antibody-induced conformational changes in hsv gd reveal...

1

Antibody-induced conformational changes in HSV gD reveal new targets for virus 1 neutralization 2

3 4 Eric Lazear1^, J. Charles Whitbeck1, Manuel Ponce-de-Leon1, Tina M. Cairns1, Sharon 5 H. Willis4, Yi Zuo1, Claude Krummenacher2, Gary H. Cohen1 and Roselyn J. Eisenberg3* 6 7 Departments of Microbiology1 and Biochemistry2, School of Dental Medicine and 8 Department of Pathobiology3, School of Veterinary Medicine, University of Pennsylvania, 9 Philadelphia, PA, USA, and Integral Molecular4, Philadelphia, PA, USA 10 11 12 Running title: Neutralization of HSV by anti-gD MAbs 13 14 15 Word count (abstract): 233 16 Word count (text): 7,311 17 18 19 ^Current address: Department of Pathology and Immunology, Washington University, St. 20 Louis, MO, USA 21 22 *Corresponding author address: Dr. Roselyn Eisenberg 23 School of Veterinary Medicine 24 University of Pennsylvania 25 240 S. 40th St, Levy Rm 215 26 Philadelphia, PA 19104 27 Phone: (215) 898-6552 28 FAX: (215) 898-8385 29 Email: [email protected] 30

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.06480-11 JVI Accepts, published online ahead of print on 30 November 2011

on May 4, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

2

31 Abstract 32

As the receptor-binding protein of HSV, gD plays an essential role in virus entry. In its 33 native state, the last 56 amino acids of the ectodomain C-terminus (C-term) occlude 34 binding to its receptors, HVEM and nectin-1. Although it is clear that movement of the C-35 term must occur to permit receptor binding, we believe that this conformational change is 36 also a key event for triggering later steps leading to fusion. Specifically, gD mutants 37 containing disulfide bonds that constrain the C-term are deficient in their ability to trigger 38 fusion following receptor binding. In this report, we show that two newly made MAbs, MC2 39 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. 40 In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to 41 receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances 42 the affinity of gD for both receptors. Several non-neutralizing MAbs also enhanced gD-43 receptor binding (MC4, MC10, MC14). While MC2 and MC5 recognized different epitopes 44 on the core of gD, these non-neutralizing MAbs recognized the gD C-term. Both the 45 neutralizing capacity and rate of neutralization of virus by MC2 is uniquely enhanced when 46 combined with MAbs MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from 47 performing a function that triggers later steps leading to fusion, and that the epitope for 48 MC2 is normally occluded by the C-term of the gD ectodomain. 49 50


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

3

Introduction 51 Herpes simplex virus (HSV) is an important human pathogen that infects epithelial 52 cells before spreading to the peripheral nervous system where it establishes a life-long 53 latent infection. Four virion envelope glycoproteins, gD, gB and gH/gL are essential for HSV 54 entry into all relevant cell types (19). Two surface proteins, nectin-1 and herpes virus entry 55 mediator (HVEM), can serve as gD receptors. Nectin-1 is an immunoglobulin (Ig) 56 superfamily member while HVEM is a tumor necrosis factor receptor family member (50). 57 A combination of crystal structures, mutagenesis and monoclonal antibody binding studies 58 has shown that the sites for HVEM and nectin-1 binding are largely distinct (19, 30, 51). 59

Crystallography studies have also shown that the C-terminus of the gD ectodomain 60 (C-term) normally occludes the binding site for nectin-1 and prevents formation of the N-61 terminal loop needed for HVEM binding (19, 30). Thus, for either receptor to bind to gD, 62 the C-term residues must be displaced. Notably, a gD mutant engineered to contain an 63 additional disulfide bond that constrained the motion of the C-term was able to bind both 64 HVEM and nectin-1 normally. However this mutant failed to trigger cell-cell fusion nor did 65 it complement a gD-null virus (31). Thus, the phenotype of this mutant dissociates receptor 66 binding from downstream post-receptor effects mediated by gD. This led us to hypothesize 67 that a common conformational change is responsible for triggering the downstream events 68 involved in virus-cell fusion. 69 The recent resolution of the structure of gB and gH/gL for both HSV and Epstein 70 Barr Virus (EBV) (4, 12, 15, 20, 33) revealed that while gB is a Class III fusion protein, the 71 structure of gH/gL does not resemble any known viral fusogen. Thus, the function of gH/gL 72


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

4

as part of the core-fusion machinery is still unclear. Some have suggested that the highly 73 conserved and highly hydrophobic C-terminal regions of the gH ectodomain may play a 74 direct role in fusion (15, 32, 33). However, even this suggestion leaves many questions 75 unanswered since this region does not contain a readily recognizable fusion loop or 76 peptide as is found in fusion proteins of known structure (18). 77 Another hypothesis is that gH/gL plays a regulatory role in promoting the fusion 78 activity of gB (12). In support of this concept, it was recently discovered that gH/gL does 79 not have to be in the same cell as gB in order for cell-cell fusion to occur (55). In fact, our 80 data suggests that the gH/gL ectodomain can function without being membrane bound at 81 all (2). We found that when nectin-1 bearing cells (called C10 cells) express gB, they can be 82 triggered to fuse by addition of a combination of soluble forms (ectodomains) of gD and 83 gH/gL (2). In addition, we found that brief exposure of C10 cells bearing gH/gL to soluble 84 gD was sufficient to make them fusion competent when co-cultured with cells expressing 85 gB. Importantly, the converse did not happen, i.e. cells expressing gB and a gD receptor that 86 were first exposed to soluble gD could not fuse with cells expressing gH/gL (2, 31). These 87 observations led us to propose that HSV induced fusion (and possibly virus entry) consists 88 of sequential steps: 1) binding of gD to an appropriate receptor; 2) a conformational 89 change in gD that allows it to activate gH/gL and leading to; 3) activation of gB into a 90 fusogenic state. 91 To test this hypothesis, we first examined the mechanism by which virus-92 neutralizing antibodies work. Here, we focused on MAbs to gD. In prior studies, we and 93 others showed that virus-neutralizing antibodies can be directed at HSV gD, gH/gL or gB (6, 94


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

5

8, 36, 39). Among those directed at gD, some blocked binding of gD to HVEM but not nectin-95 1 and others blocked gD binding to both receptors (28, 40). Here we report the 96 identification of two separate monoclonal antibodies, MC2 and MC5, that bind to gD in the 97 presence of either HVEM or nectin-1, do not block receptor binding to gD, but effectively 98 neutralize HSV infection. 99 Competition studies showed that MC2 and MC5 bind to distinct gD epitopes, 100 suggesting they may function in different ways. Surprisingly, we found that MC2 101 significantly enhances the affinity of gD for its receptors. We propose that this increased 102 affinity reflects the stable displacement of the gD C-term upon MC2 binding. At the same 103 time, binding of MC2 to its epitope likely interferes with the second function of gD (which 104 we propose is the activation of gH/gL). Moreover, both the neutralizing capacity and rate of 105 neutralization of virus by MC2 is enhanced when combined with the non-neutralizing MAbs 106 MC4, MC10 or MC14, all of which map to the same linear epitope (amino acids 262-272). In 107 contrast, MC5 did not enhance receptor binding to gD, nor was its neutralizing activity 108 enhanced by combining it with MC14. We suggest that MAbs MC2 and MC5 neutralize virus 109 by interfering with the ability of gD to activate gH/gL, thereby preventing activation of gB 110 into its fusogenically active state. As such, these antibodies can be utilized to delineate 111 essential information about the step that occurs just after receptor-binding. Additionally, 112 these MAbs may supply insights into the efficacy of gD as a vaccine component (14, 52) by 113 determining if responses to the vaccine include antibodies that target both functions of gD. 114 115 116


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

6

Materials and Methods 117 Cells and viruses. 293T cells were grown in DMEM supplemented with 10% fetal calf 118 serum (FCS). Sf9 (Spodoptera frugiperda) cells were grown in Sf900II SFM medium 119 (GIBCO). Vero cells were grown in Dulbecco's minimal essential medium (DMEM) 120 supplemented with 5% FCS. Gradient-purified HSV-2 strain 333 was propagated and 121 titered in Vero cells. Hybridoma cells were cultured in supplemented Kennett’s Hy medium. 122 Production and purification of recombinant baculovirus-produced proteins. The 123 methods for production and DL6 affinity purification of gD1(306t) and gD2(306t) and 124 gD2(285t) have been described previously (29, 41, 48). Production of HveA(200t) (HVEM) 125 and HveC(346t) (nectin-1) using nickel-agarose purification have also been described 126 previously (28, 59). Recombinant baculovirus expressing gD2(322) was obtained by first 127 cloning the 0.3 kbp Apa1 (blunted with mung bean nuclease)-BamH1 fragment from 128 plasmid D2-H20 (11) (which contains an insertion of charged amino acids into the middle 129 of the transmembrane region) into plasmid pAN243 (41) (which had been digested with 130 EcoR1 (and blunted with Klenow fragment) and BamH1. The resulting baculovirus transfer 131 vector (pCW259) which then contained full length gD2 with the insert at amino 322 was 132 then co-transfected with BaculoGold baculovirus DNA (BD Pharmingen) to generate a 133 recombinant baculovirus expressing this form of gD2. Recombinant gD2(322) was 134 purified via DL6 affinity purification as described above. 135 Production of MAbs against HSV2 gD. Mice were immunized with gD2(322) until 136 suitable serum antibody titers were achieved. Hybridoma production was performed using 137 standard procedures, and stable hybridomas secreting IgG reactive with HSV-2 gD by 138


