Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.     

Potent neutralization of botulinum neurotoxin/b by synergistic action of antibodies recognizing protein and ganglioside receptor binding domain

Background

Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.

Methods and Findings

We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.

Conclusions

The combination of two mAbs recognizing different receptors'' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.     

Recognition of class II molecules by human T cells

The HLA-D region of individuals with the DRw11, w52, DQw3 haplotype encodes multiple molecular products of three distinct subregions, DR, DP, and DQ. Since each molecule can carry multiple stimulatory epitopes, the repertoire of allogeneic T-cell responses to determinants of this haplotype can be quite large. In the present experiments, alloreactive cloned T-cell lines recognized six distinct epitopes associated with DRw11, DRw52, DQw3 haplotypes. Panel studies established that three epitopes were DRwll-like and three were DRw52-like. Blocking with monoclonal antibodies showed that two DRw11-like epitopes were carried by DR-subregion products and one DRwll-like epitope was carried by DQ-subregion molecules. DRw52-like epitopes were detected on separate DR subregion-encoded molecules. One of them carried both DRwl1-and DRw52-like epitopes, the other carried two of the DRw52-like epitopes. These epitopes, which represent functional units that trigger T-cell responses, can be detected at the present time only with the methods used in this report. Conventional allogeneic T-cell responses represent the summation of responses to multiple epitopes encoded by different D-subregion genes.     

Dissection of the poly(glu60ala30tyr10) (GAT)-specific T-cell repertoire in H-21 k mice

We examined the antigen recognition of the class II major histocompatibility complex (MHC) of 45 poly(glu60 ala30 tyr10) (GAT)-reactive T-cell clones isolated by limiting dilution cloning of a pool of in vivo-primed and in vitro-restimulated A.TL lymph-node T cells. Each clone expressed the Thy-1.2+, Lyt-1+, Lyt-2-, LFA-1+, Ia-, and H-2Dd+ cell-surface phenotype and exhibited strict specificity for GAT on syngeneic antigen-presenting cells (APCs). The monitoring of the proliferative responses of these clones in the presence or absence of GAT, using APCs from strains with 11 independent H-2 haplotypes, revealed several distinct specificity patterns: (i) most (31 of 45, 73%) T-cell clones recognized GAT in a self-I-Ak-restricted manner; (ii) other alloreactive clones (5 of 45, 11%) were stimulated to proliferate, irrespective of the presence of GAT, in response to allodeterminants expressed on H-2s, H-2d, H-2f or H-2u spleen cells; (iii) a third T-cell clone subset (4 of 45, 9%) was activated by GAT in the context of not only self-I-Ak but also nonself restriction Ia determinants; and (iv) three clones (7%) exhibited a triple specificity, i.e., they recognized GAT in the context of self and nonself Ia determinants and were alloreactive. One of the latter clones responded to GAT in an apparently non-MHC-restricted manner and recognized an I-Ab allodeterminant. These data provide direct evidence that the antigen-specific and alloreactive T-cell repertoires overlap and that the self-MHC restriction of GAT-specific T-cell responses is not absolute in A.TL mice.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Structural analysis of chick ephrin-A2 by function-blocking and non-blocking monoclonal antibodies

Ephrins, ligands for the Eph family of receptor tyrosine kinases, play key roles in diverse biological processes. In this study, we determined the epitopes and kinetic parameters of function-blocking (B3) and non-blocking (IV) monoclonal antibodies (mAbs) recognizing chick ephrin-A2. We show that the epitope for the non-blocking mAb is the residue Asp(105) of chick ephrin-A2. However, the binding of the function-blocking mAb depends mostly on residue Ser(108) and its epitope may reside within residues 105-132, which appear crucial for the receptor interaction site. Kinetic studies suggest a possible mechanism why mAb IV, despite recognizing a region very close to the mAb B3 epitope, fails to block the ligand-receptor interaction.     

CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.     

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.     

