Characterization of glycoproteins of viruses causing hemorrhagic fever with renal syndrome (HFRS) using monoclonal antibodies

Viruses causing hemorrhagic fever with renal syndrome (HFRS) encode two glycoproteins, G1 and G2. For determination of the biological functions of these glycoproteins, we isolated 15 hybridomas secreting monoclonal antibodies directed against the glycoproteins of the B-1 and Hantaan viruses (HV). From results of neutralizing and hemagglutination inhibition (HI) tests, and studies on the antigenic reactivities of the antibodies with other HV-related viruses by immunofluorescence, we classified these hybridoma clones into two groups producing antibodies to the G1 proteins of the B-1 virus, six groups producing antibodies to G2 proteins of the B-1 virus, and four groups producing antibodies to the G2 protein of HV. Of the antibodies to G2 produced by 12 clones, groups A and B had high HI activity with HV-related virus cross-reactivity and moderate neutralizing activity, group C had moderate HI activity with virus specificity but low neutralizing activity, group G had high neutralizing activity and low HI activity, and five other groups had little or no HI or neutralizing activity. Group A reacting with G1 protein had low level of both neutralizing and HI activity, while group B had no HI activity. One clone of monoclonal antibody had high neutralizing activity and no HI activity, but it did not react with either polypeptide by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by the immunoblotting method. These data suggest that both glycoproteins are the targets of neutralizing antibodies. Furthermore, the results indicate that the antigenic determinants with hemagglutination activity are mainly on the G2 protein, and that the domains related to neutralizing activity and to HI activity are separate.     

Mechanisms of neutralization by monoclonal antibodies to different antigenic sites on the bovine herpesvirus type 1 glycoproteins

Monoclonal antibodies directed to different antigenic sites on bovine herpesvirus type 1(BHV-1) glycoproteins were used to study the mechanisms of neutralization of the virus. Three nonoverlapping neutralizing antigenic sites, designated Ia, Ib, and Ic, were defined on gp87. Antibodies to site la which mediated viral neutralization without complement were effective on inhibition of virus adsorption. Antibodies to a single neutralization site on gp71, designated IIa, were able to neutralize the virus without complement even when they were incubated with the virus which had already adsorbed onto the cells. Antibodies directed against gp117 and antibodies against sites Ib and Ic on gp87 required complement for virus neutralization.     

Distinct functions of antigenic sites of the HN glycoprotein of Sendai virus

Monoclonal antibodies specific for the hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus were used to examine the antigenic structure of HN and its role in the initiation of infection and immunity. Using 10 anti-HN antibodies, four distinct antigenic sites designated I-IV were topographically mapped on the HN molecule by competitive-binding assays. To relate the biological functions of HN to its antigenic structure, anti-HN antibodies were analyzed for their inhibitory activity in neuraminidase, hemagglutination, and hemolysis inhibition tests. Antibodies to antigenic site I inhibited hemagglutination and one of these antibodies also inhibited neuraminidase activity. Antibodies to site II inhibited neither activity. However, hemolysis an F protein activity was inhibited, suggesting that these antibodies which bind to HN interfere with F-mediated fusion. Antigenic sites III and IV had different effects on the hemagglutinating and neuraminidase functions of HN: Site III antibodies inhibited hemagglutination while antibodies to site IV only inhibited neuraminidase activity. Antibodies to each antigenic site inhibited virus production. Since antibodies to sites I and III inhibited hemagglutination, it is likely that they block virus adsorption. Antibodies to HN site II only inhibited hemolysis, and therefore, may prevent virus penetration. Antibodies reacting with site IV inhibited virus production after virus penetration. Since neuraminidase activity was the only function inhibited, the viral enzyme may be involved in virus release. The fact that site IV antibodies inhibited neuraminidase but not hemagglutination suggests that these sites are distinct.     

