Native Epitopes Research Articles

Production and characterization of murine monoclonal antibodies against Haemophilus parasuis and study of their protective role in mice

Monoclonal antibodies (MAbs) against Haemophilus parasuis were obtained by the fusion of SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with a whole-bacterial-cell suspension (WC) of H. parasuis strain SW124 (serotype 4). Two MAbs showing strong reactivity in ELISA were further characterized using SDS-PAGE and Western-blot assays. Different treatments of the WC indicated that MAbs 4D5 and 4G9 identified epitopes of proteinic and polysaccharidic nature, respectively. Electron microscopic examination revealed that, unlike the proteinic epitopes, the lipopolysaccharidic epitopes were exposed on the surface of the cell. Using coagglutination, Western-blot and dot-blot assays it was found that both MAbs recognized common epitopes of all the reference strains and field isolates of H. parasuis. None of the other bacteria tested reacted with the MAbs. These results indicated that both the proteinic and polysaccharidic antigens carried species-specific epitopes. It is suggested that these MAbs may potentially be useful for identification of H. parasuis isolates as well as for developing serological diagnostic tools. MAbs 4D5 and 4G9 were unable to kill H. parasuis in vitro in the presence of complement. However, an enhanced bacterial clearance from blood was observed in mice inoculated with either of the MAbs. Highly significant protection was observed in mice using MAb 4G9. This is believed to be the first report of MAbs capable of identifying common species-specific antigens of H. parasuis and of their implication in protection against challenge infection in mice.

Immunization of HLA-A*0201 and/or HLA-DPβ1*04 Patients with Metastatic Melanoma Using Epitopes from the NY-ESO-1 Antigen

HLA class I-restricted peptides are often used in peptide vaccine regimens. There is strong evidence that many of these peptides can generate specific CD8 T-cell responses in vivo; however, only occasional objective clinical responses have been reported. To test whether provision of "help" would enhance antitumor immunity, the authors initiated a clinical trial in which patients with metastatic melanoma were immunized against the NY-ESO-1 tumor antigen, using an HLA-A2-restricted peptide (ESO-1:165V), an HLA-DP4-restricted peptide (NY-ESO-1:161-180), or both peptides given concomitantly. The first cohorts received only ESO-1:165V, using three vaccination schedules. Immunologically, most patients developed immune responses to the HLA-A2-restricted native ESO-1 epitope after vaccination. Peptide vaccine given daily for 4 days appeared to induce immunologic responses more rapidly than if given once a week or once every 3 weeks. In contrast, vaccination using the NY-ESO-1:161-180 peptide induced immune responses in only a few patients. Clinically, one patient who received NY-ESO-1:161-180 peptide alone had a partial response lasing 12 months. Concomitant vaccination with the HLA class II-restricted peptide did not alter the immune response to the HLA class I-restricted peptide form NY-ESO-1. However, vaccination with the HLA-A2-restricted epitope generated primarily T cells that did not recognize tumor after in vitro sensitization. This result raises questions about the use of synthetic peptides derived from NY-ESO-1 as a sole form of immunization.

Sequence similarity and structural homologies are involved in the autoimmune response elicited by mouse hepatitis virus A59

The features of autoantibodies (autoAb) to liver fumarylacetoacetate hydrolase (FAH) elicited in mice infected with mouse hepatitis virus (MHV) were studied by ELISA and western-blot competition assays. All sera tested contained Ab to cryptic FAH epitopes according with results from western-blot tests, whereas ELISA data indicated that some of these same sera did recognize native epitopes of the autoantigen (autoAg). Such differences were detected in individual sera from various mouse strains, and were ascribed to the fact that proteins insolubilized on solid supports expose a variety of conformational and cryptic antigenic determinants. On the other hand, whereas results from both experimental protocols showed that anti-MHV Ab did not cross-react with the soluble autoAg, the opposite situation did not show analogous results. Thus, binding of autoAb to insolubilized FAH could be inhibited by MHV depending on the mouse serum or the experimental protocol used. Additionally, a set of synthetic homologous peptides from mouse FAH and various viral proteins was employed to analyze the Ab repertoire of MHV-infected mice. Results indicated that two homologous peptides were recognized by most Ab: the N-terminal sequences (1–10) from FAH and the nucleocapside, both sharing 50% of identity, and sequence 2317–2326 of the RNA polymerase, a peptide showing 30% of identity with FAH 11–20. Results indicated that MHV-infection triggers at least three distinct Ab populations: anti-MHV, anti-FAH and cross-reacting Ab. This cross-reaction implies either sequential or conformational epitopes from both the viral proteins and the autoAg and may differ between individuals.