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

7

enzyme-linked immunosorbent assay (ELISA) were subcloned twice. IgG was purified from 139 mouse ascitic fluid by protein G chromatography (HiTrap; GE Healthcare) according to the 140 manufacturer's instructions. Following purification, MAbs were dialyzed against 141 phosphate-buffered saline (PBS). 142 Other antibodies used. Rabbit polyclonal antibody (PAb) R8 was raised against full-length 143 HSV-2 gD and cross-reacts with HSV-1 gD (22). Anti-gD MAbs used in this study include 144 BD66 and BD78 (kindly supplied by Becton Dickinson Co.) and DL6 which recognize linear 145 epitopes within residues 272-279, 284-301, and 264-275, respectively (22). We also used 146 MAbs DL11(13, 36), HD1 (42, 47), H128 (46) and LP2 (35), the latter kindly provided by A. 147 Minson and H. Browne. 148 SDS-PAGE and Western blotting. Purified glycoproteins were separated by sodium 149 dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under “native” (0.1% SDS, 150 no reducing agent, no boiling) (13) or denaturing (samples boiled for 5 min in sample 151 buffer containing DTT) conditions in precast 10% Tris-glycine gels (Invitrogen). Following 152 SDS-PAGE, separated proteins were transferred to nitrocellulose, probed with antibodies, 153 and visualized by enhanced chemiluminescence (Amersham Pharmacia). 154 Co-immunoprecipitation of gD-receptor complexes with gD MAbs. 300 ng of 155 gD2(285t) was combined with 1.5 µg of nectin-1(346t) or HVEM(200t) in 100 µL binding 156 buffer (60) and incubated for 1 hr at 4˚C. Next, 500 ng of the indicated MAb IgG was added 157 and samples were incubated for an additional hour at 4˚C. Finally, ProA-sepharose was 158 added to each sample and incubated for 1 hour at 4˚C. The resulting immune complexes 159 were pelleted, washed and subjected to Western blot analysis. Blots were probed with 160


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

8

rabbit polyclonal antisera against HSV gD (R7) and human nectin-1 (R154) or human 161 HVEM (R140). 162 Blocking of receptor binding by MAbs. ELISA plates were coated overnight with soluble 163 receptors [5 µg/ml of HVEM(200t), 10 µg/ml of nectin-1(346t)] and then blocked with 5% 164 milk/ 0.1% Tween-PBS for 1 h. Concurrently a constant amount of gD2(306t) was 165 incubated with dilutions of each MAb (whole IgG or Fab) for 1 h at R/T in 5% milk/ 0.1% 166 Tween-PBS. The MAb-gD mixtures were then added to receptor-coated plates for 1 h at RT. 167 Bound gD was detected with anti-gD PAb R8 followed by HRP-conjugated anti-rabbit 168 secondary antibody. This assay was repeated a minimum of three times for each MAb. 169 Enhancement of receptor binding by MAbs. ELISA plates were coated overnight with 170 soluble receptors [5 µg/ml of HVEM(200t), 10 µg/ml of nectin-1(346t)] (28, 59) and then 171 blocked with 5% milk/ 0.1% Tween-PBS for 1h. Concurrently 0.5 µM of each MAb was 172 incubated with dilutions of gD2(306t) or gD2(285t) starting at 0.5 µM for 1 h at RT in 5% 173 milk/ 0.1% Tween-PBS. The MAb-gD mixture was then added to the receptor-coated plates 174 for 1 h at RT. Bound gD was detected with anti-gD PAb R8 followed by HRP conjugated 175 anti-rabbit secondary antibody. This assay was carried out a minimum of three times for 176 each MAb. 177 Map competition by Surface Plasmon Resonance. Experiments were carried out on a 178 BIACORE 3000 optical biosensor (Biacore AB) at 25°C as previously described (1, 27, 60). 179 The running buffer for the experiments was HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM 180 EDTA, 0.005% polysorbate 20). An anti-His MAb (QIAGEN Inc.) was directly coupled to a 181 CM5 Biacore chip for competition studies. At the beginning of each cycle, ~600 response 182


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

9

units (RU) of gD2(306t) were captured via its C-terminal 6-His tag on flowcell 1 (Fc1) and 183 Fc2. The first competing antibody was injected at a concentration of 20 µg/ml (2 min at 184 5 µl/min) across Fc1. Finally, the second competing MAb (20 µg/ml) was injected across 185 both Fc1 and Fc2 for 2 min to monitor its binding to gD2(306t). To regenerate the anti-His 186 Ig chip surface, brief pulses of 0.2 M Na2CO3, 1M NaCl (pH 10.5) were injected until the 187 response signal returned to baseline. 188 Virus neutralization assay. Serial dilutions of IgG of each MAb were mixed with sucrose 189 gradient-purified HSV-2, and the mixtures were incubated at 37°C for 1 h. The virus-MAb 190 mixtures were the added to Vero cell monolayers grown in 48-well plates and incubated 191 for 18-24 h at 37°C. Cells were fixed with methanol:acetone (2:1) and plaques were 192 visualized via immunoperoxidase staining (black plaque) as described elsewhere (6). 193 Virus neutralization with MAb combinations. To assess whether combinations of 194 antibodies would neutralize to a greater extent than the sum of the two antibodies alone, 195 we carried out two types of experiments. In the first, MAbs were diluted serially and then 196 added to media with or without a constant amount of a second antibody. Virus and the 197 combination of MAbs were incubated for 1 hr at 37oC and then plated on Vero cell 198 monolayers for plaque assay. The second assay examined the kinetics of virus 199 neutralization (26)}(46). Here, we used fixed concentrations of MAbs and varied the time 200 during which MAbs and virus were incubated at 37oC (0 to 60 min). At each time point, the 201 virus-antibody mixture was rapidly diluted in growth medium and added to cells to 202 measure the virus neutralization over time. The results were plotted as V/V0, were V is the 203 number of plaques remaining at each time point and V0 is the number of plaques at time 204


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

10

zero. This number was plotted against time. A straight-line relationship between V/V0 and 205 time represents first order kinetics and the slope of the line represents the rate of virus 206 neutralization. This assay was carried out a minimum of 5 times for each MAb 207 combination. 208 209

Results 210 A new panel of anti-gD MAbs reveal two MAbs that neutralize HSV independently of 211 receptor binding. In previous studies of gD, we characterized gD-specific MAbs, studied 212 their properties and arranged them into antigenic groups, a small subset of which is shown 213 in Fig. 1A. Of particular interest were MAbs that targeted a biologic activity, e.g. ability to 214 block the interaction between gD and at least one receptor (Fig. 1A, MAbs shown in red) as 215 well as ability to neutralize HSV (Fig. 1A, Table 1). For example, Group Ib neutralizing 216 MAbs, exemplified by DL11, are type-common (bind both HSV-1 and -2), react with a 217 discontinuous (38) epitopes and block binding of gD to both receptors, HVEM and nectin-1 218 (28, 40, 58). Others, such as those in Group VII have virus-neutralizing activity but block 219 binding to only one of the two receptors (Fig. 1A, Table 1) (28, 36, 40). 220

As we hypothesize that gD has a post-receptor binding, fusion-promoting function 221 (19, 30), we set out to identify gD-specific antibodies that would neutralize virus but still 222 allow binding to both receptors. As we lacked gD2 specific MAbs in our collection, we 223 utilized gD2(322) as the immunogen to generate 22 new MAbs designated MC1 through 224 MC23. We characterized each antibody and constructed an antibody “tree” (Fig.1A), 225 ultimately relating them to each other and our previously characterized antibodies. The 226


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

11

majority of the MC MAbs were type-common, but five reacted either preferentially or 227 exclusively with a truncated form of gD2, gD2(306t) (Fig. 1A, Fig. 2). MAbs that reacted 228 with denatured gD were further mapped with overlapping synthetic peptides (data not 229 shown) as previously reported (6, 8). MAbs in the type-common Group IIa and Group IIc 230 recognized amino acids 262-272 (Fig. 1A) and none had virus-neutralizing activity. Among 231 the MAbs in Group IIa, MC4, MC10 and MC14 were chosen as negative controls in later 232 experiments. 233 Most importantly, only MC2, MC5 and MC23 exhibited virus neutralizing activity 234 (Fig. 1A, Table 1) and all three recognized only non-denatured gD. Since MC5 and MC23 235 were type common, but MC2 was type-2 specific (Fig. 2A), studies in this report used HSV-236 2, gD2 and gD2 peptides. We then pursued three goals with these three MAbs: 1) to define 237 the epitopes recognized by each; 2) to test whether any of them could still bind gD in the 238 presence of receptor; and 3) whether any functioned by blocking gD binding to one or both 239 receptors. 240

Mapping studies of conformation dependent MAbs. We first tested MC2, MC5 and MC23 241 against truncated forms of gD2 and linker insertion mutants (11) and then further grouped 242 them by competition studies. 243 We found that MC5 and MC23 were able to bind to all truncations tested, including 244 gD2(234t), whereas the shortest truncation seen by MC2 was gD2(250t) (Fig. 2B). This 245 identified the epitopes for MC5 and MC23 as being upstream of residue 234 in the linear gD 246 sequence. For MC2, the epitope (at least in part) is between amino acids 234 and 250. 247 Interestingly, the only amino acid difference between gD1 and gD2 in this stretch of amino 248


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

12

acids is at residue 246 (proline in gD2 and alanine in gD1). Thus this amino acid might be in 249 the MC2 epitope. We also examined binding of each of the MAbs to linker insertion 250 mutants (11) and found that MC5 failed to recognize a gD2 mutant with an insertion at 251 residue 77 (Fig. 1A, data not shown). Thus, part of the MC5 epitope may include this 252 residue. Importantly, MC2 and MC23 were able to bind to this mutant, suggesting their 253 epitopes were distinct from that seen by MC5. 254 As a further means of mapping epitopes, we attempted to isolate mutants resistant 255 to neutralization by MC2, MC5 and MC23 (21, 37). We succeeded in isolating such a 256 monoclonal antibody resistant (mar) mutant for MC23; it has a single mutation (threonine 257 to methionine) at residue 213 (data not shown). Since other Group Ia mar mutants map at 258 residue 216 (35, 38), close to 213 in the gD crystal structure (Fig. 1B) (10), we tentatively 259 assigned MC23 to this Group. 260

Biosensor analysis of MC2, MC5 and MC23. Because we only obtained limited 261 information about the epitopes for these MAbs, we used surface plasmon resonance to 262 determine the relationships of MC2, MC5, and MC23 to each other and to the control MAb 263 MC14 (Figure 3). Three sets of data will illustrate how we used the biosensor for MAb 264 competition. This assay worked well since the MAbs had slow off rates. Since MC2 is type-2 265 specific, gD2(306t) was bound to the sensor chip and we examined binding of each MAb. 266 Figure 3A shows that both MC5 and MC2 bound well to gD2(306t) when tested individually 267 (flow cells abbreviated Fc1 and Fc2). We then injected MC5 over the chip containing MC2 268 bound to gD (Fc1), and MC5 still bound well to gD. This indicated that the epitope for MC5 269 on gD was not obscured by the presence of MC2. Likewise, MC2 still bound well when 270