Identification of the pentapeptide constituting a dominant epitope common to all eukaryotic heat shock protein 90 molecular chaperones

We previously reported that, in human heat shock protein (Hsp) 90 (hHsp90), there are 4 highly immunogenic sites, designated sites Ia, Ib, Ic, and II. This study was performed to further characterize their epitopes and to identify the epitope that is potentially common to all members of the Hsp90 family. Panning of a bacterial library carrying randomized dodecapeptides revealed that Glu251-Ser-X-Asp254 constituted site Ia and Pro295-Ile-Trp-Thr-Arg299, site Ic. Site II (Asp701-Pro717) was composed of several epitopes. When 19 anti-hHsp90 monoclonal antibodies (mAbs) were subjected to immunoblotting against recombinant forms of 7 Hsp90-family members, 2 mAbs (K41110 and K41116C) that recognized site Ic bound to yeast Hsp90 with affinity identical to that for hHsp90, and 1 mAb (K3729) that recognized Glu222-Ala23, of hHsp90beta could bind to human 94-kDa glucose-regulated protein (Grp94), an endoplasmic reticulum paralog of Hsp90. Among the 5 amino acids constituting site Ic, Trp297 and Pro295 were essential for recognition by all anti-site-Ic mAbs, and Arg299 was important for most of them. The necessity of Ile296, Thr298, and Arg299, which are replaced by Leu, Met/Leu, and Lys, respectively, in some eukaryotic Hsp90, was dependent on the mAbs, and K41110 and K41116C could react with Hsp90s carrying these substitutions. From these data taken together, we propose that the pentapeptide Pro295-Ile-Trp-Thr-Arg299 of hHsp90 functions as an immunodominant epitope common to all eukaryotic Hsp90.     

Anti-Human IgE Monoclonal Antibodies Recognizing Epitopes Related to the Binding Sites of High and Low Affinity IgE Receptors

Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzyme-linked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the Cε1, Cε2, Cε2/Cε3 junction and Cε3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (FcεRI and FcεRII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-FcεRII binding was clearly inhibited by the mAb recognizing the Cε2/Cε3 junction, suggesting that FcεRII binds to a rather limited area around the Cε2/Cε3 junction. The IgE-FcεRI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in Cε2 was blocked simultaneously with that at the Cε2/Cε3 junction or with that in Cε3, indicating that these three distinct epitopes are related to the FcεRI binding sites. When these three epitopes were shown in the stereograph of human IgE, the FcεRI binding area was spread largely on the groove side between Cε2 and Cε3 domains. These results suggest that FcεRI acquires the high affinity through multiple bindings.     

Shared idiotope on monoclonal anti-Ia.7 antibodies reactive with determinants in a structural domain of the I-E molecules

In previous studies, heterologous anti-idiotypic (anti-Id) antisera against the C3H.SW 14-4-4S or the A.TH 41.A anti-Ia.7 monoclonal antibodies (mAb) were shown to identify an interstrain cross-reactive idiotypic specificity (IdX.Ia.7) expressed on monoclonal or conventional anti-Ia.7 alloantibodies. The objective of the present investigation was to characterize further this IdX at the idiotopic level. To this end, 11 hybridomas producing IgG1, IgG2a, or IgM anti-Id mAb were derived from a rat immunized with a mixture of 10 A.TH or A.BY anti-Ia.7 mAb. The specificity of the latter anti-Id mAb was determined by direct Id binding radioimmunoassay (RIA) with the use of a panel of 52 anti-Ia mAb derived from hybridomas produced in various inbred mouse strains. These rat anti-Id mAb recognized idiotopes expressed on i) all anti-Ia.7 mAb against determinants in the topographic domain I of the I-Ek molecule but not on 18 other anti-I-Ek mAb directed at epitopes in domains II or III; ii) three of 19 anti-I-Ak mAb; and iii) one A.TL-derived anti-I-As mAb. Competitive Id binding assays revealed that among the 14 IdX+ anti-Ia.7 mAb, one (81.B) was bound to a lesser extent by various rat anti-Id mAb, suggesting that heterogeneity probably exists in this antibody family. By contrast, two isologous (B10.S(7R)) anti-Id mAb to the IdX.Ia.7+ mAb 41.A displayed a specificity restricted to 41.A individual idiotopes (IdI). Rat anti-IdX.Ia.7 and mouse anti-41.A IdI mAb inhibited the binding of 125I-labeled mAb 41.A to CBA spleen cells. These two sets of mAb bound in a noncompetitive fashion to mAb 41.A-coated plates, indicating that their corresponding public or private idiotopes were spatially distinct. These data may have implications for in vivo manipulations of anti-Ia immune responses.     

Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes.

The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.     

Structure-function relationships of human apolipoprotein D an immunochemical analysis

Apolipoprotein D (apoD), a 169 amino acid member of the lipocalin family, is thought to be a transporter of small, hydrophobic ligands. A panel of 10 anti-apoD monoclonal antibodies (mAbs) was prepared and characterized in order to define apoD structure-function relationships. An apoD epitope map was constructed based on reactivity of the mAbs with apoD fragments. Three mAbs react with epitopes between apoD residues 7-78, seven mAbs with epitopes between residues 128-169, one mAb recognizes an epitope that straddles residues 99-102 and one mAb is specific for an epitope composed of non-contiguous apoD residues. Several pairs of mAbs whose respective epitopes are widely separated in apoD primary structure can compete for binding to immobilized apoD. This would be consistent with the compact beta-barrel tertiary structure that apoD is thought to adopt. None of the mAbs block the interaction of apoD with pregnenolone, a putative physiological ligand for apoD.     

Properties of anti-gp41 core structure antibodies, which compete with sera of HIV-1-infected patients

To determine the correlation between the immunoreaction against the core structure of human immunodeficiency virus type (HIV-1) transmembrane protein gp41 epitopes and the disease progression, it is essential to evaluate the anti-core structure antibody epitopes and the humoral immunity against the epitopes. For this purpose we evaluated monoclonal antibodies (mAbs) against the gp41 core structure such as mAbs 50.69, 98.6 and T26, by Western blotting (WB) and flow cytometry. WB showed mAbs 50.69 and 98.6 bound to both monomeric and oligomeric gp41, and mAb T26 exclusively bound to oligomeric gp41. We evaluated the sera from Pneumocystis pneumonia patients (PCP; n=7) and long-term survivors (LTS; n=7). Competition assay with sera and mAbs for binding to H9 cells infected with HIV-1 IIIB virus was done using flow cytometry. The results revealed that PCP sera as well as LTS sera inhibited the binding of all the three mAbs, and the PCP sera inhibited mAb T26 binding more efficiently than LTS. Therefore, PCP patients retain competing immunity to antibodies against not only the shared epitopes of the core structure (binding sites of mAbs 50.69 and 98.6) but also against oligomeric gp41 specific epitope (binding site of mAb T26).     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due to H-21-region differences. In both cases of MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. - Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in re-stimulation during MLR-II. - These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.     

Peptides of human thyroglobulin reactive with sera of patients with autoimmune thyroid disease

Autoantibodies to thyroglobulin (Tg) are a prominent feature of the two autoimmune thyroid diseases, chronic lymphocytic (Hashimoto's) thyroiditis and Graves' disease. Similar autoantibodies are found in the serum of many normal individuals without evidence of thyroid disease. Previous studies have indicated that patients with autoimmune thyroid disease recognize epitopes of Tg which are not usually recognized by normal individuals. The goal of this investigation was to identify peptide fragments of Tg bearing these disease-associated epitopes. For this purpose, we utilized a panel of mAbs that bind to different epitopes of the Tg molecule. One of these mAbs (137C1) reacted with an epitope that was also recognized by the sera of patients with autoimmune thyroiditis. In the present study, we show that two peptides (15 and 23 kDa) that reacted with mAb 137C1 are located in different parts of the Tg molecule. Each peptide inhibited the binding of mAb 137C1 to the other peptide and to the intact Tg, indicating that the same epitope was represented on the two peptides. Loops and helices of the secondary structure of the two peptides might be involved in the conformational epitope recognized by mAb 137C1. A striking finding of this study is that two apparently unrelated fragments of the Tg molecule bind to the same mAb. These findings may have important ramifications with regard to epitope spread and the progression of the autoimmune response to disease.