A phase I trial of a multi-epitope cancer vaccine (EP-2101) in non-small cell lung (NSCLC) and colon cancer patients

2525 Background: Effective vaccines that mediate clinical responses in cancer patients may require generation of broadly specific cytotoxic T lymphocytes (CTLs) directed against multiple epitopes and tumor-associated antigens (TAAs). The EP-2101 vaccine was designed to induce CTLs against epitopes in carcinoembryonic antigen (CEA), p53, HER-2/neu, and MAGE 2/3, four TAAs frequently over-expressed in NSCLC and colon cancer. Using a rational epitope identification process, 2 epitopes from each TAA were selected for vaccine development. All 8 epitopes displayed high HLA-A2 supertype binding affinity and immunogenicity in a primary in vitro induction assay. Six of the epitopes were analogs containing a single substitution which either enhanced MHC binding or stimulated heteroclitic T cell activation, and 2 epitopes were native sequences. The 8 epitopes together with CAP1–6D, a previously described heteroclitic CEA analog, and the PADRE helper T cell epitope were emulsified in Montanide ISA51 adjuvant and tested for safety and immunogenicity in HLA-A2+ NSCLC (stage IIb/IIIa) and colon (stage III) cancer patients with no detectable disease following standard treatment. Methods: Patients received 6 EP-2101 treatments at 3 wk intervals, at a dose of 5 mg total peptide (0.5 mg/epitope). CTL responses in the peripheral blood of patients were measured using a validated interferon-gamma (IFN-γ) ELISPOT assay. Results: Based on 7 patients who have completed treatment (4 colon, 3 lung), EP-2101 appears to be safe and well-tolerated. To date, IFN-γ-secreting CTL responses were observed in all three patients tested, with 1, 4, and 6 epitope responses being detected respectively. Vaccine responses against 1 native epitope and 5 analog epitopes in EP-2101 have ranged from 23–260 net spots/50,000 cells with <8 net spots detected in pre-vaccination samples. Conclusions: A cancer vaccine delivering 9 TAA epitopes can simultaneously induce CTL responses against multiple epitopes and TAAs in patients. This vaccine consisting of native and analog epitopes may provide effective immunotherapy in patients with diverse types of cancer expressing common TAAs. No significant financial relationships to disclose.

A phase I trial of a multi-epitope cancer vaccine (EP-2101) in non-small cell lung (NSCLC) and colon cancer patients

