Restricted Cytotoxic T Lymphocyte Epitopes Research Articles

Computational analysis of proteome of Foot-and-mouth disease Virus for the prediction of immunogenic epitopes

ObjectiveFoot and Mouth Disease virus (FMDV) commonly affects cloven hoofed animals. Infection with one serotype does not confer immunity to others and currently available vaccines do not provide effective T-cell immunity. The objective of this work is to predict the Cytotoxic T lymphocyte (CTL) epitopes which could be used as vaccine components against FMD virus. Material and methodsProteomes of three Indian FMDV vaccine strains, Type A IND40/00, Type A IND17/82 and Asia-1 IND63/72 were retrieved. BoLa-T2b allele restricted CTL epitopes were predicted using Immune Epitope Data Base Analysis Resource (IEDB-AR) and NetMHCpan 2.8 tool. The predicted overlapping epitopes were further analysed for antigenicity prediction, binding to other BoLA allele and consensus epitope analysis. ResultsA total of 207 epitopes were predicted by IEDB-AR tool. Among this, 72, 68, 67, epitopes were predicted in Asia 1 IND 63/72, Type A IND40/00 and Type A IND17/82 respectively. Totally 489 epitopes were predicted by NetMHCpan 2.0 tool. Of which, 164, 163 and 162 epitopes were predicted in Type A IND17/82, Asia 1 IND63/72 and Type A IND40/00 respectively. In total 29 consensus epitopes were predicted during this analysis, among this, 28 epitopes are present to occur in all the three Indian strains that were tested. ConclusionsThe 29 consensus epitopes predicted in this study could be used for developing a potent vaccine for both Asia I and Type A strains of FMD. Further in vitro and in vivo studies are needed to confirm the CTL efficacy of this peptide against FMDV.

Computational analysis of proteome of Foot-and-mouth disease Virus for the prediction of immunogenic epitopes

ObjectiveFoot and Mouth Disease virus (FMDV) commonly affects cloven hoofed animals. Infection with one serotype does not confer immunity to others and currently available vaccines do not provide effective T-cell immunity. The objective of this work is to predict the Cytotoxic T lymphocyte (CTL) epitopes which could be used as vaccine components against FMD virus. Material and methodsProteomes of three Indian FMDV vaccine strains, Type A IND40/00, Type A IND17/82 and Asia-1 IND63/72 were retrieved. BoLa-T2b allele restricted CTL epitopes were predicted using Immune Epitope Data Base Analysis Resource (IEDB-AR) and NetMHCpan 2.8 tool. The predicted overlapping epitopes were further analysed for antigenicity prediction, binding to other BoLA allele and consensus epitope analysis. ResultsA total of 207 epitopes were predicted by IEDB-AR tool. Among this, 72, 68, 67, epitopes were predicted in Asia 1 IND 63/72, Type A IND40/00 and Type A IND17/82 respectively. Totally 489 epitopes were predicted by NetMHCpan 2.0 tool. Of which, 164, 163 and 162 epitopes were predicted in Type A IND17/82, Asia 1 IND63/72 and Type A IND40/00 respectively. In total 29 consensus epitopes were predicted during this analysis, among this, 28 epitopes are present to occur in all the three Indian strains that were tested. ConclusionsThe 29 consensus epitopes predicted in this study could be used for developing a potent vaccine for both Asia I and Type A strains of FMD. Further in vitro and in vivo studies are needed to confirm the CTL efficacy of this peptide against FMDV.

Identification of HLA-A*0201-restricted CTL Epitopes for MLAA-34-specific Immunotherapy for Acute Monocytic Leukemia.

