Two major human allergenic sites on ragweed pollen allergen antigen E identified by using monoclonal antibodies

Murine monoclonal antibodies specific for antigen E (AgE), the major allergen isolated from short ragweed pollen, have been produced and characterized. These monoclonal antibodies, when coupled to Sepharose and used as immunoadsorbents, specifically bound AgE when a crude pollen extract was passed through the column. Three antigenic sites (A, B, and C) on AgE were identified by using five of these monoclonal antibodies in both inhibition and double-bind solid-phase ELISA. These three antigenic sites appear to be nonoverlapping and nonrepeated, that is, present only once on each AgE molecule. Site C on AgE could readily be bound by the monoclonal antibody specific for that site, but only when AgE was in solution or "presented" by an anti-site A or anti-site B antibody. Site C appears to be only marginally available for binding when AgE is directly adsorbed to polyvinyl chloride microtiter wells. The majority of monoclonal antibodies isolated after immunization of BALB/c mice were specific for site A on AgE. In addition, the binding to AgE of pooled BALB/c polyclonal, hyperimmune antisera against AgE was blocked approximately 80% by a monoclonal antibody directed against site A, but was only blocked approximately 20% by an anti-site B monoclonal antibody. This suggests that site A on AgE is the predominant antigenic site in the BALB/c immune response and that site B represents a less dominant site. The binding of IgE in pooled human serum from ragweed-allergic individuals is blocked approximately 50% by a monoclonal antibody directed to site A on AgE and also approximately 50% by a monoclonal antibody directed against site B. A series of individual human short ragweed allergic antisera also showed significant, although varied, inhibition of IgE binding to AgE by both anti-site A and anti-site B monoclonal antibodies. Simultaneous addition of anti-site A and anti-site B was somewhat additive and inhibited up to 80% of the binding of human IgE specific for AgE. The conclusion from these data is that site A and site B defined by two murine monoclonal antibodies represent two very major allergenic sites in the human response to this molecule.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.     

Antigenic heterogeneity of a foot-and-mouth disease virus serotype in the field is mediated by very limited sequence variation at several antigenic sites.

Antigenic variation in a major discontinuous site (site D) of foot-and-mouth disease virus (FMDV) of serotype C has been evaluated with neutralizing monoclonal antibodies. Isolates representing the major evolutionary sublines previously defined for serotype C were compared. Extensive variation, comparable to that of continuous epitopes within the hypervariable immunodominant site A (the VP1 G-H loop), was found. The amino acid sequences of the complete capsids of three antigenically highly divergent FMDVs (C1 Haute Loire-Fr/69, C5 Argentina/69, and C3 Argentina/85) have been determined and compared with the corresponding sequences previously determined for seven additional type C viruses. Differences in antigenicity are due to a very limited number of substitutions of surface amino acids accessible to antibodies and located within antigenic sites previously identified on FMDV. A significant number of residues at these positions were also replaced in monoclonal antibody escape mutants. Depending on the variants compared, replacements within site A or at site D, or at both sites, contributed significantly to their antigenic differences. Examples of divergence mediated by a few amino acid replacements were found among FMDVs of Europe and South America. The results suggest that within a serotype of FMDV, antigenically highly divergent viruses can arise in the field by very limited sequence variation at exposed key residues of each of several antigenic sites.     

Antibody responses of humans and nonhuman primates to individual antigenic sites of the hemagglutinin-neuraminidase and fusion glycoproteins after primary infection or reinfection with parainfluenza type 3 virus.