2525 Background: Effective vaccines that mediate clinical responses in cancer patients may require generation of broadly specific cytotoxic T lymphocytes (CTLs) directed against multiple epitopes and tumor-associated antigens (TAAs). The EP-2101 vaccine was designed to induce CTLs against epitopes in carcinoembryonic antigen (CEA), p53, HER-2/neu, and MAGE 2/3, four TAAs frequently over-expressed in NSCLC and colon cancer. Using a rational epitope identification process, 2 epitopes from each TAA were selected for vaccine development. All 8 epitopes displayed high HLA-A2 supertype binding affinity and immunogenicity in a primary in vitro induction assay. Six of the epitopes were analogs containing a single substitution which either enhanced MHC binding or stimulated heteroclitic T cell activation, and 2 epitopes were native sequences. The 8 epitopes together with CAP1–6D, a previously described heteroclitic CEA analog, and the PADRE helper T cell epitope were emulsified in Montanide ISA51 adjuvant and tested for safety and immunogenicity in HLA-A2+ NSCLC (stage IIb/IIIa) and colon (stage III) cancer patients with no detectable disease following standard treatment. Methods: Patients received 6 EP-2101 treatments at 3 wk intervals, at a dose of 5 mg total peptide (0.5 mg/epitope). CTL responses in the peripheral blood of patients were measured using a validated interferon-gamma (IFN-γ) ELISPOT assay. Results: Based on 7 patients who have completed treatment (4 colon, 3 lung), EP-2101 appears to be safe and well-tolerated. To date, IFN-γ-secreting CTL responses were observed in all three patients tested, with 1, 4, and 6 epitope responses being detected respectively. Vaccine responses against 1 native epitope and 5 analog epitopes in EP-2101 have ranged from 23–260 net spots/50,000 cells with <8 net spots detected in pre-vaccination samples. Conclusions: A cancer vaccine delivering 9 TAA epitopes can simultaneously induce CTL responses against multiple epitopes and TAAs in patients. This vaccine consisting of native and analog epitopes may provide effective immunotherapy in patients with diverse types of cancer expressing common TAAs. No significant financial relationships to disclose.

Cyclic peptides selected by phage display mimic the natural epitope recognized by a monoclonal anti‐colicin A antibody

A 10-mer random peptide library displayed on filamentous bacteriophage was used to determine the molecular basis of the interaction between the monoclonal anti-colicin A antibody 1C11 and its cognate epitope. Previous studies established that the putative epitope recognized by 1C11 antibody is composed of amino acid residues 19-25 (RGSGPEP) of colicin A. Using the phage display technique it was confirmed that the epitope of 1C11 antibody was indeed restricted to residues 19-25 and the consensus motif RXXXPEP was identified. Shorter consensus sequences (RXXPEP, RXXEP, KXXEP) were also selected. It was also demonstrated that the disulfide bond found in one group of the selected peptides was crucial for 1C11 antibody recognition. It was shown that cyclization of the peptides by disulfide bond formation could result in a structure that mimics the natural epitope of colicin A.

CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I–restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon γ release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter–dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.

A human cytotoxic T-lymphocyte epitope and its agonist epitope from the nonvariable number of tandem repeat sequence of MUC-1.

MUC-1/DF-3 remains an attractive target for vaccine therapy. It is overexpressed in the majority of human carcinomas and multiple myeloma. Clinical trials using MUC-1-based vaccines have demonstrated safety, clinical responses, and the induction of T-cell responses directed against MUC-1. Previous studies in experimental models and in clinical trials have demonstrated that altering the amino acid sequence of a "self" epitope can lead to the generation of an enhancer agonist epitope capable of eliciting stronger T-cell responses than the native epitope can. We describe here the identification of six novel class I HLA-A2 epitopes of MUC-1 that reside outside of the variable number of tandem repeat region. Each is shown to have the ability to activate human T cells as measured by IFN-gamma production. One epitope (ATWGQDVTSV, at amino acid position 92-101 and designated P-92), which demonstrated the highest level of binding to HLA-A2 and which induced the highest level of IFN-gamma in human T cells, was further studied for the generation of potential enhancer agonist epitopes. Of four potential agonists identified, one epitope (ALWGQDVTSV, designated P-93L) was identified as an enhancer agonist. Compared with the native P-92 peptide, the P-93L agonist (a). bound HLA-A2 at lower peptide concentrations, (b). demonstrated a higher avidity for HLA-A2 in dissociation assays, (c). when used with antigen-presenting cells, induced the production of more IFN-gamma by T cells than with the use of the native peptide, and (d). was capable of more efficiently generating MUC-1-specific human T-cell lines from normal volunteers and pancreatic cancer patients. Most importantly, the T-cell lines generated using the agonist epitope were more efficient than those generated with the native epitope in the lysis of targets pulsed with the native epitope and in the lysis of HLA-A2 human tumor cells expressing MUC-1. In addition to the identification of novel MUC-1 epitopes outside the variable number of tandem repeat region, the studies reported here describe the first agonist epitope of MUC-1. The employment of this agonist epitope in peptide-, protein-, and vector-based vaccines may well aid in the development of effective vaccines for a range of human cancers.

