Epitopes Of Class Research Articles

An Introduction to Epitope Mapping.

Antibodies are protein molecules used routinely for therapeutic, diagnostic, and research purposes due to their exquisite ability to selectively recognize and bind a given antigen. The particular area of the antigen recognized by the antibody is called the epitope, and for proteinaceous antigens the epitope can be of complex nature. Information about the binding epitope of an antibody can provide important mechanistic insights and indicate for what applications an antibody might be useful. Therefore, a variety of epitope mapping techniques have been developed to localize such regions. Although the real picture is even more complex, epitopes in protein antigens are broadly grouped into linear or discontinuous epitopes depending on the positioning of the epitope residues in the antigen sequence and the requirement of structure. Specialized methods for mapping of the two different classes of epitopes, using high-throughput or high-resolution methods, have been developed. While different in their detail, all of the experimental methods rely on assessing the binding of the antibody to the antigen or a set of antigen mimics. Early approaches utilizing sets of truncated proteins, small numbers of synthesized peptides, and structural analyses of antibody-antigen complexes have been significantly refined. Current state-of-the-art methods involve combinations of mutational scanning, protein display, and high-throughput screening in conjunction with bioinformatic analyses of large datasets.

Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, resulting in significant economic losses to the swine industry worldwide. Current vaccination approaches against this emerging coronavirus are only partially effective, though natural infection protects pigs against reinfection and provides lactogenic immunity to suckling piglets. The viral spike (S) glycoprotein, responsible for receptor binding and cell entry, is the major target for neutralizing antibodies. However, knowledge of antibody epitopes, their nature and location in the spike structure, and the mechanisms by which the antibodies interfere with infection is scarce. Here we describe the generation and characterization of 10 neutralizing and nonneutralizing mouse monoclonal antibodies raised against the S1 receptor binding subunit of the S protein. By expression of different S1 protein fragments, six antibody epitope classes distributed over the five structural domains of the S1 subunit were identified. Characterization of antibodies for cross-reactivity and cross-neutralization revealed antigenic differences among PEDV strains. The epitopes of potent neutralizing antibodies segregated into two epitope classes and mapped within the N-terminal sialic acid binding domain and in the more C-terminal receptor binding domain. Antibody neutralization escape mutants displayed single amino acid substitutions that impaired antibody binding and neutralization and defined the locations of the epitopes. Our observations picture the antibody epitope landscape of the PEDV S1 subunit and reveal that its cell attachment domains are key targets of neutralizing antibodies.IMPORTANCE Porcine epidemic diarrhea virus (PEDV), an emerging porcine coronavirus, causes an economically important enteric disease in pigs. Effective PEDV vaccines for disease control are currently lacking. The spike (S) glycoprotein on the virion surface is the key player in virus cell entry and, therefore, the main target of neutralizing antibodies. To understand the antigenic landscape of the PEDV spike protein, we developed monoclonal antibodies against the spike protein's S1 receptor binding region and characterized their epitopes, neutralizing activity, and cross-reactivity toward multiple PEDV strains. Epitopes of antibodies segregated into six epitope classes dispersed over the multidomain S1 structure. Monoclonal antibodies revealed antigenic variability in B-cell epitopes between PEDV strains. The epitopes of neutralizing antibodies mapped to two distinct domains in S1 that are involved in binding to carbohydrate and proteinaceous cell surface molecules, respectively, indicating the importance of these cell attachment sites on the PEDV spike protein in eliciting a protective humoral immune response.

