Identification Of Immunodominant Epitopes Research Articles

Comparison of Anti-Trop2 Extracellular Domain Antibodies Generated Against Peptide and Protein Immunogens for Targeting Trop2-Positive Tumour Cells.

Trophoblast antigen 2 (Trop2) is a transmembrane glycoprotein upregulated in multiple solid tumours. Trop2-based passive immunotherapies are in clinical trials, while Trop2 targeting CAR-T cell-based therapies are also reported. Information about its T- and B-cell epitopes is needed for it to be pursued as an active immunotherapeutic target. This study focused on identification of immunodominant epitopes in the Trop2 extracellular domain (ECD) that can mount an efficient anti-Trop2 antibody response. In silico analysis using various B-cell epitope prediction tools was carried out to identify linear and conformational B-cell epitopes in the ECD of Trop2. Three linear peptide immunogens were shortlisted and synthesized. Along with linear peptides, truncated Trop2 ECD that possesses combination of linear and conformational epitopes was also selected. Recombinant protein immunogen was produced in 293-F suspension culture system and affinity purified. Antisera against different immunogens were characterized by ELISA and Western blotting. Two anti-peptide antisera detected recombinant and ectopically expressed Trop2 protein; however, they were unable to recognize the endogenous Trop2 protein expressed by cancer cells. Antibodies against truncated Trop2 ECD could bind to the endogenous Trop2 expressed on the surface of cancer cells. In addition to their high avidity, these polyclonal anti-sera against truncated Trop2 protein also mediated antibody-dependent cell-mediated cytotoxicity (ADCC). In summary, our comparative analysis demonstrated the utility of truncated Trop2 ECD as a promising candidate to be pursued as an active immunotherapeutic molecule against Trop2-positive cancer cells.

Identification of immunodominant epitopes in allelic variants VK210 and VK247 of Plasmodium Vivax Circumsporozoite immunogen.

Plasmodium vivax-induced malaria is among the leading causes of morbidity and mortality in sub-tropical and tropical regions and infect 2.85 billion people globally. The continual rise and propagation of resistance against anti-malarial drugs is a prerequisite to develop a potent vaccine candidate for Plasmodium vivax (P. vivax). Circumsporozoite protein (CSP) is an important immunogen of malaria parasite that has the conserved CSP structure as an immune dominant B-cell epitope. In current study, we focused on designing multi-epitope vaccines (MEVs) using various immunoinformatics tools against Pakistani based allelic variants VK210 and VK247 of P. vivax CSP (PvCSP) gene. Antigenicity, allergic potential and physicochemical parameters of both PvCSP variants were assessed for the designed MEVs and they were within acceptable range suitable for post experimental investigations. The three-dimensional structures of both MEVs have been predicted ab initio, optimized, and validated by using different online servers. The both MEVs candidates were stable and free from aggregation-prone regions. The stability of both MEVs had been improved by a disulfide engineering approach. To estimate the binding energy and stability of the MEVs, molecular docking simulation and binding free energy calculations with TLR-4 immune receptor have been conducted. The docking score of PvCSP210 and PvCSP247 for TLR-4 was -6.34kJ/mol and-2.3kJ/mol, respectively. For PvCSP210-TLR4 system, mean RMSD was 4.96Å while PvCSP247-TLR4 system, average RMSD was 4.49Å. The binding free energy of PvCSP210-TLR4 complex and PvCSP247-TLR4 complex was -50.49/-117.15kcal/mol (MMGBSA/MMPSA) and -52.94/-96.26kcal/mol (MMGBSA/MMPSA), respectively. The expression of both MEVs produced in Escherichia coli K12 expression system by in silico cloning was significant. Immune simulation revealed that the proposed MEVs induce strong humoral and cellular immunological responses, in addition to significant production of interleukins and cytokines. In conclusions, we believed that the MEVs proposed in current research, using combine approach of immunoinformatics, structural biology and biophysical approaches, could induce protective and effective immune responses against P. vivax and the experimental validation of our findings could contribute to the development of potential malaria vaccine.