Reactivity-based one-pot synthesis of oligomannoses: defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody.

An HIV-1 vaccine may eventually be developed from oligomannoses synthesized by a reactivity-based one-pot self-condensation strategy. The compounds were tested for their ability to inhibit the binding of gp120 to the broadly neutralizing HIV-1 antibody 2G12. New epitope mimics (see structure for example) were identified that inhibit this binding as well as, or better than the natural epitope Man9GlcNAc2 (Man=mannose, Glc=glucose).

A powerful combination: the use of positional scanning libraries and biometrical analysis to identify cross-reactive T cell epitopes

Studies on the elucidation of the specificity of the T cell receptor (TCR) at the antigen and peptide level have contributed to the current understanding of T cell cross-reactivity. Historically, most studies of T cell specificity and degeneracy have relied on the determination of the effects on T cell recognition of amino acid changes at individual positions or MHC binding residues, and thus they have been limited to a small set of possible ligands. Synthetic combinatorial libraries (SCLs), and in particular positional scanning synthetic combinatorial libraries (PS-SCLs) represent collections of millions to trillions of peptides which allow the unbiased elucidation of T cell ligands that stimulate clones of both known and unknown specificity. PS-SCLs have been used successfully to study T cell recognition and to identify and optimize T cell clone (TCC) epitopes in infectious diseases, autoimmune disorders and tumor antigens. PS-SCL-based biometrical analysis represents a further refinement in the analysis of the data derived from the screening of a library with a TCC. It combines this data with information derived from protein sequence databases to identify natural peptide ligands. PS-SCL-based biometrical analysis provides a method for the determination of new microbial antigen and autoantigen sequences based solely on functional data rather than sequence homology or motifs, making the method ideally suited for the prediction and identification of both native and cross-reactive epitopes by virtue of its ability to integrate the examination of trillions of peptides in a systematic manner with all of the protein sequences in a given database. We review here the application of PS-SCLs and biometrical analysis to identify cross-reactive T cell epitopes, as well as the current efforts to refine this strategy.

Quaternary solution structures of galectins-1, -3, and -7.

Galectins are a growing family of animal lectins with functions in growth regulation and cell adhesion that bind beta-Gal residues in oligosaccharides. Evidence indicates that some of the biological properties of galectins are due to their cross-linking activities with multivalent glycoconjugate receptors. Therefore determination of the quaternary solution structures of these proteins is important in understanding their structure-function properties. The present study reports analytical sedimentation velocity and equilibrium data for galectins-1, -3, and -7 in the absence and presence of bound LacNAc, the natural ligand epitope. Galectin-1 from bovine heart and recombinant human galectin-7 were found to be stable dimers by both methods. In contrast, recombinant murine galectin-3, as well as its proteolytical derived C-terminal domain, are predominantly monomeric. The presence of LacNAc at concentrations sufficient to fully saturate the proteins had no significant effect on either the weight average molecular weight determined by sedimentation equilibrium or the hydrodynamic properties determined from sedimentation velocity experiments. These results show that binding of a monovalent ligand does not affect oligomerization of these galectins.

Detection of parvovirus B19 IgG: choice of antigens and serological tests.