Our previous research has shown that monocytic leukemia-associated antigen-34 (MLAA-34) was a novel antiapoptotic molecule with unique expression in acute monocytic leukemia (M5), making it an ideal target for T-cell-based immunotherapy. Here, we sought to identify HLA-A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope of MLAA-34 by reverse immunology. In all, 10 HLA-A*0201 restricted epitopes of MLAA-34 were predicted by bioinformatics. MLAA-34324-332, MLAA-34293-301, and MLAA-34236-244 showed the strongest HLA-A*0201-binding affinity. The percentages of HLA-A*0201-MLAA-34236-244 tetramer+ CD8+ T cells in MLAA-34236-244-induced CTLs were raised apparently. Enzyme-linked immunospot showed that MLAA-34236-244 and MLAA-34324-332-specific CTLs produced a higher amount of interferon-γ. MLAA-34236-244-induced CTLs presented a stronger cytotoxic effect on THP-1 cells (HLA-A*0201+MLAA-34+) at various effector to target ratios. MLAA-34236-244 peptide vaccine could inhibit the tumor growth and improve mean survival time of leukemia-bearing human peripheral blood lymphocyte reconstituting severe combined immunodeficient mice. Mice immunized with MLAA-34236-244 vaccine had increased percentages of MLAA-34236-244 tetramer+ CD8+ T cells in the spleen after each round of immunization. High-purity CD8+ and CD4+ T cells were sorted by Dynabeads as effector cells. The killing activity of CD8+ T cells was higher than that of CD4+ T cells. CTLs derived from the MLAA-34 peptide vaccine group were significantly higher than other therapeutic groups and showed specific cytotoxicity to THP-1 cells. Increased interferon-γ and interleukin (IL)-2 and decreased IL-10 and IL-4 were seen in the MLAA-34236-244 peptide vaccine group. MLAA-34236-244 peptide (ILDRHNFAI) is an effective HLA-A*0201-restricted CTL epitope and that it may serve as a promising strategy in designing antigen-specific immunotherapy against MLAA-34-positive acute monocytic leukemia.

Identification of Novel Cytotoxic T Lymphocyte Epitopes of Drug- Resistance Related Protein InhA from Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), especially the drug-resistant MTB, poses serious challenges to human healthcare worldwide. Cytotoxic T lymphocytes (CTLs) play a vital role in immune defense against MTB. To identify novel CTL epitopes that could induce cellular immunity against MTB infections. The HLA-A*0201 restricted CTL epitopes of the drug-resistant protein InhA from MTB were predicted by online algorisms and synthesized by the Fmoc solid phase method. The candidate peptides were used to induce CTLs from human peripheral blood mononuclear cells (PBMCs) of HLA-A*0201 healthy donors and the HLA-2.1/Kb mice. IFN-γ productions of CTLs were detected by enzyme linked immunospot assay (ELISPOT), flow cytometry and enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay. A group of 4 epitopes were screened out with high affinities to HLA-A*0201. ELISPOT and flow cytometry analysis indicated these peptides significantly induced that IFN-γ release of CTLs from the HLA-A*0201+/PPD+ donors, as the mutant analogues had more potent stimulation effects. LDH assay showed that CTLs from PPD+ donors and the immunized mice exhibited significant cytotoxicity and low cross-reactivity. ELISA analysis revealed comparative levels of IFN-γ were released by CTLs isolated from the mice spleen. Our study has identified 4 novel CTL epitopes of InhA that could elicit potent CTL immunity, establishing a foundation for the development of multivalent peptide vaccines against the drug-resistant MTB.

Identification of a novel HLA-A24-restricted cytotoxic T lymphocyte epitope peptide derived from mesothelin in pancreatic cancer.

Pancreatic cancer involves highly malignant tumors, and the development of new therapeutic strategies is critical. Mesothelin is overexpressed in infiltrating pancreatic cancer cells and plays an important role in the invasion and migration processes. In this study, we focused on mesothelin as a tumor-specific antigen target for a pancreatic cancer vaccine. We first investigated the mesothelin-derived epitope peptide restricted to HLA-A*2402. A total of 19 candidate peptides were synthesized, and we then determined their potential to induce peptide-specific cytotoxic T lymphocytes (CTLs). Peptide-specific CTLs were induced by five peptides derived from mesothelin, and these CTLs successfully exhibited peptide-specific IFN-γ production. After the expansion of each CTL, two CTL lines were established, which were induced by mesothelin-10-5 peptide (AFYPGYLCSL). These CTL lines exhibited peptide-specific cytotoxicity and IFN-γ production. Moreover, we were able to generate mesothelin-10-5 peptide-specific CTL clones. These CTL clones also had specific cytotoxic activity against HLA-A*2402-positive pancreatic cancer cells that endogenously expressed mesothelin. These results indicate that the mesothelin-10-5 peptide is a novel HLA-A*2402 restricted CTL epitope and that it is a promising candidate target for antigen-specific immunotherapy against pancreatic cancers.

