mouse IgG1 Fab preparation kit (Thermo Scientific) following the manufacturer's instructions. The products were buffer-exchanged into a buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM NaCl by an additional round of size-exclusion chromatography for crystallization.

Indirect ELISAs
Briefly, 96-well microtiter plates were coated with purified PRV-gB and PRV-gB-D_IV at 200 ng/well in a carbonate-bicarbonate coating buffer (pH 9.6) at 4˚C overnight. Plates were blocked at 37˚C for 1 h with PBST containing 5% skimmed milk. Antibodies were then added in the well and incubated for 1 h at 37˚C. After three times of washing, the wells were incubated with goat anti-mouse IgG-HRP (Santa Cruz) for 40 min at 37˚C. The plates were washed again for five times before the reaction substrate TMB was added. The reaction was conducted in dark at room temperature for 5 min and was stopped with 2 M H 2 SO 4 . The optical density at 450 nm (OD 450 ) of each well was read using a microplate reader (Thermo). Statistical presentations were generated with GraphPad Prism 5 (San Diego, CA).
Virus neutralization assays in the presence or absence of complement PK-15 cells were seeded in 96-well plates. Pseudorabies viruses (200 times of TCID 50 ) were incubated with serial two-fold dilutions of the 1H1 mAb at 37˚C for 1 h prior infecting cells. Then, the mixture was added to PK-15 monolayers in 96-well plates and incubated for 1 h. Each concentration was conducted with eight replicates. The supernatant was removed after incubation and replaced by fresh DMEM medium. The cells were cultivated for another 72 h at 37˚C before analysis. The cellular pathology was directly observed using microscopy. All experiments were conducted in three independent trials. The half maximal inhibitory concentration (IC 50 ) was measured to describe the neutralization titer of each antibody. To identify complement-dependent neutralization activities, the experiment was conducted following the same protocol as above except that fresh rabbit serum (working concentration: 5%) as an exogenous complement was added into the virus-antibody mixture before infecting the cells. The data was graphed using GraphPad Prism 5 for presentation (San Diego, CA).

Sequencing of the immunoglobulin variable regions of hybridoma cells
The V gene sequences of each mAb clone were amplified as previously described [33]. Briefly, 10 6 hybridoma cells were collected by centrifugation. Total RNA was extracted using TRIzol regent (Takara) according to the manufacturer's protocol. Reverse transcription and PCR amplification were performed using a set of primers [33]. PCR products were identified by agarose gel electrophoresis and purified using a commercial kit (Tiangen). The DNA fragments were cloned into the pMD 18-T vector (Takara) and sequenced individually.

Crystallization, data collection, and structure determination
The protein samples were concentrated to 10 mg/mL for crystallization using the sitting drop vapor diffusion method at 18˚C. PRV gB was crystallized with a reservoir solution of 34% PEG200 and 0.1 M citric acid, pH 6.5. The crystals of PRV-gB-D_IV were obtained in reservoir solution containing 0.05 M calcium chloride dihydrate and 0.1 M MES, pH 6.0. The 1H1 Fab was crystallized with a reservoir solution of 0.2 M potassium sulfate and 20% PEG3350, pH 6.8. X-ray diffraction data was collected at the Shanghai Synchrotron Radiation Facility (SSRF) BL17U at a wavelength of 0.97915 Å [34]. The datasets were processed with HKL2000 software [35]. Structures were determined by the molecular replacement method using the Phaser program [36] in the CCP4 suite [37]. The PRV gB structure was solved using the HSV-1 gB structure (PDB ID: 2GUM) as the search model. The Fab structure (PDB ID: 1SY6) was used as the search input for 1H1 Fab structure determination. Initial restrained rigid-body refinement was performed using PHENIX [38], which was followed by manual rebuilding and adjustment in COOT [39]. Further refinement was performed using PHENIX [38]. The stereochemical qualities of the final models were assessed using MOLPROBITY [40]. All the data collection and refinement statistics are summarized in S1 Table. Bio-layer interferometry (BLI) The binding affinities of wild type 1H1 Fab or mutants to PRV gB were measured by BLI at room temperature (298K) with the Octet RED96 biosensor method (ForteBio, Inc.). The runnning buffer is composed of 20 mM Hepes (pH 7.4), 150 mM NaCl and 0.005% (vol/vol) Tween 20. Soluble gB was immobilized on an Ni-NTA-coated biosensor surface and then exposed to a series of analytes at different concentrations (6.25-100 nM for 1H1_WT, 62.5-1000 nM for 1H1_NL or 1H1_SL). Background subtraction was used to correct the errors of sensor drifting. The data was processed by the ForteBio's data analysis software and plotted with Origin 8.0 program.