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

13

injected over a chip containing MC5 bound to gD (data not shown). These data suggest that 271 MC2 and MC5 recognize different sites on gD. In similar studies neither MC2 nor MC5 272 competed with any of the other neutralizing MAbs (e.g. 1D3, DL11, or LP2) for binding to 273 gD (Fig. 1, data not shown). 274 Secondly, we examined whether MC23 would compete with LP2, a prototypical 275 Group Ia MAb (35). In this case, when MC23 was bound to gD, binding of LP2 was blocked 276 by more than 90% (Fig. 3B). This blocking was reciprocal, i.e. when LP2 was bound first, 277 MC23 no longer bound to gD2(306t) (data not shown). Since the two MAbs competed with 278 each other for binding to gD, the data indicated that their epitopes overlap and that MC23 is 279 a Group Ia MAb. Moreover, these data predicted that MC23 functions by interfering with 280 nectin-1 binding to gD. As a third example, we first injected MC2 across gD2. Once binding 281 reached a plateau, we then injected MC5, then MC14 and finally MC23 (Fig. 3C). In each 282 case the newly injected MAb bound to gD2(306t), showing that these four MAbs recognize 283 distinct epitopes on gD. Since MC23 likely functions by blocking nectin-1 binding (like 284 other Group Ia MAbs), we turned our attention to MC2 and MC5 to determine if they 285 functioned independently of receptor binding. 286

Do MC2 or MC5 block gD binding to receptor? We took several approaches to address 287 this question. 288 i. Co-immunoprecipitation of gD and nectin-1 in the absence or presence of gD 289 MAbs. In previous studies we showed that the non-neutralizing MAb DL6 could co-290 precipitate gD bound to HVEM (40). In contrast the neutralizing MAbs 1D3 (Group VII) and 291 DL11 (Group Ib) did not co-precipitate the complex of gD and HVEM (40). Here, using a 292


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

14

similar approach, we pre-incubated gD2(306) with nectin-1 (Fig. 4A) or HVEM (Fig. 4B), 293 followed by addition of MAbs to these mixtures. For both receptors, we used DL6 as a 294 positive control because it co-precipitates gD with HVEM and nectin-1; DL11 was used as a 295 negative control because it fails to co-precipitate either receptor (28, 40). When gD was 296 pre-incubated with nectin-1 or HVEM, MAbs MC2, MC4 and MC5 each co-precipitated both 297 proteins showing that the epitopes for these MAbs were still accessible when gD was 298 bound to either receptor. Since both MC2 and MC5 neutralize virus (Table 1), these results 299 suggest that both MAbs block a critical gD function other than receptor binding. 300 ii. ELISA. The next question was to what extent these antibodies could prevent 301 receptors from binding to gD. To do this we incubated a set amount of gD-2 306t with 302 increasing amounts of each antibody, allowed for binding and then added the mixtures to 303 ELISA plates coated with HVEM (Fig. 5A,C) or nectin-1 (Fig. 5B,D). In this case, MAbs DL11, 304 LP2 and MC23 effectively blocked the binding of gD to HVEM or nectin-1 in a dose-305 dependent manner. However, in contrast, MC5 did not block gD binding to either receptor. 306 In fact, at higher concentrations, MC5 appeared to enhance binding of gD to both receptors. 307 MC2 significantly enhanced binding of either HVEM or nectin-1 to gD (Fig. 5C,D). To our 308 surprise, several non-neutralizing MAbs, had this property, including MC14, DL6, BD66, 309 BD78. The data suggest that binding of MC2 as well as these non-neutralizing antibodies 310 alters gD structure in some way to allow better access of receptors to associate with their 311 binding sites. 312

To what extent do MC2, MC5 and MC14 enhance gD binding to receptor? Previously, 313 we found that the affinity of gD(306t) for both HVEM and nectin-1 is in the micromolar 314


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

15

range whereas the affinity of gD(285t) for these receptors is enhanced significantly 315 (approximately 50 fold) (44, 60). The reason for this is explained by crystallographic data 316 which showed that the C-terminal residues of the gD ectodomain lie across the core of gD 317 and interact with the N-terminus, thereby impeding receptor binding (16, 30, 60). Thus, 318 removal of residues from 286-306 likely relieves the impediment to receptor binding. 319 Additionally, we showed that a mutant form of gD with a single amino acid change at 320 residue 294 (from a tryptophan to alanine) also has very high affinity for receptors, 321 suggesting it acts as a linchpin that helps to stabilize the C-term in a “closed” position 322 across the gD core (16, 30). In the crystal structure, W294 fits into a groove near the gD N-323 terminus and forms hydrogen bonds with several key residues in that region. Thus, 324 receptor binding requires that the C-term be in an “open” conformation. 325 To quantify the extent of the enhancement of receptor binding, a single concentration 326 (excess) of MAbs MC2, MC5, MC14 or MYC IgG was incubated with increasing amounts of gD-327 2(306t) or with gD-2(285t). Then each mixture was added to an ELISA plate coated with HVEM 328 (Fig. 6A,C, E) or nectin-1 (Fig. 6 B,D,F). The anti-MYC antibody control reflects the normal binding 329 capacity of each form of gD to receptor. As expected (44, 60), gD2(285t) bound to HVEM and 330 nectin-1 better than gD2(306t). Interestingly, MC2 enhanced the binding of gD2(306t) to HVEM 331 almost as well as the C-terminal truncation [gD2(285t)] (Fig. 6A). This antibody also enhanced 332 gD2(306t) binding to nectin but the effect was not as dramatic as with HVEM (Fig. 6B). These 333 results support our concept that binding of MC2 affects the position of the C-term of gD. In contrast, 334 MC5 had a much more modest enhancing effect on binding of gD2(306t) to HVEM (Fig. 6C) and had 335 no effect at all on gD2(306t) binding to nectin-1 (Fig. 6D). As predicted, neither MC2 nor MC5 336 enhanced the binding of gD(285t) to HVEM or nectin-1. Finally, MC14 enhanced binding of 337 gD2(306t) to both HVEM and nectin-1 (Fig. 6E,F) and the extent of this enhancement mirrored that 338


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

16

seen for MC2. Biosensor analysis indicated that the increased affinity of gD2(306t) for HVEM 339 and nectin-1 caused by MC2 or MC14 was due to an increase in the rate of association (kon) 340 with little change in dissociation (koff) (data not shown). This pattern was identical to that 341 seen for the kinetics of gD2(285t) binding to HVEM and nectin-1 (44, 60).These 342 experiments support the concept that binding of MC2 or MC14 to gD induces a more “open” 343 conformation of the C-term, thereby enhancing the ability of gD to bind receptor. 344 Since MC2 and MC14 recognize different epitopes, we wondered whether binding of 345 gD2(306t) to receptors would be even more enhanced if we mixed the two MAbs. If so, this 346 would suggest that there may be two different mechanisms to alter the C-term. Therefore, 347 we tested the effect of a combination of MC2+MC14 on receptor binding (Fig. 7). As shown 348 before (Fig. 6), MC2 or MC14 (each one alone) enhanced the binding of gD2(306t) to either 349 HVEM (Fig. 7A) or nectin-1 (Fig. 7B). The combination of the two MAbs enhanced 350 gD2(306t) binding to HVEM and nectin-1 even more. Overall, our data suggest that the 351 combination of MC2+MC14 has a greater enhancing effect on receptor binding to gD than 352 does either MAb alone. This supports the idea that MC2 and MC14 may affect gD 353 conformation in different ways. A similar effect was seen for MC4 or MC10 in combination 354 with MC2 (data not shown). More importantly, our observations about enhancement of 355 receptor binding led us to wonder whether the non-neutralizing MAbs MC4, MC10 and 356 MC14 would enhance either the extent or kinetics of virus neutralization by MC2 (46). 357

Effect of MC4, MC10 and MC14 on HSV-2 neutralization by MC2. Sanna et al. (46) 358 showed that the combination of H170, a Group VII MAb, and H128, a Group 1b MAb (Fig. 1) 359 was more effective at neutralizing HSV-2 infection than was either MAb alone both in terms 360


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

17

of efficiency (50% endpoint) and kinetics (the rate of neutralization as a function of time of 361 exposure of virus to MAb). However, in that case, Sanna and colleagues combined two 362 virus-neutralizing MAbs that function by interfering with receptor binding. Having shown 363 that MC2 does not interfere with receptor binding but still neutralizes HSV2, we used a 364 similar approach to investigate the extent and kinetics of neutralization by MC2 in the 365 presence or absence MAbs MC4, MC10 or MC14. Additionally, we tested two other non-366 neutralizing MAbs with epitopes in the C-term that enhanced receptor binding, BD66 and 367 DL6 (Fig. 5D). 368 First, we examined the effect of MAbs that mapped to the C-term on neutralization 369 by MC2 (Fig. 8). MAbs such as MC4, MC10 and MC14 enhanced neutralization by MC2 (Fig. 370 8A,B). These MAb combinations increased the amount of virus neutralized (to nearly 371 100%), whereas, in multiple experiments, MC2 alone never neutralized more than 80% of 372 the input virus. Interestingly, neither DL6 (22) or BD66 (22) had any enhancing effect on 373 neutralization by MC2 (Fig.8C,D). Thus the enhancing effect may be due to how each MAb 374 to a linear epitope along the C-term is oriented in relation to the orientation of MC2 (Fig. 1). 375 No MAb enhanced neutralization by MC5, again emphasizing that it neutralizes virus 376 differently than MC2 (data not shown). Thus, there is specificity among the MAbs that 377 enhance receptor binding. In addition to MC4, MC10 and MC14, several other MAbs that 378 map to this epitope in particular worked in concert with MC2 to enhance neutralization. 379 Having determined that MC4, MC10 and MC14 enhance the extent of neutralization 380 by MC2, we tested how rapidly they neutralized virus when compared with antibodies such 381 as DL11 that block receptor binding. First, we repeated the neutralization experiment 382