A modified HLA-A*0201-restricted CTL epitope from human oncoprotein (hPEBP4) induces more efficient antitumor responses.

We previously identified human phosphatidylethanolamine-binding protein 4 (hPEBP4) as an antiapoptotic protein with increased expression levels in breast, ovarian and prostate cancer cells, but low expression levels in normal tissues, which makes hPEBP4 an attractive target for immunotherapy. Here, we developed hPEBP4-derived immunogenic peptides for inducing antigen-specific cytotoxic T lymphocytes (CTLs) targeting breast cancer. A panel of hPEBP4-derived peptides predicted by peptide-MHC-binding algorithms was evaluated to characterize their HLA-A2.1 affinity and immunogenicity. We identified a novel immunogenic peptide, P40-48 (TLFCQGLEV), that was capable of eliciting specific CTL responses in HLA-A2.1/Kb transgenic mice, as well as in peripheral blood lymphocytes from breast cancer patients. Furthermore, amino-acid substitutions in the P40-48 sequence improved its immunogenicity against hPEBP4, a self-antigen, thus circumventing tolerance. We designed peptide analogs by preferred auxiliary HLA-A*0201 anchor residue replacement, which induced CTLs that were crossreactive to the native peptide. Several analogs were able to stably bind to HLA-A*0201 and elicit specific CTL responses better than the native sequence. Importantly, adoptive transfer of CTLs induced by vaccination with two analogs more effectively inhibited tumor growth than the native peptide. These data indicate that peptide analogs with high immunogenicity represent promising candidates for peptide-mediated therapeutic cancer vaccines.

Immunological tolerance to LCMV antigens differently affects control of acute and chronic virus infection in mice.

Cytotoxic T lymphocytes (CTLs) play a key role in the control of lymphocytic choriomeningitis virus (LCMV) infection. In C57BL/6 mice (H-2b ), the CTL response is mainly directed against epitopes from the LCMV glycoprotein (GP) and the nucleoprotein (NP) which represent the two major viral proteins. The role of GP- versus NP-derived epitopes for viral clearance was examined using transgenic (tg) mice ubiquitously expressing LCMV GP and NP, respectively. These mice lack GP- or NP-specific CTLs and show decreased levels of GP- or NP-specific antibodies as a result of tolerance induction. During acute LCMV infection, CTLs specific for GP- and NP-derived epitopes are generated with similar frequencies. Nonetheless, we found that lack of GP- but not of NP-specific CTLs abolished control of acute LCMV infection. In contrast, after high-dose or chronic LCMV infection, virus elimination was delayed to a similar extent in GP- and NP-tg mice. Thus, immunological tolerance to LCMV antigens differently affects virus clearance in acute and chronic infection settings. In addition, our data reveal that immunodominance of H-2b -restricted LCMV-specific CTL epitopes and their antiviral activity do not strictly correlate.

Prediction and Identification of HLA-A*0201 Restricted CTL Epitopes from Eps8

To find and identify HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes from epidermal growth factor pathway substrate number 8 (Eps8) for specific immunotherapy based on Eps8-derived epitopes in clinic. Online biological softwares involved C-proteasomal cleavage, MHC class I binding affinity and TAP transport efficiency were used for prediction of HLA-A*0201 restricted epitopes from Eps8. Then, T2-binding assays and peptide/MHC complex stability tests were used to further verify the predicted epitopes. Specific secretion of IFN-γ from human CTL was assayed using the IFN-γ ELISPOT kit, and cytolytic activity was measured by a 4-h lactate dehydrogenase (LDH) release assay. Finally, the functional effects in vivo were measured in HLA-A*0201/Kb transgenic (Tg) mice. Four natural epitopes were designed through online biological softwares. Of the four epitopes selected, p360-368 was found to have the high binding affinity to HLA-A*0201, while p101-109 and p276-284 showed moderate affinities. DC50 of peptide/MHC complexes of the natural epitopes mentioned were all longer than 8 h. In functional assays with human PBMNC in vitro and in HLA-A*0201/Kb transgenic mice in vivo, CTLs primed by each epitope (p101-109, p276-284 and p360-368) secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A*0201-restricted and Eps8-specific manner. Natural epitopes (p101-109, p276-284 and p360-368) may be the HLA-A*0201 restricted epitope derived from Eps8.

