Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli

Previous characterizations of equine infectious anemia virus (EIAV) glycoprotein variation by DNA sequence analysis and epitope mapping using monoclonal antibodies (MAbs) have revealed the presence of conserved and variable regions within the EIAV env gene. To extend these studies, fragments of the EIAV envelope proteins gp90 and gp45 were expressed in Escherichia coli and used in Western blot analysis with a diverse panel of equine immune sera to identify antigenic segments. All sera from EIAV-infected animals reacted with the carboxyl terminal portion of gp90 and the amino terminal portion of gp45, indicating the highly conserved and immunodominant nature of these regions. Other gp90 segments, both from conserved and variable env sequences, displayed variable reactivities with the panel of equine sera. A panel of MAbs was also used in Western blot assays with the recombinant protein fragments for physical localization of previously identified MAb epitopes. The binding sites of two neutralizing MAbs were localized to a highly variable region of gp90, while nonneutralizing epitopes were localized to conserved and variable regions of the envelope glycoproteins. These results, in addition to localizing important antigenic sites on EIAV glycoproteins, indicate that previously defined conserved and variable env nucleotide sequences indeed encode protein sequences constituting conserved and variable immunogens during persistent infection by EIAV.     

Epitope mapping of the gp53 envelope protein of bovine viral diarrhea virus

Epitopes recognized by nine monoclonal antibodies (mAbs) on the envelope protein, gp53, of two strains of bovine viral diarrhoea virus (NADL and Oregon C24V) were mapped by competitive binding assays and by the characterization and sequence analyses of mAb neutralization escape mutants. This defined an antigenic domain on gp53 that was shared by many BVDV strains, while other less conserved epitopes were possibly distinct. Sequencing of escape mutant viruses revealed that a cluster of three amino acids in the N-terminal half of gp53 were involved in the main antigenic domain shared by both NADL and Oregon C24V viruses, while an amino acid 31 residues further toward the N-terminus was involved in a second site present only on the NADL strain. Since other amino acids defining these epitopes were located at distant positions within the gp53 protein, it is likely that a major domain [corrected] on gp53 consist of composite, conformation-dependent epitopes.     

In vitro isolation of a neutralization escape mutant of equine infectious anemia virus (EIAV)

Summary A neutralization escape mutant (A/1 E) of equine infectious anemia virus was isolated after 13 passages in cell culture in the presence of serum containing antibodies to type- and group-specific determinants of EIAV envelope glycoproteins. Loss of neutralization by the selecting serum correlated with loss of two epitopes in the major envelope glycoprotein gp90 of A/1 E which were present in a parallel variant isolated from a persistently infected pony.     

Equine monoclonal antibodies recognize common epitopes on variants of equine infectious anaemia virus.

Equine-murine xenohybridoma cells were produced using SP2/0 murine myeloma cells and splenic lymph node cells obtained from horses infected with 10(6) TCID50 of single cloned variants of equine infectious anaemia virus (EIAV). The xenohybridomas secreted equine IgG monoclonal antibodies reactive with EIAV in enzyme immunoassays employing purified virus. Seven antibodies were studied in detail. They bound to viral glycoproteins (gp90 or gp45) in radioimmunoprecipitation assays, and reacted with homologous EIAV as well as five other cloned variants of EIAV. When evaluated against a single cloned variant of EIAV (EIAV-WSU5), two antibodies bound to different epitopes on gp90. The five remaining antibodies reacted with the same or overlapping epitopes on gp45. None of the antibodies exhibited viral neutralizing activity.     

Monoclonal antibodies to bovine coronavirus: characteristics and topographical mapping of neutralizing epitopes on the E2 and E3 glycoproteins

Monoclonal antibodies to the Quebec isolate of bovine coronavirus were produced and characterized. Monoclonal antibodies to both the E2 and the E3 glycoproteins were found to efficiently neutralize virus in vitro. None of the monoclonal antibodies directed against the E1 glycoprotein neutralized virus infectivity. Neutralizing monoclonal antibodies to the E2 glycoprotein were all found to immunoprecipitate gp190, gp100, and their intracellular precursor protein gp170. Neutralizing monoclonal antibodies to the E3 glycoprotein immunoprecipitated gp124 and showed differential reactivity to its precursor proteins gp59 and gp118. These monoclonal antibodies also showed differential reactivity to an apparent degradation product of E3. Neutralizing monoclonal antibodies to E2 bound to two distinct nonoverlapping antigenic domains as defined by competitive binding assays. Neutralizing monoclonal antibodies to the E3 glycoprotein also bound to two distinct antigenic sites as defined by competitive binding assays plus a third site which overlapped these regions. Other results indicated that one domain on the E3 glycoprotein could be further subdivided into two epitopes. Thus four epitopes could be defined by E3-specific monoclonal antibodies.     