Experimental Identification of Immuno- dominant B-cell Epitopes from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current public health crisis with devastating consequences to our societies. This COVID-19 pandemic has become the most serious threat to global public health in recent history. Given the unprecedented economic and social impact that it is causing, identification of immunodominant epitopes from SARS-CoV-2 is of great interest, not only to gain better insight into the adaptive immune response, but also for the development of vaccines, treatments and diagnostic tools. In this review, we summarize the already published or preprinted reports on the experimental identification of B-cell linear epitopes of SARS-CoV-2 proteins. Six different epitopes leading to neutralizing antibodies have been identified. Moreover, a summary of peptide candidates to be used for diagnostic tools is also included.

Evaluation of a cystatin-like protein of Trichinella spiralis for serodiagnosis and identification of immunodominant epitopes using monoclonal antibodies

An antigenic cystatin-like protein (Ts-CLP) selected from cDNA library of intestinal infective larvae at 6 h post-infection, was expressed by prokaryotes in the form of a histidine-tagged protein (rTs-CLP). The fusion protein was purified by an on-column refolding procedure using Ni-NTA affinity chromatography. An indirect rTs-CLP ELISA was developed using 270 known negative serum samples from commercial swine maintained under non-special pathogen free conditions. Based on the distribution of the signal-to-positive (S/P) ratio, a cut-off value was set at 0.30. Using this cut-off value, rTs-CLP ELISA was evaluated using sera from swine experimentally infected with 1000 and 50,000 muscle larvae of Trichinella spiralis. Specific IgG antibodies were detectable by rTs-CLP ELISA as soon as 17 days post-infection (dpi), but the commercial ELISA kit based on excretory-secretory (ES) antigens did not permit detection before 21 dpi. Three monoclonal antibodies (McAbs) against Ts-CLP (designated 1H9, 6B5 and 7F8) were obtained by screening with both rTs-CLP ELISA and ES ELISA methods. Two dominant epitopes recognized by McAbs were determined by analysis with overlapping fusion peptides and synthetic peptides. One epitope 39 HEALFSSDLKQESGV 53 was recognized by 1H9 and 6B5, and the other epitope 178 REALFSSDSKEQSGV 192 was recognized by 7F8. The generation of McAbs against Ts-CLP and the characterization of the two dominant epitopes provide a foundation for the development of a specific early serodiagnostic strategy for T. spiralis infection.