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

18

shown in Fig. 8, testing whether MC14 would enhance neutralization by DL11 (Fig. 9A,B). 383 Again, we varied the IgG concentration and measured plaque reduction at 1h post addition 384 of DL11 alone or in combination with MC14 (Fig. 9A,B). In contrast to what was seen with 385 MC2 (Fig. 9B), MC14 had no effect on virus neutralization by DL11. We next compared the 386 kinetics of neutralization by DL11 and MC2 in the presence or absence of MC14 (Fig. 9C,D). 387 For this experiment, the amount of antibody (IgG) was held constant at 10 nM for both MC2 388 and DL11 and plaques were counted over time once the antibody virus mixture was added 389 to the cells (46). We used 80 nM of MC14 to be certain that any enhancement of kinetics 390 would not be limited by MC14 concentration. We then varied the time of incubation of the 391 MAbs with virus (from 5 min to 1h) before adding the mixture to cells. We plotted the data 392 as the number of plaques at each time point divided by the number of plaques at time zero 393 (no MAb) on the Y axis (V/Vo) against time on the X-axis (46). Thus, we could determine 394 the rate from the slope of each line; the steeper the slope, the faster the rate of 395 neutralization. As expected, antibody neutralization followed first order kinetics in each 396 case (i.e. a straight line). For DL11, the slopes (rate) were nearly identical in the presence 397 or absence of MC14 (Fig. 9C). Similar results were found for MC23 or MC5 plus or minus 398 MC14 (data not shown). However, in the case of MC2, the rate of neutralization was twice 399 as fast when both MC14 and MC2 were present as compared with MC2 alone (Fig. 9D). We 400 found the same enhancement in the kinetics of neutralization when we used different 401 concentrations of MC2 (data not shown). This suggests that the kinetics of neutralization by 402 MC2 and the enhancing effect of MC14 are independent of MC2 concentration over a wide 403 range. Together, these data suggest that MAb MC14 which is directed at residues 262-272 404 enhances both the extent and the rate of neutralization by MC2. 405


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

19

406 Discussion 407

In previous studies of MAbs to HSV gD, we identified a number that have virus-408 neutralizing activity. In all cases, the antibodies prevented binding of gD to the HSV 409 receptors, HVEM or nectin-1. We concluded that this activity accounted for neutralization 410 of HSV by gD antibodies. However, by solving the structures of gD bound to HVEM (10, 30) 411 and gD bound to nectin-1 (16), we found that approximately 50 of the C-terminal amino 412 acids (the C-term) of the gD ectodomain occluded the binding site for nectin-1 and also 413 prevented formation of the N-terminal hairpin of gD needed for HVEM binding (16, 30). We 414 hypothesized that in both cases, receptor binding necessitated a common conformational 415 change in gD whereby the C-term moved away from the core, allowing receptors to bind. 416 We postulated that gD with the C-term in this “open” position is an activated form of gD 417 that is responsible for triggering events carried out by gH/gL and/or gB. These events then 418 result in fusion of the virion envelope with a cell membrane to allow exposure of the 419 capsid. Indeed, a gD mutant, which locked much but not all of the C-term of the gD 420 ectodomain against the gD core, was still able to bind both gD receptors yet was unable to 421 trigger cell-cell or virus-cell fusion (31). This hypothesis was ultimately supported by 422 ultrastructural studies which revealed the normal position of the C-term and proved that 423 binding of either HVEM or nectin-1 to gD requires the same movement of the C-term to 424 reveal the receptor binding sites for both HVEM and nectin-1 (10, 16, 30). Presumably, 425 these “open” sites on gD were the target of virus neutralizing antibodies that blocked 426 binding of gD to receptor. 427


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

20

As another means of testing our hypothesis, we searched for anti-gD MAbs that 428 would not interfere with receptor binding but still neutralized virus infection. In screening 429 a newly made panel of anti-gD2 MAbs, we found three that had virus-neutralizing activity. 430 Of these, MC23 blocked gD binding to receptors and competed with another Group Ia MAb 431 (LP2). However, two others MAbs in the new panel, MC2 and MC5, bound to gD in the 432 presence of the receptors HVEM and nectin-1 and neither MAb prevented these receptors 433 from binding to gD. Thus, MAbs MC2 and MC5 had unique properties that might interfere 434 with a gD function other than receptor binding. 435 We have modeled antibody-induced changes to gD in Fig. 10, using MC2 and MC14 436 sites as examples of what happens when MAbs such as MC2 and MC14 bind. We did not 437 include MC5 or MAbs to the C-term such as MC4 and MC10 for the sake of simplicity. We 438 hypothesize that MC2 and MC5 neutralize virus by occluding residues needed to trigger 439 later events. To test our hypothesis, we studied the properties of these MAbs in more detail. 440 First, we found that binding of receptors to gD was modestly enhanced by binding of MC5 441 to gD, but was dramatically enhanced by binding of MC2 to gD. This led us to propose that 442 gD is normally in a “closed conformation” with the C-term bound to the core of gD (Fig. 443 10A). Binding of MC2 induces the C-term of gD2(306) to assume the “open” conformation 444 similar to that caused by receptor (Fig. 10B). MC2 interferes with post-receptor binding 445 events that are critical for triggering virus-cell fusion. Based on previous results (2), we 446 think that MC2 binds to and masks the gH/gL interaction domain (Fig. 10A,B,C). Antibodies 447 to residues along the C-term such as MC14 also cause gD to assume an open conformation, 448 thereby enhancing the binding of both HVEM and nectin-1. We believe this is the first 449 report of anti-gD antibodies that neutralize HSV without blocking the binding of one or 450


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

21

both of its receptors. It is also the first report that MAbs to gD can enhance receptor 451 binding. 452 Second, we found that the epitopes for MC2 and MC5 are distinct from each other as 453 shown by three kinds of data: 1) MC2 is type2-specific and MC5 is type-common, MC2 can 454 bind a form of gD truncated to amino acid 234, whereas MC2 cannot bind this form, but 455 does bind a form truncated at amino acid 250; and 3) biosensor data show that both MC2 456 and MC5 can bind to gD at the same time (no competition). We cannot precisely map where 457 they bind since both are conformation dependent and we were unable to generate mar 458 mutants for either MAb. Thus far, our data suggest that a portion of the MC5 epitope is 459 found downstream of residue 250 but may involve amino acids surrounding amino acid 77 460 which is near residue 250 in the 3-dimensional structure. In the case of MC2, a portion of 461 its epitope is between amino acids 234-250. Because MC2 is type-2 specific and residue 462 246 is the only residue that differs between 234-250 of gD1 and gD2, we speculate that it is 463 critical for the MC2 epitope. However, further experiments need to be done to define other 464 amino acids involved in binding of both MC2 and MC5. 465

Enhancement of receptor binding by MC14 and other MAbs. To our surprise, we found 466 that binding of gD to receptor is significantly enhanced not only by MC2 but also by several 467 non-neutralizing MAbs, including MC14 as well as its sister MAbs MC4 and MC10, all of 468 which recognize and bind to residues 262-272 in the C-term (Fig. 10B,C). In addition, 469 receptor binding is enhanced by other MAbs, such as DL6, whose epitopes are along the C-470 term of gD, but these MAbs do not enhance the effect of MC2. It should be noted that none 471 of these MAbs would be predicted to interfere with receptor binding and the fact that they 472


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

22

do not neutralize virus infection or neutralize only weakly means that they do not block a 473 downstream function. However, we believe our general hypothesis explains why particular 474 MAbs enhance receptor binding, i.e., they all cause the C-term to move away from gD and 475 “open up” the receptor binding sites (Fig. 10C). 476 Which function in the pathway to fusion is blocked by MC2? Previously, we proposed 477 that HSV induced cell fusion occurs in a series of steps that ultimately activate gB into a 478 fusogenic state (2). Movement of the gD C-term also reveals a domain that activates 479 downstream steps, the first of which we proposed to be an interaction with gH/gL. This 480 interaction with gD activates gH/gL into a form that enables it to help convert gB into a 481 fusogenic state. Briefly, cells engineered to express nectin-1 and transfected to express gB 482 could be triggered to fuse by addition of soluble forms (ectodomains) of gD and gH/gL. This 483 experiment showed that neither gD nor gH/gL need to be membrane bound to function. We 484 further showed that brief exposure of nectin-1 bearing cells transfected to express gH/gL 485 to soluble gD enabled those cells to fuse with cells transfected to express gB. Together, 486 these results suggested that the pathway to fusion that consists of three major steps: 1) 487 binding of gD to receptor; 2) activation of gH/gL by receptor activated gD; 3) activation of 488 gB into a fusogen by activated gH/gL. Although MC2 could interfere with either step 2 or 3, 489 we currently favor the idea that it interferes with the interaction between gD and gH/gL. 490 Thus far, using the technique of bimolecular complementation (24, 25), we found that MC2 491 blocks the interaction between half-EYFP tagged forms of gB and gH/gL as well as fusion 492 (data not shown and (3)). However, when we used half-EYFP tagged forms of gD and gH 493 along with untagged gB and gL, we detected an interaction between gD and gH/gL as 494 evidenced by EYFP fluorescence, but fusion was blocked (3). This might imply that there is 495


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

23

an unstable interaction between gD and gH/gL and that forming the irreversible EYFP-496 complemented complex might block the ability of gH/gL to activate gB. However, it could 497 also mean that the complex formed in this setting did not represent the appropriate one to 498 trigger gB to cause fusion. In any event we were unable to use this technique to assess the 499 ability of MC2 to block an interaction between gH/gL and gD that would lead to fusion. 500 Alternative approaches are being explored to determine if MC2 blocks a functional 501 interaction between gD and gH/gL or another downstream event that normally leads to 502 fusion. 503 It is of some interest that those MAbs that neutralize by blocking receptor binding 504 appear to cluster on one face of the three-dimensional structure of gD, the same face where 505 receptors bind, while MC2 and MC5 bind to epitopes on the opposing face (Fig. 1B and Fig. 506 10). We suggest that the uncovered residues of the gD core directly contact a site on gH/gL 507 that upregulate its activity (2). 508

The combination of MC2 with MC14 enhances neutralization by MC2. It was previously 509 noted that antibodies to two distinct epitopes on gD can behave synergistically, but in that 510 case both antibodies blocked receptor binding, i.e. H170 (Group VII) blocks binding of 511 HVEM and H128 (Group 1b) blocks both. We know from earlier studies that Group VII and 512 1b MAbs do not compete with each other (11, 13, 17, 36). Here, we found an enhancing 513 effect on both the extent and rate of neutralization by combining the neutralizing MAb 514 MC2 with non-neutralizing MAbs such as MC4, MC10 or MC14. This indicates that non-515 neutralizing MAbs can contribute, albeit indirectly, to the overall neutralization seen in a 516 polyclonal response to gD. The data suggest that binding of these non-neutralizing 517