All naturally occurring autoantibodies against the NMDA receptor subunit NR1 have pathogenic potential irrespective of epitope and immunoglobulin class

Autoantibodies of the IgG class against N-methyl-d-aspartate-receptor subunit NR1 (NMDAR1) were first described in anti-NMDAR encephalitis and seen as disease indicators. Recent work on together over 5000 individuals challenged this exclusive view by showing age-dependently up to >20% NMDAR1-autoantibody seroprevalence with comparable immunoglobulin class and titer distribution across health and disease. The key question therefore is to understand the properties of these autoantibodies, also in healthy carriers, in order to assess secondary complications and possible contributions to neuropsychiatric disease. Here, we believe we provide for human NMDAR1-autoantibodies the first comprehensive analysis of their target epitopes and functionality. We selected sera of representative carriers, healthy or diagnosed with very diverse conditions, that is, schizophrenia, age-related disorders like hypertension and diabetes, or anti-NMDAR encephalitis. We show that all positive sera investigated, regardless of source (ill or healthy donor) and immunoglobulin class, provoked NMDAR1 internalization in human-induced pluripotent stem cell-derived neurons and reduction of glutamate-evoked currents in NR1-1b/NR2A-expressing Xenopus oocytes. They displayed frequently polyclonal/polyspecific epitope recognition in the extracellular or intracellular NMDAR1 domains and some additionally in NR2A. We conclude that all circulating NMDAR1-autoantibodies have pathogenic potential regarding the whole spectrum of neuronal NMDAR-mediated effects upon access to the brain in situations of increased blood–brain–barrier permeability.

Abstract 595: Next-generation engineered toxin bodies: CD38, PD-L1 and HER2 targeted ETBs

Abstract Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit to specifically kill target expressing cells. Once delivered to appropriate cells, the Shiga toxin A subunit inhibits protein synthesis and promotes apoptosis of tumor cells. Bacterial or plant derived toxin moieties have the potential to induce an immune response, commonly limiting repeat dosing of immunotoxins. Our next-generation ETB scaffold has been modified to overcome this limitation through genetic engineering to systematically and comprehensively reduce B and CD4+T cell epitopes. Molecular Templates has developed a proprietary epitope class switching technology designed to both reduce the anti-drug response and promote the anti-tumor response by replacing naturally occurring CD4+ T cell epitopes with CD8+ T cell epitopes. We have found that a combination of surface remodeling and epitope class switching combined in one protein results in powerful reductions in the anti-drug response against ETBs after repeat administration. This de-immunized next-generation scaffold retains the potency and specificity of the unmodified ETB. Additionally, the engineering of CD8+ T cell epitopes on the ETB scaffold can allow for foreign antigen presentation in complex with MHC class I on the tumor cell surface. This antigen presentation may recruit activated cytotoxic T lymphocytes (CTLs) to the tumor microenvironment thereby adding an additional mechanism of action to the direct cell kill activity of the ETBs. The second generation ETB scaffold has been combined with multiple target binding domains, including scFvs that target CD38, PD-L1 and HER2. CD38 is a validated target for multiple myeloma, and our CD38 targeted ETB has shown potent activity in vitro and in vivo. Pre-clinical studies have shown effective combination of the CD38 targeted ETB with standard of care agents such as IMiDs. Additionally, we have developed a PD-L1 targeted ETB that can be used against various PD-L1 expressing malignancies including Hodgkin's lymphoma and melanoma. HER2 is a well characterized target in breast cancer that persists in many patients after HER2 targeted treatment relapse; a novel MOA to this target may impart additional clinical benefit. The potency, reduced immunogenicity, unique mechanism of action and immune modulating activities of ETBs in this second generation scaffold allows for these agents to fit in well in a refractory setting as well as in combination with other agents in the growing field of immuno-oncology. Citation Format: Sangeetha Rajagopalan, Garrett L. Robinson, Brigitte Brieschke, Jennifer Erdman, Jane Neill, Jack P. Higgins, Erin K. Willert. Next-generation engineered toxin bodies: CD38, PD-L1 and HER2 targeted ETBs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 595.

Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies.

A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose–response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120–gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations.

Fel d 1-derived synthetic peptide immuno-regulatory epitopes show a long-term treatment effect in cat allergic subjects.