An unusual feature of human parainfluenza virus type 3 (PIV3) is ita ability to cause reinfection with high efficiency. The antibody responses of 45 humans and 9 rhesus monkeys to primary infection or subsequent reinfection with PIV3 were examined to identify deficiencies in host immunologic responses that might contribute to the ability of the virus to cause reinfection with high frequency. Antibody responses in serum were tested by using neutralization and hemagglutination inhibition (HI) assays and a monoclonal antibody blocking immunoassay able to detect antibodies to epitopes within six antigenic sites on the PIV3 hemagglutinin-neuraminidase (HN) glycoprotein and eight antigenic sites on the fusion (F) protein. Primary infection of seronegative infants or children with PIV3 stimulated strong and rather uniform HI and neutralizing antibody responses. More than 90% of the individuals developed antibodies to four of the six HN antigenic sites (including three of the four neutralization sites), but the responses to F antigenic sites were of lesser magnitude and varied considerably from person to person. Young infants who possessed maternally derived antibodies in their sera developed lower levels and less frequent HI, neutralizing, and antigenic site-specific responses to the HN and F glycoproteins than did seronegative infants and children. In contrast, children reinfected with PIV3 developed even higher HI and neutralizing antibody responses than those observed during primary infection. Reinfection broadened the HN and F antigenic site-specific responses, but the latter remained relatively restricted. Adults possessed lower levels of HI, neutralizing, and antigenic site-specific antibodies in their sera than did children who had been reinfected, suggesting that these antibodies decay with time. Rhesus monkeys developed more vigorous primary and secondary antibody responses than did humans, but even in these highly responsive animals, response to the F glycoprotein was relatively restricted following primary infection. Bovine PIV3 induced a broader response to human PIV3 in monkeys than was anticipated on the basis of their known relatedness as defined by using monoclonal antibodies to human PIV3. These observations suggest that the restricted antibody responses to multiple antigenic sites on the F glycoprotein in young seronegative infants and children and the decreased responses to both the F and HN glycoproteins in young infants and children with maternally derived antibodies may play a role in the susceptibility of human infants and young children to reinfection with PIV3.     

Competitive inhibition by human sera of mouse monoclonal antibody binding to glycoproteins C and D of herpes simplex virus types 1 and 2.

A competitive enzyme-linked immunosorbent assay was used to test for human antibodies to antigenic sites on herpes simplex virus (HSV) glycoproteins C and D, which are recognized by mouse monoclonal antibodies. Antibodies capable of blocking the monoclonal antibodies were detected in the human sera, and the inhibition of binding correlated with the histories of herpetic infections. The binding of monoclonal antibody to glycoprotein C of HSV type 2 was inhibited primarily by sera from patients with recurrent herpes genitalis; however, the binding of the monoclonal antibodies to gC of HSV type 1 was inhibited by sera from patients previously infected with either HSV type 1 or HSV type 2. The observations suggest that the antigenic sites defined by the mouse monoclonal antibodies are recognized by the human host.     

Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin.     

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.     

Antigenic sites on foot-and-mouth disease virus type A10.

A set of monoclonal antibodies was used to isolate nonneutralizable foot-and-mouth disease virus variants, and the RNAs of the variants were sequenced. Cross-neutralization studies and mapping of the amino acid changes indicated two major antigenic sites. The first site was trypsin sensitive and included the VP1 140 to 160 sequence. The second site was trypsin insensitive and included mainly VP3 residues. Two minor sites were located near VP1 169 and on the C terminus of VP1. Comparison with poliovirus type 1 and human rhinovirus 14 showed a similarity in the immunogenicity of comparable sites on the viruses.     

Nonoverlapping antigenic sites of woodchuck hepatitis virus surface antigen and their cross-reactivity with ground squirrel hepatitis virus and hepatitis B virus surface antigens.

Five nonoverlapping antigenic sites (sites I through V) of woodchuck hepatitis virus surface antigen were identified with competitive binding assays involving monoclonal antibodies. Site I contributed to cross-reactions among surface antigens of hepatitis B-like viruses infecting woodchucks, ground squirrels, and humans. At least three distinct sites (sites I, II, and III) are responsible for cross-reactions between woodchuck and ground squirrel hepatitis virus surface antigens. Sites IV and V of woodchuck hepatitis virus surface antigen are not major cross-reactive sites, suggesting that these elicit virus-specific antibodies. There were no cross-reactions with duck hepatitis B virus surface antigen.     

Availability to monoclonal antibodies of antigenic sites of the alpha and beta subunits in active, denatured or membrane-bound mitochondrial F1-ATPase

The binding of five monoclonal antibodies to mitochondrial F1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F1 integrated in the mitochondrial membrane, for purified beta-subunit and for purified F1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-beta monoclonal antibodies bound more easily to the beta-subunit than to active F1, and recognized equally active F1 and F1 integrated in the membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated beta-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-beta monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the beta-subunit. One anti-alpha monoclonal antibody (20 D6) bound poorly to F1 integrated in the membrane, while the other (7 B3) had a higher affinity for F1 integrated in the membrane than for soluble F1. Therefore, 20 D6 recognizes an epitope of the alpha-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the alpha-subunit well exposed at the surface of the inner face of the mitochondrial membrane.     