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

24

antibodies alters gD structure so that access of receptors is enhanced (Fig. 10). Since the 518 MC2 epitope appears to be located away from receptor binding sites, we suggest that MC14 519 allows for better exposure of the MC2 epitope on gD, thereby accounting for the enhanced 520 effect on the extent and rate of neutralization. 521 Virus neutralization by antibodies that block receptor binding. Interference with 522 receptor binding is a common mechanism by which antibodies block virus infection (49, 523 61). For instance, a combination of neutralizing human MAbs against SARS coronavirus 524 acted synergistically by targeting different epitopes of the spike (S) protein (61). However, 525 the authors did not determine whether any MAbs allowed receptor binding, nor did they 526 examine whether the MAbs interfered with a pre-attachment step or one that occurred 527 after attachment. In a recent study, Sabo et al. studied a large panel of MAbs to the 528 attachment protein of hepatitis C virus (45), and found some that blocked pre-attachment 529 and others that blocked at a post-attachment step. In that case, the receptor is not known, 530 so one does not know which of the MAbs interfered with receptor binding itself. In our 531 studies, we have been able to determine which MAbs to gD interfere with receptor binding 532 and which do not. We did not test pre- versus post-attachment effects of these MAbs 533 because attachment to the cell surface, as opposed to receptor binding, is largely due to 534 interaction of the virus with heparan sulfate (5, 23, 53). However, since the neutralization 535 assays were done on Vero cells, the effect of these MAbs does not depend on a step that 536 occurs only in endosomes since virus entry into Vero cells occurs at the plasma membrane 537 (34). 538


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

25

Neutralization of other viruses by other antibodies that block at a post-receptor-539 binding step. For other enveloped viruses, there are a number of reports of MAbs that 540 block entry at a post receptor-binding step, e.g. ones directed at the MuLV receptor binding 541 protein, SU (7). In this case, the antibodies are thought to block interactions between the 542 receptor binding protein and the fusion protein that are important for triggering fusion. 543 Indeed, it has been recognized for a number of years that virus neutralization can occur 544 both at the receptor binding step and any other step that precedes “the first major 545 biosynthetic event in the viral replicative cycle” (26). Since many viruses are taken into 546 endocytic compartments, bound antibodies are also taken into the cell, allowing them to act 547 not just at the cell surface but “inside” the cell. For example, Vogt et al. (56, 57) have shown 548 that certain MAbs to West Nile virus (WNV) neutralize at a post attachment. Moreover, as 549 Klasse and Sattentau stress (26), the mechanisms of neutralization should block an event in 550 the replicative cycle and that the kinetics of neutralization may vary due to differences in 551 which event is blocked. 552

Recently, a MAb (E53) to WNV envelope protein E was discovered to block the acid-553 catalyzed fusion step required for release of the nucleocapsid from the endocytic 554 compartment it is taken into (54). It does so by binding to the fusion loop of the E protein 555 thereby preventing WNV from binding to the membrane bilayer. In another study with this 556 MAb, it was discovered that the kinetics of neutralization increased over time of incubation 557 between virus and MAb (43). The authors suggested that the MAb actually engages an 558 epitope (the fusion loop) that is not well exposed on the virion surface. In their case, they 559 examined kinetics of neutralization by incubating virus and MAb for many hours. 560 According to our hypothesis, MAbs that enhance receptor binding to gD cause the C-term of 561


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

26

gD to move, thereby making receptor binding sites more accessible. Perhaps we may find 562 that the kinetics of MC2 or the combination of MC2+MC14 is no longer linear if we extend 563 the time of incubation from 1h to a longer period. 564 Some gD mutants can bind alternative receptors. How can we reconcile them with 565 our hypothesis regarding open and closed forms of the C-term? Other studies show 566 that large portions of gD can be deleted and replaced by domains that bind new receptors 567 (9). This has led to some skepticism about our model for receptor-induced activation of gD. 568 The recently published study of gD bound to nectin-1 (16) points out that even when a 569 large portion of the gD core was replaced with a single chain antibody, the N- and C-570 terminal extensions to the Ig core are maintained. Thus, the regions of gD that are needed 571 for its activation and binding to other viral glycoproteins may still be maintained in this 572 hybrid construct. Thus we predict that even in these cases, receptor binding would 573 necessitate displacement of the C-term. This could be tested by measuring the affinity of 574 these mutated forms of gD for their receptors when the C-term is present and when it is 575 absent. The resulting virus was highly attenuated and that “HSV evolved to unmask the gD 576 C-terminus only upon receptor binding to ensure efficient tropism in the host”. 577

Such studies are beyond the scope of the present report but are ones that should be 578 considered, particularly by those who are interested in using gD with altered receptor 579 tropism for gene therapy. By knowing if displacement of the C-term is always needed for 580 gD to activate downstream events, one could predict which type of receptor binding 581 residues would be likely to produce a form of gD that was functional. 582


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

27

Finally, we believe that our hypothesis of how MC2 functions is well supported by 583 ultrastructural studies that imply that the C-term of gD must move out of the way in order 584 for nectin-1 to bind and for gD to form the loop that binds HVEM (10, 16, 30). One caveat is 585 that MC2 might prevent binding of the C-term to gH/gL or gB, not necessarily linked to the 586 displacement of the C-term, but as a consequence of a local perturbation. Likewise, MC14 587 might alter the capacity of receptor-bound gD to bind gH/gL and/or gB. The latter 588 possibility, though not yet tested will be important to explore. Ultimately, however, it 589 would be of great value to know how the structure of gD bound to neutralizing MAbs that 590 block receptor binding, e.g. DL11, compares with the structure of gD2 bound to MC2 and 591 how the latter structure compares with those of gD bound to nectin-1 and gD bound to 592 HVEM. Such structures are clearly ones we would be greatly interested in knowing and 593 would help us understand much more about how MC2 works. 594 595

Acknowledgments 596 We thank all the members of our lab for helpful discussions of the work reported 597 here. We particularly thank Huan Lou for providing purified soluble glycoproteins. Special 598 thanks to Leslie B. King of the School of Veterinary Medicine at the University of 599 Pennsylvania for careful editing of the manuscript. 600 This work was supported by NIH grants R01-AI-056045 and R01-AI-076231 to R.J.E 601 and by NIH grant R37-AI-18289 to G.H.C. E.L. was supported by NIH T32-07234. C.K. was 602 supported by the NIH grant R21-AI-073384 and by the Joseph and Josephine Rabinowitz 603 award for Research Excellence at the UPENN School of Dental Medicine. 604


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

28

Experiments were carried out by E.L., C.W, S.W., M.P., T.M.C and Y.Z. 605 Intellectual input and manuscript preparation were done by E.L., R.J.E., G.H.C., C.K., T.M.C 606 and C.W. 607 608

References 609 610 1. Aldaz-Carroll, L., J. C. Whitbeck, M. Ponce de Leon, H. Lou, L. Hirao, S. N. Isaacs, 611

B. Moss, R. J. Eisenberg, and G. H. Cohen. 2005. Epitope-mapping studies define two 612 major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein 613 B5R. J Virol 79:6260-6271. 614

2. Atanasiu, D., W. T. Saw, G. H. Cohen, and R. J. Eisenberg. 2010. Cascade of events 615 governing cell-cell fusion induced by herpes simplex virus glycoproteins gD, gH/gL, and 616 gB. J Virol 84:12292-12299. 617

3. Atanasiu, D., J. C. Whitbeck, T. M. Cairns, B. Reilly, G. H. Cohen, and R. J. 618 Eisenberg. 2007. Bimolecular complementation reveals that glycoproteins gB and gH/gL 619 of herpes simplex virus interact with each other during cell fusion. Proc Natl Acad Sci U 620 S A 104:18718-18723. 621

4. Backovic, M., R. Longnecker, and T. S. Jardetzky. 2009. Structure of a trimeric 622 variant of the Epstein-Barr virus glycoprotein B. Proc Natl Acad Sci U S A 106:2880-623 2885. 624


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

29

5. Banfield, B. W., Y. Leduc, L. Esford, K. Schubert, and F. Tufaro. 1995. Sequential 625 isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective 626 agent: evidence for a proteoglycan-independent virus entry pathway. J Virol 69:3290-627 3298. 628

6. Bender, F. C., M. Samanta, E. E. Heldwein, M. P. de Leon, E. Bilman, H. Lou, J. C. 629 Whitbeck, R. J. Eisenberg, and G. H. Cohen. 2007. Antigenic and mutational analyses 630 of herpes simplex virus glycoprotein B reveal four functional regions. J Virol 81:3827-631 3841. 632

7. Burkhart, M. D., S. C. Kayman, Y. He, and A. Pinter. 2003. Distinct mechanisms of 633 neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal 634 domain of murine leukemia virus SU. J Virol 77:3993-4003. 635

8. Cairns, T. M., M. S. Shaner, Y. Zuo, M. Ponce-de-Leon, I. Baribaud, R. J. 636 Eisenberg, G. H. Cohen, and J. C. Whitbeck. 2006. Epitope mapping of herpes 637 simplex virus type 2 gH/gL defines distinct antigenic sites, including some associated 638 with biological function. J Virol 80:2596-2608. 639

9. Campadelli-Fiume, G., C. De Giovanni, V. Gatta, P. Nanni, P. L. Lollini, and L. 640 Menotti. 2011. Rethinking herpes simplex virus: the way to oncolytic agents. Rev Med 641 Virol 21:213-226. 642

10. Carfi, A., S. H. Willis, J. C. Whitbeck, C. Krummenacher, G. H. Cohen, R. J. 643 Eisenberg, and D. C. Wiley. 2001. Herpes simplex virus glycoprotein D bound to the 644 human receptor HveA. Mol Cell 8:169-179. 645


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

30

11. Chiang, H. Y., G. H. Cohen, and R. J. Eisenberg. 1994. Identification of functional 646 regions of herpes simplex virus glycoprotein gD by using linker-insertion mutagenesis. J 647 Virol 68:2529-2543. 648

12. Chowdary, T. K., T. M. Cairns, D. Atanasiu, G. H. Cohen, R. J. Eisenberg, and E. 649 E. Heldwein. 2010. Crystal structure of the conserved herpesvirus fusion regulator 650 complex gH-gL. Nat Struct Mol Biol 17:882-888. 651

13. Cohen, G. H., V. J. Isola, J. Kuhns, P. W. Berman, and R. J. Eisenberg. 1986. 652 Localization of discontinuous epitopes of herpes simplex virus glycoprotein D: use of a 653 nondenaturing ("native" gel) system of polyacrylamide gel electrophoresis coupled with 654 Western blotting. J Virol 60:157-166. 655

14. Cohen, J. 2010. Immunology. Painful failure of promising genital herpes vaccine. 656 Science 330:304. 657

15. Connolly, S. A., J. O. Jackson, T. S. Jardetzky, and R. Longnecker. 2011. Fusing 658 structure and function: a structural view of the herpesvirus entry machinery. Nature 659 reviews. Microbiol 9:369-381. 660