Cat-PAD, the first in a new class of synthetic peptide immuno-regulatory epitopes (SPIREs), was shown to significantly improve rhinoconjunctivitis symptoms in subjects with cat allergy up to 1 year after the start of a short course of treatment. To evaluate the long-term effects of Cat-PAD on rhinoconjunctivitis symptoms following standardized allergen challenge 2 years after treatment. In a randomized, double-blind, placebo-controlled, parallel group study, subjects were exposed to cat allergen in an environmental exposure chamber (EEC) before and after treatment with two regimens of Cat-PAD (either eight doses of 3 nmol or four doses of 6 nmol) given intradermally over a 3-month period. In this follow-up study, changes from baseline in rhinoconjunctivitis symptoms were reassessed 2 years after the start of treatment. The primary endpoint showed a mean reduction in total rhinoconjunctivitis symptom scores of 3.85 units in the 4 × 6 nmol Cat-PAD group compared to placebo 2 years after the start of treatment (P = 0.13), and this difference was statistically significant in the secondary endpoint at the end of day 4 when the cumulative allergen challenge was greatest (P = 0.02). Consistent reductions in nasal symptoms of between 2 and 3 units were observed for 4 × 6 nmol Cat-PAD compared to placebo between the 2 and 3 h time points on days 1-4 of EEC challenge at 2 years (P < 0.05). The 8 × 3 nmol dose did not show a meaningful effect in this study. A persistent, clinically meaningful reduction in rhinoconjunctivitis symptoms was observed on EEC challenge 2 years after the start of a short course of treatment with 4 × 6 nmol Cat-PAD. This study is the first to provide evidence of a long-term therapeutic effect with this new class of SPIREs.

Prediction of common epitopes on hemagglutinin of the influenza A virus (H1 subtype)

Influenza A virus infection is a persistent threat to public health worldwide due to hemagglutinin (HA) variation. Current vaccines against influenza A virus provide immunity to viral isolates similar to vaccine strains. Antibodies against common epitopes provide immunity to diverse influenza virus strains and protect against future pandemic influenza. Therefore, it is vital to analyze common HA antigenic epitopes of influenza virus. In this study, 14 strains of monoclonal antibodies with high sensitivity to common epitopes of influenza virus antigens identified in our previous study were selected as the tool to predict common HA epitopes. The common HA antigenic epitopes were divided into four categories by ELISA blocking experiments, and separately, into three categories according to the preliminary results of computer simulation. Comparison between the results of computer simulations and ELISA blocking experiments indicated that at least two classes of common epitopes are present in influenza virus HA. This study provides experimental data for improving the prediction of HA epitopes of influenza virus (H1 subtype) and the development of a potential universal vaccine as well as a novel approach for the prediction of epitopes on other pathogenic microorganisms.

The link between CD8+ T-cell antigen-sensitivity and HIV-suppressive capacity depends on HLA restriction, target epitope and viral isolate

Although it is established that CD8 T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8⁺ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8⁺ T-cell responses against the Gag₂₆₃₋₂₇₂ KK10 epitope restricted by human leukocyte antigen (HLA)-B27. To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8⁺ T-cell clones specific for Nef₇₃₋₈₂ QK10 and Gag₂₀₋₂₉ RY10, both restricted by HLA-A3, alongside CD8⁺ T-cell clones specific for Gag₂₆₃₋₂₇₂ KK10. For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. Collectively, these data emphasize the central role of the TCR as a determinant of CD8⁺ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

Molecular characterization of human T-cell leukemia virus type 1 gp46 glycoprotein from healthy carriers and tropical spastic paraparesis/HTLV-associated myelopathy infected individuals

The present study attempts to assess the molecular diversity of gp46 glycoprotein in HAM/TSP patients and Health Carrier (HC) individuals. The median HTLV-1 proviral load of HC (n=5) and HAM/TSP (n=5) patients was similar (average 316,227 copies/106 PBMCs). The gp46 molecular characterization of 146 clones (70 HC and 76 HAM/TSP) revealed an overall diversity, within HC and HAM/TSP clones, of 0.4% and 0.6%, respectively. A single amino acid (aa) substitution (S35L) was exclusive for the HC group, and three gp46 substitutions (F14S, N42H, G72S) were exclusive for the HAM/TSP group. The remaining frequent mutation (V247I) was present in both groups (p = 0.0014). The in silico protein analysis revealed that the mutated alleles F14S and N42H represent more hydrophilic and flexible protein domains that are likely to be less antigenic. The Receptor Binding Domain is quite variable in the HAM/TSP group. Two other domains (aa 53-75 and 175-209) that contain multiple linear T-cell epitopes showed genetic diversity in both HAM/TSP and HC groups. Further analysis revealed 27 and 13 T-cell epitopes for class I HLA alleles and class II HLA alleles, when analyzing the entire gp46. We still cannot associate any of the gp46 found mutations with the clinical profile.

