B-Cell and T-Cell Epitopes in Anti-factor VIII Immune Responses

Adequate hemostasis is achieved for many hemophilia A patients by infusion of plasma-derived or recombinant factor VIII (FVIII), but unfortunately, a significant subset of patients develop an immune response in which anti-FVIII antibodies, referred to clinically as “inhibitors,” interfere with its procoagulant activity. Inhibitors are the subset of anti-FVIII antibodies that bind to surfaces on FVIII (B-cell epitopes) that are important for its proper functioning in coagulation. Less antigenic FVIII molecules may be designed by identifying and then modifying the amino acid sequences of inhibitor B-cell epitopes. Conversely, characterization of these epitopes can yield important information regarding functionally important surfaces on FVIII. The production of inhibitor antibodies is driven by T cells. T cells recognize FVIII as foreign when FVIII-derived peptides bind to major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells. The class II–peptide complexes must then be recognized by T-cell receptors (TCRs). T-cell stimulation requires sustained association of antigen-presenting cells and T cells through formation of a class II–peptide–TCR complex, and peptide sequences that mediate this association are termed “T-cell epitopes.” MHC class II tetramers that bind FVIII-derived peptides and recognize antigen-specific TCRs are proving useful in the characterization of human leukocyte antigen-restricted T-cell responses to FVIII.     

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.     

A pattern search method for putative anchor residues in T cell epitopes

The binding affinity between an antigenic peptide and its particular major histocompatibility complex (MHC) molecule seems to be largely determined by only a few residues. These residues have been called “anchors” because of their property of fitting into “pockets” inside the groove of the MHC molecule. To predict natural antigenic epitopes within a longer sequence, it therefore appears to be important to know the motif or pattern describing the anchors, i.e. the anchors amino acid residue preference and the distance between anchor residues. A large set of MHC class I-restricted peptides has been described. Peptide sequences vary in length and lack an obvious common sequence motif. For a list of peptides belonging to one type of MHC class I molecule, we describe a method to find the most prominent sequence motif with at least two anchor residues. Briefly, antigenic sequences are aligned, and two anchor positions are searched for, where all anchor residues share a high similarity. The alignments are scored according to the similarity of their anchor residues. We show that the motifs predicted for the MHC alleles A2.1, B27, K^b, K^d, D^b are in substantial agreement with experimental data. We derive binding motifs for the MHC class I alleles HLA-A1, All, B8, B14, H-2L^d and for the MHC class II alleles I-A^b and I-A^s. In some cases, higher scores were obtained by allowing a slight variation in the number of residues between anchors. Therefore, we support the view that the length of epitopes belonging to a particular class I MHC is not uniform. This method can be used to predict the natural short epitope inside longer antigenic peptides and to predict the epitopes anchor residues. Anchor motifs can be used to search for antigenic regions in sequences of infectious viruses, bacteria and parasites.     

Implications of available design space for identification of non-immunogenic protein therapeutics

Immunogenicity/antibody responses are major issues for parenteral proteins and nanotherapeutics (nanovectors, diagnostics, theranostics, etc.), and robust antibody responses require T-helper epitopes. T-helper epitopes consist of specific amino acids at specific positions (anchor positions) in immunogens which contact the major histocompatibility complex (MHC), provide most of the energy for MHC binding and constitute the binding motif for the corresponding MHC alleles. We developed an algorithm that considers motifs to design vaccines lacking unwanted T-cell epitopes, and found numbers of such vaccines can be astronomical (Lee et al. 2009). The algorithm can be used to design reduced immunogenicity proteins, and numbers of predicted proteins are also immense. Reducing T-helper epitope content reduces protein immunogenicity, but the depth of mutagensis needed to eliminate immunogenicity is commonly assumed to be too great for retention of protein bioactivity. However, very deep, but successful substitution, insertion and deletion mutagenesis have been reported. These reports and design space the algorithm reveals suggest development of non-immunogenic therapeutics might be more feasible than commonly assumed.     

Proteasome and peptidase function in MHC-class-I-mediated antigen presentation

MHC-class-I-presented peptides are predominantly generated by the proteasome system. IFN-gamma strongly influences the processing efficiency by inducing immunoproteasome formation and proteasome activator PA28 synthesis. Depending on the protein substrate, the presence of immunoproteasomes and PA28 influence epitope liberation either positively or negatively. Abundantly occurring defective ribosomal products are a major source for proteasome-dependent antigen processing; however, antigen presentation is relatively inefficient. This is in part due to the existence of a panel of cytosolic aminopeptidases, such as bleomycin hydrolase (BH), puromycin-sensitive aminopeptidase (PSA) and thimet oligoendopeptidase (TOP), that can destroy epitopes or their precursors. Other aminopeptidases, such as leucine aminopeptidase (LAP) and endoplasmic reticulum aminopeptidase 1 (ERAP 1), can trim epitope precursors from the amino terminus to their correct size for MHC class I binding to enhance antigen presentation. Recent evidence suggests that tripeptidyl peptidase II (TPPII), a large peptidase with exo-and endo-proteolytic activities, is also involved in antigen processing and may generate a specific set of MHC class I epitopes.     

Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes

To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5-45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830-844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L(4)T(4))(2) and (L(2)T(2))(4)] and the helix conformation in one [(H(2)T(2))(4)] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L(2)T(2))(4), were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L(4)T(4))(2), (L(2)T(2))(4), (H(2)T(2))(4)] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.     

B and T Cell Epitope‐Based Peptides Predicted from Evolutionarily Conserved and Whole Protein Sequences of Ebola Virus as Vaccine Targets

Ebola virus (EBV) has become a serious threat to public health. Different approaches were applied to predict continuous and discontinuous B cell epitopes as well as T cell epitopes from the sequence‐based and available three‐dimensional structural analyses of each protein of EBV. Peptides ‘⁷⁹VPSATKRWGFRSGVPP⁹⁴’ from GP1 and ‘⁵¹⁵LHYWTTQDEGAAIGLA⁵³⁰’ from GP2 of Ebola were found to be the consensus peptidic sequences predicted as linear B cell epitope of which the latter contains a region ⁵¹⁹TTQDEG⁵²⁴ that fulfilled all the criteria of accessibility, hydrophilicity, flexibility and beta turn region for becoming an ideal B cell epitope. Different nonamers as T cell epitopes were obtained that interacted with different numbers of MHC class I and class II alleles with a binding affinity of <100 nm . Interestingly, these alleles also bound to the MHC class I alleles mostly prevalent in African and South Asian regions. Of these, ‘LANETTQAL’ and ‘FLYDRLAST’ nonamers were predicted to be the most potent T cell epitopes and they, respectively, interacted with eight and twelve class I alleles that covered 63.79% and 54.16% of world population, respectively. These nonamers were found to be the core sequences of 15mer peptides that interacted with the most common class II allele, HLA‐DRB1*01:01. They were further validated for their binding to specific class I alleles using docking technique. Thus, these predicted epitopes may be used as vaccine targets against EBV and can be validated in model hosts to verify their efficacy as vaccine.     

Inhibition of HLA B8-restricted recognition by unrelated peptides: evidence for allosteric inhibition

A panel of synthetic peptides representing human lymphocyte antigen (HLA) B8, other class I and class II restricted T cell epitopes and two B cell epitopes, were all able to compete with recognition of a HLA B8 restricted epitope by a cytotoxic T cell clone. Competition was obtained when the competitor peptides were added either before or after the target epitope. The target epitope also had a slow off rate, implicating allosteric inhibition. The presence of non-specific, allosteric binding sites may interfere with experiments attempting to define immunologically relevant MHC binding specificities.     

The interface between tapasin and MHC class I: identification of amino acid residues in both proteins that influence their interaction

Prior to the binding of antigenic peptide, a complex of chaperone proteins associates with the Major Histocompatibility Complex (MHC) class I heavy chain/β₂m heterodimer. Although each dornain of the MHC class I heavy chain contains amino acid resid uses that influence chaperone binding, there are several pieces of evidence that point to an interaction between the MHC clas 1α2/α3 domains and tapasin. In egard to the site on tapasin involved in the tapasin/MHC interface, we have found that a particular region of tapasin (containing amino acid residues 334–342) is necessary for the binding of tapasin to the MHC class I heavy chain. Our results also indicate that amino acids in this region of tapasin also affect the proportion of MHC class I open forms expressed at the cell surface and MHC class I egress from the endoplasmic reticulurn. Based on these results and those obtained by other laboratories, a model for MHC class I/tapasin interaction is proposed.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Need for cellular and humoral immune responses in bovines to ensure protection from foot-and-mouth disease virus (FMDV)—A point of view

The published studies on immunization of experimental animals, cattle, and sheep with synthetic peptides containing the antigenic domains in FMDV structural protein VP1 were analyzed. The results obtained with various FMDV synthetic peptides designed to stimulate the humoral immune response in bovines were compared to the current knowledge on MHC class I and class II, and the properties of the peptide binding grooves in each of them. X-ray crystallography of MHC class I proteins provided the three-dimensional structure of the peptide binding groove and led to the isolation and identification of self and viral peptides that naturally associate with the peptide binding grooves of both types of MHC and HLA molecules. The available knowledge of the amino acid motifs in MHC and HLA class I-bound viral peptides priming the CD8⁺ cytotoxic T cell responses must be coupled with the understanding of the three-dimensional structure of BoLA class I. This would aid in the development of an experimental approach to induce bovine anti-FMDV CD8⁺ cytotoxic cells to complement the humoral immune response to FMDV, which is currently achieved by a killed virus vaccine and, at the experimental level, by a peptide vaccine. Stimulation of both cellular and humoral immune responses against FMDV in cattle may reduce the risk of disease and virus shedding.     