Early treatment outcomes of patients with extensively drug resistant tuberculosis in South Africa

Background: Mycobacterium tuberculosis (Mtb) infects approximately one third of the world’s population, 10% of which develop disease during lifetime. Beijing genotype strains are the most predominant strains in China. The aim of this study was to screen and validate the possible HLA*0201 restricted specific T cell epitopes of latent phase Mtb strains H37Rv and Beijing genotype. Methods & Materials: MTB DNA microarray gene expression analysis was performed to screen the Mtb genes which expressed up-regulated under hypoxia. SYFPEITHI and NetCTLpan databases were used to predict the HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes on Mtb, followed by peptide/HLA-A*0201 affinity and complex stability assays using the T2 cells. IFN-gammaproducing T cells were detected by enzyme-linked immunospot assay (ELISPOT) and a LDH release assay were performed to detect peptide-specific CTL activity using PBMC derived from HLAA*0201-positive human donors latently infected with Mtb (LTBI). Results: Using a whole genome microarray, we identified 130 genes of Mtb strain H37Rv and Beijing genotype whose expression was up-regulated under hypoxic conditions, 37 genes were responsible for encoding membrane protein, cell wall proteins or exported proteins. Of selected four proteins coded by upregulated genes, Six potential epitopes on Rv0350, Rv0351 and Rv0440were selected as candidate HLA-A*0201 restricted CTL epitopes by SYFPEITHI and NetCTLpan prediction. Four of these 6 study epitopes (Rv0440 p416-424, Rv0440 p362-370, Rv0351 p122-130 and Rv0350 p363-371) were showed high binding affinity and stabilization to HLA-A*0201 molecules by T2 binding studies. Synthesis of multi-epitope peptides Rv0351-A-T (containing Rv0351 p122-130, APDRE and Trojan peptide) and Rv0350-A-T (containing Rv0350 p363-371, APDRE and Trojan peptide) via ufrinsensitive linker VRKR. Cytotoxic activity showed that significant lysis of T2cellspulsedwithRv0351-A-TorRv0350-A-Twas induced by peptide-specific T cells derived fromHLA-A*0201-positive LTBI. IFNgamma was released by PBMC in vitro from HLA-A*0201positive LTBI when challenged with the peptides by an ELISPOT assay. Conclusion: Our data suggest that Rv0351p122-130 and Rv0350p363-371 are HLA-A*0201-restricted CTL epitopes and could be useful in the design of an Mtb vaccine for LTBI. Acknowledgments: This investigation was funded by a grant from Key Projects in the National Science & Technology Program (2009ZX10004-305).

Detection of respiratory syncytial virus fusion protein variants between 2009 and 2012 in China

Respiratory syncytial virus (RSV) causes respiratory tract infection, particularly acute lower respiratory tract infection (ALRTI), in early childhood. The RSV fusion protein (F protein) is an important surface protein, and it is the target of both cytotoxic T lymphocytes (CTL) and neutralizing antibodies; thus, it may be useful as a candidate for vaccine research. This study investigated the genetic diversity of the RSV F protein. To this end, a total of 1800 nasopharyngeal aspirates from hospitalized children with ALRTI were collected for virus isolation between June 2009 and March 2012. There were 333 RSV-positive cases (277 cases of RSV A, 55 of RSV B, and 1 with both RSV A and RSV B), accounting for 18.5 % of the total cases. Next, 130 clinical strains (107 of RSV A, 23 of RSV B) were selected for F gene sequencing. Phylogenetic analysis revealed that the F gene sequence is highly conserved, with significant amino acid changes at residues 16, 25, 45, 102, 122, 124, 209, and 447. Mutations in human histocompatibility leukocyte antigen (HLA)-restricted CTL epitopes were also observed. Variations in RSV A F protein at the palivizumab binding site 276 (N→S) increased between 2009 and 2012 and became predominant. Western blot analysis and microneutralization data showed a substitution at residue 276 (N→S) in RSV A that did not cause resistance to palivizumab. In conclusion, the RSV F gene is geographically and temporally conserved, but limited genetic variations were still observed. These data could be helpful for the development of vaccines against RSV infection.