Structural differences around hormonogenic sites on thyroglobulins from different species detected by monoclonal antibodies

Thyroxine remains attached to its synthetic site in thyroglobulin until it is released by proteolysis. Strong homology in the primary sequence surrounding thyroxine-forming residues in thyroglobulins from various species suggests a unique three-dimensional structure at hormonogenic sites. To examine this, two thyroxine-binding mouse anti-(chicken thyroglobulin) monoclonal antibodies, 1A10 and 5F6, were used as probes for this region in an enzyme-linked immunosorbent inhibition assay. The thyroxine content of thyroglobulins had a marked positive influence on the monoclonal antibody binding: when the thyroxine content of human thyroglobulin rose by 6.6-fold, cross-reactivities rose 25-fold for the 1A10 monoclonal antibody and 17.6-fold for the 5F6 monoclonal antibody. However, interspecies comparison of thyroglobulin preparations with similar thyroxine content showed lower than expected cross-reactivities for human, pig and sheep thyroglobulins when compared with chicken thyroglobulin. Only when the thyroxine content of heterologous thyroglobulin preparations was two or three times higher did the cross-reactivities equal or surpass that of chicken thyroglobulin. It is concluded that in thyroglobulin there are structural differences in the different animal species near the thyroxine-forming sites bound by these monoclonal antibodies. The known primary sequence similarity does not seem to result, therefore, in identical three-dimensional structures about this site. These differences may reflect species-specific variations in distant regions brought close as a result of chain folding to form the hormonogenic site, such as those around the donor diiodotyrosine residue or in polysaccharide structures. These monoclonal antibodies provide information about the structure of thyroglobulin, which cannot be obtained from knowledge of the amino acid sequence alone.     

Antibodies against preselected peptides to map functional sites on the acetylcholine receptor

Rabbit immune sera and mouse monoclonal antibodies were raised against the synthetic peptide Tyr-Cys-Glu-Ile-Ile-Val matching in sequence residues 127-132 of the alpha-subunit of all nicotinic acetylcholine receptors sequenced so far. Representative cholinergic ligands did not interfere with the binding of these antibodies to the receptor from Torpedo marmorata, indicating that this sequence is not part of the binding sites for cholinergic ligands. The applicability of antigenic sites analysis to the mapping of functional sites on receptor proteins is discussed.     

Topographical separation of the catalytic sites of asparagine synthetase explored with monoclonal antibodies

Monoclonal antibodies which inhibited the enzymatic activity of bovine pancreatic asparagine synthetase were mapped to two topographically separate regions of the enzyme surface using competitive binding assays. Three antibodies which all inhibited glutamine- and NH3-dependent synthesis of asparagine bound to a common antigenic region. A fourth monoclonal antibody which interfered with glutamine binding or cleavage but not with NH3-dependent synthesis of asparagine was mapped to a separate region of the enzyme surface. These findings suggest a topographical separation between the aspartyl-AMP and glutamine-binding sites of bovine pancreatic asparagine synthetase. Three noninhibitory antibodies exhibited conformation-dependent binding and were mapped to a third region of the enzyme. Binding assays were used to demonstrate extensive cross-reaction of these antibodies with asparagine synthetases isolated from bovine liver and sheep pancreas. Substantial cross-reactions were also seen with the enzyme isolated from rat liver or pancreas, a human tumor cell line, and a mouse tumor cell line. Of the four antibodies that inhibited glutamine- and NH3-dependent synthesis of asparagine from ruminant species, only one bound to and inhibited the enzyme from rat liver or mouse cells, which suggests significant structural differences between the ruminant and rodent enzymes.     

Localization of an immunodominant domain on baculovirus-produced parvovirus B19 capsids: correlation to a major surface region on the native virus particle.

An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.     

Respiratory syncytial virus fusion glycoprotein: further characterization of a major epitope involved in virus neutralization

Competition experiments and biological assays with a panel of 15 monoclonal antibodies confirmed the presence of at least four antigenic sites on the fusion protein of human respiratory syncytial virus, three of which were involved in virus neutralization. One antigenic site, recognized by two strongly neutralizing antibodies, was conserved after reduction and denaturation and shown by immunoblotting to be localized on the F1 fragment of the fusion protein. Cleavage of this protein with staphylococcal protease V8 or papain produced a series of smaller peptides from 11 to 7 kilodaltons that retained this important neutralization determinant. Compared with the other neutralization sites, the epitope defined by monoclonal antibody 7C2 thus appears as the major neutralization epitope. Our peptide mapping results support the hypothesis that this major epitope is composed of a continuous sequence on the viral genome.     

