Regulation of polyclonal T cell responses by an MHC anchor-substituted variant of myelin oligodendrocyte glycoprotein 35-55

Analogs of immunogenic peptides containing substitutions at TCR contact residues (altered peptide ligands (APLs)) have been used to manipulate Ag-specific T cell responses in models of autoimmunity, including experimental autoimmune encephalomyelitis. However, recent clinical trials with APL of a myelin basic protein epitope revealed limitations of this therapy. In this study, we demonstrate that individual myelin oligodendrocyte glycoprotein (MOG) 35-55-specific T cell clones responded differentially to a MOG 35-55 APL, raising questions about the ability of peptide analogs containing amino acid substitutions at TCR contact residues to control polyclonal populations of T cells. In contrast, we found that a variant peptide containing a substitution at an MHC anchor residue uniformly affected multiple MOG 35-55-specific clones and polyclonal lines. Stimulation of polyclonal MOG 35-55-specific T cells with an MHC variant peptide resulted in the induction of anergy, as defined by a dramatic reduction in proliferation and IL-2 production upon challenge with wild-type peptide. Furthermore, treatment of T cell lines with this peptide in vitro resulted in a significant reduction in their encephalitogenicity upon adoptive transfer. These results indicate that the use of MHC anchor-substituted peptides may be efficacious in the regulation of polyclonal T cell responses such as those found in EAE.     

CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses

MHC class I-restricted T cell epitopes lack immunogenicity unless aided by IFA or CFA. In an attempt to circumvent the known inflammatory side effects of IFA and CFA, we analyzed the ability of immunostimulatory CpG-DNA to act as an adjuvant for MHC class I-restricted peptide epitopes. Using the immunodominant CD8 T cell epitopes, SIINFEKL from OVA or KAVYNFATM (gp33) from lymphocytic choriomeningitis virus glycoprotein, we observed that CpG-DNA conveyed immunogenicity to these epitopes leading to primary induction of peptide-specific CTL. Furthermore, vaccination with the lymphocytic choriomeningitis virus gp33 peptide triggered not only CTL but also protective antiviral defense. We also showed that MHC class I-restricted peptides are constitutively presented by immature dendritic cells (DC) within the draining lymph nodes but failed to induce CTL responses. The use of CpG-DNA as an adjuvant, however, initiated peptide presenting immature DC progression to professional licensed APC. Activated DC induced cytolytic CD8 T cells in wild-type mice and also mice deficient of Th cells or CD40 ligand. CpG-DNA thus incites CTL responses toward MHC class I-restricted T cell epitopes in a Th cell-independent manner. Overall, these results provide new insights into CpG-DNA-mediated adjuvanticity and may influence future vaccination strategies for infectious and perhaps tumor diseases.     

Ii-Key/HER-2/<Emphasis Type="Italic">neu</Emphasis> MHC class-II antigenic epitope vaccine peptide for breast cancer

Purpose Cytotoxic T lymphocytes (CTL)- and T-helper cell-specific, and major histocompatibility complex (MHC) class-I and class-II peptides, respectively, of the HER-2/neu protein, induce immune responses in patients. A major challenge in developing cancer peptide vaccines is breaking tolerance to tumor-associated antigens which are functionally self-proteins. An adequate CD4+ T-helper response is required for effective and lasting responses.Methods Stimulating anti-cancer CD4+ T cell responses by MHC class-II epitope peptides has been limited by their weak potency, at least compared with tight-binding MHC class-I epitope peptides. Previously, a potent T-cell response to a MHC class-II epitope was engineered by coupling the N-terminus of the pigeon cytochrome C [PGCC(95–104)] MHC class-II epitope to the C-terminus of an immunoregulatory segment of the Ii protein (hIi77–81, the Ii-Key peptide) through a polymethylene spacer.Results In vitro presentation of the MHC class-II epitope to a T hybridoma was enhanced greatly (>250 times). Now, an Ii-Key/HER-2/neu (777–789) MHC class-II epitope hybrid peptide stimulated lymphocytes from both a healthy donor and a patient with metastatic breast carcinoma. The in vitro primary stimulation with the hybrid peptide strongly activated IFN- release, whereas the epitope-only peptide was weakly active. In fact, the hybrid stimulated IFN- release as well as the wild-type peptide when augmented with IL-12; however, the hybrid was comparable to free peptide in stimulating IL-4 release. This pattern is consistent with preferential activation along a non-tolerogenic Th1 pathway.Conclusion Such Ii-Key/MHC class-II epitope hybrid peptides have both diagnostic and therapeutic applications.     

Efficient delivery of Antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8+ T cells.

The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.     

DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines

DNA vaccination offers a strategy to induce immune attack on cancer cells, but tumor Ags are often weak. Inclusion of a "foreign" protein increases immunogenicity, and we found previously that fusion of the fragment C (FrC) of tetanus toxin to the tumor Ag sequence promotes Ab and CD4(+) responses against B cell tumors. For CTL responses, use of the full two-domain FrC may be less helpful, because known immunogenic MHC class I-binding peptides in the second domain could compete with attached tumor-derived epitopes. Therefore, we removed the second domain, retaining the N-terminal domain, which contains a "universal" helper epitope. We investigated the ability to induce CTL responses of candidate peptides placed at the C terminus of this domain. As test peptides, we repositioned the two known CTL motifs from the second domain to this site. Strong CTL responses to each peptide were induced by the engineered construct, as compared with the native FrC construct. Induced CTLs were able to specifically kill tumor cells transfected with FrC as a surrogate tumor Ag both in vitro and in vivo. Further reduction of the domain to a short helper epitope generated only weak CTL responses against fused peptides, and synthetic peptides mixed with the plasmid containing the first domain were ineffective. The single FrC domain-peptide vaccine design also was able to induce high levels of CTLs against a known epitope from carcinoembryonic Ag. Response to peptide was suppressed if two FrC domains were present, consistent with immunodominance. These principles and designs may have relevance for cancer vaccines delivered via DNA.     

Mimetics of a T cell epitope based on poly-N-acylated amine backbone structures induce T cells in vitro and in vivo

Peptidomimetics of the major histocompatibility complex (MHC) class I-restricted ovalbumin-derived T cell epitope SIINFEKL were generated by replacing parts of the peptide backbone by a poly-N-acylated amine (PAA) backbone with aromatic, heteroaromatic, and pseudoaromatic side chains that branch off of the main chain at the amine nitrogen. The structure of the PAAs was designed to position this side chain in the central epitope anchor pocket of the MHC molecule. A number of biologically active PAAs were found that induced cytolysis by the mouse cytotoxic T cell clone 4G3. Competition experiments with independent peptides that are known to bind to the restricting MHC molecule H-2K(b) suggest that the PAAs are bound by the MHC molecules at the same site as conventional peptide epitopes. The PAAs were active also in vivo and induced primary cytotoxic T cell responses in mice.     

Peptide vaccines incorporating a ‘promiscuous’ T-cell epitope bypass certain haplotype restricted immune responses and provide broad spectrum immunogenicity

An ideal peptide vaccine should contain both B- and T-cell epitopes. Recognition of antigen by B cells is highly dependent on the three-dimensional conformation of the antigen whereas T cells recognize antigen only after it has been processed to release a peptide fragment which is bound to the major histocompatibility complex (MHC) class II molecule. However, T cells provide ‘help’ to B cells displaying the same processed, MHC-restricted from of the antigen, demonstrating that the T-cell response to a protein antigen is under genetic control. Thus, strategies for co-inclusion of T cell ‘helper’ epitopes with the B-cell determinant elicit immune responses that are in most cases genetically restricted to only one or a few alleles of the MHC with limited activity across divergent MHC class II haplotypes. This genetically restricted T cell stimulatory activity of peptides is a serious obstacle and consequently such constructs would be of limited practical value as a vaccine targeted to a majority of an outbred population. In the study described here, we have engineered tow peptides to encompass the sequences from the universally immunogenic tetanus toxoid (TT) epitope and the contraceptive vaccine candidate lactate dehydrogenase C₄ (LDH-C₄). We demonstrate the feasibility of using ‘promiscuous’ T-Cell epitopes colinearly constructed with a defined B-cell epitope to induce high titer antipeptide IgG antibodies specific for native protein antigen LDH-C₄ in several inbred strains of mice, outbred mice and rabbits. There appears to be a strong correlation between the capacity for the hybrid peptides to be stimulatory for the corresponding T cells in C57BL/6 (H-2^b) and C3H/HeJ (H-2^k) mice and their ability to be immunogenic. This correlation, however, appears to break down in H-2^d strains of mice since no antibodies were detected in BALB/c and barely detectable levels of antibodies in B10.D2 although activated T cells were detectable. Conversely, high titers of antipeptide antibodies are elicited in some strains (B10.BR) (H-2^k); C57BL/10 (H-2^k) without detectable IL-2 responses. Finally, we show that a determinant which was previously restricted to H-2^k can be rendered immunogenic in H-2^b with the ‘promiscuous’ TT epitope. Thus, certain haplotype-restricted immune responses can be bypassed, setting forth the ground work for the design of a universal vaccine by broadening the effective response in a larger number of individuals typically of the genetically diverse outbred human population.     

