Discontinuous Antigenic Site Research Articles

Rational Dissection of Binding Surfaces for Mimicking of Discontinuous Antigenic Sites

Peptide-based approaches to mimicking protein interactive regions have relied mainly on linear peptides; however, most binding sites are discontinuous and thus not easily reproducible by a linear sequence. Any attempt to replicate those sites by chemical means must not only integrate all residues involved in the recognition but also provide structural organization to native-like levels. Here we describe a surface mimic approach to the reconstruction of such complex molecular architectures, using as a model a discontinuous antigenic site of foot-and-mouth disease virus that is defined by residues belonging to three different capsid proteins. Our surface mimics are synthetic cyclic peptides, designed in silico, capable of binding antibodies directed to this site, and with demonstrated functional capabilities as vaccines in guinea pigs. Further, by saturation transfer difference NMR, we have determined several antibody binding residues on these peptides.

Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide.

Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulfide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194) (VP=viral protein). Guinea pig antisera to the peptides recognized the viral particle and competed with site D-specific monoclonal antibodies, while inoculation with a simple (not covalently joined to one another) admixture of the three VP1-VP3 sequences yielded no detectable virus-specific serum conversion. Similar results have been reproduced in two bovines. Antisera to the peptides also moderately neutralize FMDV in cell cultures and partially protect guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent, peptide-based vaccine against FMDV.

Synthetic Peptides as Functional Mimics of a Viral Discontinuous Antigenic Site

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71–84), followed by a polyproline module and by VP3(52–62), and a smaller unit corresponding to VP1(188–194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1–VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.

A Rationally Designed Synthetic Peptide Mimic of a Discontinuous Viral Antigenic Site Elicits Neutralizing Antibodies

A Rationally Designed Synthetic Peptide Mimic of a Discontinuous Viral Antigenic Site Elicits Neutralizing Antibodies

Deduced structural model for animal rhabdovirus glycoproteins.

The G protein sequences of fourteen animal rhabdoviruses, representing all four recognized genera (Vesiculovirus, Lyssavirus, Ephemerovirus and Novirhabdovirus) and the ungrouped sigma virus, were aligned using CLUSTAL W and adjusted to account for obvious sequence similarities not detected by the algorithm. Analysis of the alignment indicated remarkable preservation of G protein structural features including cysteine residues, antigenic sites and significant elements of secondary structure (alpha-helices, beta-strands and loops). Twelve highly conserved cysteine residues were assigned numbers (C(I) to C(XII)) according to their location in the alignment. Other cysteine residues were assigned numbers (C0 to C(XIIe)) according to their position relative to the conserved cysteines. The pattern of conservation of cysteine residues and the structural characteristics of identified discontinuous antigenic sites were used to deduce a model for G protein structure. Six absolutely conserved cysteines are predicted to associate in three disulphide bridges (C(I)-C(XII); C(VIII)-C(XI); C(IX)-C(X)) that form the core of the G protein structure and define the common discontinuous antigenic site. The associations of six other highly conserved cysteines (C(II)-C(IV); C(III)-C(V); C(VI)-C(VII)) are predicted by the absence of a specific pair in all viruses within a genus. Of the other cysteines, one pair occurs only in ephemeroviruses and novirhabdoviruses (C0-C(XIIa)); two pairs occur only in ephemeroviruses (C(Ib)-C(VIIIa); C(XIIb)-C(XIIe)); and two pairs occur only in lyssaviruses (C(Ia)-C(VIIIb); C(XIIc)-C(XIId)). The structures predicted by the model account for the preservation of conformational antigenic sites, accommodate genus-specific variations, and are generally consistent with previous observations of G protein structure.

Fine characterization of a V3-region neutralizing epitope in human immunodeficiency virus type 2