A Sampling of Highlights from the Literature

Inflamed tumor (by Y. Rosen via Wikimedia Commons)The use of 300 patient samples across 22 cancer types from PD-1 blockade trials revealed that inflammatory gene expression profiling and the tumor mutational burden are independent biomarkers for objective response rates to anti–PD-1. This finding was validated in a set of more than 6,000 tumors from TCGA database, in which biomarker-defined patterns identified potential targets to overcome resistance to therapy.Cristescu R, …, Kaufman D. Science 2018 Oct 12;362:eaar3593.Clustered macrophages from hyperprogression (from Fig. 2 of Lo Russo et al., Clin Cancer Res 2018)Blockade of immune checkpoints in some NSCLC patients causes a hyperprogression that correlates with the presence of myeloperoxidase+ myeloid cells and CD163+CD33+PD-L1+ macrophages. When mice with patient-derived xenografts are treated with anti–PD-1, tumors hyperprogress and increase their numbers of M2 macrophages. Treatment with anti–PD-1 F(ab)2 fragments, however, does not induce hyperprogression. Thus, stimulation of innate cell Fc receptors within tumors can sustain hyperprogression.Lo Russo G, …, Garassino MC. Clin Cancer Res 2018 Sep 11. DOI: 10.1158/1078-0432.CCR-18-1390.Blocking phagocytosis inhibition (from Ahkâm via Free Icons PNG)Tumor-promoting macrophages can express CD47, which inhibits phagocytosis. A phase Ib trial tested blockage of the CD47-SIRPα interaction, combined with rituxibab (anti-CD20), in 22 patients with relapsed/refractory non-Hodgkin lymphoma. Refractory lymphoma cells are eliminated via macrophage-mediated phagocytosis. Half of patients achieved objective responses, with manageable grade 1 or 2 adverse effects. This combination boosts antitumor immunity and shows promise for patients with aggressive lymphomas.Advani R, …, Smith SM. N Engl J Med 2018 Nov 1;379:1711–21.Macrophage-surrounded tumor (by R. Weissleder via NCI).Complement is key to inflammation and may promote protumoral microenvironments. Urokinase+ macrophages in squamous carcinomas produce C5a, which attracts other phagocytes and induces their degranulation while promoting secretion of proangiogenic and tissue remodeling cytokines. PMX-53 blocks C5a from activating its receptor and causes an increase in M1 macrophages, setting up chemokine and cytokine conditions that, with chemotherapy, recruit CD8+ T cells and inhibit tumor growth.Medler TR, …, Coussens LM. Cancer Cell 2018 Oct 8;34:561–78.Identifying standout epitopes (by Trajaner via Pixabay)Identification of immunodominant epitopes in disease can be accomplished with a deep neural network for genome-scale predictions. The described "bacteria originated T cell antigen" (BOTA) predictor identifies MHC class II–restricted epitopes from model pathogens. The CD4+ T-cell epitopes were validated through high-throughput screening that could identify reactive TCRs based on the genomic sequence of bacterial epitopes. Application of this technique to cancer may help identify immunodominant epitopes in tumors.Graham DB, …, Xavier RJ. Nat Med 2018 Oct 22;24:1762–72.Protein barcodes for CRISPR (by by Marius Walter via Wikimedia Commons)CRISPR can be used to produce unique nucleotide sequences (barcodes), and usually these are “read” on the DNA level. A barcoding system at the protein level called Pro-codes has been developed. Synthesized sequences encoding 3 combinations of 14 different linear epitopes, results in more than 300 Pro-codes. Each Pro-code was paired to CRISPR single-guide RNA and used to screen for genes affecting breast cancer sensitivity to antigen-specific T-cell responses. Identification of Rtp4, Psmb8, and Socs1 indicates this method can identify mechanisms of immune evasion.Wroblewska A, …, Brown BD. Cell 2018 Nov 1;175:1141–55.

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

Computational Prediction of Immunodominant Epitopes on Outer Membrane Protein (Omp) H of Pasteurella multocida Toward Designing of a Peptide Vaccine.

Contemporary vaccine design necessitates discrimination between the immunogenic and non-immunogenic components within a pathogen. To successfully target a humoral immune response, the vaccine antigen should contain not only B-cell epitopes but abounding Th-cell agretopes and MHC-II binding regions as well. No single computational method is available that allows the identification of such regions on antigens with good reliability. A consensus approach based on several prediction methods can be adopted to overcome this problem.Targeting the outer membrane protein (Omp) H as a candidate, a comprehensive work flow is described for the computational identification of immunodominant epitopes toward the designing of a peptide vaccine against Pasteurella multocida.

Characterization of peanut-reactive monoclonal antibodies (HYP7P.265)

Abstract Peanut allergies are a rising concern in Western society. Incidence and prevalence of affected children has tripled from 1997 to 2010. The exact cause for this increase is unknown, but lack of exposure to pathogens and increasing hygienic conditions has been implicated. Exposure to peanuts causes the immune system to become sensitized to peanut proteins. Subsequent exposure to peanuts causes hypersensitivity reactions which may escalate to anaphylaxis and death. The only course of prevention is complete avoidance. Limiting peanut exposure is difficult as even trace amounts of peanut (100µg-50mg; 1 peanut ≈ 1g) consumed can cause a reaction. Immunotherapies aim to increase the tolerance of these severely allergic individuals. Current approaches are limited to certain populations and are potentially dangerous. Better therapeutics require thorough characterization of peanut proteins responsible for peanut allergies. In this study, C3H/HeJ mice were sensitized to crude peanut extract (CPE). The mice were then exposed to CPE to induce a hypersensitive reaction. Hybridomas were generated , characterized for antibody production, subtype, and screened for peanut-specific immunoreactivity, as well as specific peanut proteins believed to be involved with peanut allergies. Characterization of peanut-specific monoclonal antibodies (MAb) may aid in the identification of immunodominant epitopes and lead to/aid in the development of immunotherapeutic interventions.