Dendritic cell-based immunotherapy for colon cancer using an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope from tumor-associated antigen 90K

Tumor-associated antigen 90K is implicated in cell-cell and cell-extracellular matrix adhesion through its interaction with galectin-3 and integrin-β1 and is highly expressed in malignant tissues, making it a novel target for the development of new immunotherapies. We investigated a potential immunotherapy treatment for colon cancer using 90K-specific cytotoxic T lymphocytes induced by autologous dendritic cells and pulsed with 90K peptides. We selected three peptides (90K351, 90K5 and 90K523) that bind to HLA-A*0201 molecules on the basis of their binding affinity, as determined by a peptide-T2 binding assay. Dendritic cells pulsed with 90K peptides resulted in the efficient generation of mature dendritic cells and exhibited enhanced T-cell stimulation and polarization of naive T cells toward Th1. Dendritic cells pulsed with 90K peptides generated potent cytotoxic T-lymphocytes that lysed T2 cells loaded with each 90K peptide, and 90K(+)/HLA-A2(+) colon cancer cell lines, including HCT116 and SW480, in a dose-dependent and HLA-A*0201-restricted manner. No killing was observed in 90K(+)/HLA-A2(-) DLD1 or 90K(-)/HLA-A2(-) K562 cells. Therefore, we believe that cytotoxic T-lymphocytes stimulated by 90K peptide-pulsed dendritic cells naturally recognize the 90K peptide presented by colon cancer cells in the context of HLA-A2, and kill 90K-positive tumor cells. Dendritic cells pulsed with 90K peptides led to the induction of granzyme B and perforin positive CD8(+) T cells against HCT116 and SW480 cells, but not DLD1 cells. In conclusion, 90K-specific cytotoxic T lymphocytes, generated by stimulating T cells with 90K peptide-pulsed dendritic cells, could be useful effector cells for the immunotherapy treatment of colon cancer.

Identification of novel HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from Zinc Transporter 8

Numerous evidences demonstrated that type 1 diabetes (T1D) is due to a loss of immune tolerance to islet antigens, and CD8+ T cells play an important role in the development of T1D. Zinc Transporter 8 (ZnT8) has emerged in recent years as a target of disease-associated autoreactive T cells in human T1D. However, ZnT8-associated CTL specific-peptides have not been identified. In this study, we predicted and identified HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from ZnT8, and utilized it to immunize HLA-A2.1/Kb transgenic (Tg) mice. The results demonstrated that peptides of ZnT8 containing residues 107–115, 115–123 and 145–153 could elicit specific CTLs in vitro, and induce diabetes in mice. The results suggest that these specific peptides are novel HLA-A*0201-restricted CTL epitopes, and could have therapeutic potential in preventing of T1D disease.

Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund’s adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8+ T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One Db-restricted and three HLA-A*0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use.

Identification of three H-2Kd restricted CTL epitopes of NS4A and NS4B protein from Yellow fever 17D vaccine

In this study, 12 nonamers derived from the nonstructural protein NS4A and NS4B of a Chinese attenuated Yellow fever 17D (YF-17D) vaccine strain were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus expressing NS4A or NS4B protein. Three peptides (amino acid residues S(106)YIMLIFFV from NS4A protein and V(43)YVGIVTML and L(174)YLLLALSL from NS4B protein) were identified as CD8(+) cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited a response from a significant numbers of IFN-γ secreting cells compared with the other nonamers of NS4A and NS4B and compared to one irrelevant nonamer derived from infectious bronchitis virus (IBV) H52 strain. Bioinformatics analysis demonstrated that these three nonamers are H-2K(d)-restricted CTL epitopes. The multiple amino acid sequence alignments among different YFV NS4A, and NS4B sequences submitted to GenBank indicated that these three CTL epitopes are strongly conserved; this novel finding will potentially aid further studies on cellular immunity against YF virus and YFV-based expression vectors.