Serum samples were analysed for the presence of (a) IgG against VP1+VP2 using recombinant native conformational antigens by ELISA test (b) IgG against VP2 using recombinant native conformational antigens by ELISA test and (c) IgG against VP1 and against VP2 using denatured linear antigens by Western blot. Out of the 446 samples examined, 353 were positive for specific B19 IgG and out of these, 98.6 % proved positive in the ELISA assay using conformational VP1+VP2 antigens, 94.6% proved positive in the ELISA assay using conformational VP2 antigens, 89.5% were positive at the Western blot assay using denatured linear VP1 and VP2 antigens, with all proving positive for linear VP1 and only 29.5% out of the positive samples proving positive for linear VP2. Since all samples positive by Western blot proved positive by ELISA, our data show that recombinant capsids obtained either with VP1+VP2 or with VP2 alone, used in ELISA, are very useful for detecting the immune response against both conformational and native linear epitopes of B19 structural proteins although some sera may have antibodies directed exclusively against VP1+VP2 antigens and few may have antibodies directed exclusively against VP2 antigens alone.

Functional fine-mapping and molecular modeling of a conserved loop epitope of the measles virus hemagglutinin protein.

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine-map the highly conserved hemagglutinin noose epitope (H379-410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus-neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729-735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild-type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent-exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent-hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.

Recombinant anti-P protein autoantibodies isolated from a human autoimmune library: reactivity, specificity and epitope recognition.

The ribosomal P proteins are specific and important autoantigens in patients affected by systemic lupus erythematosus. In this study, we describe for the first time the selection and characterization of recombinant human monoclonal anti-P protein (auto)-antibody fragments from an autoimmune patient-derived phage display antibody library. The selected recombinant anti-P antibodies specifically recognize the P proteins in immunofluorescence assays on HEp-2 cells and in immunoblotting assays, and they immunoprecipitate the P proteins under native conditions. Using both anti-P-positive patient sera and the selected recombinant anti-P antibodies, the immunodominant epitope was determined and shown to be located at the C-terminal end of the P proteins (amino acids 111-115). Inhibition of in vitro protein translation demonstrated that interaction of the monoclonal patient-derived anti-P antibodies with their native epitope functionally inhibits the activity of the P proteins on the ribosome, confirming the notion that patient autoantibodies are often directed to the functional centre of their autoantigenic target.

Effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen.

An immunization protocol that induces antibodies (Abs) directed to cryptic epitopes of a protein antigen (Ag) reduces the efficacy of vaccines that ideally should induce Abs against native epitopes. We have shown earlier that viral infections concomitant with immunization against a protein tend to shift the Ab specificity toward cryptic epitopes and tend to induce the production of autoantibodies (autoAbs). Here, we show the effects of three adjuvants on the Ab specificity in the absence or presence of a viral infection (lactate dehydrogenase-elevating virus or LDV), with human growth hormone (hGH) being, as before, the protein Ag. Pathogen-free CBA/Ht and BALB/c mice were immunized with hGH in the presence of complete Freund's adjuvant (CFA), monophosphoryl lipid A (MPL) or alum, with the animals being either infected with LDV or not infected with LDV. Conventional and competition enzyme-linked immunosorbent assays (ELISAs) indicated that in noninfected mice, CFA induced higher titres of anti-hGH Ab than did MPL or alum, with the Ab being almost totally directed to cryptic hGH epitopes. Strikingly, CFA plus LDV infection in CBA/Ht mice shifted the specificity of the anti-hGH Ab toward native epitopes, whereas the virus decreased the Ab titre when MPL or alum was used. Our Western blot results showed that 70% of mice immunized with hGH in the presence of any adjuvant produced autoAbs against a variety of tissue Ags. The amount of autoAb and the concentration of Ab to hGH cryptic epitopes did correlate, suggesting a relationship between both kinds of Ab. Significant differences were observed in the various effects of adjuvants and the viral infection between the two mouse strains used in this work.

Successful identification of novel agents to control infectious diseases from screening mixture-based peptide combinatorial libraries in complex cell-based bioassays.