Identification of a MHC class-II restricted epitope in carcinoembryonic antigen

Purpose The carcinoembryonic antigen (CEA) is extensively expressed on the vast majority of colorectal, gastric, and pancreatic carcinomas, and, therefore, is a good target for tumor immunotherapy. CD4+ T-helper (Th) cells play a critical role in initiation, regulation, and maintenance of immune responses. In this study, we sought to identify Th epitopes derived from CEA which can induce CEA-specific Th responses. The combined application with cytotoxic T lymphocyte (CTL) epitopes would be more potent than tumor vaccines that primarily activate CTL alone.Methods We utilized a combined approach of using a computer-based algorithm analysis TEPITOPE and in vitro biological analysis to identify Th epitopes in CEA.Results Initial screening of healthy donors showed that all five predicted peptides derived from CEA could induce peptide-specific T-cell proliferation in vitro. We characterized these CEA epitopes by establishing and analyzing peptide-specific T-cell clones. It was shown that CD4+ T-cells specific for the CEA₁₁₆ epitope can recognize and respond to naturally processed CEA protein and CEA₁₁₆ epitope can be promiscuously presented by commonly found major histocompatibility complex (MHC) alleles. Furthermore, it was demonstrated that immunization of human leukocyte antigen (HLA)-DR4 transgenic mice with CEA₁₁₆ peptide elicited antigen-specific Th responses which can recognize the antigenic peptides derived from CEA protein and CEA-positive tumors.Conclusion The MHC class II-restricted epitope CEA₁₁₆ could be used in the design of peptide-based tumor vaccine against several common cancers expressing CEA.     

Identification of a novel HLA-A2-restricted mutated Survivin epitope and induction of specific anti-HCC CTLs that could effectively cross-recognize wild-type Survivin antigen

Peptide vaccine based on tumor-associated antigen (TAA), which usually belongs to self-antigen with poor immunogenicity, has been considered as an attractive option for treatment of malignant tumors. The ideal TAA epitopes should have stable affinity to major histocompatibility complex (MHC) molecules and elicit strong anti-tumor immune response. Although point-mutation technology of TAA peptide may increase the binding capability to MHC molecules, some previous studies have revealed that part of the variant peptides results in lymphocyte not to effectively cross-recognize and kill the target tumor expressed wild-type TAA. Here, we designed a novel HLA-A2-restricted mutated TAA Survivin epitope nonapeptide Sur79L2 (KLSSGCAFL) that showed higher binding ability compared to wild-type peptide Sur79 (KHSSGCAFL) in T2-binding assays. To investigate whether Sur79L2 can induce Survivin-specific anti-hepatocellular carcinoma (HCC) response, we stimulated tumor-associated lymphocytes from a HCC patient with Sur79L2 in vitro. IFN-γ release and cytotoxicity assays showed Sur79L2 could effectively cross-recognize and lysis T2 cell plus peptide Sur79 and HCC cell lines (expression of wild-type Survivin antigen) in an HLA-A2-restricted manner. In contrast, peptide Sur95 (ELTLGEFLKL) that has been reported as a very promising anti-tumor epitope in a variety of tumors except HCC were not able to generate detectable cytotoxic immune responses against HCC in this study. Our results suggest that point-mutated peptide Sur79L2 is a new HLA-A2-restricted CTL epitope and may be useful for the immunotherapy for patients with HCC.     

Amino acid modifications in the wild type sequence p53 232-240 overcome the poor immunogenicity of this self tumour epitope.

A major limitation in antigen-specific cancer vaccines is that most of the tumour antigens that are potent candidates for broad applicability originate from self proteins. The peptides presented by tumour cells are derived from tissue-specific differentiation proteins, from proteins altered by genetic mutation or by non mutated proteins that are normally silent in most adult tissues. As a consequence, T-cell responses elicited against those antigens are rather weak. Several data showed that amino acid modifications could enhance the immunogenicity of such antigens by priming T-cells that have escaped central tolerance based on a poor avidity. In this regard, this strategy could be powerful for inducing immunity against tumours. The present report focuses on the murine wild type epitope p53 232-240 that is poorly immunogenic. It shows that substitution of the two cysteine residues by serine or amino butyric acid derivatives and substitution of the two methionine residues by norleucine residues resulted in enhanced stability of the MHC/peptide complex. The MHC binding affinity of analogue peptides was enhanced between 10 and 100 fold. They were also potent immunogens, stronger than was the original wild type epitope; T-cell responses were increased up to 50 times. Moreover, the effector T-cells elicited by three of these peptides cross reacted with the natural epitope. These observations have important implications for strategies that use the modified-peptide epitope.     

Generation of T cell help through a MHC class I-restricted TCR

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.     