We have previously identified two distinct antigenic sites in the third variable region (V3) of human immunodeficiency virus type 2 (HIV-2) corresponding to the principal neutralizing determinant (PND) of HIV-1, the conserved Phe-His-Ser-Gln and Trp-Cys-Arg motifs (positions 315–318 and 329–331), which possibly interact to form a discontinuous antigenic site. The aim of this study was to further identify and characterize the immunogenic sites in the V3-loop of HIV-2 that are important in the binding of neutralizing antibodies and to study in detail the importance of different configurations of peptides corresponding to this region. Peptides representing modifications of the V3-region of HIV-2 SBL6669-ISY were used for immunization of guinea pigs. With one exception, both the Phe-His-Ser-Gln and the Trp-Cys-Arg motifs were required in the peptide sequences to obtain neutralizing hyperimmune guinea pig sera, and the highest titers were obtained after immunization with 20–27 amino acids (aa) long peptides. Neither substitutions nor deletions of residues between the two motifs, nor the addition of peptide sequences representing a T-helper epitope improved the induction of neutralizing antibodies. Computer simulation modeling revealed that the Phe-315, His-316, Trp-329 and Cys-330 are likely to participate in the formation of a discontinuous epitope. Taken together, these data support the hypothesis that the well conserved motifs FHSQ (positions 315–318) and WCR (positions 329–331) of the HIV-2 SBL6669 V3 region are important targets for neutralizing antibodies, and this may have implications for the design of a future HIV-2 vaccine.

Immunochemical mapping of human lutropin: II. Characterization of two monoclonal antipeptide antibodies reacting with the native β-subunit

To investigate the epitopes present on the β-subunit of the human lutropin (hLHβ) and their topographical relationship at the surface of the molecule, we produced two monoclonal antipeptide antibodies, designated LHP03 and LHP04, capable of binding to the radiolabeled 125I-hLHβ and directed to the 43–52 and 110–117 regions of the hLHβ, respectively. Analysis of the accessibility of the epitopes on hLH and on the β-subunit of human chorionic gonadotropin (hCGβ), equine LH (eLHβ) and ovine LH (oLHβ) indicated that: (i) LHP03 binds to both the free hLHβ subunit and dimeric hLH whereas LHP04 binds preferentially to the free hLHβ, (ii) LHP03 recognizes weakly the hCGβ and oLHβ in comparison to hLHβ and (iii) LHP04 binds oLHβ as well as hLHβ but does not bind to hCGβ and eLHβ. The topographical relationship of epitopes recognized by LHP03 and monoclonal antibodies recognizing dimer specific epitopes on hLH allowed us to localize discontinuous antigenic sites that overlaps or are located outside the hHLβ(43–52) region. Together, our results demonstrated that the hHLβ(43–52) portion is accessible on both the free hLHβ subunit and hLH whereas the COOH-terminal portion, hHLβ(110–117), is probably buried at the α/β interface of the hormone.

Two neutralizing domains in the V3 region in the envelope glycoprotein gp125 of HIV type 2.

The purposes of this study were to map the targets for neutralizing Abs in the HIV-2 glycoproteins with particular emphasis on the role of the V3 region. Sera obtained from guinea pig immunized with peptides representing five immunogenic regions of the envelope proteins were used in cross-neutralization experiments with nine HIV-2 isolates. Broad cross-neutralizing activity was elicited by immunization with two peptides representing the central and COOH-terminal portions of the HIV-2 V3 loop. Murine mAbs were established from animals immunized with two corresponding overlapping peptides. Six mAbs showed neutralizing activity against the homologous virus isolate SBL-6669. Peptide absorption experiments were performed to define the target regions for human neutralizing Abs in the HIV-2 envelope glycoproteins. A significant blocking of neutralizing activity of five HIV-2 Ab-positive sera was seen in the presence of two peptides corresponding to the V3 region. Two overlapping deletion sets of peptides, representing amino acids Ser311 and Arg337, were used to identify the role of individual HIV-2 V3 amino acids in the binding of polyclonal and mAbs. Two distinct antigenic sites were identified in this region, the first corresponding to a region including the conserved motif Phe-His-Ser (amino acid 315-317) and the second in proximity of the COOH-terminal cysteine Trp-Cys-Arg (amino acid 329-331). Potentially these two sites can interact to represent a single discontinuous antigenic site. Taken together, these results indicate that V3 is an important neutralizing domain of HIV-2.

Lysine residues in bee venom phospholipase A2 are important for binding to human monoclonal or polyclonal antibodies of the IgG4 isotope.

Discontinuous antigenic sites on bee venom phospholipase A2 (PLA) have been mapped using human monoclonal antibodies or human polyclonal serum antibodies (hpAbs) of the IgG4 isotype from beekeepers or of the IgE isotype from individuals allergic to PLA. Lysine residues of PLA have been specifically modified by acetylation or acylation by treatment with citraconic anhydride of their epsilon-amino groups to analyze their role in antigen-antibody binding. After the modifications, the binding of PLA to the human monoclonal antibodies is lost, whereas the binding to IgG4 hpAbs is significantly decreased. In contrast, the effect on the binding of PLA to IgE hpAbs appears to be more heterogeneous. The data indicate the importance of lysine residues as being part of B cell epitopes in PLA-specific antibodies of the IgG4 isotype, but less so for those of the IgE isotype.

