Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies.

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level.     

Analysis of neutralizing epitopes on foot-and-mouth disease virus.

For the investigation of the antigenic determinant structure of foot-and-mouth disease virus (FMDV), neutralizing monoclonal antibodies (MAbs) against complete virus were characterized by Western blot (immunoblot), enzyme immunoassay, and competition experiments with a synthetic peptide, isolated coat protein VP1, and viral particles as antigens. Two of the four MAbs reacted with each of these antigens, while the other two MAbs recognized only complete viral particles and reacted only very poorly with the peptide. The four MAbs showed different neutralization patterns with a panel of 11 different FMDV strains. cDNA-derived VP1 protein sequences of the different strains were compared to find correlations between the primary structure of the protein and the ability of virus to be neutralized. Based on this analysis, it appears that the first two MAbs recognized overlapping sequential epitopes in the known antigenic site represented by the peptide, whereas the two other MAbs recognized conformational epitopes. These conclusions were supported and extended by structural analyses of FMDV mutants resistant to neutralization by an MAb specific for a conformational epitope. These results demonstrate that no amino acid exchanges had occurred in the primary antigenic site of VP1 but instead in the other coat proteins VP2 and VP3, which by themselves do not induce neutralizing antibodies.     

Polyoma viruses with mutations at endonuclease HindII site 1: alterations at the COOH terminus of VP1.

Four mutants of polyoma virus lacking endonuclease HindII site 1 were isolated and characterized with respect to the VP1 coding sequence. Three of these mutants had deletions that removed 0.2 to 0.3% of the genome. All three deletion mutants encoded VP1 proteins that were smaller than wild type and that lacked one or more tryptic peptides normally found in the wild-type VP1 protein. Our results suggest the HindII site 1 is at, or very near, the carboxy terminal end of the coding sequence for VP1. A model for the peptide organization in that region is presented.     

Hepatitis A virus mutant spectra under the selective pressure of monoclonal antibodies: codon usage constraints limit capsid variability

Severe structural constraints in the hepatitis A virus (HAV) capsid have been suggested as the reason for the lack of emergence of new serotypes in spite of the occurrence of complex distributions of mutants or quasispecies. Analysis of the HAV mutant spectra under immune pressure by the monoclonal antibodies (MAbs) K34C8 (immunodominant site) and H7C27 (glycophorin binding site) has revealed different evolutionary dynamics. Populations composed of complex ensembles of mutants with very low fitness or single dominant mutants with high fitness permit the acquisition of resistance to each of the MAbs, respectively. Deletion mutants were detected as components of the mutant spectra: up to 61 residues, with an average of 19, and up to 83 residues, with an average of 45, in VP3 and VP1 proteins, respectively. A clear negative selection of those replacements affecting the residues encoded by rare codons of the capsid surface has been detected through the present quasispecies analysis, confirming a certain beneficial role of such clusters. Since these clusters are located near or at the epitope regions, the need to maintain such clusters might prevent the emergence of new serotypes.     

Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization.

A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens.     

Evidence for a complex structure of neutralization antigenic site I of poliovirus type 1 Mahoney

We have selected neutralization escape mutants by using a monoclonal antibody (nt-MAb) against a sequential epitope between amino acids 93 through 104 (neutralization antigenic site I) of poliovirus type 1 Mahoney. The majority of mutants were also resistant against five strain-specific nt-MAbs which recognized conformation-dependent epitopes, suggesting that the neutralization antigenic site I must be involved in the formation of such epitopes. An analysis of all mutants by the binding of nt-MAbs and by isoelectric focusing of VP1 allowed discrimination of five classes of mutants. Sequence analysis of mutant RNAs revealed point mutations and deletions in the antibody-binding site.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes.

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1.     

Topographical rearrangements of visna virus envelope glycoprotein during antigenic drift.

