Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients

Background

TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide.

Methodology/Principal Findings

From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8⁺ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8⁺ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8⁺ T cells with frequencies ranging from 0.07–0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8⁺ T cells producing at least one cytokine after TcTLE peptide stimulation.

Conclusion/Significance

These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy.     

STEAP, a prostate tumor antigen, is a target of human CD8+ T cells

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8⁺ T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP_86–94 and STEAP_262–270). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201⁺ STEAP⁺ human tumor cells. Furthermore, STEAP_86–94 and STEAP_262–270 stimulated specific CD8⁺ T cells from HLA-A*0201⁺ healthy donors, and these peptide specific CD8⁺ T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP_86–94-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8⁺ T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

Human kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8⁺ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201⁺ KLK4 ⁺ cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.     

Identification of an HLA-A*0201-restricted CD8+ T-cell epitope encoded within Leptospiral immunoglobulin-like protein A

Leptospirosis is an important zoonosis in humans. Immunity against leptospiral infection was thought to be primarily humoral, and limited studies have addressed the role of CD8⁺ T cells. Leptospiral immunoglobulin-like protein A (LigA) is an important protective antigen of Leptospira and a potential target for Leptospira-specific cell-mediated immunity. In this study, twenty LigA-derived peptides were tested their binding affinity and stability for the HLA-A*0201 molecule. Peptides with high binding affinity and stability for HLA-A*0201 were then assessed their capacity to elicit specific cytotoxic T-lymphocyte (CTL) responses using cytotoxicity, ELISPOT assays for IFN-γ and HLA-A*0201-peptide tetramer assays. We identified a HLA-A*0201-restricted epitope, LigA_305–313 KLIVTPAAL in Leptospira LigA. CTLs specific for LigA_305–313 were elicited both in HLA-A2.1/K^b transgenic mice and in patients with a clinical and/or laboratory diagnosis of leptospirosis. Staining of the HLA-A*0201–LigA_305–313 tetramer revealed the presence of LigA_305–313-specific CTLs in peripheral blood mononuclear cells (PBMCs) sourced from five patients infected with three different serovars of Leptospira. In conclusion, we report the existence of specific cytotoxic CD8⁺ T cells in patients with leptospirosis and we suggest that the newly identified epitope, LigA_305–313, will be helpful in enhancing the understanding of the mechanism of immunity to leptospirosis.     

Low TCR avidity and lack of tumor cell recognition in CD8<Superscript>+</Superscript> T cells primed with the CEA-analogue CAP1-6D peptide

The use of “altered peptide ligands” (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8⁺ T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201⁺ healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- γ release CEA⁺CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.     

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828–836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu ⁺ tumor cell lines. HER-2/neu(828-836), [HER-2(9₈₂₈)], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9₈₂₈) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8⁺ T-lymphocytes specifically recognizing HER-2(9₈₂₈) in 8 out of 20 HLA-A*0201⁺ HER-2/neu ⁺ breast cancer patients. Moreover, HER-2(9₈₂₈)-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9₈₂₈) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9₈₂₈) as a promising candidate for peptide-based cancer vaccines.     

Immunization with a Peptide Containing MHC Class I and II Epitopes Derived from the Tumor Antigen SIM2 Induces an Effective CD4 and CD8 T-Cell Response

Here, we sought to determine whether peptide vaccines designed harbor both class I as well as class II restricted antigenic motifs could concurrently induce CD4 and CD8 T cell activation against autologous tumor antigens. Based on our prior genome-wide interrogation of human prostate cancer tissues to identify genes over-expressed in cancer and absent in the periphery, we targeted SIM2 as a prototype autologous tumor antigen for these studies. Using humanized transgenic mice we found that the 9aa HLA-A*0201 epitope, SIM2_237–245, was effective at inducing an antigen specific response against SIM2-expressing prostate cancer cell line, PC3. Immunization with a multi-epitope peptide harboring both MHC-I and MHC-II restricted epitopes induced an IFN-γ response in CD8 T cells to the HLA-A*0201-restricted SIM2_237–245 epitope, and an IL-2 response by CD4 T cells to the SIM2_240–254 epitope. This peptide was also effective at inducing CD8⁺ T-cells that responded specifically to SIM2-expressing tumor cells. Collectively, the data presented in this study suggest that a single peptide containing multiple SIM2 epitopes can be used to induce both a CD4 and CD8 T cell response, providing a peptide-based vaccine formulation for potential use in immunotherapy of various cancers.     

Identification of Special AT-Rich Sequence Binding Protein 1 as a Novel Tumor Antigen Recognized by CD8+ T Cells: Implication for Cancer Immunotherapy

Background

A large number of human tumor-associated antigens that are recognized by CD8⁺ T cells in a human leukocyte antigen class I (HLA-I)-restricted fashion have been identified. Special AT-rich sequence binding protein 1 (SATB1) is highly expressed in many types of human cancers as part of their neoplastic phenotype, and up-regulation of SATB1 expression is essential for tumor survival and metastasis, thus this protein may serve as a rational target for cancer vaccines.