Epitope affinity for MHC class I determines helper requirement for CTL priming

We show here that priming and memory generation of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) does not require help if the immunogen binds major histocompatibility complex (MHC) class I molecules with high affinity. This conclusion was based on the study of three chemically distinct optimal length CTL epitopes with high affinity for the restriction element Kb. In contrast, when two subdominant epitopes with intermediate MHC binding affinity were studied, either a class II MHC-restricted T helper cell epitope or administration of antibody to CD40 was required to obtain significant CTL priming. Depending on the epitope, one source of help was much more efficient than the other.     

Antigen three-dimensional structure guides the processing and presentation of helper T-cell epitopes

Antigen three-dimensional structure potentially controls presentation of CD4(+) T-cell epitopes by limiting the access of proteolytic enzymes and MHC class II antigen-presenting proteins. The protease-sensitive mobile loops of Hsp10s from mycobacteria, Escherichia coli, and bacteriophage T4 (T4Hsp10) are associated with adjacent immunodominant helper T-cell epitopes, and a mobile-loop deletion in T4Hsp10 eliminated the protease sensitivity and the associated epitope immunodominance. In the present work, protease-sensitivity and epitope presentation was analyzed in a group of T4Hsp10 variants. Two mobile-loop sequence variants of T4Hsp10 were constructed by replacing different segments of the mobile loop with an irrelevant sequence from hen egg lysozyme. The variant proteins retained native-like structure, and the mobile loops retained protease sensitivity. Mobile-loop deletion and reconstruction affected the presentation of two epitopes according to whether the epitope was protease-independent or protease-dependent. The protease-independent epitope lies within the mobile loop, and the protease-dependent epitope lies in a well-ordered segment on the carboxy-terminal flank of the mobile loop. The results are consistent with a model for processing of the protease-dependent epitope in which an endoproteolytic nick in the mobile-loop unlocks T4Hsp10 three-dimensional structure, and then the epitope becomes available for binding to the MHC protein.     

Characterization of intertype specific epitopes on adenovirus hexons

Summary.  The specification and differentiation of eighteen intertype specific (IT) epitopes of adenovirus hexons defined by monoclonal antibodies are given by two groups of hexon types: on which they are andon which they are not present. The close specificity relationship among some groups of epitopes determined by pairwise analysis according to their presence on the hexon types indicate that they could be continuous (sequential), overlapping or discontinuous (topographic) epitopes. Based on the identification of the hydrophilic regions and the localization of β-turns sixteen IT epitope sites were predicted on human adenovirus (HAdV) type 2 and nineteen on HAdV-41 hexon’s amino acid (aa) sequences beside the type and genus specific ones. The 16 predicted IT epitopes on HAdV-2 hexon show good coincidency with the 14 IT epitopes demonstrated with monoclonal antibodies (MAbs) on this hexon type. The predicted number of common epitopes between HAdV types 2 and 41 also corresponds well with the eight IT epitopes determined by MAbs, but the 17 predicted non-common epitopes indicate the possibility of the existence of more epitopes on these hexon types. The location of the predicted epitopes of HAdV-2 were determined by alignment of their sequence numbers on the three dimensional ribbon representation of the hexon subunit. Most of the predicted IT epitopes were found between the type and genus specific epitopes i.e. in the “upper” regions of pedestal domains P1 and P2 orientated toward the virion surface and in the “lower” part of loop 1 region orientated inside the virion. Two peptides representing potential IT epitopes were synthesized corresponding to residues 309–320 from the “lower” part of loop 1 and 399–408 from the “upper” part of P1 region of HAdV-2 hexon. Antibodies raised against the peptide-carrier conjugates recognized different purified native hexon types in ELISA. Received November 3, 1997 Accepted April 24, 1998     

Efficient MHC class I-peptide binding is required but does not ensure MHC class I-restricted immunogenicity

