Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

Epitopes of herpes simplex virus type 1 glycoprotein gC are clustered in two distinct antigenic sites.

Epitopes of herpes simplex virus type 1 (HSV-1) strain KOS glycoprotein gC were identified by using a panel of gC-specific, virus-neutralizing monoclonal antibodies and a series of antigenic variants selected for resistance to neutralization with individual members of the antibody panel. Variants that were resistant to neutralization and expressed an antigenically altered form of gC were designated monoclonal antibody-resistant (mar) mutants. mar mutants were isolated at frequencies of 10(-3) to 10(-5), depending on the antibody used for selection. The epitopes on gC were operationally grouped into antigenic sites by evaluating the patterns of neutralization observed when a panel of 22 antibodies was tested against 22 mar mutants. A minimum of nine epitopes was identified by this process. Three epitopes were assigned to one antigenic site (I), and six were clustered in a second complex site (II) composed of three distinct subsites, IIa, IIb, and IIc. The two antigenic sites were shown to reside in physically distinct domains of the glycoprotein, by radioimmunoprecipitation of truncated forms of gC. These polypeptides lacked portions of the carboxy terminus and ranged in size from approximately one-half that of the wild-type molecule to nearly full size. Antibodies recognizing epitopes in site II immunoprecipitated the entire series of truncated polypeptides and thereby demonstrated that site II resided in the N-terminal half of gC. Antibodies reactive with site I, however, did not immunoprecipitate fragments smaller than at least two-thirds the size of the wild-type polypeptide, suggesting that site I was located in the C-terminal portion. Sites I and II were also shown to be spatially separate on the gC polypeptide by competition enzyme-linked immunosorbent assay with monoclonal antibodies representative of different site I and site II epitopes.     

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity.

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.     

Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.     

Alterations in the antigenic structure of two major HSV-1 glycoproteins, gC and gB, influence immune regulation and susceptibility to murine herpes keratitis

We previously demonstrated that anterior chamber (AC) injection of HSV-1 before or simultaneous with topical corneal HSV-1 infection resulted in cellular immune tolerance of HSV-1 Ag and a reduced frequency of corneal stromal lesions. In the present study, we have investigated the role of the HSV-1 cell-surface glycoproteins gC and gB in the induction of tolerance, and the resulting reduced susceptibility to HSV-1 corneal stromal disease. These studies utilized mutant strains of HSV-1 with deletion or point mutations in the gene coding for gC or gB. Groups of mice received topical corneal infections with wild-type HSV-1, followed by AC injection of the same eye with wild-type HSV-1 or a mutant strain. Varying the antigenic composition of the virus injected into the AC resulted in three distinct patterns of immune responsiveness. In agreement with our previous findings, AC injection of wild-type HSV-1 induced a state of HSV-1 specific tolerance that extended to both the delayed type hypersensitivity (DTH) and CTL responses. A mutant strain lacking gC (gC-) induced partial tolerance characterized by undetectable CTL activity but a normal DTH response. A mutant strain lacking gB (gB-) caused partial suppression of the CTL response and no reduction of the DTH response. Thus, whereas gB may be involved in CTL tolerance induction in this model, gC clearly is not involved. In contrast, both gC and gB must be present in the AC to induce detectable DTH tolerance. The latter interpretation was strengthened by the observation that AC injection of a mixture of gC- (expressing normal gB) and gB- (expressing normal gC) effectively suppressed the DTH response to wild-type HSV-1. A panel of mar mutants with individual point mutations affecting gC and gB was used to identify the epitopes responsible for induction of DTH tolerance. Two of the gC mutants failed to induce DTH tolerance to wild-type HSV-1 when injected into the AC, suggesting that the sites on the gC molecule that are altered by these mutations are important for the induction of DTH tolerance. Similarly, one of the mar mutants for gB uniformly failed to suppress the DTH response, while another had a variable effect. The unique pattern of cellular immune reactivity exhibited by the mice receiving simultaneous topical corneal infection with wild-type HSV-1 and AC injection of gC- (no CTL but normal DTH) was associated with significantly reduced susceptibility to HSV-1 corneal stromal lesions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface.

To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.     

Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice.