Methodology/Principal Findings

Twelve SATB1-derived peptides were predicted by an immuno-informatics approach based on the HLA-A*02 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from HLA-A*02⁺ healthy donors and/or HLA-A*02⁺ cancer patients. The recognition of HLA-A*02⁺ SATB1-expressing cancer cells was also tested. Among the twelve SATB1-derived peptides, SATB1_565–574 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and cancer patients. Importantly, SATB1_565–574-specific T cells recognized and killed HLA-A*02⁺ SATB1⁺ cancer cells in an HLA-I-restricted manner.

Conclusions/Significance

We have identified a novel HLA-A*02-restricted SATB1-derived peptide epitope recognized by CD8⁺ T cells, which, in turn, recognizes and kills HLA-A*02⁺ SATB1⁺ tumor cells. The SATB1-derived epitope identified may be used as a diagnostic marker as well as an immune target for development of cancer vaccines.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Identification of HLA‐A*11:01‐restricted Mycobacterium tuberculosis CD8+ T cell epitopes

New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette‐Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug‐resistant tuberculosis (MDR‐TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8⁺ T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8⁺ T cell epitopes specific for MTB is essential for the design of peptide‐based vaccines. To identify CD8⁺ T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01‐binding motifs. HLA‐A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon‐γ release and CD8⁺ T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8⁺ T cells from active pulmonary TB patients. In addition, a significant level of epitope‐specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA‐A*11:01 dextramer carrying the peptide Rv3130c_194‐204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8⁺ T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB.     

Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects

Leishmaniases are vector-borne diseases for which no vaccine exists. These diseases are caused by the Leishmania species complex. Activation of the CD8⁺ T cell is crucial for protection against intracellular pathogens, and peptide antigens are attractive strategies for the precise activation of CD8⁺ T in vaccine development against intracellular infections. The traditional approach to mine the epitopes is an arduous task. However, with the advent of immunoinformatics, in silico epitope prediction tools are available to expedite epitope identification. In this study, we employ different immunoinformatics tools to predict CD8⁺ T cell specific 9 mer epitopes presented by HLA-A*02 and HLA-B40 within the highly conserved 3′-ectonucleotidase of Leishmania donovani. We identify five promiscuous epitopes, which have no homologs in humans, theoretically cover 85% of the world's population and are highly conserved (100%) among Leishmania species. Presentation of selected peptides was confirmed by T2 cell line based HLA-stabilization assay, and three of them were found to be strong binders. The in vitro peptide stimulation of peripheral blood mononuclear cells (PBMC) from cured HLA-A02⁺ visceral leishmaniasis (VL) subjects produced significantly higher IFN-γ, IL-2 and IL-12 compared to no peptide control healthy subjects. Further, CD8⁺ cells from treated VL subjects produced significantly higher intracellular IFN-γ, lymphocyte proliferation and cytotoxic activity against selected peptides from the PBMCs of treated HLA-A02⁺ VL subjects. Thus, the CD8⁺ T cell specific epitopes shown in this study will speed up the development of polytope vaccines for leishmaniasis.     

A quantitative analysis of the variables affecting the repertoire of T cell specificities recognized after vaccinia virus infection

Many components contribute to immunodominance in the response to a complex virus, but their relative importance is unclear. This was addressed using vaccinia virus and HLA-A*0201 as the model system. A comprehensive analysis of 18 viral proteins recognized by CD8(+) T cell responses demonstrated that approximately one-fortieth of all possible 9- to 10-mer peptides were high-affinity HLA-A*0201 binders. Peptide immunization and T cell recognition data generated from 90 peptides indicated that about one-half of the binders were capable of eliciting T cell responses, and that one-seventh of immunogenic peptides are generated by natural processing. Based on these results, we estimate that vaccinia virus encodes approximately 150 dominant and subdominant epitopes restricted in by HLA-A*0201. However, of all these potential epitopes, only 15 are immunodominant and actually recognized in vivo during vaccinia virus infection of HLA-A*0201 transgenic mice. Neither peptide-binding affinity, nor complex stability, nor TCR avidity, nor amount of processed epitope appeared to strictly correlate with immunodominance status. Additional experiments suggested that vaccinia infection impairs the development of responses directed against subdominant epitopes. This suggested that additional factors, including immunoregulatory mechanisms, restrict the repertoire of T cell specificities after vaccinia infection by a factor of at least 10.     