T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5− DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.

Identification of Immunodominant Epitopes in N-Acetylgalactosamine 6-Sulfate Sulfatase (GALNS) for Designing an Effective Peptide-Based Immunotherapy

Identification of Immunodominant Epitopes in N-Acetylgalactosamine 6-Sulfate Sulfatase (GALNS) for Designing an Effective Peptide-Based Immunotherapy

Identification of immunodominant epitopes of alpha-crystallins recognized by antibodies in sera of patients with uveitis

A high incidence of autoantibodies to lens proteins has been found in sera of patients with uveitis. We showed previously that the anti-lens antibodies reacted predominantly with α-crystallins. The aim of the present study was to identify immunodominant epitopes within the protein chains of human αA- and αB-crystallin. Epitope specificities of antibodies to αA- and αB-crystallin were examined by ELISA using synthetic overlapping peptides, spanning the entire length of both α-crystallins. The peptides consisted of 25 amino acid residues, with an overlap of at least eight amino acids each. The synthetic peptides were tested against sera of 110 patients with different uveitis forms, classified according to anatomical location of intraocular inflammation. Four immunodominant regions within the protein chains of αA- and αB-crystallin could be identified. These regions were recognized by antibodies in sera of 56% of uveitis patients. Anti-lens antibodies of IgG-type reacted preferentially with regions located at amino acid (aa) residues aa:69-93 and aa:137-161 of αA-crystallin as well as aa:69-110 and aa:137-161 of αB-crystallin. IgM antibodies recognized predominantly region aa:149-173 of αA-crystallin, and aa:69-110 and aa:151-175 of αB-crystallin. IgM antibodies directed to peptide aa:69-93 of αB-crystallin were found in sera of 30% of patients with intermediate uveitis. Four immunodominant B-cell epitopes within the protein chains of αA- and αB-crystallin have been identified; however, no clear correlation with the anatomically defined uveitis subtypes has been found except for intermediate uveitis. Whether there may be a correlation with uveitis forms with similar etiopathogenesis has to be evaluated in further studies.

Epitope spreading of the anti-CYP2D6 antibody response in patients with autoimmune hepatitis and in the CYP2D6 mouse model

Autoimmune hepatitis (AIH) is a serious chronic inflammatory disease of the liver with yet unknown etiology and largely uncertain immunopathology. The hallmark of type 2 AIH is the generation of liver kidney microsomal-1 (LKM-1) autoantibodies, which predominantly react to cytochrome P450 2D6 (CYP2D6). The identification of disease initiating factors has been hampered in the past, since antibody epitope mapping was mostly performed using serum samples collected late during disease resulting in the identification of immunodominant epitopes not necessarily representing those involved in disease initiation. In order to identify possible environmental triggers for AIH, we analyzed for the first time the spreading of the anti-CYP2D6 antibody response over a prolonged period of time in AIH patients and in the CYP2D6 mouse model, in which mice infected with Adenovirus-human CYP2D6 (Ad-h2D6) develop antibodies with a similar specificity than AIH patients. Epitope spreading was analyzed in six AIH-2-patients and in the CYP2D6 mouse model using SPOTs membranes containing peptides covering the entire CYP2D6 protein. Despite of a considerable variation, both mice and AIH patients largely focus their humoral immune response on an immunodominant epitope early after infection (mice) or diagnosis (patients). The CYP2D6 mouse model revealed that epitope spreading is initiated at the immunodominant epitope and later expands to neighboring and remote regions. Sequence homologies to human pathogens have been detected for all identified epitopes. Our study demonstrates that epitope spreading does indeed occur during the pathogenesis of AIH and supports the concept of molecular mimicry as a possible initiating mechanism for AIH.