Cytotoxic T lymphocyte (CTL) epitopes are naturally processed peptides bound and presented by major histocompatibility (MHC) class I molecules. Since they are expressed at the cell surface in sufficient amounts to be recognized by CTL, it is generally believed, and in some cases demonstrated, that they bind efficiently to MHC class I molecules in vivo. Based on this knowledge, candidate CTL epitopes are now searched for by identifying peptides that efficiently bind to MHC class I molecules in vitro. We analysed whether this approach is valid by systematically investigating the relationship between MHC class I-peptide binding and peptide immunogenicity. Fifteen peptides that represent known CTL epitopes were tested for their MHC class I binding ability. In a comparative study with 83 peptides that bear the appropriate MHC class I allele-specific motifs but do not contain known CTL epitopes, the CTL epitope-bearing peptides showed the highest binding affinity for MHC class I. This was true for two MHC class I alleles in two different assay systems that monitor peptide-MHC class I binding. Furthermore, selected motif-bearing K^b binding peptides were used to induce peptide-specific CTL responses in mice. Only a subset of the high affinity K^b binding peptides induced reproducible peptide-specific CTL responses, whereas none of the low affinity K^b binding peptides induced a response. Taken together, these results indicate that efficient peptide-MHC class I binding is required for immunogenicity. Vice versa, immunogenicity is not guaranteed by efficient peptide-MHC class I binding, implying that additional factors are involved. Nevertheless, selection of candidate CTL epitopes on the basis of MHC class I binding seems valid. Our data indicate that, although an excess of peptides might be selected, the chance of missing immunogenic peptides is minimal.     

Comparison of class I- and II-restricted T cell recognition of the identical peptide.

There is structural and functional evidence that both class I- and II-restricted T cells recognize short processed peptides bound to MHC molecules. Although the structural conformation of bound peptides remains unknown, no evidence of distinct structural motifs of class I- or class II-restricted peptides has been described. Conversely, two algorithms proposed to predict T cell epitopes, and based on primary amino acid sequence or tertiary structure, are both compatible with many observed class I- and class II-restricted peptides. We previously identified eight class I-restricted peptides which were also recognized by class II-restricted T cells. Based on functional and direct binding studies, additional examples of peptides with both class I and II restrictions have been identified. In this study, we have directly compared the fine specificity of T cell recognition of a single epitope in a single mouse strain in the context of both class I- and class II-restricted responses. Based on a panel of analogue peptides with amino acid substitutions and peptides of various lengths, we observed several striking similarities in the recognition patterns of both class I- and class II-restricted T cells. In addition, some characteristics of recognition were different in the two systems indicating that the recognition processes were similar but not identical.     

Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

Comprehensive analysis of T cell epitope discovery strategies using 17DD yellow fever virus structural proteins and BALB/c (H2d) mice model

Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity. PREDBALB/c prediction software partially agreed with the ELISPOT and binding assay results, but presented low specificity. The use of prediction software to exclude peptides containing no epitopes, followed by high throughput screening of the remaining peptides by ELISPOT, and the use of MHC-biding assays to characterize the MHC restrictions demonstrated to be an efficient strategy. The results allowed the characterization of 2 MHC class I and 17 class II epitopes in the envelope protein of the YF virus in BALB/c (H2d) mice.     

No essential role for tripeptidyl peptidase II for the processing of LCMV-derived T cell epitopes

The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Approximately 20% of all peptides generated by the proteasome are too large for direct presentation by MHC class I molecules. Reits et al. (Immunity 2004. 20: 495-506) suggested that a major portion of proteasomal products are larger than 15 amino acids and require further degradation by the tripeptidyl peptidase II (TPPII) before becoming ligands of MHC class I molecules. Using the well-characterized lymphocytic choriomeningitis virus (LCMV) model, the role of TPPII in the processing of several LCMV-derived T cell epitopes was investigated. In contrast to Reits' proposal, TPPII inhibition and TPPII overexpression experiments revealed that five out of six LCMV-derived CD8(+) T cell epitopes were not affected by inhibition of TPPII, while one epitope (GP276) was slightly reduced upon TPPII overexpression. Additionally, we demonstrated that the processing of two epitopes derived from ovalbumin and murine cytomegalovirus were not altered by TPPII inhibition. We propose that TPPII is not generally required for the production of MHC class I peptides, but the presentation of some peptides can be negatively affected by TPPII.     

基于氨基酸结构信息的MHCⅠ类抗原表位的定量构效关系建模

目的主要组织相容复合物(MHC)在细胞免疫中具有重要作用,MHC与表位亲和力的大小直接影响T细胞免疫响应的强弱,因此准确预测MHC-表位的结合亲和力可以提高表位的筛选效率并且降低实验成本。方法筛选89种天然氨基酸的理化性质用于15个MHCⅠ类分子亚型表位的结构表征,然后使用逐步线性回归(STR)方法优选对亲和力具有重要贡献结构参数建立预测模型,最终用多元线形回归法(MLR)方法建立了15个MHC亚型的QSAR模型。结果建立的QSAR模型具有稳定性高、预测能力强等优点,能确定表位中各氨基酸及其相应理化性质队对亲和力的贡献,并可很好解释MHC与表位的相互作用机理。结论 STR-MLR建模方法具有计算简便、易于解释、性能优异、物化意义明确等优势。