Equine infectious anemia virus (EIAV) Humoral responses of recipient ponies and antigenic variation during persistent infection

Summary Three ponies were inoculated with plasma containing 10^4.8 TCID₅₀ of equine infectious anemia virus (EIAV) and observed for 165 to 440 days. Each pony developed a febrile response within 3 weeks of infection during which a plasma viremia 10^3.5 TCID₅₀/ml was observed. Analyses of four isolates from sequential febrile episodes in a single pony were conducted by two-dimensional tryptic peptide maps and with monoclonal antibodies in immunoblots. Structural and antigenic alterations were observed in the envelope glycoproteins gp90 and gp45, with greatest variation in gp90. Specific IgG to EIAV gp90, gp45, and p26 of homologous and heterologous isolates was detectable by immunoblots within one month after infection although IgG levels to gp45 at this time were relatively low. The group-specific determinants of gp90 and gp45 were more antigenic than those of p26; however, binding of IgG to these determinants did not correlate with neutralization of EIAV as assayed in fetal equine kidney cells. Neutralizing antibodies were first detectable within two months of infection and only neutralized viruses isolated prior to serum collection. Neutralizing activity of sera collected later in the infection was broadly reactive regardless of the number of clinical episodes the donor had suffered.     

Epitopes functional in neutralization of varicella-zoster virus.

By competition neutralization assay using monoclonal antibodies (MAbs) to varicella-zoster virus (VZV) glycoproteins (gps), we attempted to determine the topographical relationship of epitopes which are functional in VZV neutralization. MAbs against gpI interfered moderately to strongly with neutralization of MAbs against gpIII, and one antigenic domain with two distinct epitopes was identified on gpIII. Competition neutralization assays performed with MAbs to gpI revealed at least three distinct antigenic domains: the first contained two complement-dependent neutralizing epitopes; the second contained five complement-dependent neutralizing, overlapping epitopes and one nonneutralizing, nonoverlapping epitope; and the third contained one complement-enhanced neutralizing epitope. Competition neutralization assays performed with MAbs to gpIV showed one antigenic domain with two distinct epitopes which competed with nonneutralizing gpI MAbs. gpII did not interfere with neutralization of gpI, gpIII, or gpIV. Our data suggest that neutralizing and nonneutralizing MAbs can interfere with the action of viral neutralization either by inhibition or by enhancement. This report describes the epitope mapping of VZV gps by a functional biological assay.     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

Equine infectious anemia virus, a putative lentivirus, contains polypeptides analogous to prototype-C oncornaviruses

The polypeptide composition of purified radioactive leucine or glucosamine-labeled equine infectious anemia virus (EIAV) was investigated using guanidine hydrochloride gel filtration (GHCI-GF) and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to Friend murine leukemia virus (FLV), a prototype-C oncornavirus. The apparent molecular weights and relative abundance of each EIAV polypeptide were calculated. The results of these studies indicate that EIAV contains four major nonglycosylated proteins (p26, p15, p11, and p9) and two glycoproteins (gp90 and gp45), which together account for greater than 95% of the total virion protein. Four minor polypeptides of unknown significance were also detected reproducibly. EIAV gp90 appears to be the more heavily glycosylated polypeptide, while the gp45 component aggregates in 6 M GHCI, evidently reflecting a hydrophobic character. No disulfide linkages were detected between the EIAV glycoproteins. These observations demonstrate for the first time that EIAV contains polypeptides analogous to prototype-C oncornaviruses, such as FLV. However, the demonstrated serological unrelatedness between EIAV and FLV was reflected in biochemical differences in protein apparent molecular weights and by the resistance of EIAV nonglycosylated proteins to dissociation in GHCI, a property shared by visna virus.     

Characterization of monoclonal antibodies to bovine enteric coronavirus and antigenic variability among Quebec isolates

Summary Twenty monoclonal antibodies (MAbs) were prepared against the Mebus strain of bovine enteric coronavirus, 14 of them reacting with the peplomeric S (gp 100) glycoprotein. Competition binding assays allowed the definition of at least 4 distinct antigenic domains for the S glycoprotein, designated as A, B, C, and D; epitopes associated to neutralizing activity being located in sites A, B, and C. One MAb directed to the hemagglutinin HE (gp 140/gp 65) glycoprotein inhibited the hemagglutinating activity of the virus, but had no neutralizing activity. Comparison of Quebec enteropathogenic BCV isolates using polyclonal antiserum and MAbs directed to the S glycoprotein confirmed their close antigenic relationship, but also revealed the occurrence of at least three distinct antigenic subgroups. Antigenic domain D was highly conserved among BCV isolates, as well as non-neutralizing epitopes assigned to antigenic domains A and C. The Minnesota strain of turkey enteric coronavirus could be distinguished from BCV isolates by MAbs directed to epitopes of antigenic domain C, whereas human coronavirus HCV-OC 43 could be distinguished by MAbs directed to epitopes of antigenic domain A. The porcine hemagglutinating encephalomyelitis virus could be distinguished from the other hemagglutinating coronaviruses by neutralizing epitopes located on antigenic domains A, B, and C.     