16. Di Giovine, P., E. C. Settembre, A. K. Bhargava, M. A. Luftig, H. Lou, G. H. Cohen, 661 R. J. Eisenberg, C. Krummenacher, and A. Carfi. 2011. Structure of herpes simplex 662 virus glycoprotein D bound to the human receptor nectin-1. PLoS Pathog 7:e1002277. 663

17. Eisenberg, R. J., D. Long, M. Ponce de Leon, J. T. Matthews, P. G. Spear, M. G. 664 Gibson, L. A. Lasky, P. Berman, E. Golub, and G. H. Cohen. 1985. Localization of 665 epitopes of herpes simplex virus type 1 glycoprotein D. J Virol 53:634-644. 666


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

31

18. Harrison, S. C. 2008. Viral membrane fusion. Nat Struct Mol Biol 15:690-698. 667 19. Heldwein, E. E., and C. Krummenacher. 2008. Entry of herpesviruses into mammalian 668

cells. Cell Mol Life Sci 65:1653-1668. 669 20. Heldwein, E. E., H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. 670

Harrison. 2006. Crystal structure of glycoprotein B from herpes simplex virus 1. Science 671 313:217-220. 672

21. Highlander, S. L., S. L. Sutherland, P. J. Gage, D. C. Johnson, M. Levine, and J. C. 673 Glorioso. 1987. Neutralizing monoclonal antibodies specific for herpes simplex virus 674 glycoprotein D inhibit virus penetration. J Virol 61:3356-3364. 675

22. Isola, V. J., R. J. Eisenberg, G. R. Siebert, C. J. Heilman, W. C. Wilcox, and G. H. 676 Cohen. 1989. Fine mapping of antigenic site II of herpes simplex virus glycoprotein D. J 677 Virol 63:2325-2334. 678

23. Karger, A., A. Saalmuller, F. Tufaro, B. W. Banfield, and T. C. Mettenleiter. 1995. 679 Cell surface proteoglycans are not essential for infection by pseudorabies virus. J Virol 680 69:3482-3489. 681

24. Kerppola, T. K. 2008. Bimolecular fluorescence complementation (BiFC) analysis as a 682 probe of protein interactions in living cells. Annu Rev Biophys 37:465-487. 683

25. Kerppola, T. K. 2008. Bimolecular fluorescence complementation: visualization of 684 molecular interactions in living cells. Methods Cell Biol 85:431-470. 685

26. Klasse, P. J., and Q. J. Sattentau. 2002. Occupancy and mechanism in antibody-686 mediated neutralization of animal viruses. J Gen Virol 83:2091-2108. 687


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

32

27. Krummenacher, C., I. Baribaud, M. Ponce de Leon, J. C. Whitbeck, H. Lou, G. H. 688 Cohen, and R. J. Eisenberg. 2000. Localization of a binding site for herpes simplex 689 virus glycoprotein D on herpesvirus entry mediator C by using antireceptor monoclonal 690 antibodies. J Virol 74:10863-10872. 691

28. Krummenacher, C., A. V. Nicola, J. C. Whitbeck, H. Lou, W. Hou, J. D. Lambris, 692 R. J. Geraghty, P. G. Spear, G. H. Cohen, and R. J. Eisenberg. 1998. Herpes simplex 693 virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry 694 mediator, two structurally unrelated mediators of virus entry. J Virol 72:7064-7074. 695

29. Krummenacher, C., A. H. Rux, J. C. Whitbeck, M. Ponce-de-Leon, H. Lou, I. 696 Baribaud, W. Hou, C. Zou, R. J. Geraghty, P. G. Spear, R. J. Eisenberg, and G. H. 697 Cohen. 1999. The first immunoglobulin-like domain of HveC is sufficient to bind herpes 698 simplex virus gD with full affinity, while the third domain is involved in oligomerization 699 of HveC. J Virol 73:8127-8137. 700

30. Krummenacher, C., V. M. Supekar, J. C. Whitbeck, E. Lazear, S. A. Connolly, R. J. 701 Eisenberg, G. H. Cohen, D. C. Wiley, and A. Carfi. 2005. Structure of unliganded 702 HSV gD reveals a mechanism for receptor-mediated activation of virus entry. EMBO J 703 24:4144-4153. 704

31. Lazear, E., A. Carfi, J. C. Whitbeck, T. M. Cairns, C. Krummenacher, G. H. Cohen, 705 and R. J. Eisenberg. 2008. Engineered disulfide bonds in herpes simplex virus type 1 706 gD separate receptor binding from fusion initiation and viral entry. J Virol 82:700-709. 707

32. Luftig, M. A., M. Mattu, P. Di Giovine, R. Geleziunas, R. Hrin, G. Barbato, E. 708 Bianchi, M. D. Miller, A. Pessi, and A. Carfi. 2006. Structural basis for HIV-1 709


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

33

neutralization by a gp41 fusion intermediate-directed antibody. Nat Struct Mol Biol 710 13:740-747. 711

33. Matsuura, H., A. N. Kirschner, R. Longnecker, and T. S. Jardetzky. 2010. Crystal 712 structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) 713 complex. Proc Natl Acad Sci U S A 107:22641-22646. 714

34. Milne, R. S., A. V. Nicola, J. C. Whitbeck, R. J. Eisenberg, and G. H. Cohen. 2005. 715 Glycoprotein D receptor-dependent, low-pH-independent endocytic entry of herpes 716 simplex virus type 1. J Virol 79:6655-6663. 717

35. Minson, A. C., T. C. Hodgman, P. Digard, D. C. Hancock, S. E. Bell, and E. A. 718 Buckmaster. 1986. An analysis of the biological properties of monoclonal antibodies 719 against glycoprotein D of herpes simplex virus and identification of amino acid 720 substitutions that confer resistance to neutralization. J Gen Virol 67:1001-1013. 721

36. Muggeridge, M. I., V. J. Isola, R. A. Byrn, T. J. Tucker, A. C. Minson, J. C. 722 Glorioso, G. H. Cohen, and R. J. Eisenberg. 1988. Antigenic analysis of a major 723 neutralization site of herpes simplex virus glycoprotein D, using deletion mutants and 724 monoclonal antibody-resistant mutants. J Virol 62:3274-3280. 725

37. Muggeridge, M. I., S. R. Roberts, V. J. Isola, G. H. Cohen, and R. J. Eisenberg. 726 1990. Herpes simplex virus., p. 459-481. In M. H. V. Van Regenmortel and A. R. 727 Neurath (ed.), Immunochemistry of viruses, vol. II. The basis for serodiagnosis and 728 vaccines. Elsevier Biochemical Press, Amsterdam, The Netherlands. 729


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

34

38. Muggeridge, M. I., W. C. Wilcox, G. H. Cohen, and R. J. Eisenberg. 1990. 730 Identification of a site on herpes simplex virus type 1 glycoprotein D that is essential for 731 infectivity. J Virol 64:3617-3626. 732

39. Muggeridge, M. I., T. T. Wu, D. C. Johnson, J. C. Glorioso, R. J. Eisenberg, and G. 733 H. Cohen. 1990. Antigenic and functional analysis of a neutralization site of HSV-1 734 glycoprotein D. Virology 174:375-387. 735

40. Nicola, A. V., M. Ponce de Leon, R. Xu, W. Hou, J. C. Whitbeck, C. 736 Krummenacher, R. I. Montgomery, P. G. Spear, R. J. Eisenberg, and G. H. Cohen. 737 1998. Monoclonal antibodies to distinct sites on herpes simplex virus (HSV) glycoprotein 738 D block HSV binding to HVEM. J Virol 72:3595-3601. 739

41. Nicola, A. V., S. H. Willis, N. N. Naidoo, R. J. Eisenberg, and G. H. Cohen. 1996. 740 Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D. J 741 Virol 70:3815-3822. 742

42. Pereira, L., T. Klassen, and J. R. Baringer. 1980. Type-common and type-specific 743 monoclonal antibody to herpes simplex virus type 1. Infect Immun 29:724-732. 744

43. Pierson, T. C., and R. W. Doms. 2003. HIV-1 entry and its inhibition. Curr Top 745 Microbiol Immunol 281:1-27. 746

44. Rux, A. H., S. H. Willis, A. V. Nicola, W. Hou, C. Peng, H. Lou, G. H. Cohen, and R. 747 J. Eisenberg. 1998. Functional region IV of glycoprotein D from herpes simplex virus 748 modulates glycoprotein binding to the herpesvirus entry mediator. J Virol 72:7091-7098. 749


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

35

45. Sabo, M. C., V. C. Luca, J. Prentoe, S. E. Hopcraft, K. J. Blight, M. Yi, S. M. 750 Lemon, J. K. Ball, J. Bukh, M. J. Evans, D. H. Fremont, and M. S. Diamond. 2011. 751 Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind 752 discontinuous epitopes and inhibit infection at a postattachment step. J Virol 85:7005-753 7019. 754

46. Sanna, P. P., F. Ramiro-Ibanez, and A. De Logu. 2000. Synergistic interactions of 755 antibodies in rate of virus neutralization. Virology 270:386-396. 756

47. Serafini-Cessi, F., F. Dall'Olio, N. Malagolini, L. Pereira, and G. Campadelli-Fiume. 757 1988. Comparative study on O-linked oligosaccharides of glycoprotein D of herpes 758 simplex virus types 1 and 2. J Gen Virol 69:869-877. 759

48. Sisk, W. P., J. D. Bradley, R. J. Leipold, A. M. Stoltzfus, M. Ponce de Leon, M. Hilf, 760 C. Peng, G. H. Cohen, and R. J. Eisenberg. 1994. High-level expression and 761 purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized 762 by baculovirus-infected insect cells. J Virol 68:766-775. 763

49. Skehel, J. J., and D. C. Wiley. 2000. Receptor binding and membrane fusion in virus 764 entry: the influenza hemagglutinin. Annu Rev Biochem 69:531-569. 765

50. Spear, P. G., R. J. Eisenberg, and G. H. Cohen. 2000. Three classes of cell surface 766 receptors for alphaherpesvirus entry. Virology 275:1-8. 767

51. Spear, P. G., S. Manoj, M. Yoon, C. R. Jogger, A. Zago, and D. Myscofski. 2006. 768 Different receptors binding to distinct interfaces on herpes simplex virus gD can trigger 769 events leading to cell fusion and viral entry. Virology 344:17-24. 770