An altered peptide ligand for naïve cytotoxic T lymphocyte epitope of TRP-2<Subscript>(180–188)</Subscript> enhanced immunogenicity

Tyrosinase-related protein-2 (TRP-2) is a non-mutated melanocyte differentiation antigen. The TRP-2-recognizing CD8⁺ T cells can evoke immune responses to melanoma in both humans and mice. Developing epitopes with amino acid replacements in their sequences might improve the low immunogenicity against this ‘self’ tumor antigen. We designed altered peptide ligands (APLs) of TRP-2_(180–188) (SVYDFFVWL) with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. These APLs were screened for MHC-affinity by affinity prediction plots and molecular dynamics simulation, and analyzed in vitro for stability and binding-affinity to molecular HLA-A*0201. We also investigated the CTLs activities induced by TRP-2 wild-type epitope and the APLs both in vitro in human PBMCs and HLA-A2.1/K^b transgenic mice. The results indicate that TRP-2 2M analog simultaneously had stronger binding-affinity and a lower dissociation rate to HLA-A*0201, than wild-type peptide. In addition, the analog 2M was superior to other APLs and wild-type epitope in terms of immunological efficacy ex vivo as measured by the ELISPOT assays of IFN-γ and granzyme B. These results demonstrate that TRP-2 2M is an agonist epitope that can induce anti-tumor immunity superior to its wild-type epitope, and has potential application in peptide-mediated immunotherapy.     

Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with N^ε-(5-carboxyfluorescein)-l -lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (K_a) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.     

Structural basis of biased T cell receptor recognition of an immunodominant HLA-A2 epitope of the SARS-CoV-2 spike protein

CD8⁺ T cells play an important role in vaccination and immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although numerous SARS-CoV-2 CD8⁺ T cell epitopes have been identified, the molecular basis underpinning T cell receptor (TCR) recognition of SARS-CoV-2-specific T cells remains unknown. The T cell response directed toward SARS-CoV-2 spike protein–derived S^269–277 peptide presented by the human leukocyte antigen (HLA)-A∗02:01 allomorph (hereafter the HLA-A2^S269–277 epitope) is, to date, the most immunodominant SARS-CoV-2 epitope found in individuals bearing this allele. As HLA-A2^S269–277-specific CD8⁺ T cells utilize biased TRAV12 gene usage within the TCR α-chain, we sought to understand the molecular basis underpinning this TRAV12 dominance. We expressed four TRAV12⁺ TCRs which bound the HLA-A2^S269–277 complex with low micromolar affinity and determined the crystal structure of the HLA-A2^S269–277 binary complex, and subsequently a ternary structure of the TRAV12⁺ TCR complexed to HLA-A2^S269–277. We found that the TCR made extensive contacts along the entire length of the S^269–277 peptide, suggesting that the TRAV12⁺ TCRs would be sensitive to sequence variation within this epitope. To examine this, we investigated cross-reactivity toward analogous peptides from existing SARS-CoV-2 variants and closely related coronaviruses. We show via surface plasmon resonance and tetramer studies that the TRAV12⁺ T cell repertoire cross-reacts poorly with these analogous epitopes. Overall, we defined the structural basis underpinning biased TCR recognition of CD8⁺ T cells directed at an immunodominant epitope and provide a framework for understanding TCR cross-reactivity toward viral variants within the S^269–277 peptide.     

Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

Background

There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.

Methods and Findings

HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.

Conclusions

Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.     

A similarity in peptide cross-reactivity between alloantigen- and nominal antigen-induced CD8+ T cell responses in vitro

Raising tumor-specific allorestricted T cells in vitro for adoptive transfusion is expected to circumvent host tumor tolerance. However, it has been assumed that alloreactive T cell clones activated in vitro ranges from peptide-specific with high avidity to peptide-degenerate with low avidity. In this study, we examined the peptide specificity and cross-reactivity of T cell responses in vitro to an allogeneic epitope and a nominal epitope with a modified co-culture of lymphocytes and autologous monocytes. After binding to the monocyte via the interaction of its Fc part and the cell surface IgG Fc receptor type I (FcγRI), a fusion protein consisting of the extracellular domains of HLA-A2 molecule and the Fc region of IgG1 (the dimer) introduced a single epitope into the co-culture. The dimer-coated monocytes stimulated the proliferation of autologous CD8⁺ T cells after co-culturing. The CD8⁺ T cell responses were self-HLA-restricted for HLA-A2-positive (HLA-A2+ve) samples and allo-HLA-restricted for HLA-A2-negative (HLA-A2-ve) samples, since the co-cultural bulks stained with HLA-A2 tetramers, human interferon-gamma (IFN-γ) production in response to T cell receptor (TCR) ligands, and cytotoxicity against a panel of target cells exhibited peptide-specific properties. Two HLA-A2-restricted peptides with sequence homology were included, allowing the comparison of cross-reactivity between allo-antigen- and nominal antigen-induced CD8⁺ T cell responses. Interestingly, the allo- and self-HLA-restricted CD8⁺ T cell responses were similar in the peptide cross-reactivity, although the allorestricted T cell response seemed, overall, more intensive and had higher binding affinity to specific tetramer. Our findings indicated the alloreactive T cells raised by the co-culture in vitro were as peptide specific and cross-reactive as the self-HLA-restricted ones.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.