We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Vaccination of mice with gB, gC, gD, gE, or gI resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (90 to 100% survival). In contrast, vaccination with gH produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gG produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gG did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gG-, gH-, and gE-vaccinated mice, while on day 6 mice vaccinated with gC, gE, and gI had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gG. The same five glycoproteins (gB, gC, gD, gE, and gI) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity.     

Oligomeric structure of glycoproteins in herpes simplex virus type 1.

A number of herpes simplex virus (HSV) glycoproteins are found in oligomeric states: glycoprotein E (gE)-gI and gH-gL form heterodimers, and both gB and gC have been detected as homodimers. We have further explored the organization of glycoproteins in the virion envelope by using both purified virions to quantitate glycoprotein amounts and proportions and chemical cross-linkers to detect oligomers. We purified gB, gC, gD, and gH from cells infected with HSV type 1 and used these as immunological standards. Glycoproteins present in sucrose gradient-purified preparations of two strains of HSV type 1, KOS and NS, were detected with antibodies to each of the purified proteins. From these data, glycoprotein molar ratios of 1:2:11:16 and 1:1:14:9 were calculated for gB/gC/gD/gH in KOS and NS, respectively. gL was also detected in virions, although we lacked a purified gL standard for quantitation. We then asked whether complexes of these glycoproteins could be identified, and if they existed as homo- or hetero-oligomers. Purified KOS was incubated at 4 degrees C with bis (sulfosuccinimidyl) suberate (BS3), an 11.4 A (1A = 0.1 mm) noncleavable, water-soluble cross-linker. Virus extracts were examined by Western blotting (immunoblotting), or immunoprecipitation followed by Western blotting, to assay for homo- and hetero-oligomers. Homodimers of gB, gC, and gD were detected, and hetero-oligomers containing gB cross-linked to gC, gC to gD, and gD to gB were also identified. gH and gL were detected as a hetero-oligomeric pair and could be cross-linked to gD or gC but not to gB. We conclude that these glycoproteins are capable of forming associations with one another. These studies suggest that glycoproteins are closely associated in virions and have the potential to function as oligomeric complexes.     

Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies.

Polyclonal and monoclonal antibodies to individual herpes simplex virus (HSV) glycoproteins were tested for ability to inhibit adsorption of radiolabeled HSV type 1 (HSV-1) strain HFEMsyn [HSV-1(HFEM)syn] to HEp-2 cell monolayers. Polyclonal rabbit antibodies specific for glycoprotein D (gD) or gC and three monoclonal mouse antibodies specific for gD-1 or gC-1 most effectively inhibited HSV-1 adsorption. Antibodies of other specificities had less or no inhibitory activity despite demonstrable binding of the antibodies to virions. Nonimmune rabbit immunoglobulin G and Fc fragments partially inhibited adsorption when used at relatively high concentrations. These results suggest involvement of gD, gC, and perhaps gE (the Fc-binding glycoprotein) in adsorption. The monoclonal anti-gD antibodies that were most effective at inhibiting HSV-1 adsorption had only weak neutralizing activity. The most potent anti-gD neutralizing antibodies had little effect on adsorption at concentrations significantly higher than those required for neutralization. This suggests that, although some anti-gD antibodies can neutralize virus by blocking adsorption, a more important mechanism of neutralization by anti-gD antibodies may be interference with a step subsequent to adsorption, possibly penetration.     

Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.     

Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC(-)39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.     

Mutations conferring resistance to neutralization with monoclonal antibodies in type 1 poliovirus can be located outside or inside the antibody-binding site.