Proteins of lymphocytic choriomeningitis virus: antigenic topography of the viral glycoproteins

Topographical relationships among antigenic sites on the envelope glycoproteins of lymphocytic choriomeningitis virus (LCMV) were established using a panel of monoclonal antibodies (MAb) directed against viral GP-1 and GP-2. Purified MAb were radioiodinated and used as probes in a solid phase competitive binding assay. Epitopes on LCMV GP-1 were found to cluster in four antigenic sites. Five neutralizing MAb raised by immunization with the WE strain of LCMV reacted with a single topographic site, termed GP-1A, which was present on four strains of LCMV examined in this study. A second site, GP-1B, was characterized by two MAb which partially competed with one another and with a subset of neutralizing antibodies. This site appeared to be close to site A and was found to be nonneutralizing. The third site, GP-1C, contained sequential epitopes and was also nonneutralizing. Antibodies binding to site B enhanced the binding of MAb at site C, presumably through a conformational change. In addition to the common neutralizing site A, LCMV Armstrong strain (LCMV-Arm) GP-1 contained a second topographically related neutralizing site, GP-1D, which was specific for LCMV-Arm, absent in WE, and appeared to be the major immunogenic epitope on GP-1 of this virus. Analysis of MAb binding to LCMV GP-2 demonstrated the presence of three overlapping binding sites. GP-2A was defined by two antibodies while GP-2B and C represented binding sites of one antibody each. Guinea pigs primed with LCMV-Arm and challenged with LCMV-WE developed a significant immune response which was directed toward the common major neutralizing site, GP-1A, but had poor responses to the LCMV-Arm specific neutralizing site GP-1D. Immune sera contained antibody to site GP-1B but lacked detectable antibody to GP-1C.     

Biological activities of monoclonal antibodies reactive with antigenic sites mapped on the G1 glycoprotein of La Crosse virus

Monoclonal antibodies have been prepared which are specific for the G1 glycoprotein of La Crosse virus. By competitive radioimmunoassay, 20 IgG-producing clones were found to map in eight antigenic sites; three distinct and five which showed individual patterns of partial competition indicating they may be in close proximity. Unique in situ trypsin cleavage sites on G1 have helped in orienting these defined epitopes relative to the viral membrane. Antibody molecules belonging to one epitope (H) mapped on the trypsin-resistant part of G1 and had negative or extremely low neutralizing and hemagglutination inhibition activities. Seven epitopes were located on the trypsin-sensitive part of G1, a 25,000-Da region which is probably the amino terminus of the protein. Antibodies binding to six of these seven epitopes (A, B, D, E, F, and G) were positive for neutralization and inhibition of hemagglutination, but exhibited a wide range of activities. Epitopes A, F, and G seem to be in an immunodominant region containing the primary site(s) for attachment to cell receptors. Antibody specific for the remaining epitope (C) was unique in that it bound to a site closely adjacent to neutralizing antibody sites, enhanced antibody binding to epitopes A and G, but lacked the capacity to neutralize viral infectivity or inhibit hemagglutination. Enhancement of antibody binding also occurred between two other closely adjacent sites (B and D) and one other distinct epitope (G). In addition, antibody from an IgM-producing clone competed with antibodies to these same four epitopes (B, C, D, and G), indicating they are in close proximity. These data have been used to construct an antigenic map that may now be used as a working model for the study of virus neutralization.     

The antigenic structure of the influenza B virus hemagglutinin: operational and topological mapping with monoclonal antibodies