Identification of B-cell epitopes in an antigen for inducing specific class of antibodies

BackgroundIn the past, numerous methods have been developed for predicting antigenic regions or B-cell epitopes that can induce B-cell response. To the best of authors’ knowledge, no method has been developed for predicting B-cell epitopes that can induce a specific class of antibody (e.g., IgA, IgG) except allergenic epitopes (IgE). In this study, an attempt has been made to understand the relation between primary sequence of epitopes and the class of antibodies generated.ResultsThe dataset used in this study has been derived from Immune Epitope Database and consists of 14725 B-cell epitopes that include 11981 IgG, 2341 IgE, 403 IgA specific epitopes and 22835 non-B-cell epitopes. In order to understand the preference of residues or motifs in these epitopes, we computed and compared amino acid and dipeptide composition of IgG, IgE, IgA inducing epitopes and non-B-cell epitopes. Differences in composition profiles of different classes of epitopes were observed, and few residues were found to be preferred. Based on these observations, we developed models for predicting antibody class-specific B-cell epitopes using various features like amino acid composition, dipeptide composition, and binary profiles. Among these, dipeptide composition-based support vector machine model achieved maximum Matthews correlation coefficient of 0.44, 0.70 and 0.45 for IgG, IgE and IgA specific epitopes respectively. All models were developed on experimentally validated non-redundant dataset and evaluated using five-fold cross validation. In addition, the performance of dipeptide-based model was also evaluated on independent dataset.ConclusionPresent study utilizes the amino acid sequence information for predicting the tendencies of antigens to induce different classes of antibodies. For the first time, in silico models have been developed for predicting B-cell epitopes, which can induce specific class of antibodies. A web service called IgPred has been developed to serve the scientific community. This server will be useful for researchers working in the field of subunit/epitope/peptide-based vaccines and immunotherapy (http://crdd.osdd.net/raghava/igpred/).ReviewersThis article was reviewed by Dr. M Michael Gromiha, Dr Christopher Langmead (nominated by Dr Robert Murphy) and Dr Lina Ma (nominated by Dr Zhang Zhang).

Two year persistent treatment effect in reducing nasal symptoms of cat allergy after 4 doses of Cat‐PAD, the first in a new class of synthetic peptide immuno‐regulatory epitopes

Background Treatment with Cat-PAD (also known as ToleroMune Cat, the first in a new class of synthetic peptide immuno-regulatory epitopes), in an Environmental Exposure Chamber (EEC) model of cat allergy showed a persistent treatment effect one year [1] and two years [2] after administration of only 4 injections over 12 weeks. Here we report the differences in Total Nasal Symptom Scores (TNSS) between Cat-PAD treatment arms and placebo two years after treatment started.

Identification of an HIV-1 Clade A Envelope That Exhibits Broad Antigenicity and Neutralization Sensitivity and Elicits Antibodies Targeting Three Distinct Epitopes

Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gp120, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (L111A) enabled more efficient production of a stable gp120 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gp120 protein elicited neutralizing antibodies in rabbits (following a gp120 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector.