Visna virus undergoes antigenic drift during persistent infection in sheep and thus eludes neutralizing antibodies directed against its major envelope glycoprotein, gp135. Antigenic variants contain point mutations in the 3' end of the genome, presumably within the envelope glycoprotein gene. To localize the changes in the viral proteins of antigenic mutants, we isolated 35 monoclonal antibodies (MAbs) against the envelope glycoprotein gp135 or the major core protein p27 of visna virus. The MAbs defined five partially overlapping epitopes on gp135. We used the MAbs and polyclonal immune sera directed against visna virus, gp135, or p27 in enzyme-linked immunosorbent assays to compare visna virus (strain 1514) with antigenic mutants (LV1-1 to LV1-6) previously isolated from a single sheep persistently infected with plaque-purified strain 1514. Polyclonal immune sera and anti-core p27 MAbs failed to distinguish antigenic differences among the viruses. By contrast, the anti-gp135 MAbs detected changes in all five epitopes of the envelope glycoprotein. Three gp135 epitopes, prominently exposed on strain 1514, were lost or obscured on the mutants; two covert gp135 epitopes, poorly exposed on strain 1514, were reciprocally revealed on the mutants. Even virus LV1-2, which is indistinguishable from parental strain 1514 by serum neutralization tests and which differs from it by only two unique oligonucleotides on RNase-T1 fingerprinting, displayed global changes in gp135. Our data suggest that visna virus variants may emerge more frequently during persistent infection than can be detected by serological tests involving the use of polyclonal immune sera, and the extent of phenotypic changes in their envelope glycoproteins may be greater than predicted by the small number of genetic changes previously observed. We suggest that topographical rearrangements in the three-dimensional structure of gp135 may magnify the primary amino acid sequence changes caused by point mutations in the env gene. This may complicate strategies to construct lentiviral vaccines by using the envelope glycoprotein.     

Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection

The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341-1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.     

Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins.

The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.     

Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.     

Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

Heterogeneity of apolipoprotein E epitope expression on human lipoproteins: importance for apolipoprotein E function

The conformations of apolipoproteins on the surfaces of lipoprotein particles affect their physiologic functions. The conformations of apoE on plasma lipoproteins were examined using a panel of eight anti-apoE monoclonal antibodies (MAbs). The antibodies, which reacted with the major isoforms of apoE (E2, E3, and E4), defined at least five epitopes on apoE. Proteolytic fragments and synthetic peptides of apoE were used in binding assays to assign antibody epitopes; the epitopes were all localized to the middle third of the apoE molecule. The expression of apoE epitopes on isolated apoE and on lipoproteins was probed in competitive microtiter plate immunoassays using the anti-apoE MAbs, 125I-labeled apoE as tracer, and isolated apoE, intermediate density (IDL), very low density (VLDL1-3), and high density (HDL2 and HDL3) lipoproteins as competitors. The antibodies determined the patterns of competition exhibited by the lipoprotein preparations. Antibodies of the IgM class (WU E-1, WU E-2, WU E-3) defined two sets of conformation-dependent epitopes that were assigned towards the middle and the carboxyl terminal of the middle third of apoE. Competition curves using these antibodies, apoE, and lipoproteins showed a large variability in ED50 values. MAbs WU E-4, WU E-7, and WU E-10 defined epitopes near the receptor recognition site on apoE. Competition curves demonstrated small ranges of ED50 values. MAbs WU E-11 and WU E-12, which defined epitopes toward the amino-terminal region of apoE, exhibited competition curves for apoE and lipoproteins that had consistent, but wider ranges of ED50 values. There was no strict relationship between lipoprotein flotation rates and epitope expression for any of the MAbs. Immunoaffinity chromatography of VLDL subfractions on four different MAb columns indicated that the differences in the competitive abilities of VLDL subfractions were partly due to heterogeneity of apoE epitope expression within any population of particles. VLDL particles specifically retained on two different anti-apoE MAb columns were better competitors than unretained fractions for 125I-labeled LDL binding to the apoB, E-receptor of cultured human fibroblasts, suggesting that increased accessibility of apoE on the surface of VLDL is associated with increased receptor recognition. These data suggest that individual epitopes of apoE can be modulated; epitope expressions are not determined solely by the sizes and/or densities of lipoprotein particles; and differences in apoE conformation have significant metabolic consequences.     