Negative stain electron microscopy and 3D reconstruction
To prepare the PRV gB-1H1 Fab complex, soluble PRV gB and 1H1 Fab samples were mixed with a molar ratio of 1:1.5 and incubated at 4˚C for 2 h. The mixture was then separated by size-exclusion chromatography using a Superose 6 10/300 GL column (GE Healthcare). The complex sample at a concentration of 0.02 mg/mL was applied to glow-discharged copper grids coated with continuous carbon films and stained with 2% uranyl acetate. The excessive stain liquor was blotted with a filter paper and let the grid to air-dry. The specimen was then loaded onto a Tecnai F20 transmission electron microscope (FEI) equipped with a field emission gun for data collection, which was operated at 200 kV acceleration voltage and with a defocus range of-(1-3) μm. Images were recorded with a 4k×4k BM-Eagle CCD camera with a calibrated pixel size of 1.36 Å.
A total of~8000 particles were semi-automatically picked from 200 micrographs using e2boxer.py in EMAN2 [41] package. The contrast transfer function (CTF) parameters were estimated by e2ctffit.py [41] and applied to correct the images by phase-flipping [42] method. All the subsequent classification and reconstruction processes were conducted with relion-2.0 [43] using the phase-flipped particles without further CTF corrections. After several rounds of iterative 2D and 3D classifications, a stack of~3000 particles was selected with 3 copies of 1H1 Fab bound to a gB trimer. The stack was subjected to 3D refinement with 3-fold symmetry applied, which resulted in a reconstruction of 35 Å resolution as determined by the gold-standard fourier shell correlation (FSC) 0.5 cut-off value (S6 Fig).

Map interpretation and atomic model fitting
Though with low resolution, the reconstructed map clearly shows the feature of gB trimer in the center and three copies of 1H1 Fab density branching out at one end. We first fitted the crystal structure of PRV gB into the density map using CHIMERA [44], which showed a high degree of matching. The atomic structure of 1H1 Fab was further fitted into the remaining density using SITUS [45] with several rounds of orientation search. The special hinged structure of the Fab density allowed us to distinguish the relative orientation of heavy chain and light chain of Fab molecules despite the low resolution of the reconstruction. All the fitting processes were performed following the rigid-docking protocols without local adjustments. No obvious clash or close contact was observed in the final pseudo-atomic model, which was used for further structural analysis. All the EM density related figures were rendered using CHIMERA [44].

Mutagenesis and flow cytometric analysis
The gene sequence encoding the full length wild type PRV gB (amino acids 1-914) was cloned into the vector pEGFP-N1 to generate a gB expression vector with an EGFP tag fused at the Cterminus. A QuickChange site-directed mutagensis kit was used to obtain the mutants with the indicated mutations (Q206A, D210A, R214A, R215A or Mut4). Mut4 denotes the quadruple mutant with all four amino acids replaced by Alanine. Protein expression was verified by fluorescence microscopy.
The binding between the 1H1 mAb and gB/mutants was analyzed by flow cytometry. Briefly, Human Embryonic Kidney 293 cells with large T antigen (293T cells, obtained from cell resource center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were transfected with these plasmids above separately. After 24 h, the transfected cells were incubated with 1H1 mAb at room temperature for 30 min. The cells were then washed 3 times with 1×PBS to remove the unbound antibodies. Subsequently, the cells were further incubated with APC-linked goat anti-mouse IgG (minimal x-reactivity) (Biolegend, U.S.A) secondary antibody for 30 min at room temperature (avoiding light). Again, discard the liquid and wash 3 times with 1×PBS. Finally, the cells were loaded onto the flow cytometry (BD FACSCalibur) to detect the APC fluorescence signals.