Cytotoxic T Lymphocyte Epitope Peptide Research Articles

Novel epitopes identified from Mycobacterium tuberculosis antigen Rv2629induces cytotoxic T lymphocyte response

There is an urgent need for a more effective vaccine against tuberculosis (TB). Cytotoxic T lymphocytes (CTLs) play a critical role in combating Mycobacterium tuberculosis (M.tb). The identification of novel CTL epitopes is essential for the design of peptide-based vaccines. In this study, we predicted CTL epitope peptides of M.tb antigen Rv2629 restricted by HLA-A2, using bioinformatics methods. The affinity and stability of binding of these peptides with HLA-A2 molecules were detected by flow cytometry. Their ability to induce CTLs generation was determined in peripheral blood mononuclear cells (PBMCs) from healthy uninfected subjects, Latent tuberculosis infection (LTBI) subjects, and TB patients ex vivo. The cytotoxic activity induced by the epitope peptides was tested by lactate dehydrogenase (LDH) release assay. Finally, we found four novel CTL epitope peptides, Rv2629-p190-2L, Rv2629-p190-1Y2L, Rv2629-p274, and Rv2629-p315, which had high-affinity and stability of binding with T2 cells. Their ability of inducing CTLs was highest in PBMCs from TB patients (P < 0.05). In addition, these peptides could induce the CTLs to generate specific cytotoxic activity. They showed higher immunogenicity in TB patients and had the potential to become candidate vaccines for TB therapy.

Specificity Characterization of SLA Class I Molecules Binding to Swine-Origin Viral Cytotoxic T Lymphocyte Epitope Peptides in Vitro.

Swine leukocyte antigen (SLA) class I molecules play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They mainly bind and present antigens of intracellular origin to circulating MHC I-restricted cytotoxic T lymphocytes (CTLs). Binding of an appropriate epitope to an SLA class I molecule is the single most selective event in antigen presentation and the first step in the killing of infected cells by CD8+ CTLs. Moreover, the antigen epitopes are strictly restricted to specific SLA molecules. In this study, we constructed SLA class I complexes in vitro comprising viral epitope peptides, the extracellular region of the SLA-1 molecules, and β2-microglobulin (β2m) using splicing overlap extension polymerase chain reaction (SOE-PCR). The protein complexes were induced and expressed in an Escherichia coli prokaryotic expression system and subsequently purified and refolded. Specific binding of seven SLA-1 proteins to one classical swine fever virus (CSFV) and four porcine reproductive and respiratory syndrome virus (PRRSV) epitope peptides was detected by enzyme-linked immunosorbent assay (ELISA)-based method. The SLA-1∗13:01, SLA-1∗11:10, and SLA-1∗11:01:02 proteins were able to bind specifically to different CTL epitopes of CSFV and PRRSV and the MHC restrictions of the five epitopes were identified. The fixed combination of Asn151Val152 residues was identified as the potentially key amino acid residues influencing the binding of viral several CTL epitope peptides to SLA-1∗13:01 and SLA-1∗04:01:01 proteins. The more flexible pocket E in the SLA-1∗13:01 protein might have fewer steric limitations and therefore be able to accommodate more residues of viral CTL epitope peptides, and may thus play a critical biochemical role in determining the peptide-binding motif of SLA-1∗13:01. Characterization of the binding specificity of peptides to SLA class I molecules provides an important basis for epitope studies of infectious diseases in swine, and for the rational development of novel porcine vaccines, as well as for detailed studies of CTL responses in pigs used as animal models.

Direct Loading of iTEP-Delivered CTL Epitope onto MHC Class I Complexes on the Dendritic Cell Surface.

Cytotoxic T lymphocyte (CTL)-mediated immune responses are the primary defense mechanism against cancer and infection. CTL epitope peptides have been used as vaccines to boost CTL responses; however, the efficacy of these peptides is suboptimal. Under current vaccine formulation and delivery strategies, these vaccines are delivered into and processed inside antigen-presenting cells such as dendritic cells (DCs). However, the intracellular process is not efficient, which at least partially contributes to the suboptimal efficacy of the vaccines. Thus, we hypothesized that directly loading epitopes onto MHC class I complexes (MHC-Is) on the DC surface would significantly improve the efficacy of the epitopes because the direct loading bypasses inefficient intra-DC vaccine processing. To test the hypothesis, we designed an immune-tolerant elastin-like polypeptide (iTEP)-delivered CTL vaccine containing a metalloproteinase-9 (MMP-9)-sensitive peptide and an CTL epitope peptide. We found that the epitope was released from this MMP-sensitive vaccine through cleavage by DC-secreted MMP-9 outside of the DCs. The released epitopes were directly loaded onto MHC-Is on the DC surface. Ultimately, the MMP-sensitive vaccine strikingly increased epitope presentation by DCs by 7-fold and enhanced the epitope-specific CD8+ T-cell response by as high as 9.6-fold compared to the vaccine that was uncleavable by MMP. In summary, this novel direct-loading strategy drastically boosted vaccine efficacy. This study offered a new avenue to enhance CTL vaccines.

Immunogenicity of dominant epitopes of complex antigen rhoptry protein 2-sunface protein 1 derived from Toxoplasma gondii

Objective To analyze the immunogenicity of dominant epitope of complex antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) derived from Toxoplasma gondii (T. gondii). Methods Dominant epitope of ROP2-SAG1 containing both dominant T- and B-cell epitopes was predicted and selected from T. gondii with bioinformatics methods. The gene fragment cloned into pET32a expression vector was transformed into the competent cell (Escherichia coli strain Rosetta) and expressed under the induction. The protein purified by nitrilotriacetic acid (Ni-NTA) agarose resin were finally identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Japanese rabbits were immunized subcutaneously with purified epitope protein in contrast with control group immunized with pET32a protein or phosphate buffevred saline (PBS). The sera from immunized rabbits were collected at week 0, week 2 and week 4 for determination of epitope-specific antibody IgG using enzyme-linked immunosorbent assay, and immunodot assay was used to further confirm the specificity of antibody. After BALB/c mice were immunized with purified epitope proteins, the capacity of production of interferon-γ(IFN-γ) in splenocytes was detected by enzyme-linked immunospot assay. Results Relative molecular weight 30 000 of dominant epitope was derived from prokaryotic system. Then the rabbits immunized with purified dominant epitope could produce corresponding epitope-specific antibody IgG. And with the increased frequency of immunization, the level of antibody gradually increased. At week 2 and 4, higher antibody response were observed in group of rabbit immunized with dominant epitope than those of control group (1.454±0.098 vs 0.616±0.084, F=0.000, P<0.05; 2.299±0.224 vs 1.580±0.192, F=0.112, P<0.05). The antibody titer at week 4 was as high as 1∶40 960. Immunodot assay further confirmed the antibody specificity against the dominant epitope. The level of IFN-γ in splenocytes from mice immunized with dominant epitope after stimulation with three epitope specific CTL peptides (epitope peptides 1 [19.333±1.528]/100 000 cells, epitope peptides 2 ([40.333±1.528]/100 000 cells), epitope peptides 3 ([70.667±1.890]/100 000 cells) was significantly higher than that of PBS control group (epitope peptides 1 [1.033±0.150]/100 000 cells, epitope peptides 2 [1.045±0.110]/100 000 cells, epitope peptides 3 [1.041±0.120]/100 000 cells, F=0.284, 0.000 and 0.284, respectively; all P<0.05). The level of IFN-γ from splenocytes stimulated with combined peptide with cytotoxic T lymphocyte (CTL) epitope peptide and helper T cell epitope peptide (epitope peptides 3) was significantly higher than that with single CTL epitope peptides (epitope peptides 1 and epitope peptides 2, F=5.796 and 0.000, respectively; both P<0.05). Conclusion Screened dominant epitopes of ROP2-SAG1 from T. gondii derived from prokaryotic expression system exhibit remarkable immunogenicity. Key words: Toxoplasma; ROP2-SAG1; Epitopes; Immunogenicity