Molecular characterization of HTLV-1 gp46 glycoprotein from health carriers and HAM/TSP infected individuals

BackgroundHuman T-cell Leukemia Virus type 1 (HTLV-1) is the etiological agent of tropical spastic paraparesis/HTLV-associated myelopathy (HAM/TSP) that can be identified in around 0.25%–3.8% of the infected population. Disease progression can be monitored by the proviral load and may depend on genetic factors, however, it is not well understood why some HTLV-1 infected people develop the disease while others do not. The present study attempts to assess the molecular diversity of gp46 glycoprotein in HAM/TSP patients and Health Carrier (HC) individuals.MethodsBlood samples were collected from 10 individuals, and DNA was extracted from PBMCs to measure the HTLV-1 proviral load. The gp46 coding sequences were amplified PCR, cloned and sequenced. The molecular characterization was performed using bioinformatics tools.ResultsThe median HTLV-1 proviral load of HC (n = 5) and HAM/TSP (n = 5) patients was similar (average 316,227 copies/106 PBMCs). The gp46 molecular characterization of 146 clones (70 HC and 76 HAM/TSP) revealed an overall diversity, within HC and HAM/TSP clones, of 0.4% and 0.6%, respectively. Five frequent mutations were detected among groups (HAM/TSP and HC clone sequences). A single amino acid (aa) substitution (S35L) was exclusive for the HC group, and three gp46 substitutions (F14S, N42H, G72S) were exclusive for the HAM/TSP group. The remaining frequent mutation (V247I) was present in both groups (p = 0.0014). The in silico protein analysis revealed that the mutated alleles F14S and N42H represent more hydrophilic and flexible protein domains that are likely to be less antigenic. The Receptor Binding Domain is quite variable in the HAM/TSP group. Two other domains (aa 53–75 and 175–209) that contain multiple linear T-cell epitopes showed genetic diversity in both HAM/TSP and HC groups. Further analysis revealed 27 and 13 T-cell epitopes for class I HLA alleles and class II HLA alleles, when analyzing the entire gp46.ConclusionsThe most common gp46 mutations were not associated clinical status because they were found in only one individual, except for the V247I mutation, that was found at viral clones from HAM/TSP ad HC individuals. Because of this, we cannot associate any of the gp46 found mutations with the clinical profile.

Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.

Characterization of Structural Features and Diversity of Variable-Region Determinants of Related Quaternary Epitopes Recognized by Human and Rhesus Macaque Monoclonal Antibodies Possessing Unusually Potent Neutralizing Activities

A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909. These studies showed that determinants for the epitopes defined by the RhMAbs differed in both the V2 (positions 160, 167, and 169) and V3 (positions 313 and 315) regions from 2909, and in a number of cases, from each other. Attempts to reconstitute expression of these epitopes on the cell surface by cotransfecting Envs containing either the V2 or the V3 determinant of the epitope were not successful, suggesting that these epitopes were expressed on individual protomers in a trimer-dependent manner. Several of the V2 positions found to be critical for expression of these quaternary epitopes also significantly affected exposure and neutralization sensitivity of targets in the V3 and CD4-binding domains. These results demonstrated a considerable diversity in the fine structure of this class of epitopes and further suggested a potentially important relationship between the expression of such quaternary epitopes and V1/V2-mediated masking of immunodominant epitopes.

Monoclonal antibodies against human CD34 antigens do not cross-react with ovine umbilical cord blood cells

CD34 is a cell surface glycoprotein expressed by hematopoietic progenitors and endothelial cells. It is widely used in the clinic for isolation of human hematopoietic stem cells. In recent years large animals are gaining increasing importance in biomedical research for the study and therapy of human diseases. Sheep has proved to be an useful experimental model for preclinical trials in transplantation procedures. Unfortunately, the lack of specie-specific monoclonal antibodies (MABS) recognizing hemopoietic progenitor cells hampers the use of this animal in experimental hematology. The aim of this paper was to determine whether commercial monoclonal antibodies specific for human CD34 molecule could cross-react with hematopoietic progenitor cells (HPC) present in sheep umbilical cord blood (UCB). Six antihuman CD34 MABS, recognizing the three different epitope classes, were tested in flow cytometry on purified mononuclear cells (MNC) isolated from cord blood of both species. None of the MABS used in this trial seemed to be able to identify HPC from sheep UCB. These data suggest that the panel of monoclonal antibodies used for cross reactivity detection has to be expanded with recently produced reagents. Further studies should be directed towards the production of ovine specific anti CD34 MABS.

Reduced Immunogenicity of FVIII Peptides by Rational Modifications of An Immunodominant T-Cell Epitope.