Mixture-based peptide synthetic combinatorial libraries (SCLs) represent a valuable source for the development of novel agents to control infectious diseases. Indeed, a number of studies have now proven the ability of identifying active peptides from libraries composed of thousands to millions of peptides in cell-based biosystems of varying complexity. Furthermore, progressing knowledge on the importance of endogenous peptides in various immune responses lead to a regain in importance for peptides as potential therapeutic agents. This article is aimed at providing recent studies in our laboratory for the development of antimicrobial or antiviral peptides derived from mixture-based SCLs using cell-based assays, as well as a short review of the importance of such peptides in the control of infectious diseases. Furthermore, the use of positional scanning (PS) SCL-based biometrical analyses for the identification of native optimal epitopes specific to HIV-1 proteins is also presented.

Anti-plasminogen autoantibodies from plasma of patients with systemic lupus erythematosus having anti-phospholipid antibody syndrome: isolation and some immunochemical properties.

Blood plasma samples from patients with systemic lupus erythematosus having the anti-phospholipid antibody syndrome were found to contain anti-plasminogen antibodies of the IgG class. The titers of anti-plasminogen autoantibodies of the IgG class were elevated in these patients compared with normal controls. Part of the pool of IgG anti-plasminogen antibodies reacts with an epitope in the lysine-binding sites of plasminogen. Anti-plasminogen IgG isolated from patients' blood plasma is specific only for a native epitope of human plasminogen passively adsorbed on immunosorbent micro-titration plate. As shown by enzyme immunoassay, autoantibodies to plasminogen of the IgG class cross-react with human fibrinogen.

Specific modulation of the anti-DNA autoantibody–nucleic acids interaction by the high affinity RNA aptamer

Anti-DNA autoantibodies are one of the frequently found autoantibodies in systemic lupus erythematosus patient sera. RNA aptamers for the monoclonal G6-9 anti-DNA autoantibody were selected from a random pool of RNA library. Binding affinity of the best aptamer is around 2 nM, which is at least 100-fold higher than that of cognate DNA antigen to the autoantibody. Aptamer binds specifically to the G6-9 autoantibody but not to other similar autoantibodies. Minimal binding motif of the aptamer was mapped, providing a hint for a natural epitope of the autoantibody. DNA binding to the G6-9 autoantibody is shown to be efficiently inhibited by the aptamer. Such binding property of the RNA aptamer could be used not only as a modulator for the pathogenic anti-DNA autoantibody, but also as a useful biochemical reagent for elucidating a fine specificity of the autoantibody–nucleic acid interaction.

Molecular recognition of oligosaccharide epitopes by a monoclonal Fab specific for Shigella flexneri Y lipopolysaccharide: X-ray structures and thermodynamics.

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.

Solid-phase epitope recovery: a high throughput method for antigen identification and epitope optimization.

Self tolerance to MHC class I-restricted nonmutated self Ags is a significant hurdle to effective cancer immunotherapy. Compelling evidence is emerging that altered peptide ligands can be far more immunogenic than their corresponding native epitopes; however, there is no way to reliably predict which modifications will lead to enhanced native epitope-specific immune responses. We reasoned that this limitation could be overcome by devising an empirical screen in which the nearly complete combinatorial spectrum of peptides of optimal length can be rapidly assayed for reactivity with a MHC class I-restricted cytotoxic T cell clone. This method, solid-phase epitope recovery, quantitatively ranks all reactive peptides in the library and allows selection of altered peptide ligands having desirable immunogenic properties of interest. In contrast to rationally designed MHC anchor-modified peptides, peptides identified by the present method are highly substituted in predicted TCR contact residues and can reliably activate and expand effector cell populations in vitro which lyse target cells presenting the wild-type epitope. We demonstrate that solid-phase epitope recovery peptides corresponding to a poorly immunogenic epitope of the melanoma Ag, gp100, can reliably induce wild-type peptide-specific CTL using normal donor T cells in vitro. Furthermore, these peptides can complement one another to induce these responses in an overwhelming majority of normal individuals in vitro. These data provide a rationale for the design of superior vaccines comprising a mixture of structurally diverse yet functionally convergent peptides.