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

36

52. Stanberry, L. R., S. L. Spruance, A. L. Cunningham, D. I. Bernstein, A. Mindel, S. 771 Sacks, S. Tyring, F. Y. Aoki, M. Slaoui, M. Denis, P. Vandepapeliere, and G. Dubin. 772 2002. Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med 773 347:1652-1661. 774

53. Tal-Singer, R., C. Peng, M. Ponce De Leon, W. R. Abrams, B. W. Banfield, F. 775 Tufaro, G. H. Cohen, and R. J. Eisenberg. 1995. Interaction of herpes simplex virus 776 glycoprotein gC with mammalian cell surface molecules. J Virol 69:4471-4483. 777

54. Thompson, B. S., B. Moesker, J. M. Smit, J. Wilschut, M. S. Diamond, and D. H. 778 Fremont. 2009. A therapeutic antibody against west nile virus neutralizes infection by 779 blocking fusion within endosomes. PLoS Pathog 5:e1000453. 780

55. Vanarsdall, A. L., B. J. Ryckman, M. C. Chase, and D. C. Johnson. 2008. Human 781 cytomegalovirus glycoproteins gB and gH/gL mediate epithelial cell-cell fusion when 782 expressed either in cis or in trans. J Virol 82:11837-11850. 783

56. Vogt, M. R., K. A. Dowd, M. Engle, R. B. Tesh, S. Johnson, T. C. Pierson, and M. S. 784 Diamond. 2011. Poorly neutralizing cross-reactive antibodies against the fusion loop of 785 West Nile virus envelope protein protect in vivo via Fc-{gamma} receptor and 786 complement-dependent effector mechanisms. J Virol 85:11567-11580. 787

57. Vogt, M. R., B. Moesker, J. Goudsmit, M. Jongeneelen, S. K. Austin, T. Oliphant, S. 788 Nelson, T. C. Pierson, J. Wilschut, M. Throsby, and M. S. Diamond. 2009. Human 789 monoclonal antibodies against West Nile virus induced by natural infection neutralize at 790 a postattachment step. J Virol 83:6494-6507. 791


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

37

58. Whitbeck, J. C., M. I. Muggeridge, A. H. Rux, W. Hou, C. Krummenacher, H. Lou, 792 A. van Geelen, R. J. Eisenberg, and G. H. Cohen. 1999. The major neutralizing 793 antigenic site on herpes simplex virus glycoprotein D overlaps a receptor-binding 794 domain. J Virol 73:9879-9890. 795

59. Whitbeck, J. C., C. Peng, H. Lou, R. Xu, S. H. Willis, M. Ponce de Leon, T. Peng, A. 796 V. Nicola, R. I. Montgomery, M. S. Warner, A. M. Soulika, L. A. Spruce, W. T. 797 Moore, J. D. Lambris, P. G. Spear, G. H. Cohen, and R. J. Eisenberg. 1997. 798 Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the 799 tumor necrosis factor receptor superfamily and a mediator of HSV entry. J Virol 800 71:6083-6093. 801

60. Willis, S. H., A. H. Rux, C. Peng, J. C. Whitbeck, A. V. Nicola, H. Lou, W. Hou, L. 802 Salvador, R. J. Eisenberg, and G. H. Cohen. 1998. Examination of the kinetics of 803 herpes simplex virus glycoprotein D binding to the herpesvirus entry mediator, using 804 surface plasmon resonance. J Virol 72:5937-5947. 805

61. Zeng, F., C. C. Hon, C. W. Yip, K. M. Law, Y. S. Yeung, K. H. Chan, J. S. Malik 806 Peiris, and F. C. Leung. 2006. Quantitative comparison of the efficiency of antibodies 807 against S1 and S2 subunit of SARS coronavirus spike protein in virus neutralization and 808 blocking of receptor binding: implications for the functional roles of S2 subunit. FEBS 809 Lett 580:5612-5620. 810

811 812


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

38

Table 1. Virus neutralization by anti-gD MAbs* 813 814 MAb 50% Neut. of HSV-1 (KOS) 50% Neut. of HSV-2 (333) MC2 >25 0.781 MC5 3.1 6.2 MC23 1.6 0.260 DL11 0.004 0.312 1D3 0.390 6.2 MC4 >25 >25 MC10 >25 >25 MC14 >25 >25 DL6 >25 >25 BD66 >25 >25 BD78 >25 >25 815 *Data are expressed as amount of IgG (µg /ml) 816 needed to neutralize 50% of visible plaques 817 formed by HSV on Vero cells at 48h P.I. 818 The starting number of plaques was 100. 819 820 821


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

39

Figure legends 822 Fig. 1. Grouping of MC series of MAbs to gD and their neutralization properties. (A) 823 Tree-like diagram shows how the MC MAbs relate to each other and to previously 824 characterized anti-gD MAbs (22, 37). MC MAbs were grouped according to several criteria, 825 starting with whether they react with any of a series of overlapping peptides to gD2. 826 Additionally, we used truncated forms of gD to delineate the C-terminal end of the 827 discontinuous epitopes. In some cases, the numbers refer to the amino acid changed in 828 monoclonal antibody resistant (mar) mutants as well as to the position of linker insertion 829 mutations found to affect MAb binding to gD (11). They were then subjected to non-830 denaturing SDS-PAGE and western blotting to determine type specificity. They were 831 compared to monoclonal antibodies that had previously been characterized and grouped 832 using biosensor competition as well as which peptides they recognized. Previous groups 833 from Group I to Group XI were maintained and new groups (XII to XVI) were added to 834 accommodate MAbs that did not fit into known groups (37). Those bolded and in red have 835 virus neutralizing activity. MC12 recognizes the same amino acids as the previously 836 mapped MAb BD66 and was placed in Group XI. MC1 recognized the synthetic peptide 1-20 837 and was placed in Group VII. Eleven type-common MC MAbs were added to Group IIa 838 (previously defined only by BD78). Five new groups (IIc, XIV, XV, XVI and XVII) accounted 839 for the remaining MC MAbs. (B) Structures of gD showing the receptor binding face and the 840 fusion activation face. 841

842


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

40

Fig. 2. Mapping of antibody epitopes on gD for the MC MAbs. (A) Western blot analysis of 843 MC2, MC5, MC14 and MC23. Purified truncated gD1(306), indicated as 1, or gD2(306), 844 indicated as 2, were subjected to SDS-PAGE under non-denaturing conditions. Western 845 blots were probed with the indicated MAbs. (B) ELISA. Truncated gD1(306t), as well as gD2 846 truncated either at amino acid 306, 285, 250 or 234 were each reacted with the same IgG 847 concentration of MC2, MC5 or MC23 for 1h at RT. Bound antibody was detected by an anti-848 mouse secondary antibody and ABTS substrate. Absorbance values were normalized by 849 considering the values obtained for each MAb binding to gD2(306t) as 100%. 850 851 Fig. 3. MAb blocking of gD binding to receptors measured by SPR. Anti-His MAb was 852 coupled to a CM5 biosensor chip and gD2(306t) was captured via its C-terminal His tag on 853 flowcell 1 (Fc1) and Fc2. The first MAb was injected across Fc1 for 2 min at 5 μl/min 854 (control flow cell). The next MAb was then injected across both Fc1 and Fc2 for 2 min. (A) 855 Sensorgram obtained using MAbs MC2 (1st MAb) and MC5 (2nd MAb). Since both bind to gD, 856 there is no competition. (B) Sensorgram obtained using MC23 (1st MAb) and LP2 (2nd MAb). 857 In this case, when MC23 was injected first, LP2 failed to bind, indicating they competed for 858 the same epitope on gD. (C) Sensorgram of MC2, MC5, MC14 and MC23, each added 859 sequentially to gD in Fc1. Not shown is binding of gD2(306t) to the anti-His MAb. In this 860 case, each MAb bound to gD independently of whether the other MAbs were present, 861 indicating that all four recognize distinct epitopes on gD. 862 Fig. 4. Co-immunoprecipitation of HVEM(200t) and nectin-1(346t) by anti-gD MAbs. 863 Reaction mixtures of gD2(285t) in the presence (+) or absence (−) of purified truncated 864


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

41

nectin-1(346t) (A) or HVEM(200t) (B) were immunoprecipitated by the indicated MAbs. 865 DL6 and DL11 were used as positive and negative controls, respectively. For nectin-1, the 866 Western blots were probed with anti-gD and anti-nectin-1 polyclonal Abs. For HVEM, two 867 gels were run after the co-immunoprecipitation and one blot was probed, one for HVEM 868 and the other for gD. Secondary Abs were then added, and enhanced chemiluminescence 869 was used to visualize the bands. 870 871 Fig. 5. Effect of MAbs on binding of gD2(306t) to HVEM and nectin-1. Serial two-fold 872 dilutions of each MAb IgG were incubated for 1h with a constant amount of gD2(306t). The 873 gD-IgG mixture was then added to an ELISA plate to which either HVEM (A and C) or 874 nectin-1 (B and D) had been previously immobilized. Binding of gD was then detected with 875 the polyclonal antibody R8 followed by secondary antibody and substrate. The results are 876 presented in separate panels for clarity (note the difference in y-axis scale). Results were 877 normalized to the amount of gD bound following incubation with a control IgG (anti-myc). 878 A representative experiment is shown; each antibody was tested a minimum of three times. 879 880 Fig. 6. Effect of MAbs MC2 and MC5 on receptor binding to gD2(306t) or gD2(285t). Serial 881 two fold dilutions of the two forms of gD-2 were mixed with antibody at a concentration of 882 0.5 μM of (A-B) MC2, (C-D) MC5, or (E-F) MC14 for 1h. The gD-IgG mixture was then 883 incubated with purified receptor that had been immobilized to an ELISA plate. Binding of 884 gD was detected with the polyclonal anti-gD antibody R8 and anti-rabbit secondary 885