Comparison of serum antibody reactivities to a conformational and to linear antigenic sites in the external envelope glycoprotein of simian immunodeficiency virus (SIVmac) induced by infection and vaccination

Forty-six overlapping peptides (20-mers) representing the amino acid sequence of the external envelope glycoprotein of simian immunodeficiency virus (SIVmac; 32H isolate) were used to investigate linear antigenic sites recognized by antibodies in sera from SIV-infected rhesus macaques and in animals vaccinated with formalin-inactivated SIV. The reactivity to a discontinuous antigenic site as defined by a neutralizing monoclonal antibody was measured by competition assay. The majority of infected macaques recognized three linear antigenic determinants within the V1, V3 and C5 regions of the external glycoprotein. Animals infected with virus derived from the molecular clone SIVmac 32H (pJ5) showed broader reactivity to peptides with half of these animals having antibodies to the V2 region in addition to the V1, V3 and C5 regions. The majority of animals produced antibodies in response to the discontinuous epitope although these responses were weaker in animals infected with molecularly cloned virus. Seven of eight animals given vaccine in syntex adjuvant formulation (saf-1) produced antibodies in response to the discontinuous epitope and all reacted with peptides from the V1, V2 and V3 regions but only half recognized the C5 region. Animals receiving vaccine in alum adjuvant generally showed weaker responses to the discontinuous and linear determinants than those receiving saf-1 adjuvanted vaccine.

Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti-liver-kidney microsome type-1 antibodies (LKM1).

Anti-liver-kidney microsome type-1 antibodies (LKM1), present in sera from a group of patients with autoimmune hepatitis, are directed against P450IID6. Previous work, using cDNA constructions spanning most of the P450IID6 protein defined the main immunogenic site between the amino acids (aa), 254-271 and predicted the presence of other putative immunogenic sites in the molecule. Fusion proteins from new cDNA constructions, spanning so-far-untested regions between aa 1-125 and 431-522, were not recognized by LKM1-positive sera. Synthetic peptides, representing sequences from putative immunogenic regions or previously untested regions, allowed a precise definition of four antigenic sites located between peptides 257-269, 321-351, 373-389 and 410-429, which were recognized, respectively, by 14, 8, 1 and 2 out of 15 LKM1-positive sera tested. The minimal sequence of the main antigenic site (peptide 257-269) recognized by the autoantibody was established to be WDPAQPPRD (peptide 262-270). In addition, deletion and replacement experiments showed that aa 263 (Asp) was essential for the binding of the autoantibody to peptide 262-270. Analysis of the second most frequently recognized peptide between aa 321-351, was performed using peptides 321-339 and 340-351 in competitive inhibition studies. Complete elimination of antibody binding to peptide 321-351 obtained by absorption of both shorter peptides indicated that peptide 321-351 is a discontinuous antigenic site. LKM1-positive sera reacting against peptide 321-351 recognized either both the shorter peptides or just one of them preferentially. Results of the present study suggest that the production of LKM1 antibodies is an antigen-driven, poly- or oligoclonal B cell response. The identification of antigenic sites will allow: (i) the development of specific diagnostic tests and (ii) further studies on the pathogenic value of LKM1 antibodies in autoimmune hepatitis.

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22–58), and complex-specific (amino acids 304–332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60–97 and 298–397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60–135; amino acids 60–205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298–397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.

Binding with lysozyme of antibodies against surface-simulation peptides representing the lysozyme antigenic sites.

Previously it had been shown that native lysozyme has three discontinuous antigenic sites (comprising spatially adjacent residues that may be distant in sequence) that were mimicked by surface-simulation synthetic peptides that had the capacity to bind the bulk (97-99%) of the antibody response against native lysozyme. In the present work these three surface-simulation synthetic peptides were coupled to succinoylated bovine serum albumin, and the conjugates were injected into rabbits. Antibodies against each peptide reacted, as expected, only with that peptide, but it was also found that the antibodies could bind with lysozyme, and the complete specificity of this binding was rigorously established. The advantages of these findings in conformational and immunological investigations are outlined.