We have probed the antigenic structure of the influenza B virus hemagglutinin (HA) with monoclonal antibodies specific for the HA of influenza B virus, B/Oregon/5/80. Seventeen laboratory-selected antigenic variants of this virus were analyzed by hemagglutination-inhibition (HI) assays or ELISA and an operational antigenic map was constructed. In addition, the monoclonal antibodies were tested in a competitive binding assay to construct a topological map of the antigenic sites. In contrast to the influenza A virus HA, only a single immunodominant antigenic site composed of several overlapping clusters of epitopes was defined by the HI-positive antibodies. Three variants could be distinguished from the parental virus with polyclonal antisera by HI and infectivity reduction assays suggesting that changes in this antigenic site may be sufficient to provide an epidemiological advantage to influenza B viruses in nature. In addition, two nonoverlapping epitopes of unknown biological significance were identified in the competitive binding analysis by two monoclonal antibodies with no HI activity and little or no neutralizing activity. We previously identified single amino acid substitutions in the HAs of the antigenic variants used in this study (M. T. Berton, C. W. Naeve, and R. G. Webster (1984), J. Virol. 52, 919-927). These changes occurred in regions of the molecule which, by amino acid sequence alignment, appeared to correspond to proposed antigenic sites A and B on the H3 HA of influenza A virus. Correlation with the antigenic map established in this report, however, demonstrates that the amino acid residues actually contribute to a single antigenic site on the influenza B virus HA and suggests significant differences in the antigenic structures of the influenza A and B virus HAs.     

Evidence for functional domains on the reovirus type 3 hemagglutinin

The reovirus σ1 protein (HA) is the polypeptide responsible for specific cell surface binding and hemagglutination. In order to define the biochemical basis of these properties, we have isolated a number of hybridomas secreting monoclonal antibodies directed against the serotype 1 and 3 HA proteins. Using neutralization and hemagglutination inhibition (HI) testing, we have been able to define at least four distinct antigenic regions (epitopes) on the type 3 HA. One class of antibodies had exclusively neutralizing activity; a second class had only HI activity. One antibody had neutralizing and HI activity and the fourth class of monoclonal antibodies had no detectable neutralization or HI activity. Thus there appears to be marked functional specialization within regions of the reovirus type 3 HA.     

Detailed immunologic analysis of the structural polypeptides of rubella virus using monoclonal antibodies

A panel of murine monoclonal antibodies prepared against rubella virus is described. Fourteen of these monoclonal antibodies react with the E1 glycoprotein of rubella virus and define a total of six spacially separate epitopes in competitive inhibition assays. Antibodies binding to epitopes E1(a), E1(b), E1(c), or E1(e) inhibit the hemagglutinin function of the virus, while antibodies binding to epitopes E1(d) or E1(f) do not. Monoclonal antibodies binding to epitopes E1(c) or E1(d) prevent virus infectivity and identify antigen in distinct intracytoplasmic vacuoles of rubella virus-infected Vero cells by indirect immunofluorescence. Monoclonal antibody to epitope E1(f) localizes antigen primarily to the plasma membrane of infected cells, while antibodies binding to epitopes E1(a), E1(b), or E1(e) localize antigen throughout the infected cell's cytoplasm. A single monoclonal antibody is described which only reacts with the mature form of the virion E2 glycoprotein after rubella virus is treated with a disulfide-bond reducing agent. This antibody immunoprecipitates a 43,000 MW precursor to the E2 glycoprotein from lysates of infected cells and localizes its antigen throughout the cytoplasm of infected cells. The five remaining monoclonal antibodies react with the rubella virus C polypeptide. They define four topographically separate epitopes on the C polypeptide, C(a), C(b), C(c), and C(d), each of which is diffusely distributed throughout the cytoplasm of rubella virus-infected cells.     

Topographical mapping of epitopes on the glycoproteins of murine hepatitis virus-4 (strain JHM): correlation with biological activities

Monoclonal hybridoma antibodies (MAb) of defined polypeptide specificity and biological activity were used in a competition binding assay to identify antibody binding sites (epitopes) on the glycoproteins of murine hepatitis virus-4 strain JHM (MHV-4). Individual MAb were labeled with horseradish peroxidase (HRP) and used as probes in a competition enzyme immunoassay (EIA). Four topographically distinct antigenic sites were detected on the E2 glycoprotein of MHV-4. Antibodies reacting with these four determinants provisionally designated A(E2), B(E2), C(E2), and D(E2) had corresponding biological activities (M. J. Buchmeier, H. A. Lewicki, P. J. Talbot, and R. L. Knobler (1984) Virology 132, 261-270). Antibodies to sites A(E2) and B(E2) mediated virus neutralization in vitro and passively protected mice against lethal virus challenge in vivo. Antibody to site C(E2) neutralized virus efficiently in vitro but did not alter disease in vivo, while antibody to site D(E2) neither neutralized nor protected. Two major nonoverlapping antigenic sites were defined on the E1 glycoprotein. Overlapping epitopes A(E1) and B(E1) constituted one site and epitope C(E1) the other.     

Immune responses to wild and vaccine rubella viruses after rubella vaccination

Summary Antibody responses to individual structural proteins (E1, E2, and C) of the M33 wild rubella virus and the RA 27/3 live attenuated rubella strain were assayed by immunoblotting in 11 girls, following immunization with RA 27/3 rubella vaccine. Serum samples were drawn before immunization and at 10 days, 1 month, 1 year, 2 years, and 3 years afterwards. All the subjects showed antibodies to E1 glycoprotein of both the virus strains up to three years after immunization, indicating the importance of E1 in immunity. Antibodies to E1 were always present when with neutralizing activity was observed. Antibodies to E2 protein of both the viruses and to the C protein of the M33 virus gradually disappeared with time in some samples, while antibodies to C protein of the RA 27/3 virus strain were found persistently in all the sera.     

Biochemical and biological characteristics of epitopes on the E1 glycoprotein of western equine encephalitis virus

Antigenic determinants identified by monoclonal antibodies (Mabs) on the E1 glycoprotein of western equine encephalitis (WEE) virus have been characterized by their serological activity, requirements for secondary structure, expression on the mature virion, and their role in protecting animals from WEE virus challenge. On the basis of a cross-reactivity enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition assay, eight antigenic determinants (epitopes) on the E1 glycoprotein have been identified, ranging in reactivity from WEE-specific to alphavirus group reactive. No neutralization of virus infectivity was demonstrable with any of the Mabs. An alphavirus group-reactive hemagglutination (HA) site, a WEE complex-reactive HA site, and a WEE virus-specific HA site were identified. Spatial arrangement of these epitopes was determined by a competitive binding ELISA. Four competition groups defining three distinct antigenic domains were identified. Antibodies directed against four E1 epitopes were capable of precipitating the E1/E2 heterodimer from infected cells or purified virus disrupted with nonionic detergents. These same antibodies precipitated only E1 in the presence of 0.1% SDS. That E1 conformation was important was shown by the inability of antibodies specific for seven of the epitopes to bind to virus denatured in 0.5% SDS. As determined by equilibrium gradient analysis of virus-antibody mixtures, four epitopes were found to be fully accessible on the mature virion, three epitopes were inaccessible, and one epitope was partially accessible to antibody binding. Antibodies specific for three epitopes were able to passively protect mice from WEE virus challenge.     

Characterization of epitopes on the measles virus hemagglutinin

Measles virus hemagglutinin epitopes were analyzed using nine monoclonal antibodies (MAbs) in competitive binding assays (CBA) and by in vitro selection of antigenic variants. In CBA the nine MAbs formed four different but partially overlapping binding groups. In vitro selected variants were analyzed by radioimmune precipitation assay, hemagglutination inhibition (HI), and neutralization assays. Seven operationally distinct MAb antigenic sites could be delineated: two sites were defined by two MAbs to each and five sites by single MAbs. Variants which had lost reactivity to different combinations of MAbs were generated by a branched sequential selection procedure. The most epitopically modified variants had lost reactivity in all assays to eight MAbs. None of these variants showed any major changes in hemagglutination nor neurotropism per se. Only minor changes were detected by HI in reactivity of these variants with human measles antiserum indicating that the epitopic changes were not of major epidemiological significance at least in terms of HI.     

