Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.     

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.     

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.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates.

The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo.     

Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.     

Immunological characterization of herpes simplex virus type 1 and 2 polypeptide(s) involved in viral ribonucleotide reductase activity

Mammalian cells infected with herpes simplex virus (HSV) express a novel ribonucleotide reductase which is biochemically and immunologically distinct from the uninfected-cell enzyme. Using polyvalent rabbit antiserum raised against partially purified HSV type 2 reductase as well as monoclonal antibodies to HSV type 1 and HSV type 2 early antigens, we have been able to show that in both serotypes reductase activity is associated with phosphoproteins of molecular weights 144,000 and 38,000 encoded between map units 0.566 and 0.602 in the viral genomes. The major antigenic species (144,000) have been tentatively identified as HSV type 1 ICP6 and HSV type 2 ICP10.     

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Mechanism of interferon action. Production and characterization of monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1

Monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1 were prepared using protein P1 purified from human amnion U cells as the immunogen. Rabbit antiserum to protein P1 recognized with comparable efficiency P1 both from human U cells and from mouse L929 cells. Immunoprecipitates that contained protein P1 also possessed a protein kinase activity that catalyzed the phosphorylation of protein P1 and the alpha subunit of initiation factor eIF-2. Three BALB/C mouse monoclonal antibodies efficiently recognized human protein P1, but either did not recognize or recognized very poorly P1 from mouse cells. A fourth monoclonal antibody against human P1 recognized mouse P1 with nearly equal efficiency. Immunoprecipitation of human P1 with different sequential combinations of the monoclonal antibodies suggest that two antigenic classes of protein P1 may exist.     

Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies.

Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced by polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected-cell extracts. Seven hybridomas producing antibodies specific for gC, one for gB, and one for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, one specific for gB and one specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 X 10(-4) in nonmutagenized stocks to 1 X 10(-2) in mutagenized stocks. Four gC and four gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The two other gC antigenic variants and the four gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The two mar C mutants were tested for resistance to neutralization with a panel of seven gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least two antigenic sites on glycoprotein gC.     

Prevalence of Herpes Simplex Virus Type 1- and 2- Specific Antibodies among the Acute,Recurrent, and Provoked Types of Female Genital Herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P< 0.005), and 55% for the recurrent type (P<0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

p185, an immunodominant epitope, is an autoantigen mimotope

An immunodominant peptide (p185(378-394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378-394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378-394). Representative mouse and human sera with anti-p185(378-394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.     

Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

Herpes simplex virus type 1 infection of endothelial, epithelial, and fibroblast cells induces a receptor for C3b

We recently demonstrated that herpes simplex virus type 1 (HSV 1) induces a receptor on human umbilical vein endothelial cells for complement component C3b (C3bR). We assigned this receptor function to HSV 1 viral glycoprotein C (gC) based on several observations: tunicamycin, which prevents glycosylation and expression of N-linked glycoproteins on the surface of infected cells, markedly reduced expression of the C3bR; monoclonal antibodies to HSV 1 gC blocked detection of the C3bR, whereas monoclonal antibodies to other HSV 1 glycoproteins (gB, gD, gE) had no effect; and the MP mutant of HSV 1, which fails to express gC, did not induce C3bR. We now report that HSV 1 induces C3bR on a wide variety of cell types including bovine thoracic aorta and pulmonary artery endothelial cells, human embryonic lung and embryonic foreskin fibroblasts, and human embryonic kidney cells. To date, all cells studied that are permissive to HSV 1 express C3bR, although the pattern of rosetting of C3b-coated erythrocytes varies among the cell strains examined. We also demonstrate that C3bR expression is not a general response of human umbilical vein endothelial cells to injury, because three other viruses (adenovirus 7, measles, and mumps) do not induce C3bR after infection of these cells. Previously we had shown that among herpes simplex viruses, a variety of HSV 1 strains induce C3bR, whereas HSV 2 strains do not. We now demonstrate that other herpes family viruses (CMV and VZV) do not express C3bR. Therefore, C3bR expression appears to be unique for HSV 1 and occurs on a wide variety of cells permissive to this virus.     

Anti-alpha-galactosyl antibodies recognizing epitopes terminating with alpha1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

The preparation and characterization of monoclonal antibodies to human complement component C8 and their use in purification of C8 and C8 subunits.

1. Ten mouse monoclonal antibodies to human complement component C8 were prepared. It was found that six of these antibodies reacted with the alpha-subunit, two with the beta-subunit and two with the gamma-subunit, when assessed by immunoblotting after separation of C8 subunits by SDS/polyacrylamide-gel electrophoresis. 2. Epitope analysis of the ten monoclonal antibodies in a competitive binding assay showed that the six antibodies to the alpha-subunit could be classified in four overlapping epitope groups. The antibodies to the beta- and gamma-subunits bound to a single antigenic site on each, but also cross-reacted with the antigenic sites on the alpha-subunit. 3. Monoclonal anti-C8 immunoaffinity columns were used to purify C8 from fresh human plasma and to prepare C8-depleted serum. Immunoaffinity purified C8 was biologically active when assessed by using haemolysis assays of sheep and rabbit erythrocytes. 4. Salt elution was used to purify either alpha gamma- or beta-subunits when C8 was respectively bound to an anti-beta or anti-alpha immunoaffinity column. The purified subunits reconstituted C8-depleted serum when added together in a haemolysis assay.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

B-cell epitopes of canine parvovirus: distribution on the primary structure and exposure on the viral surface.

Ten antigenic sites on canine parvovirus (CPV) were mapped with a complete set of overlapping nonapeptides of the capsid proteins VP1 and VP2: five of these sites were recognized by sera from CPV-infected dogs, three were recognized by a rabbit anti-CPV antiserum, and two were recognized by murine monoclonal anti-CPV antibodies. A region covering the first 21 amino-terminal amino acid residues of VP2 was recognized by three sera from infected dogs, one neutralizing rabbit antiserum, and one neutralizing murine monoclonal antibody. Immunoabsorption experiments with full virions indicated that at least 6 of the 10 antigenic sites are located on the surface. Of these six, three sites occur in the amino terminus of VP2. When superimposed on the three-dimensional structure of canine parvovirus (J. Tsao, M. S. Chapman, M. Agbandje, W. Keller, K. Smith, H. Wu, M. Luo, T. J. Smith, M. G. Rossmann, R. W. Compans, and C. R. Parrish, Science 251:1456-1464, 1991), the other three epitopes are located on two loops of VP2 which form the highly exposed "spike" around the threefold-symmetry axis of the virus. Thus, these regions (amino terminus and loops 1 and 3) are of interest as major target sites for induction of neutralizing antibodies.     

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.