85 IL28B GENETIC STATUS IN ASSOCIATION WITH HCV SPECIFIC T-CELL IMMUNITY

Introduction: Hepatitis E virus (HEV) infection usually results in an acute, self-limiting hepatitis in immunocompetent individuals while it may rarely progress to fulminant hepatic failure. Several cases of chronic evolution of HEV infections have been described recently in French, Dutch and German immunosuppressed patients. However immune correlates of clearance or persistence of HEV have not been described so far. Aims of this study were to i. investigate whether HEV persistence is associated with impaired immune responses and ii. identify potential epitopic regions in HEV genome that may elicit strong immune cellular responses to be considered for a potential vaccine. Methods: Immune responses against HEV were studied in a total of 29 subjects including 21 healthy immunocompetent individuals (anti-HEV-positive; n = 13 (“exposed”); anti-HEV-negative n =8 (“no exposure”)) and 8 anti-HEV-positive patients after organ transplantation. 4 transplanted patients evolved to chronic hepatitis while 4 subjects were followed and cleared HEV spontaneously. PBMCs were stimulated with HEV overlapping peptide pools (spanning ORF2 and ORF3) or individual peptides. T-cell proliferation was measured after 7 days by CFSE (5, 6-carboxyfluorescein diacetate succinimidyl ester) assay. Cytokine secretion was investigated by intra-cellular cytokine staining (IFN-gamma, MIP-1beta and TNF-alpha). Results: Multispecific (positive responses for more at least 2 peptide pools) and strong HEV-specific CD4/CD8 T-cell were detectable in 77%/62% of HEV-seropositive healthy subjects respectively. No HEV-specific responses were observed in any of the HEV-unexposed individuals. In transplant recipients, multispecific but moderate T-cell responses were detectable in resolved individuals while chronic patients did not show any specific T cell response. Using individual peptides, immune responses were narrowed-down to two epitopes that presented very strong CD8 responses. These results were further corroborated by cytokine production, where IFN-gamma and MIP-1beta secretion was observed only in seropositive healthy individuals. Conclusion: These results suggest that chronic HEV infection is associated with impaired HEV-specific adaptive immune responses. Identification of immunodominant epitopes may have implications for vaccine design. 85 IL28B GENETIC STATUS IN ASSOCIATION WITH HCV SPECIFIC T-CELL IMMUNITY C. Bucci, M. Rahman, I. Humphreys, A.C. Brown, M. Lucas, K. Pfafferott, J. Collier, D. O’Donnell, E. Stafford, P. Klenerman, S. Gaudieri, E. Barnes. Department of Hepatology, Nuffield Department of Medicine & Oxford NIHR BRC, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Institute of Immunology and Infectious Diseases, Murdoch University, Perth, WA, Australia; Hepatology & Oxford NIHR BRC, University of Oxford, Oxford, UK; School of Anatomy and Human Biology and Centre for Forensic Science, University of Western Australia, Perth, WA, Australia E-mail: cristinabucci@hotmail.it

Stepwise identification of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus type 1 genome boosted by a steprank scheme

Identification of immunodominant epitopes is the first step in the rational design of peptide vaccines aimed at T-cell immunity. To date, however, it is yet a great challenge for accurately predicting the potent epitope peptides from a pool of large-scale candidates with an efficient manner. In this study, a method that we named StepRank has been developed for the reliable and rapid prediction of binding capabilities/affinities between proteins and genome-wide peptides. In this procedure, instead of single strategy used in most traditional epitope identification algorithms, four steps with different purposes and thus different computational demands are employed in turn to screen the large-scale peptide candidates that are normally generated from, for example, pathogenic genome. The steps 1 and 2 aim at qualitative exclusion of typical nonbinders by using empirical rule and linear statistical approach, while the steps 3 and 4 focus on quantitative examination and prediction of the interaction energy profile and binding affinity of peptide to target protein via quantitative structure-activity relationship (QSAR) and structure-based free energy analysis. We exemplify this method through its application to binding predictions of the peptide segments derived from the 76 known open-reading frames (ORFs) of herpes simplex virus type 1 (HSV-1) genome with or without affinity to human major histocompatibility complex class I (MHC I) molecule HLA-A*0201, and find that the predictive results are well compatible with the classical anchor residue theory and perfectly match for the extended motif pattern of MHC I-binding peptides. The putative epitopes are further confirmed by comparisons with 11 experimentally measured HLA-A*0201-restrcited peptides from the HSV-1 glycoproteins D and K. We expect that this well-designed scheme can be applied in the computational screening of other viral genomes as well.

Identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus.

Similar to other coronaviruses, the membrane (M) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a major transmembrane glycoprotein with multiple biological functions. To date, limited information is available about its antigenic properties. In this study, we identified two major immunodominant epitopes on the M protein located in the extreme N-terminal region (residues 1 to 31) and the interior C-terminal region (residues 132 to 161), respectively, by Pepscan analyses against convalescent-phase sera from SARS patients and antisera from virus-immunized mice and rabbits. Synthetic peptides M1-31 derived from the N-terminal epitope and M132-161 derived from the C-terminal epitope were highly reactive with all of the convalescent-phase sera from 40 SARS patients but not with 30 control serum samples from healthy blood donors, suggesting their potential application for serologic diagnosis of SARS. We showed that both peptides (M1-31 and M132-161) were able to induce high titers of antibody responses in the immunized rabbits, highlighting their antigenicity and immunogenicity. These findings provide important information for developing SARS diagnostics and vaccines.

HLA-B7 β-pleated sheet-derived synthetic peptides are immunodominant T-cell epitopes regulating alloresponses

AbstractChronic rejection of transplanted allografts is the major cause of graft loss after clinical solid organ transplantation. Recent data link the indirect presentation of allopeptides to chronic graft loss; thus, identification of immunodominant epitopes on major histocompatibility complex (MHC) antigens could significantly contribute to establishing novel ways for monitoring and managing chronic rejection. Here, we show that synthetic allo-MHC–derived peptides covering the polymorphic region 56 to120 of HLA-B7 modulate alloresponses. In particular, the 2 β-pleated sheet-derived peptides covering residues 91 to 105 and 96 to 120, respectively, but not sequences from the α1 helix, were presented by autologous peripheral blood lymphocytes to induce T-cell proliferation. In addition, the 2 β-pleated sheet-derived peptides and the α1-derived peptide residues 60 to 75 abrogated lysis of HLA-B7 target cells by anti–HLA-B7 cytotoxic T lymphocytes (CTLs). Although most residues between 91 and 120 are normally not directly accessible to T cells, our results indicate that peptides derived from the lower surface of the peptide-binding groove of HLA-B7 are immunodominant in HLA-B7 alloresponses. To characterize the binding and stability of allopeptides to T cells, the 62-70 peptide—derived from the 60-75 allopeptide that blocked cytotoxicity of anti–HLA-B7 CTL—was synthesized and coupled with fluorescein isothiocyanate. The peptide specifically labeled anti-B7 CTL, but not anti–HLA-A2 CTL as measured by flow cytometry. Peptide binding to CTL was specific at 4°C and remained stable for 12 hours, whereas it remained stable for less than 2 hours at 37°C. These studies allow the identification of HLA-B7 T-cell epitopes and reveal for the first time a novel, previously unrecognized application of synthetic HLA-derived allopeptides to visualize alloreactive T cells.

Hierarchies of antigen‐specific cytotoxic T‐cell responses

Studies carried out using either mice or humans have shown that cytotoxic T-lymphocyte (CTL) responses to many different pathogenic organisms often comprise CTL specific for multiple class I-restricted peptide epitopes. Differences in the magnitude of epitope-specific CTL responses appear to arise mainly from differences in the expression level of the corresponding class I/peptide complex on the surface of the antigen-presenting cell. The size of the CTL response may be limited by the frequency and possibly by the affinity of specific CTL precursors in the naive T-cell pool. Thus, both the efficiency of antigen processing and the composition of the peripheral T-cell pool impose direct limitations on the extent of a T-cell response to a given peptide epitope. Studies of CTL hierarchies have resulted in the identification of immunodominant epitopes i.e. peptide epitopes which stimulate the largest number of specific CTL and which are therefore generally believed to offer the best level of protection against the pathogen from which they were derived. It is also thought that CTL responses to non-dominant epitopes mediate protection against pathogenic challenge. These ideas are considered here with respect to experimental data collected following infection of mice with lymphocytic choriomeningitis virus.