Antigenic variants resistant to eight neutralizing monoclonal antibodies were selected from wild (Mahoney) and attenuated (Sabin) type 1 infectious poliovirions. Cross-immunoprecipitation revealed interrelationships between epitopes which were not detected by cross-neutralization. Operational analysis of antigenic variants showed that seven of eight neutralization epitopes studied were interrelated. Only one neutralization epitope, named Kc, varied independently from all the others. This latter, recognized by C3 neutralizing monoclonal antibody, was present not only on infectious virions but also on heat-denatured (C-antigenic) particles and on isolated capsid protein VP1. Loss of the neutralization function of an epitope did not necessary result from the loss of its antibody-binding capacity. Such potential, but not functional, neutralization epitopes exist naturally on Mahoney and Sabin 1 viruses. Their antibody-binding property could be disrupted by isolating antigenic variants in the presence of the nonneutralizing monoclonal antibody and anti-mouse immunoglobulin antibodies. Single-point mutations responsible for the acquisition of resistance to neutralization in the antigenic variants were located by sequence analyses of their genomes. Mutants selected in the presence of C3 neutralizing monoclonal antibody always had the mutation located inside the antibody-binding site (residues 93 through 103 of VP1) at the amino acid position 100 of VP1. On the contrary, antigenic variants selected in the presence of neutralizing monoclonal antibodies reacting only with D-antigenic particles had mutations situated in VP3, outside the antibody-binding site (residues 93 through 103 of VP1). The complete conversion of the Mahoney to the Sabin 1 epitope map resulted from a threonine-to-lysine substitution at position 60 of VP3.     

Herpes simplex virus type 1 glycoprotein C-negative mutants exhibit multiple phenotypes, including secretion of truncated glycoproteins

A virus-neutralizing monoclonal antibody specific for glycoprotein C (gC) of herpes simplex virus type 1 strain KOS was used to select a number of neutralization-resistant mutants. A total of 103 of these mutants also were resistant to neutralization by a pool of gC-specific antibodies and thus were operationally defined as gC-. Analysis of mutant-infected cell mRNA showed that a 2.7-kilobase mRNA, comparable in size to the wild-type gC mRNA, was produced by nearly all mutants. However, six mutants, gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98, did not produce the normal-size gC mRNA but rather synthesized a novel 1.1-kilobase RNA species. These mutants had deletions of 1.6 kilobases in the coding sequence of the gC structural gene, which explains their gC- phenotype. Despite the production of an apparently normal mRNA by the remaining 97 mutants, only 7 mutants produced a detectable gC polypeptide. In contrast to wild-type gC, which is a membrane-bound glycoprotein with an apparent molecular weight of 130,000 (130K), five of these mutants quantitatively secreted proteins of lower molecular weight into the culture medium. These were synLD70 (101K), gC-8 (109K), gC-49 (112K), gC-53 (108K), and gC-85 (106K). The mutant gC-3 secreted a protein that was indistinguishable in molecular weight from wild-type KOS gC. Another mutant, gC-44, produced a gC protein which also was indistinguishable from wild-type gC by molecular weight and which remained cell associated. Pulse-labeling of infected cells in the presence and absence of the glycosylation inhibitor tunicamycin demonstrated that these proteins were glycosylated and provided estimates of the molecular weights of the nonglycosylated primary translation products. The smallest of these proteins was produced by synLD70 and was 48K, about two-thirds the size of the wild-type polypeptide precursor (73K). Physical mapping of the mutations in synLD70 and gC-8 by marker rescue placed these mutations in the middle third of the gC coding sequence. Mapping of the mutations in other gC- mutants, including two in which no protein product was detected, also placed these mutations within or very close to the gC gene. The biochemical and genetic data available on mutants secreting gC gene products suggest that secretion is due to the lack of a functional transmembrane anchor sequence on these mutant glycoproteins.     

Antigenic variation (mar mutations) in herpes simplex virus glycoprotein B can induce temperature-dependent alterations in gB processing and virus production.