Neutralizing determinants of canine herpesvirus as defined by monoclonal antibodies

Summary Monoclonal antibodies (MoAbs) against canine herpesvirus (CHV) were produced to identify the immunogenic proteins of the virus carrying neutralizing determinants. A panel of 24 MoAbs showing neutralizing activities was obtained and tentatively classified into 3 different groups based on their reactivity patterns in immunoblotting analysis. Group I consisting of 10 clones was specific for 145/112 kDa; Group II of 9 clones, for 80 kDa; and Group III of 5 clones, for 41 kDa glycoproteins (gps). Complement-requirement for neutralizing activities of the MoAbs suggests that gp 145/112 and gp 80 elicit mainly complement-requiring and -enhanced neutralizing antibodies, while gp 41 elicits complement-independent ones. In addition, these MoAbs were used in ELISA additivity tests for functional and topographical mapping of epitopes in each of the CHV gp. The results indicated that antigenic reactivities of gp 145/112 and gp 80 were, respectively, localized on at least 5 and 7 overlapping epitopes. On the other hand, 4 epitopes were identified on gp 41.     

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.     

Topographical analysis of viral epitopes using monoclonal antibodies: Mechanism of virus neutralization

The mouse mammary tumor virus (MMTV) was used in neutralization studies with monoclonal antibodies (monoclones) to the major external glycoprotein gp52. Monoclones to MMTV gp52 showed that epitopes with different antigenic specificities were targets for virus neutralization. These epitopes were group specific (common to C3H, GR, RIII, and C3Hf MMTVs), class specific (common to C3H and GR MMTVs), and type specific (unique to C3H MMTV). Competition antibody binding assays using a radioiodinated C3H MMTV type-specific neutralizing monoclone revealed that all the monoclones that neutralized virus infectivity also blocked the binding of the type-specific monoclone to C3H MMTV. A topographically distinct site from this determinant was found using other monoclones which did not compete for the binding of the type-specific monoclone and their epitopes did not function as targets for neutralizing antibodies. However, these nonneutralizing monoclones could neutralize virus infectivity in the presence of antimouse IgG sera. Therefore, two topographically distinct sites could be identified on a single protein of the virus. One site functioned as a target for neutralization and the second site bound antibody but did not affect neutralization. The observation that virus bound to cells was no longer susceptible to neutralization by monoclones showed that neutralization affected virus prior to its binding to cell surface receptors. This result further indicated that antibody prevented virus adsorption to cells and that neutralization was mediated by steric hindrance from antibodies binding to epitopes adjacent to those determinants which functioned in virus binding to cell surface receptors.     

Recombinant gp135 envelope glycoproteins of caprine arthritis-encephalitis lentivirus variants inhibit homologous and heterologous variant-specific neutralizing antibodies.

The envelope (env) genes of two antigenic variants of caprine arthritis-encephalitis virus (CAEV), defined by serum neutralization, were expressed in vaccinia virus. Recombinant gp135 envelope glycoprotein competitively inhibited neutralizing activity of serum from CAEV-infected goats, indicating gp135 is a dominant target antigen of CAEV neutralizing antibody. In addition, type-specific neutralizing activity of goat serum directed against one variant was inhibited by both homologous and heterologous variant recombinant gp135. Hence, a CAEV variant env gene encodes type-specific neutralization epitopes of both variants. The results indicate that antigenic variation of CAEV involves env gene mutations encoding amino acid differences outside conserved neutralization epitopes affecting epitope exposure to the immune system.     

Antigenic analysis of the surface glycoproteins of a Venezuelan equine encephalomyelitis virus (TC-83) using monoclonal antibodies

Splenic lymphocytes from BALB/c mice immunized with the TC-83 vaccine strain of Venezuelan equine encephalomyelitis virus (VEE) were fused to the nonsecreting myeloma cell line Sp2/0-Ag 14. Fusion products were screened for antibody synthesis using an enzyme-linked immunosorbent assay (ELISA) with purified TC-83 virus. Antigenic specificity of cloned hybridoma cells was determined by ELISA or radioimmune precipitation using purified E2 (gp56), El (gp50), and capsid. Antibodies were charcterized by chromatography on protein A-Sepharose and by isoelectric focusing. Analysis of antigenic epitopes by cross-reactivity panels using closely related VEE strains and competitive binding assays indicated the presence of three epitopes on the gp56 and four epitopes on the gp50. Close spatial arrangement of gp56 and gp50 in the native virion could be demonstrated. The biologic functions of hemagglutination and virus neutralization were primarily associated with only one antigenic epitope present on the gp56.     

Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: the envelope of the human immunodeficiency virus (HIV-1).

Synthetic peptides and anti-peptide antibodies have been widely used as probes to map B- and T-cell epitopes on proteins. Such probes also have the potential to delineate contact sites involved generally in protein-protein interactions or in association of domains within a protein. We applied peptide/anti-peptide probes to define: (1) regions on the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41 involved in the association between these two glycoproteins; and (2) sites on gp120/gp41, essential for the association of HIV-1 with the CD4 cell receptor. Results of this examination suggested the following: (1) two segments on gp120, encompassing residues (102-126) and (425-452), contribute to the binding site for CD4 and are expected to be juxtaposed in the folded gp120 chain; (2) portions of immunodominant gp120 and gp41 epitopes, encompassing residues (303-338) and (579-611), respectively, appeared to be involved in the gp120-gp41 association, as suggested by direct binding studies and by the limited accessibility of these epitopes on HIV-1 virions: other portions of gp120 also appeared to contribute to the association between these two glycoproteins; (3) there is a partial overlap between gp41 and CD4 binding sites on gp120; (4) the fusion domain and a segment (637-666) of gp41 are not accessible to antibodies after oligomerization of gp41; and 5) the gp120-gp41 association was blocked by aurintricarboxylic acid, suggesting the possibility of developing antiviral compounds interfering with HIV-1 assembly.     

Biologically active epitopes of bovine leukemia virus glycoprotein gp51: their dependence on protein glycosylation and genetic variability

A panel of monoclonal antibodies to the bovine leukemia virus envelope glycoprotein (BLV gp51) has previously demonstrated the association of the biological activities of the virus (infectivity, syncytia induction) with three out of eight epitopes of gp51. In BLV-infected cells, the unglycosylated homolog of the precursor to the BLV envelope glycoproteins (gPr72env) is a 47,000-MW polypeptide. Immunoprecipitation studies with monoclonal antibodies show that the neutralizing antibody-inducing sites, although present in gPr72env, are not conserved in the 47,000-MW unglycosylated homolog. Finally, it is demonstrated that the neutralizing antibody-inducing sites of gp51 are subject to antigenic variation among BLV isolates of the same or different geographical origins.     

Characterization of neutralizing sites in the second variable and fourth variable region in gp125 and a conserved region in gp36 of human immunodeficiency virus type 2.

Several determinants of human immunodeficiency virus (HIV) have been suggested to harbor sites important for neutralization. The third variable region (V3) of the envelope glycoprotein (gp) is an important neutralizing determinant for both serotypes of HIV. The localization of additional neutralizing regions is an urgent task because the virus appears to mutate to phenotypes that escape neutralizing antibodies. Therefore, we have focused on the possibility of finding other immunodominant regions in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2). By immunization of guinea pigs with peptides corresponding to different selected regions of gp125 and gp36, we have found three antigenic determinants located in the V2 and V4 regions of the envelope protein gp125, and one region in the glycoprotein gp36, which are important for human antibody binding and also as targets for neutralization. The peptide representing the V2 region had the most pronounced capacity to induce neutralizing anti-HIV-2 antibodies in guinea pigs. Neutralizing activity was also detected in an antipeptide guinea pig sera representing a linear site in gp36, amino acids 644-658. A substitution set of peptides representing the conserved antigenic site in the central part of gp36 was used to identify the role of individual amino acids important for human antibody binding.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4- binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.      

Acute infectious bursal disease in poultry: Immunological and molecular basis of antigenicity of a highly virulent strain

This study has confirmed, by the use of immunological and molecular tools, that the recent failures of vaccination against infectious bursal disease (IBD) encountered in Europe were not related to antigenic variation, but to increased virulence of the circulating IBD virus strains. Neutralizing monoclonal antibodies (Mabs) showed that the vaccines of intermediate virulence and the pathogenic strain 849VB had a similar pattern of reactivity in ss neutralization tests. Four distinct epitopes could be defined in seroneutralization and addition ELISA tests. All neutralizing Mabs bound to the structural VP2 protein only in its native form. Moreover, Mabs which did not neutralize some strains precipitated well the VP2 protein from extracts of cells infected with the same virus. This suggests that slight changes in the conformation of the epitope were sufficient to allow the virus to escape to neutralization. VP2 sequencing results confirmed that the neutralizing epitopes are clustered in the variable domain which is highly hydrophobic and flanked by two major hy-drophilic peaks. Three potential 'minor' antigenic sites were identified within the hydrophobic region. Comparison of the VP2 sequence of 849VB strain with other highly virulent isolates showed that they are close together and clearly distinct from 'classical' strains. Moreover, sequencing of IBD vaccines revealed that some of them had not been cloned.