Detection of antibodies to individual proteins of rubella virus

Individual rubella virus structural polypeptides were electroeluted from SDS-polyacrylamide gels. The eluted polypeptides were used, without further purification, as antigens in ELISA assays for the detection of rubella-specific antibodies in patients' sera. This provided a more sensitive detection method than that involving classical serological assays such as HI or VN or that using immunoprecipitation. Antisera against individual viral polypeptides were raised in mice. No haemagglutination inhibition activity was observed in any of these sera and weak virus neutralizing activity was only detected with antiserum to the E1 protein. Antisera to either the E1 or E2(a,b) complex proteins cross-reacted with both the E1 and E2(a,b) complex proteins.     

Effects of specific monoclonal antibodies on La crosse virus neutralization: Aggregation, inactivation by Fab fragments, and inhibition of attachment to baby hamster kidney cells

At high concentrations, several monoclonal antibodies to the G1 glycoprotein of La Crosse (LAC) virus aggregated the virus. To determine whether this accounted for the neutralization, the monoclonal antibodies were digested to make Fab fragments. With one exception, each monovalent antibody neutralized LAC virus to the same extent that bivalent antibody did, although higher concentrations were needed. Fab fragments of synergistic pairs of antibodies also exhibited enhanced binding in a competition binding assay but did not increase neutralization. To determine specific mechanisms for neutralization, the effects of polyclonal or monoclonal antibodies on virus attachment were examined. Polyclonal antibody to LAC virus reduced virus attachment by only 68% although it neutralized 99.99% of the virus. When virus was preincubated with a neutralizing monoclonal antibody to each of seven antigenic regions on G1, only antibody to one region reduced attachment of virus by as much as 92%. Antibodies to two regions that neutralize virus by 90-98% only inhibited attachment by 9 and 13%, respectively. The other antibodies showed intermediate degrees of neutralization and inhibition of attachment. Pairs of antibodies previously shown to be synergistic in neutralizing activity did not inhibit attachment any more than the single antibodies did.     

Virus-like particles as a source for obtaining antigens for diagnosing rubella

Rubella diagnostic agents for hemagglutination inhibition (HI) and enzyme immunoassay (EIA) based on rubella virus-like particles (RVLP) have been developed. Noninfectious RVLPs containing three structural E1, E2, and C proteins were expressed in transfected CHO24S cell culture. HI titer in culture medium was 1:256. Tween-80 treatment and ether increased HI titer 4-6-fold and rendered the antigen higher stability. Immunogenic properties of RVLPs were similar to the native rubella virus in HI test with international reference human rubella serum and sera from convalescents after rubella. Serum of mice immunized with RVLP reacted similarly with RVLP antigens and native virus. Antigen for EIA from RVLP was prepared by concentrating RVLP from culture fluid and partial purification by ultracentrifugation. The results of human sera testing by HI and EIA with RVLP and native virus antigens coincided. RVLP are identical to antigenic structure of the virus, are stable and easily purified, and can therefore be used for commercial production of HI and EIA antigens.     

Biological activities of the structural proteins of Japanese encephalitis virus

Structural proteins V1, V2 and V3 of Japanese encephalitis virus (JEV) were isolated and purified by means of polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and extracted from the sliced gel. The purified and renatured V3 envelope protein was found to bind to receptors for JEV on red blood cells. V3 also bound to haemagglutination inhibition (HI) antibodies from anti-JEV sera. In addition, V3 protein induced neutralizing antibodies against JEV when injected into mice. These facts strongly suggest that V3 protein carries the antigenic epitope(s) that elicit neutralizing antibodies and react with HI antibodies. We conclude the V3 plays an important role in virus infection. V2 protein did not exhibit these biological activities, but antibody against V1 protein, not contaminated with V3 protein, showed slight virus neutralizing activity. Possible implications of these results are discussed.     