Monoclonal antibody-resistant (mar) mutants altered in the antigenic structure of glycoprotein B (gB) of herpes simplex virus type 1, strain KOS-321, were selected by neutralization with each of six independently derived gB-specific monoclonal antibodies. Analysis of the reactivity patterns of these mar mutants with a panel of 16 virus-neutralizing monoclonal antibodies identified at least five nonoverlapping epitopes on this antigen, designated groups I through V. Multiple mar mutations were also introduced into the gB structural gene by recombination and sequential antibody selection to produce a set of mar mutants with double, triple, and quadruple epitope alterations. Group II (B2) and group III (B4) antibodies were used to select the corresponding mutants, mar B2.1 and mar B4.1, which in addition to carrying the mar phenotype were temperature sensitive (ts) for processing of the major partially glycosylated precursor of gB, pgB (Mr = 107,000), to mature gB (Mr = 126,000) and showed reduced levels of gB on the cell surface at high temperature (39 degrees C). These mutants were not, however, ts for production of infectious progeny. A recombinant virus, mar B2/4.1, carrying both of these alterations was ts for virus production and failed to produce and transport any detectable mature gB to the cell surface at 39 degrees C. Rather, pgB accumulated in the infected cell. Revertants of the ts phenotype, isolated from virus plaques at 39 degrees C, regained the B2 but not the B4 epitope and were phenotypically indistinguishable from the mar B4.1 parent. Finally, it was shown that group II (B5) and group III (B4) antibodies failed to immunoprecipitate pgB (39 degrees C) produced by ts gB mutants of herpes simplex virus type 1 which were not selected with monoclonal antibodies. Taken together, our findings indicate that (i) mar mutations can alter antigenic as well as other functional domains of gB, namely, the domain(s) involved in processing and infectivity, and (ii) group II and group III epitopes lie within an essential functional domain of gB which is a target for ts gB mutations.     

Demonstration of three major species of pseudorabies virus glycoproteins and identification of a disulfide-linked glycoprotein complex.

The glycoproteins of pseudorabies virus (PRV) Phylaxia were characterized with monoclonal antibodies as specific reagents. Three major structural glycoproteins with molecular weights of 155,000 (155K) (gC), 122K (gA), and 90K (gB) could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We investigated the processing of glycoproteins gA, gB, and gC by in vitro translation, pulse-chase experiments, and in the presence of the ionophore monensin which inhibits glycosylation. gA and gB were found to compose a single polypeptide, whereas gC was found to be a disulfide-linked glycoprotein complex. Immunoprecipitates formed with the aid of anti-gC monoclonal antibodies gave rise to three glycoprotein bands (gC0 [120K], gC1 [67K], and gC2 [58K]) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Limited proteolysis of gC0, gC1, and gC2 resulted in peptide maps of gC0 related to those of both gC1 and gC2. No common peptide bands between gC1 and gC2, however, were seen. We suggest that (i) gC1 and gC2 arise by proteolytic cleavage from the same precursor molecule and stay joined via disulfide bridges and (ii) gC0 is an uncleaved precursor.     

A novel function of the herpes simplex virus type 1 Fc receptor: participation in bipolar bridging of antiviral immunoglobulin G

We describe a novel function of the Fc receptor of herpes simplex virus type 1 (HSV-1), its ability to participate in antibody bipolar bridging. This refers to the binding of a single immunoglobulin G (IgG) molecule by its Fab end to its antigenic target and by its Fc end to an Fc receptor (FcR). We demonstrate that various immune IgG antibodies, including polyclonal rabbit antibodies to HSV-1 glycoproteins gC1 and gD1 and monoclonal human antibody to gD1 blocked rosetting of IgG-coated erythrocytes at IgG concentrations 100- to 2,000-fold lower than required for rosette inhibition with nonimmune IgG. Steric hindrance did not account for the observed differences between immune and nonimmune IgG since rabbit anti-gC1 F(ab')2 fragments did not block rosetting. Murine anti-gC1 or anti-gD1 IgG, a species of IgG incapable of binding by its Fc end to the HSV-1 FcR, also did not block rosetting. When cells were infected with a gC1-deficient mutant, anti-gC1 IgG inhibited rosetting to the same extent as nonimmune IgG. This indicates that binding by the Fab end of the IgG molecule was required for maximum inhibition of rosetting. Bipolar bridging was shown to occur even when small concentrations of immune IgG were present in physiologic concentrations of nonimmune IgG. The biologic relevance of antibody bipolar bridging was evaluated by comparing antibody- and complement-dependent virus neutralization of an FcR-negative mutant and its parent HSV-1 strain. By engaging the Fc end of antiviral IgG, the parent strain resisted neutralization mediated by the classical complement pathway. These observations provide insight into the role of the HSV-1 FcR in pathogenesis and may help explain the function of FcR detected on other microorganisms.     

Antibody-selected variation and reversion in Sindbis virus neutralization epitopes.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).     

Impact of valency of a glycoprotein B-specific monoclonal antibody on neutralization of herpes simplex virus

Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab')(2)] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.     

Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding.