Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans.

UNLABELLED: Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE: Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally directed against gD, gB, and, to a lesser extent, gC. While several key HSV-neutralizing epitopes within gD and gB are commonly targeted by human serum IgG, others fail to induce consistent responses. These data are particularly relevant to the design of future HSV vaccines.

Mechanism of neutralization of herpes simplex virus by antibodies directed at the fusion domain of glycoprotein B.

UNLABELLED: Glycoprotein B (gB), the fusogen of herpes simplex virus (HSV), is a class III fusion protein with a trimeric ectodomain of known structure for the postfusion state. Seen by negative-staining electron microscopy, it presents as a rod with three lobes (base, middle, and crown). gB has four functional regions (FR), defined by the physical location of epitopes recognized by anti-gB neutralizing monoclonal antibodies (MAbs). Located in the base, FR1 contains two internal fusion loops (FLs) and is the site of gB-lipid interaction (the fusion domain). Many of the MAbs to FR1 are neutralizing, block cell-cell fusion, and prevent the association of gB with lipid, suggesting that these MAbs affect FL function. Here we characterize FR1 epitopes by using electron microscopy to visualize purified Fab-gB ectodomain complexes, thus confirming the locations of several epitopes and localizing those of MAbs DL16 and SS63. We also generated MAb-resistant viruses in order to localize the SS55 epitope precisely. Because none of the epitopes of our anti-FR1 MAbs mapped to the FLs, we hyperimmunized rabbits with FL1 or FL2 peptides to generate polyclonal antibodies (PAbs). While the anti-FL1 PAb failed to bind gB, the anti-FL2 PAb had neutralizing activity, implying that the FLs become exposed during virus entry. Unexpectedly, the anti-FL2 PAb (and the anti-FR1 MAbs) bound to liposome-associated gB, suggesting that their epitopes are accessible even when the FLs engage lipid. These studies provide possible mechanisms of action for HSV neutralization and insight into how gB FR1 contributes to viral fusion. IMPORTANCE: For herpesviruses, such as HSV, entry into a target cell involves transfer of the capsid-encased genome of the virus to the target cell after fusion of the lipid envelope of the virus with a lipid membrane of the host. Virus-encoded glycoproteins in the envelope are responsible for fusion. Antibodies to these glycoproteins are important biological tools, providing a way of examining how fusion works. Here we used electron microscopy and other techniques to study a panel of anti-gB antibodies. Some, with virus-neutralizing activity, impair gB-lipid association. We also generated a peptide antibody against one of the gB fusion loops; its properties provide insight into the way the fusion loops function as gB transits from its prefusion form to an active fusogen.

Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes simplex virus 2 glycoprotein D.

The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366: 34-43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. Importance: Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2). Although several distinct, well-characterized virus-neutralizing epitopes on gD2 are targeted by murine monoclonal antibodies, it is not known whether the same epitopes are targeted by the humoral response to gD2 in humans. We have developed a novel, biosensor-based competition assay to directly address this important question. Using this approach, we identified epitopes that elicit strong humoral responses in humans, as well as other epitopes that elicit much weaker responses. These data provide new insight into the human response to known neutralizing gD2 epitopes and reveal characteristics of this response that may guide future vaccine development.

The myristate moiety and amino terminus of vaccinia virus l1 constitute a bipartite functional region needed for entry.

Vaccinia virus (VACV) L1 is a myristoylated envelope protein which is required for cell entry and the fusion of infected cells. L1 associates with members of the entry-fusion complex (EFC), but its specific role in entry has not been delineated. We recently demonstrated (Foo CH, et al., Virology 385:368-382, 2009) that soluble L1 binds to cells and blocks entry, suggesting that L1 serves as the receptor-binding protein for entry. Our goal is to identify the structural domains of L1 which are essential for its functions in VACV entry. We hypothesized that the myristate and the conserved residues at the N terminus of L1 are critical for entry. To test our hypothesis, we generated mutants in the N terminus of L1 and used a complementation assay to evaluate their ability to rescue infectivity. We also assessed the myristoylation efficiency of the mutants and their ability to interact with the EFC. We found that the N terminus of L1 constitutes a region that is critical for the infectivity of VACV and for myristoylation. At the same time, the nonmyristoylated mutants were incorporated into mature virions, suggesting that the myristate is not required for the association of L1 with the viral membrane. Although some of the mutants exhibited altered structural conformations, two mutants with impaired infectivity were similar in conformation to wild-type L1. Importantly, these two mutants, with changes at A4 and A5, undergo myristoylation. Overall, our results imply dual differential roles for myristate and the amino acids at the N terminus of L1. We propose a myristoyl switch model to describe how L1 functions.

Antibody-induced conformational changes in herpes simplex virus glycoprotein gD reveal new targets for virus neutralization.