Immunological characterisation of the rubella E 1 glycoprotein

Summary Three epitopes have been identified on rubella virion envelope polypeptide E 1 using monoclonal antibodies. Antibodies to two of the epitopes, E 1_EP1 and E 1_EP2, show both haemagglutination inhibition and neutralization activities whereas antibodies to the remaining epitope, E 1_EP3, show neutralizing activity only.With 2 Figures     

The neutralization site on the E2 glycoprotein of Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple conformationally stable epitopes

The neutralization (N) site on the gp56 (E2) surface glycoprotein of the TC-83 vaccine strain of Venezuelan equine encephalomyelitis (VEE) virus has been characterized using monoclonal antibodies. Five new epitopes (E2d-h) were identified three of which could be mapped into the critical N site by using a competitive binding assay (CBA). Antibodies reactive with these three epitopes had either N or N and hemagglutination-inhibition activity. All epitopes contained within this N site elicited monoclonal antibodies that could protect mice from peripheral virus challenge. Antibodies reactive with the N site on other subtypes of VEE virus (IC and II) bound to, but failed to neutralize, TC-83 virus. Epitopes defined by these antibodies could be located outside of the N site on TC-83 virus by CBA. Antigenic activity of all epitopes except E2d was resistant to treatment with 2% SDS, 3% beta-mercaptoethanol, or cleavage with Staphylococcus aureus V8 protease. Those antibodies which defined epitopes located within the N site of TC-83 with CBA bound the same V8 fragments in immunoblots. Those antibodies which defined epitopes not located within the N site bound a different set of fragments than neutralizing antibodies. These results indicate that there is a specific N site on the E2 of VEE virus which undergoes significant antigenic drift while maintaining structural and functional integrity.     

Biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of A/Seal/Massachusetts/1 /80 (H7N7) influenza virus

Monoclonal antibodies to the hemagglutinin (HA) molecule of A/Seal/Mass/1/80 (H7N7) have been prepared and used to establish an operational antigenic map. Four nonoverlapping antigenic areas on the HA of seal influenza viruses were defined. Monoclonal antibodies belonging to two of the groups (III and IV) failed to inhibit hemagglutination of intact virus yet effectively neutralized viral infectivity. These findings could not be explained by differences in affinities and were interpreted in terms of functional differences between the assays. Antibodies that failed to inhibit hemagglutination may bind nearer to the hydrophobic end of the HA molecule and might not block the receptor binding site for erythrocytes. Antibodies to these areas may inhibit infectivity by interference with cell fusion or viral replication. The monoclonal antibodies that failed to inhibit hemagglutination of intact virus nevertheless did inhibit HA activity of purified isolated rosettes of hemagglutinin. The mechanism of inhibition is not resolved but may involve the access of a larger number of antibody molecules to the HA when it is in the form of rosettes compared to when it is located on the viral membrane. The reactivity of seal influenza virus and other H7 viruses from birds with the monoclonal antibodies show that some avian strains possess HA molecules that are closely related to those of seal virus and may therefore be related to viruses that were important in the evolution of this strain. Since HI tests do not necessarily detect all antibodies that neutralize viral infectivity, the question is raised as to whether HI assays alone can be used for determining the efficacy of all influenza virus vaccines.     

A comparative study of monoclonal and monospecific antibodies in determining the immunodominant antigenic sites of influenza virus A (H3N2) hemagglutinin]

Comparative studies of monospecific (MSA) and monoclonal (MCA) antibodies showed MSA to detect three non-overlapping immunodominant sites on the surface of hemagglutinin (HA) molecule whereas MCA established more subtle differences in HA antigenic structure on the level of epitopes with different immunological significance. The activity of MSA and MCA differed in various tests. While MCA were more active in HI and EIA tests, MSA had a higher neutralizing activity, reducing the infectious virus titre by 5.0-7.5 Ig. Similar reduction of the virus biologic activity was observed only with two MCA whereas the other 20 MCA had a poor neutralizing effect (the virus titre reduction not more than by 2.5 Ig). The employment of MSA and MCA gives most complete information on antigenic restructuring of influenza virus HA at the site level and more subtle structures, epitopes, in the process of evolutionary variability.