Modification of peptide interaction with MHC creates TCR partial agonists

We report the creation of TCR partial agonists by the novel approach of manipulating the interaction between immunogenic peptide and MHC. Amino acids at MHC anchor positions of the I-E(k)-restricted hemoglobin (64-76) and moth cytochrome c (88-103) peptides were exchanged with MHC anchor residues from the low affinity class II invariant chain peptide (CLIP), resulting in antigenic peptides with altered affinity for MHC class II. Several low affinity peptides were identified as TCR partial agonists, as defined by the ability to stimulate cytolytic function but not proliferation. For example, a peptide containing methionine substitutions at positions one and nine of the I-E(k) binding motif acted as a partial agonist for two hemoglobin-reactive T cell clones (PL.17 and 3.L2). The identical MHC anchor substitutions in moth cytochrome c (88-103) also created a partial agonist for a mCC-reactive T cell (A.E7). Thus, peptides containing MHC anchor modifications mediated similar T cell responses regardless of TCR fine specificity or antigen reactivity. This data contrasts with the unique specificity among individual clones demonstrated using traditional altered peptide ligands containing substitutions at TCR contact residues. In conclusion, we demonstrate that altering the MHC anchor residues of the immunogenic peptide can be a powerful method to create TCR partial agonists.     

Chimeric epitopes delivered by polymeric synthetic linear peptides induce protective immunity to malaria

Polymeric linear peptide chimeras (LPCs) that incorporate Plasmodium vivax promiscuous T cell epitopes and the P. falciparum circumsporozoite protein B cell epitope have been shown to induce a high level of immunogenicity and overcome genetic restriction when tested as vaccine immunogens in BALB/c mice. The present study evaluates the biological relevance of several LPCs using a well characterized rodent malaria model. Polymeric peptide constructs based on P. berghei and P. yoelii sequences, and orthologous to the human malaria sequences included in the original LPCs, were designed and tested for immunogenicity in mice of different H-2 haplotypes. We demonstrate that robust immune responses are induced and that peptides containing the orthologous rodent Plasmodium sequences exhibited similar immunogenic capabilities. Unique to this report, we show that LPCs can also prime MHC class I-restricted cytotoxic T lymphocytes (CTLs) and, most relevantly, that a peptide construct prototype incorporating single B, T and CTL epitopes induced protection against an experimental challenge with P. berghei or P. yoelii sporozoites. Collectively, these results suggest that polymeric polypeptide chimeras can be used as a platform to deliver subunit vaccines.     

Immunologic hierarchy,class II MHC promiscuity,and epitope spreading of a melanoma helper peptide vaccine

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4⁺ T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8⁺ T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4⁺ and CD8⁺ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4⁺ T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3_281–295 (49 %) and tyrosinase_386–406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8⁺ T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4⁺ and CD8⁺ T cell responses against melanoma antigens. CD4⁺ T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8⁺ T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.     

Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen

Ran is considered to be a promising target for tumor-specific immunotherapy because its protein is exclusively expressed in tumor tissues, though its mRNA can be expressed in most normal tissues. In our study, we obtained four candidate wild-type epitopes designated Ran1, Ran2, Ran3, and Ran4, derived from the Ran antigen with the highest predicted affinity with MHC-I, indicated by affinity prediction plots and molecular dynamics simulation. However, in vitro affinity assays of these epitopes showed only a moderate affinity with MHC-I. Thus, we designed altered peptide ligands (APLs) derived from Ran wild-type epitopes with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. Of the eight tested peptides, the 1Y analog had the strongest binding-affinity and lowest-dissociation rate to HLA-A*0201. Additionally, we investigated the CTLs activities induced by Ran wild-type peptides and the APLs in human PBMCs and in HLA-A*0201/K^b transgenic mice. Ran1 1Y was superior to other APLs and wild-type peptides in eliciting epitope-specific CTL immune responses both in vitro and in vivo. In summary, a wild-type epitope of the tumor-specific antigen Ran, expressed broadly in many tumors, was identified and designated Ran1. An APL of Ran1, Ran1 1Y, was further designed and verified in vitro and in vivo and found to elicit a stronger Ran-specific CTL response, indicating a potential anti-tumor application in the future.     

Supra-agonist peptides enhance the reactivation of memory CTL responses

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Mutations in a dominant Nef epitope of simian immunodeficiency virus diminish TCR:epitope peptide affinity but not epitope peptide:MHC class I binding

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.     

Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.     

Immunogenically fit subunit vaccine components via epitope discovery from natural peptide libraries

Antigenic peptides that bind pathogen-specific Abs are a potential source of subunit vaccine components. To be effective the peptides must be immunogenically fit: when used as immunogens they must elicit Abs that cross-react with native intact pathogen. In this study, antigenic peptides obtained from phage display libraries through epitope discovery were systematically examined for immunogenic fitness. Peptides selected from random peptide libraries, in which the phage-displayed peptides are encoded by synthetic degenerate oligonucleotides, had marginal immunogenic fitness. In contrast, 50% of the peptides selected from a natural peptide library, in which phage display segments of actual pathogen polypeptides, proved very successful. Epitope discovery from natural peptide libraries is a promising route to subunit vaccines.