Abstract 220 Introduction.The development of antibodies that interfere with factor VIII (FVIII) pro-coagulant activity, often referred to as “inhibitors”, can complicate the treatment of hemophilia A. These alloimmune responses, as well as the rare development of autoimmune FVIII inhibitors, are associated with significant morbidity and mortality. The production of anti-FVIII antibodies follows stimulation of helper T cells by epitopes in FVIII. An immunodominant HLA-DRB1*0101-restricted T-cell epitope was recognized by CD4+ T cells from a mild hemophilia A inhibitor subject and from his brother, who had a sub-clinical inhibitor (James et al., J Thromb Haemost 5: 2399-2407, 2007). Their CD4+ T cells recognized overlapping synthetic peptides with sequences corresponding to FVIII residues 2186-2205, 2187-2205 and 2194-2213. Nineteen T-cell clones recognizing this epitope were isolated, with phenotypes representing four distinct T-cell lineages. Aims:(1) to evaluate the promiscuity/immunodominance of an HLA-DRB1*0101-restricted T-cell epitope in FVIII; (2) to introduce amino acid substitutions that will prevent presentation of this epitope to the immune system by DR0101 and by other DR alleles. Methods.The minimal epitope and MHC Class II (DR0101) “anchor” residues were determined using a competition assay measuring displacement of a labeled peptide having high affinity for recombinant DR0101 by a series of FVIII peptides. Peptide concentrations at which 50% inhibition of the labeled peptide binding occurred (IC50) were obtained by regression analysis. Binding of the peptides to five additional DR alleles was evaluated directly using recombinant proteins; predicted binding of peptides to additional DR alleles was evaluated using the program ProPred. Proliferation and cytokine production by the clones in response to wild-type and modified peptides were measured, and the concentrations at which half-maximal T-cell responses (EC50) to the FVIII peptides occurred were determined. Results.Binding of truncated peptides to DR0101 identified FVIII2194-2205 as the minimal epitope. Binding of FVIII2194-2205 peptides with single Arg substitutions identified F2196, M2199, A2201 and S2204 as anchor residues at positions 1, 4, 6 and 9, respectively, corresponding to peptide-binding pockets seen in the crystal structure of a DR0101-peptide complex. The relative binding of Ala-substituted peptides confirmed that F2196 and M2199 are anchor residues. T-cell stimulation requires recognition of peptides by both the Class II receptor and the T-cell receptor (TCR). Sequences of TCR variable regions (TCRBVs) expressed by the clones were identified as TCRBV20-1*01 (3 VDJ combinations), TCRBV6-6*01, TCRBV5-1*01, and TCRBV6-1*01, indicating at least six different T-cell progenitors recognized this epitope. The clones were next stimulated with peptides having modified epitopes. Strikingly, none proliferated or secreted cytokines when stimulated by FVIII2194-2205, F2196A, which also showed an IC50 > 10 μM when tested for binding to DR0101, DR0301, DR0401, DR1101, DR1104, and DR1501. Substitutions at other anchor positions affected binding to some but not all of the DR proteins. Predicted binding of the F2196A variant to 51 DR alleles was analyzed using ProPred; none bound at a threshold stringency of 10% (low stringency, thus the predicted epitopes included those with lower calculated affinities). In preparation for directly testing the immunogenicity of additional substitutions, all possible amino acid substitutions at position 2196 were evaluated using ProPred. 13 of 19 possible substitutions were predicted to prevent FVIII2194-2205 binding to all 51 DR alleles included in the algorithm (with a 3% threshold = intermediate stringency). Conclusions.MHC class II anchor residues and TCR contact sites for an immunodominant HLA-DRB1*0101-restricted T-cell epitope have been mapped precisely. Both measured and predicted effects of amino acid substitutions indicated that this F2196 is essential for effective presentation of this epitope by multiple DR alleles. Effects of various sequence modifications on FVIII function, conformation and immunogenicity are currently being evaluated using recombinant FVIII and FVIII C2 domain proteins to indicate their possible therapeutic potential. Disclosures:Pratt:CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxter: Honoraria; Grifols: Honoraria.