Accessibility of antigenic sites recognized by AUA1, HMFG1 and HMFG2 monoclonal antibodies: its influence on antibody binding of live cells

We assessed the immunoreactivity of live and alcohol-fixed monolayers of HRA-19, a rectal adenocarcinoma cell line, to the monoclonal antibodies AUA1, HMFG1 and HMFG2. Differences in staining patterns between live and alcohol-fixed colonies were found. The well-polarized cells forming the centers of the monolayer colonies showed strong membrane staining when the cells were alcohol-fixed prior to AUA1 incubation, but showed no staining when the cells were alive during the incubation. When AUA1 incubation was done both before and after alcohol fixation, membrane staining was again seen, ruling out the possibility of antigenic modulation. Incubation of live cells with AUA1 together with EDTA showed strong staining of dissociating cells. It is concluded that AUA1 antigenic sites, which on polarized cells are basolateral in location, are inaccessible to the antibody-containing culture fluid, which bathes the apical aspects of the cells, but they become accessible after alcohol fixation, or treatment with EDTA. HMFG1 antigenic sites are located on the apical cell membrane, and accordingly, no differences were seen between incubation of live and alcohol-fixed cells when incubated with HMFG1. The antigenic sites of HMFG2 are partly intracellular, and in our monolayer model, the staining of live cells was weaker and more scarce than on alcohol-fixed cells. It is concluded that immunostaining of cytological and histological material of tumours may not adequately predict antibody binding on live cells, and thus, these findings are of importance in the context of selection of monoclonal antibodies for clinical radio-immunotargeting.     

Antigenic Structure of Human Respiratory Syncytial Virus Fusion Glycoprotein

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.     

Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides

Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.     

Radioimmunological analysis of the antigenic variability of influenza virus nucleoprotein

Influenza A virus ability to bind anti-NP monoclonal antibodies to two viral strains has been studied by radioimmunoassay on polyethylene film with the subsequent autoradiographic registration of results. Monoclonal antibodies were obtained to the viral strains differing in antigenic formula of outer glycoproteids and isolated at different time. The studied influenza viruses were divided into seven groups due to their ability to bind monoclonal antibodies. The absence of correlation between the antigenic properties of nucleoprotein and glycoproteids has been registered. Variability of some antigenic sites has been analyzed. The human epidemic strains of influenza virus are different in ability to bind monoclonal antibodies from the viral strains that are connected with animals in nature or laboratory practice.     

Organization of ligand binding sites at the acetylcholine receptor: a study with monoclonal antibodies

We have studied 20 monoclonal antibodies directed against both the solubilized and the membrane-bound receptor from Torpedo marmorata. We find the following: (i) Six of the antibodies compete with cholinergic ligands for receptor binding and, hence, are directed against the ligand binding regions. (ii) Of these six antibodies, two cross-react with receptor from Electrophorus electricus, rat myotubes, and chicken sympathetic ganglia. These two antibodies therefore define a preserved structure within the ligand binding regions. The other four antibodies bind to structures not common between the receptor preparations tested. (iii) From competition binding studies using internally 3H-labeled antibodies, nine nonoverlapping antigenic regions were defined at the surface of the receptor. Three of these regions overlap with the ligand binding regions. Since two of these three regions do not overlap with each other, two structurally distinct ligand binding regions must exist at the receptor. (iv) From competition binding studies with representative cholinergic ligands, the antibodies directed against the ligand binding regions can be subdivided into three groups: one group competes with all ligands tested; the second group competes with all ligands except the bismethonium compounds; the third group competes with all ligands except the bismethonium compounds and tubocurarine. The results are summarized in a model of the organization of ligand binding sites at the receptor: There are two ligand binding regions differing in their antigenic properties. Furthermore, either there exists separate sites for distinct groups of ligands within each of these binding regions or some ligands produce conformational changes of the receptor that reversibly abolish some antigenic sites. In any case, the cholinergic ligands must interact with the receptor by more and/or other structural determinants than are provided by the structure of acetylcholine.