Computational prediction and experimental assessment of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from neutral endopeptidase

Neutral endopeptidase (NEP) is the first target antigen identified on podocytes in human membranous nephropathy (MN). Cytotoxic T lymphocytes (CTLs) are considered essential for glomerular destruction in MN model. The aim of this study was to show that the CTL epitopes of NEP could be used to design more effective and better tolerated therapies. The CTL epitopes of NEP were screened using the long-distance prediction system SYFPEITHI and the Bioinformatics and Molecular Analysis Section of the MHC Peptide Binding Predictions program. Peptides were synthesized and immunoreactivity was assessed by peptide-MHC-binding affinity assay, cytotoxicity assay and HLA-A2.1/Kb transgenic mice immunization. Five candidates were identified according to the high scores generated by the computer predicting system. Peptide NEP(375-383) (FIMDLVSSL), which up-regulated HLA-A2.1 molecular expression, showed a high affinity to HLA-A2.1, whereas NEP(268-276), NEP(297--305) and NEP(492-500) (QLALEMNKV, MLLYNKMRL and KLNNEYLEL) showed a moderate affinity and NEP(559-567) (ILQPPFFSA) only had a low affinity. Cytotoxicity assay further showed that NEP(268-276) and NEP(375-383) could induce NEP-specific CTL responses in vitro. Unexpectedly, we found that a single CTL epitope, NEP(375-383), could induce proteinuria and glomerular injury in HLA-A2.1/K(b) transgenic mice in vivo. HLA-A*0201-restricted CTL epitope NEP(375-383) can serve as a potential candidate for designing MN vaccine.

Identification of HLA-A*0201-restricted CD8+ T-cell epitope C64–72 from hepatitis B virus core protein

The efficacy of a potential therapeutic vaccine against chronic hepatitis B virus (HBV) infection depends on the development of strong and multi-specific T cell responses. The potency of CD8+ cytotoxic T lymphocyte (CTL) responses toward HBV core antigen (HBcAg) has been shown to be critical for the outcomes of HBV chronic infection. In this study we have identified a previously undescribed HLA-A*0201-restricted HBcAg-specific CTL epitope (HBcAg64–72, C64–72, ELMTLATWV). T2 binding assay showed that C64–72 had high affinity to HLA-A*0201 molecule. Functionally, the peptide C64–72 could induce peptide-specific CTLs both in vivo (HLA-A2.1/Kb transgenic mice) and in vitro (PBLs of healthy HLA-A2.1+ donors), as demonstrated by interferon-γ (IFN-γ) secretion upon stimulation with C64–72-pulsed T2 cells or autologous human dendritic cells (DCs) respectively. HLA-A*0201-C64–72 tetramer staining revealed the presence of a significant population of C64–72-specific CTLs in C64–72-stimulated CD8+ T cells. Furthermore, the peptide-specific cytotoxic reactivity and the production of perforin and granzyme B of CTLs also increased after stimulation with C64–72-pulsed autologous DCs. These results indicate that the newly identified epitope C64–72 has potential to be used in the development of immunotherapeutic approaches to HBV infection.

A Novel HLA-A2–restricted CTL Epitope of Tumor-associated Antigen L6 can Inhibit Tumor Growth In Vivo

Vaccines utilizing cytotoxic T lymphocyte (CTL) epitopes are promising for the treatment of cancer and chronic infectious diseases. Tumor-associated antigen L6 (TAL6) is overexpressed in some epithelial cancer cells. In this report, we detected TAL6 expression in breast cancer tissue using quantitative reverse-transcriptase-polymerase chain reaction. We found that >80% of breast tumor tissue highly expressed TAL6 compared with adjacent normal breast tissue. To identify CTL epitopes from TAL6, we synthesized 18 peptides for HLA-A2-binding assay based on the MHC-binding motif using 4 computer prediction programs. Positive binders identified by ELISA were immunized in HLA-A2 transgenic (A2 Tg) mice. Two peptides, peptide 2 and peptide 5, induced T-cell responses in A2 Tg mice. To confirm whether these peptides could be processed and presented to induce T-cell responses in vivo, A2 Tg mice were immunized with plasmid DNA encoding TAL6. We found that both peptides 2 and 5 stimulated splenocytes from TAL6-immunized mice to secrete interferon-γ. However, only peptide 5 could induce expression of the cytolytic molecule CD107a on CD8+ T cells after immunization. Furthermore, peptide 5-immunized A2 Tg mice could inhibit the growth of TAL6-positive tumors (EL4/TAL6/HLA-A2) in A2 Tg mice but not in wild-type mice. These results demonstrate that the TAL6-derived CTL epitope could induce HLA-A2-restricted immunity against TAL6-expressing tumor cells.