Molecular and structural bases for the antigenicity of VP2 of infectious bursal disease virus

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is responsible for a highly contagious and economically important disease causing immunosuppression in chickens. IBDV variants isolated in the United States exhibit antigenic drift affecting neutralizing epitopes in the capsid protein VP2. To understand antigenic determinants of the virus, we have used a reverse-genetics approach to introduce selected amino acid changes-individually or in combination-into the VP2 gene of the classical IBDV strain D78. We thus generated a total of 42 mutants with changes in 8 amino acids selected by sequence comparison and their locations on loops P(BC) and P(HI) at the tip of the VP2 spikes, as shown by the crystal structure of the virion. The antibody reactivities of the mutants generated were assessed using a panel of five monoclonal antibodies (MAbs). Our results show that a few amino acids of the projecting domain of VP2 control the reactivity pattern. Indeed, the binding of four out of the five MAbs analyzed here is affected by mutations in these loops. Furthermore, their importance is highlighted by the fact that some of the engineered mutants display identical reactivity patterns but have different growth phenotypes. Finally, this analysis shows that a new field strain isolated from a chicken flock in Belgium (Bel-IBDV) represents an IBDV variant with a hitherto unobserved antigenic profile, involving one change (P222S) in the P(BC) loop. Overall, our data provide important new insights for devising efficient vaccines that protect against circulating IBDV strains.     

Monoclonal antibodies against human immunodeficiency virus type 1 integrase: epitope mapping and differential effects on integrase activities in vitro.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN.     

Exploration of antigenic variation in gp120 from clades A through F of human immunodeficiency virus type 1 by using monoclonal antibodies.

The reactivities of a panel of 14 monoclonal antibodies (MAbs) with monomeric gp120 derived from 67 isolates of human immunodeficiency virus type 1 of clades A through F were assessed by using an antigen-capture enzyme-linked immunosorbent assay. The MAbs used were all raised against gp120 or gp120 peptides from clade B viruses and were directed at a range of epitopes relevant to human immunodeficiency virus type 1 neutralization: the V2 and V3 loops, discontinuous epitopes overlapping the CD4-binding site, and two other discontinuous epitopes. Four of the five V3 MAbs showed modest cross-reactivity within clade B but very limited reactivity with gp120s from other clades. These reactivity patterns are consistent with the known primary sequence requirements for the binding of these MAbs. One V3 human MAb (19b), however, was much more broadly reactive than the others, binding to 19 of 29 clade B and 10 of 12 clade E gp120s. The 19b epitope is confined to the flanks of the V3 loop, and these sequences are relatively conserved in clade B and E viruses. In contrast to the limited reactivity of V3 MAbs, CD4-binding site MAbs were much more broadly reactive across clades, two of these MAbs (205-46-9 and 21h) being virtually pan-reactive across clades A through F. Another human MAb (A-32) to a discontinuous epitope was also pan-reactive. The CD4-binding site is strongly conserved between clades; but when considering the epitopes near the CD4-binding site, clade D gp120 appears to be the most closely related to clade B and clade E appears to be the least related. A tentative rank order for these epitopes is B/D-A/C-E/F. V2 MAbs reacted sporadically within and between clades, and no clear pattern was observable. While results from binding assays do not predict neutralization serotypes, they suggest that there may be antigenic subtypes related, but not identical, to the genetic subtypes.     

Mapping and characterization of antigenic epitopes and the nucleic acid-binding domains of the VP6 protein of bluetongue viruses.

Heterologously expressed VP6 and truncated VP6 proteins of bluetongue virus (BTV) serotype 11 purified to near homogeneity were used for structure and function analyses. The yield of the expressed VP6 was host cell dependent. Six antigenic epitopes of VP6 of BTV were identified and mapped by immunoblot analyses and enzyme-linked immunosorbent assay with oligoclonal antibodies. These determinants were surface accessible and conserved among the cognate VP6 proteins of five U.S. BTV serotypes. The amino acid sequences and sizes of these six antigenic epitopes were determined, and their precise locations were also mapped and confirmed by deletion analyses. The nucleic acid binding activities of VP6, confirmed by electrophoretic mobility shift assay, were concentration dependent. The binding activities and affinities of the purified expressed VP6 protein towards double-stranded RNA and double-stranded DNA were similar. Two domains of VP6, corresponding to three of the six antigenic epitopes, were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6 by electrophoretic mobility shift assay and deletion mutant analyses. Synthetic oligopeptides corresponding to these three regions also exhibited similar concentration-dependent nucleic acid binding activities.