Intraspecific Epitopic Variation in a Carbohydrate Antigen Exposed on the Surface of Trichostrongylus colubriformis Infective L3 Larvae

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

B-Cell Chronic Lymphocytic Leukemia (B-CLL) Cells Express Antibodies Reactive with Antigenic Epitopes Expressed on the Surface of Common Bacteria.

B-CLL cells of the unmutated (UM) subgroup frequently express polyreactive autoantibodies, similar to “natural” IgM autoantibodies, whereas most cells of the mutated (M) subgroup are oligo- or mono-reactive. Natural IgM autoantibodies cross-react with epitopes on microbes and, therefore, are positively selected to protect against common infections. In this study, we report reactivity profiles of recombinant antibodies cloned from B-CLL cells against three Gram-positive [Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis] and two Gram-negative bacterial strains [Enterobacter cloacae, strains CBAN and MK7]. Twenty-five B-CLL mAbs were tested: 13 expressed VH1 family genes (11/13 UM), of which 7 utilized VH1-69 (6/7 UM); 5 expressed VH3 genes (1/5 UM), while 7 expressed VH4 genes (4/7 UM). In general, UM-CLL mAbs exhibited the highest level of reactivity. The UM-VH1-69 mAbs showed the strongest binding: five VH1-69 mAbs were highly reactive with all bacterial strains, while two VH1-69 mAbs exhibited lower reactivity. Among other VH1 mAbs, stronger binding was observed for antibodies that belong to a set of quasi-identical BCRs. Interestingly, one of these mAbs bound exclusively S.pyogenes, although mutated and not polyreactive. Among VH3 mAbs, only a UM-VH3-21 mAb showed strong reactivity against all bacterial strains. One VH4 mAb, UM-VH4-39, bound well, reacting with all the bacteria tested. Other VH4 mAbs were also reactive, albeit their reactivity was weaker. Two M-VH4-34 antibodies reacted with some strains, although they did not react with common autoantigens. In all cases, mAbs reacted with significantly lower affinity with a1,3 dextran-positive E.cloacae strain MK7. In order to define the nature of the reactive bacterial antigens, S.pyogenes were subjected to treatment with pronase to digest proteins and with sodium periodate to oxidize carbohydrates. Reactivity of 5/7 VH1-69 mAbs and the UM-VH3-21 mAb was significantly suppressed by periodate oxidation, suggesting a carbohydrate epitope as the target for these mAbs. Pronase treatment enhanced binding of VH1-69 mAbs, further supporting recognition of a carbohydrate epitope. In contrast, binding of other (non-VH1-69) VH1 mAbs was significantly inhibited by pronase treatment, indicating a possible protein epitope. The VH4-39 mAb was sensitive to both treatments, suggesting recognition of either more than one epitope or a glycoprotein. The same pattern was observed for the non-polyreactive cases that bound to bacteria, indicating a glycoprotein as the target epitope. In conclusion, our data show that self/poly-reactive mAbs, especially those from U-CLL cases as well a few non-polyreactive mutated B-CLL mAbs, can react with antigenic epitopes on the surface of common bacteria. B-CLL mAbs encoded by different genes appear to react with different classes of antigenic epitopes. Thus, bacterial infections could intermittently stimulate B-CLL precursors, and possibly B-CLL cells themselves, allowing the interplay between autoreactivity and exoreactivity to promote survival, expansion, and malignant transformation, and potentially clonal evolution to a more aggressive state.

The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules.

The endosomes and lysosomes of antigen-presenting cells host the processing and assembly reactions that result in the display of peptides on major histocompatibility complex (MHC) class II molecules and lipid-linked products on CD1 molecules. This environment is potentially hostile for T cell epitope and MHC class II survival, and the influence of regulators of protease activity and specialized chaperones that assist MHC class II assembly is crucial. At present, evidence indicates that individual proteases make both constructive and destructive contributions to antigen processing for MHC class II presentation to CD4 T cells. Some features of CD1 antigen capture within the endocytic pathway are also discussed.