Identification of novel HLA-A∗0201-restricted CTL Epitopes from Pokemon

Pokemon is a member of the POK family of transcriptional repressors and aberrant overexpressed in various human cancers. Therefore, the related peptide epitopes derived from Pokemon is essential for the development of specific immunotherapy of malignant tumors. In this study, we predicted and identified HLA-A∗0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from Pokemon with computer-based epitope prediction, peptide-binding assay and testing of the induced CTLs toward different kinds of carcinoma cells. The results demonstrated that effectors induced by peptides of Pokemon containing residues 32–40, 61–69, 87–95, and 319–327 could specifically secrete IFN-γ and lyse tumor cell lines of Pokemon-positive and HLA-A2-matched. The results suggest that Pokemon32, Pokemon61, Pokemon87, and Pokemon319 peptides are novel HLA-A∗0201-restricted restricted CTL epitopes, and could be utilized in the cancer immunotherapy against a broad spectrum of tumors.

Cross-Protective Peptide Vaccine against Influenza A Viruses Developed in HLA-A*2402 Human Immunity Model

BackgroundThe virus-specific cytotoxic T lymphocyte (CTL) induction is an important target for the development of a broadly protective human influenza vaccine, since most CTL epitopes are found on internal viral proteins and relatively conserved. In this study, the possibility of developing a strain/subtype-independent human influenza vaccine was explored by taking a bioinformatics approach to establish an immunogenic HLA-A24 restricted CTL epitope screening system in HLA-transgenic mice.Methodology/Principal FindingsHLA-A24 restricted CTL epitope peptides derived from internal proteins of the H5N1 highly pathogenic avian influenza A virus were predicted by CTL epitope peptide prediction programs. Of 35 predicted peptides, six peptides exhibited remarkable cytotoxic activity in vivo. More than half of the mice which were subcutaneously vaccinated with the three most immunogenic and highly conserved epitopes among three different influenza A virus subtypes (H1N1, H3N2 and H5N1) survived lethal influenza virus challenge during both effector and memory CTL phases. Furthermore, mice that were intranasally vaccinated with these peptides remained free of clinical signs after lethal virus challenge during the effector phase.Conclusions/SignificanceThis CTL epitope peptide selection system can be used as an effective tool for the development of a cross-protective human influenza vaccine. Furthermore this vaccine strategy can be applicable to the development of all intracellular pathogens vaccines to induce epitope-specific CTL that effectively eliminate infected cells.

Efficient induction of a Her2-specific anti-tumor response by dendritic cells pulsed with a Hsp70L1–Her2341–456 fusion protein

Heat shock proteins (HSPs) have been shown to interact with antigen-presenting cells (APCs), especially dendritic cells (DCs). HSPs act as potent adjuvants, inducing a Th1 response, as well as antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) via cross-presentation. Our previous work has demonstrated that Hsp70-like protein 1 (Hsp70L1), a new member of the Hsp70 subfamily, can act as a powerful Th1 adjuvant in a DC-based vaccine. Here we report the efficient induction of tumor antigen-specific T cell immune response by DCs pulsed with recombinant fusion protein of Hsp70L1 and Her2(341-456), the latter of which is a fragment of Her2/neu (Her2) containing E75 (a HLA-A2 restricted CTL epitope). The fusion protein Hsp70L1-Her2(341-456) promotes the maturation of DCs and activates them to produce cytokines, such as IL-12 and TNF-α, and chemokines, such as MIP-1α, MIP-1β and RANTES. Taken together, these results indicate that the adjuvant activity of Hsp70L1 is maintained in the fusion protein. Her2-specific HLA-A2.1-restricted CD8(+) CTLs can be generated efficiently either from the Peripheral blood lymphocytes (PBL) of healthy donors or from the splenocytes of immunized HLA-A2.1/K(b) transgenic mice by in vitro stimulation or immunization with DCs pulsed with the Hsp70L1-Her2(341-456) fusion protein. This results in more potent target cell killing in an antigen-specific and HLA-A2.1-restricted manner. Adoptive transfer of splenocytes from transgenic mice immunized with Hsp70L1-Her2(341-456)-pulsed DCs can markedly inhibit tumor growth and prolong the survival of nude mice with Her2(+)/HLA-A2.1(+) human carcinomas. These results suggest that Hsp70L1-Her2(341-456)-pulsed DCs could be a new therapeutic vaccine for patients with Her2(+) cancer.