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

Vaccinia virus exhibits cell-type-dependent entry characteristics.

Differing and sometimes conflicting data have been reported regarding several aspects of vaccinia virus (VV) entry. To address this, we used a beta-galactosidase reporter virus to monitor virus entry into multiple cell types under varying conditions. Entry into HeLa, B78H1 and L cells was strongly inhibited by heparin whereas entry into Vero and BSC-1 cells was unaffected. Bafilomycin also exhibited variable and cell-type-specific effects on VV entry. Entry into B78H1 and BSC-1 cells was strongly inhibited by bafilomycin whereas entry into Vero and HeLa cells was only partially inhibited suggesting the co-existence of both pH-dependent and pH-independent VV entry pathways in these cell types. Finally, entry into HeLa, B78H1, L and BSC-1 cells exhibited a lag of 6-9 min whereas this delay was undetectable in Vero cells. Our results suggest that VV exploits multiple cell attachment and entry pathways allowing it to infect a broad range of cells.

Vaccinia virus L1 binds to cell surfaces and blocks virus entry independently of glycosaminoglycans.

L1 and A28 are vaccinia virus (VACV) envelope proteins which are essential for cellular entry. However, their specific roles during entry are unknown. We tested whether one or both of these proteins might serve as receptor binding proteins (RBP). We found that a soluble, truncated form of L1, but not A28, bound to cell surfaces independently of glycosaminoglycans (GAGs). Hence, VACV A28 is not likely to be a RBP and functions after attachment during entry. Importantly, soluble L1 inhibited both binding and entry of VACV in GAG-deficient cells, suggesting that soluble L1 blocks entry at the binding step by competing with the virions for non-GAG receptors on cells. In contrast, soluble A27, a VACV protein which attaches to GAGs but is non-essential for virus entry, inhibited binding and entry of VACV in a GAG-dependent manner. To our knowledge, this is the first report of a VACV envelope protein that blocks virus binding and entry independently of GAGs.

Epitope mapping of herpes simplex virus type 2 gH/gL defines distinct antigenic sites, including some associated with biological function.

The gH/gL complex plays an essential role in virus entry and cell-cell spread of herpes simplex virus (HSV). Very few immunologic reagents were previously available to either identify important functional regions or gain information about structural features of this complex. Therefore, we generated and characterized a panel of 31 monoclonal antibodies (MAbs) against HSV type 2 (HSV-2) gH/gL. Fourteen MAbs bound to a conformation-dependent epitope of the gH2/gL2 complex, and all blocked virus spread. The other 17 MAbs recognized linear epitopes of gH (12) or gL (5). Interestingly, two of the gL MAbs and six of the gH MAbs were type common. Overlapping synthetic peptides were used to map MAbs against linear epitopes. These data, along with results of competition analyses and functional assays, assigned the MAbs to groups representing eight distinct antigenic sites on gH (I to VIII) and three sites on gL (A, B, and C). Of most importance, the MAbs with biological activity mapped either to site I of gH2 (amino acids 19 to 38) or to sites B and C of gL2 (residues 191 to 210). Thus, these MAbs constitute a novel set of reagents, including the first such reagents against gH2 and gL2 as well as some that recognize both serotypes of each protein. Several recognize important functional domains of gH2, gL2, or the complex. We suggest a common grouping scheme for all of the known MAbs against gH/gL of both HSV-1 and HSV-2.

Epitope-mapping studies define two major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein B5R.

Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.

Physical and immunological characterization of a recombinant secreted form of the membrane protein encoded by the vaccinia virus L1R gene.

We reported that immunization with recombinant proteins derived from vaccinia virus (VV) particles could provide protection against infection. Here we describe the physical and antigenic properties of the L1R membrane protein. The recombinant protein (L1R(185t)) was secreted as a monomer and correct folding was suggested by the presence of three intramolecular disulfide bonds and binding to conformation-specific monoclonal antibodies (MAbs). Furthermore, anti-L1R(185t) rabbit antisera exhibited potent virus-neutralizing activity against the IMV form of VV. We raised six MAbs against L1R(185t). Three recognized linear epitopes (residues 118--128) and neutralized IMV infectivity. These MAbs blocked binding of each other to L1R(185t) but failed to block binding of two previously described neutralizing anti-L1R MAbs, 7D11 and 2D5. The latter two antibodies blocked each other in binding L1R(185t). Thus, two antigenic sites on L1R overlap functional domains and based on recent structural studies these are found in accessible regions of the IMV L1R protein.

Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry.

Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts.

Comparative usage of herpesvirus entry mediator A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus.

The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.

Structure-function analysis of herpes simplex virus type 1 gD and gH-gL: clues from gDgH chimeras.

In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replication-competent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.