Identification of immunodominant epitopes in the core and non-structural region of hepatitis C virus by enzyme immunoassay using synthetic peptides.

Thirty-two synthetic peptides, components of the core and non-structural protein of Hepatitis C virus (HCV), were tested for their reactivities against antibodies in sera of healthy, HCV antibody positive of chronic liver disease patients. Among them, 8 of the core peptides, 4 of the NS4 peptides and 3 of the NS5 peptides reacted with the HCV infected sera. In particular, C22 (core peptide) and NS4-1924 (NS4 peptide) were most reactive with the serum samples giving a positive signal with commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our results indicate that the immunodominant regions of the HCV-derived proteins are located at three regions in the core protein, three regions in the NS4 protein, and one region in the NS5 protein. These results indicate that the selected peptides are useful antigens in detecting antibodies in the sera from individuals infected with HCV.

Autoantigens in thyroid diseases.

The autoantigens involved in autoimmune thyroid disease have now been extensively characterised, and the autoantibodies they evoke provide important aids to diagnosis, leading to early treatment of thyroid autoimmunity. The next stage in the puzzle is to determine towards which epitopes on the autoantigens the immune response is directed. We have already come a long way in the identification of immunodominant epitopes and have been able to identify one T cell epitope which has pathogenic capabilities. Identification of other T cell and B cell epitopes will help us understand the cell-mediated and humoral responses in greater detail and in time lead to more specific therapeutic intervention. A greater understanding of the mechanisms underlying one particular autoimmune disease will give us insights into other diseases, due to the belief that there may well be common underlying defects that, due to a multitude of factors, manifest as different diseases. The susceptibility factors in autoimmune thyroidits and autoimmune disease in general are very complex. A greater understanding is required of HLA associations and how particular peptides are presented in vivo. Are susceptible MHC types the ones capable of presenting the pathogenic peptides? Our major T cell thyroiditogenic epitope contains a T4 residue which accounts for over half the molecular weight of the peptide. Its structure is large and consists of a double benzene ring structure with four iodine atoms. It will be interesting to see how such a peptide can be presented and which residues bind T cell receptor or MHC. In summary we can say that autoimmune disease is due to a cocktail of factors which all contrive to tip the delicate balance of the immune system into an autoimmune state. HLA association may play a role in conferring an enhanced ability to select from a restricted repertoire of pathogenic epitopes, those epitopes perhaps only becoming available for presentation after interaction with environmental agents, whatever they may be. Following this, the normal regulation of self presentation and tolerance mechanisms break down and autoimmunity supervenes.

Identification of immunodominant epitopes in envelope glycoprotein of human T Lymphotropic virus type II

A series of synthetic peptides derived from the envelope glycoprotein of human T lymphotropic virus type II (HTLV-II) was used in an enzyme immunoassay to determine the immunodominant epitopes of envelope glycoprotein. Of the 11 synthetic peptides spanning the external glycoprotein of HTLV-II (gp52) and the 3 from the transmembrane protein (gp21), 3 peptides from gp52 (termed Env-20(85-102), Env-202(173-209), and Env-203(219-256] reacted with most of the polymerase chain reaction-confirmed HTLV-II specimens (83, 95, and 76%, respectively); all other peptides reacted minimally with these specimens. Env-202(173-209) reacted with a greater percentage (91 to 100%) of specimens from different risk groups, including intravenous drug users (n = 30), North American Indians (n = 13), Guaymi Indians from Panama (n = 22), and routine U.S. blood donors (n = 34), when compared with Env-20(85-102) (73 to 100%) or Env-203(219-256) (68 to 83%). Furthermore, Env-20(85-102) and Env-202(173-209) had some reactivity (8-25%) with sera from HTLV-I-infected individuals, whereas Env-203(219-256) reacted with 58% of HTLV-I specimens. We conclude that peptides Env-20(85-102) and Env-202(173-209) represent the type-specific immunodominant epitopes of HTLV-II external glycoprotein.