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

42

antibody. A representative experiment is shown; each antibody was tested a minimum of 886 three times. 887 888 Fig. 7. Effect of mixtures of MAbs MC2 and MC14 on receptor binding of gD2(306t) or 889 gD2(285t). (A) Serial two fold dilutions of the two forms of gD2 were mixed with either 890 MYC (control antibody), MC2, MC14 or a mixture of MC2 and MC14 and then incubated 891 with purified HVEM that had been immobilized to an ELISA plate. (B) The procedure was 892 the same as in panel A, except that nectin-1 was bound to the ELISA plate. Binding of gD 893 was detected with the polyclonal anti-gD antibody R8 and anti-rabbit secondary antibody. 894 A representative experiment is shown; each antibody was tested a minimum of three times. 895 896 Fig. 8. Effect of MAbs to the C-term of gD on virus neutralization by MC2. In each case serial 897 two fold dilutions of MC2 were either mixed with antibody IgGs or with no Ig control and 898 then incubated with 100 PFU of virus for 1h at 37°C. The virus-antibody mixtures were 899 added to Vero cells and plaques were allowed to develop. (A) Neutralization by MC4, MC2 900 or a combination of MC4 (33.3 nM) plus MC2. (B) Neutralization by MC10, MC2 or a 901 combination of MC10 (33.3 nM) plus MC2. (C) Neutralization by BD66, MC2 or a 902 combination of BD66 (80 nM) plus MC2. (D) Neutralization by DL6, MC2 or a combination 903 of DL6 (80 nM) plus MC2. The results are expressed as % of the no Ig control. A 904 representative experiment is shown; each antibody was tested a minimum of three times. 905 906


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

43

Fig. 9. Effect of MAb MC14 on the rate of virus neutralization by DL11 or MC2. (A,B) 907 Neutralization with antibody concentrations. Comparison of DL11 plus or minus 80 nM 908 MC14 (panel A) versus MC2 plus or minus 80 nM MC14 (panel B). In each case serial two 909 fold dilutions of DL11 or MC2 were either mixed with MC14 or with no Ig control and then 910 incubated with 100 PFU of virus for 1h at 37°C. The virus-antibody mixtures were added to 911 Vero cells and plaques were allowed to develop. The results are expressed as % of the no Ig 912 control. (C,D) Kinetics of neutralization. For this assay, the concentrations of MAbs were 913 fixed and the time during which MAbs and virus were incubated was varied (0 to 60 min). 914 At each time point, the virus-antibody mixture was rapidly diluted in growth medium and 915 added to cells to measure the virus neutralization over time. The results were plotted as 916 V/V0, were V is the number of plaques remaining at each time point and V0 is the number of 917 plaques at time zero. This number was plotted against time. A straight-line relationship 918 between V/V0 and time represents first order kinetics and the slope of the line represents 919 the rate of virus neutralization. A representative experiment is shown; each antibody was 920 tested a minimum of three times. 921 922 Fig. 10. Model depicting the receptor binding and fusion activation domains of gD relative 923 to the MC2 and MC14 epitopes. The Ig-like core of gD (residues 56-184) is represented as a 924 disc. Each side of the disc presents a distinct protein interaction site. One side of gD 925 interacts with cellular receptors (i.e. nectin-1) while the opposite side interacts with 926 downstream elements of the HSV membrane fusion machinery (i.e. gH/gL). The C-927 terminus (residues 240-306) is depicted as a red line extending from one side of gD to the 928


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

44

other around the base of the Ig-like gD core. The MC2 and MC14 epitopes are represented 929 by a blue oval and a green rectangle, respectively. (A) Native gD with the C-terminus 930 positioned across the nectin-1 binding site. In this conformation, receptor access is 931 competed by the gD C-term. (B) The binding of either MC2 (shown) or MC14 to gD alters 932 gD conformation such that receptor binding is enhanced. The most likely explanation is 933 that the C-term is displaced from its native conformation (movement is shown by the blue 934 arrow). (C) When MC2 and MC14 are both bound to gD, the effect on gD structure is more 935 profound. One manifestation of this structural alteration is an even greater increase in 936 nectin-1 binding. We hypothesize that the neutralizing activity of MC2 is a result of the 937 proximity of its epitope to the fusion activation site. Perhaps MC14 augments MC2-938 mediated neutralization by imposing additional conformational constraints on the fusion 939 activation domain. Alternatively, MC14 may simply enhance MC2 binding or Ab:Ag 940 complex stability. 941 942


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

A HSV gD-2 MAbs

Discontinuous Continuous

Type

Type 2 Specific Type

CommonType 2

Specific

MC2262-27210-20 280-316262-272235-245Group XVII

Group VII Group XIV Group IIa Group XI Group IIc

272-279

Group IIb

246

CommonMC6

MC11MC13MC18

MC5MC20

BD78MC3MC4MC8MC9

MC10MC14

Group XVI

Group VII Group XIV Group IIa Group XI Group IIc1D3MC1

MC16 BD66MC12

Group IbDL11

Group IIbDL6

25,27277 310

213,216

132,140 75-79

MC23LP2HD1

Group IaGroup XII

AP7

Receptor binding face Fusion activation face

277-310

B

306MC23

ecep o b d g ace

306

MC5

Fusion activation face

DL11MC5

27

MC14

MC2285

Figure 1


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

MC2

1 2

MC5 MC23MC14A

1 2 1 2 1 2

100

120 MC2MC5MC23

B

40

60

80

erce

nt o

f gD

2(30

6t)

0

20

Bin

ding

(pe

D1(

306t

)

D2(

306t

)

D2(

285t

)

D2(

250t

)

D2(

234t

)

gD gD gD gD gD

Figure 2


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

2500

A

gD-2 injection

MC5 injection500

1000

1500

2000

2500

esp

on

se u

nit

s Fc1

Fc2

MC2 injection (Fc1 only)

0

0 200 400 600 800 1000 1200Time (s)

s

Re

B

100

200

300

400

500

600

Res

po

nse

un

its

LP2 injection

gD-2 injection

MC23

Fc1Fc2

0

0 200 400 600 800 1000 1200 1400Time (s)

R MC23 injection (Fc1 only)

1000C

250

500

750

MC5 injection

MC14 injection

MC23 injection

Res

po

nse

un

its

0

1

250

499

748

997

1246

1495

1744

1993

2242

2491

2740

2989

MC2 injection

Time (s)

Figure 3


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

nectin-1:

DL6 DL11

+- +-

MC2 MC4 MC5

+- +- +-

AkDa

gD

nectin-1 49

37

DL6 DL11

+- +-HVEM:

HVEM

MC2 MC4 MC5

+- +- +-

B

2619

kDa

gD 37

Figure 4


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

A C200

250

ng

(%

con

tro

l)

ing

(%

con

tro

l)

DL11LP2MC5

MC2MC14DL6BD66

100

150

200

100

150

200

HVEM

Bin

din

Antibody (nM)

Bin

di

Antibody (nM)

HVEM

MC23 BD78

0

50

0.1 1 10 100 10000

50

100

0.1 1 10 100 1000

DL11con

tro

l)

B%

con

tro

l)

MC2

D

200

250

300

350

150

200

DL11LP2MC5MC23

Bin

din

g (

%c

Bin

din

g (

% MC14DL6BD66BD78

nectin-1nectin-10

50

100

150

200

0 1 1 10 100 10000

50

100

0 1 1 10 100 1000

Antibody (nMl) Antibody (nM)

0.1 1 10 100 10000.1 1 10 100 1000

Figure 5


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

HVEMA 0.4nectin-1B

0.2

0.3

0.4

MYC-306MC2-306MYC-285MC2-285A

bs

405

0 1

0.2

0.3

MYC-306MC2-306MYC-285MC2-285

Ab

s 40

5

0

0.1

0.1 1 10 100 1000gD (nM)

0

0.1

0.01 1 100 10000gD (nM)

C D

0.2

0.3

0.4

MYC-306MC5-306MYC-285

Ab

s 40

5

C

0.2

0.3

0.4

MYC-306MC5-306MYC-285MC5 285

Ab

s 40

5D

0

0.1

0.1 1 10 100 1000

MC5-285A

gD (nM)

0

0.1

0.01 1 100 10000

MC5-285

gD (nM)

0 2

0.3

0.4

MYC-306MC14-306

bs

405

E

0.3

0.4

0.5

MYC-306MC14-306MYC-285A

bs

405

F

D ( M)

0

0.1

0.2

0.1 1 10 100 1000

MYC-285MC14-285

Ab

gD (nM)

0

0.1

0.2

0.01 1 100 10000

MYC-285MC14-285

A

gD (nM)

Figure 6


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

2 5HVEM

A

1

1.5

2

2.5306 MC2

306 MC14

306 2+14

306 MYC

285 MYC

Ab

s 40

5

0

0.5

0.0

2.0

7.8

31.3

125.

0

500.

0

gD (nM)

2

2.5306 MC2

306 MC14

306 2+14

306 MYC

Nectin-1B

5

0

0.5

1

1.5306 MYC

285 MYC

Ab

s 40

5

0

0.0

2.0

7.8

31.3

125.

0

500.

0

gD (nM)

Figure 7


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

100

120

100

120

IgG

)A B

60

80

100

60

80

100

er (p

erce

nt o

f no

MC10MC4

0

20

40

0 5 0 2 3 7 3

0

20

40

0 5 0 2 3 7 3

Pla

que

num

be

MC2

MC2+MC4 MC2+MC10

MC2

0

0.5

1.0

2.1

4.2

8.3

16.7

33.3 0

0.5

1.0

2.1

4.2

8.3

16.7

33.3

IgG (nM) IgG (nM)

120

C D120

80

100

erce

nt o

f no

IgG

)

BD66 DL680

100

20

40

60

laqu

e nu

mbe

r (pe

MC2 (o) MC2 (o)

MC2+BD66 (*) MC2+DL6 (*)20

40

60

0

0

1.25 2.

5 5 10 20 40 80

IgG (nM) IgG (nM)

Pl

0

0

1.25 2.5 5 10 20 40 80

Figure 8


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

120120A B

60

80

100

ber

% o

f co

ntr

ol

60

80

100

ber

% o

f co

ntr

ol

20

40

60

Pla

qu

e n

um

bMC2+MC14

MC2

20

40

60

Pla

qu

e n

um

b

DL11+MC14

DL11

11

00 2.5 5 10 20 40 80

MC2 IgG (nM)

MC2+MC140

DL11 IgG (nM)0 2.5 5 10 20 40 801.25

C D

DL11+MC14

V/V

o

MC2

V/V

o

DL11

0.10 20 40 60 80

MC2+MC14

0.10 20 40 60 80 0 20 40 60 80

Time (min)

0 20 40 60 80Time (min)

Figure 9


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

A CB

gH/L-interaction domain

306

A CB

306

Native gD(306)

C-terminus occludesNectin-1 binding site

MC2-gD(306)

C-term. moves awayfrom Nectin-1 site

306

MC2/14-gD(306)

C-term. displacedfrom Nectin-1 site

Nectin-1 binding site

MC2 MAbMC2 epitopeMC14 MAbMC14 epitope

Figure 10


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

antibody-induced conformational changes in hsv gd reveal...

Documents