Vaccination with liposome-coupled glypican-3-derived epitope peptide stimulates cytotoxic T lymphocytes and inhibits GPC3-expressing tumor growth in mice

Because therapeutic manipulation of immunity can induce tumor regression, anti-cancer immunotherapy is considered a promising treatment modality. We previously reported that glypican-3 (GPC3), an oncofetal antigen overexpressed in hepatocellular carcinoma (HCC), is a useful target for cytotoxic T lymphocyte (CTL)-mediated cancer immunotherapy, and we have performed clinical trials using the GPC3-derived peptide vaccine. Although vaccine-induced GPC3-peptide-specific CTLs were often tumor reactive in vitro and were correlated with overall survival, no complete response was observed. In the current study, we synthesized liposome-coupled GPC3-derived CTL epitope peptide (pGPC3-lipsome) and investigated its antitumor potential. Vaccination with pGPC3-liposome induced peptide-specific CTLs at a lower dose than conventional vaccine emulsified in incomplete Freund's adjuvant. Coupling of pGPC3 to liposomes was essential for effective priming of GPC3-specific CTLs. In addition, immunization with pGPC3-liposome inhibited GPC3-expressing tumor growth. Thus, vaccination with tumor-associated antigen-derived epitope peptides coupled to the surfaces of liposomes may be a novel therapeutic strategy for cancer.

Abstract B29: Improving the efficacy of dendritic cell-based human papillomavirus vaccines in mice using a natural lignin-carbohydrate complex and/or synthetic lignin-like polyphenol polymers

Abstract Immunization with dendritic cell (DC)-based vaccines is a promising vaccination strategy to provide an effective personalized immunotherapy against cancer and other diseases. However, the results of clinical cancer immunotherapy trials carried out using DC-based vaccines to date have shown only modest clinical outcomes. We have previously demonstrated that a polyphenylpropenoid-polysaccharide complex (PPC), a lignin-carbohydrate complex derived from the cones of Pinus sylvestris, induces in vitro the differentiation and maturation of human mononuclear cell-derived DCs (Bradley et al., Int Immunopharmacol 2003;3) and murine bone marrow-derived DCs (BMDCs) (An et al., Anticancer Res 2010;30). Furthermore, oral administration of PPC enhanced a DC vaccine-induced melanoma antigen-specific CD8+ T-cell response (Burrows et al., BMC Complement Altern Med 2009;9) and the proliferative phase of a primary T cell response (Bradley et al., BMC Complement Altern Med 2014;14). In the present study, using a human papillomavirus (HPV)-induced mouse tumor model, we investigated whether ex vivo treatment of BMDCs with the synthetic lignin-like polyphenol polymers poly-caffeic acid (pCA) and poly-coumaric acid (pCoA), as well as PPC, improves the efficacy of DC-based vaccines. Polymerization of the phenylpropenoic acids caffeic acid (CA) and poly-coumaric acid (CoA) was performed by horseradish peroxidase/hydrogen peroxide-mediated enzymatic reactions. BMDCs were prepared by culturing murine bone marrow cells in the presence of GM-CSF for 8-10 days. These BMDCs were pulsed with synthetic tumor antigen peptides HPV16 E743-62 or HPV16 E743-77 that contain both a cytotoxic T lymphocyte (CTL) and a helper T cell epitope. Peptide-pulsed DCs were treated further ex vivo with synthetic polyphenol polymers or PPC and then injected twice into C57BL/6 mice. In addition, splenocytes obtained from immunized mice were restimulated with HPV16 E749-57 peptide (E7 CTL epitope) for 5 days. Tumor antigen-specific CD8+ T cell responses were assessed by HPV16 E749-57/H-2Db pentamer staining. We observed that immunization with PPC-treated HPV16 E743-77 peptide-loaded BMDCs significantly enhanced the induction of pentamer-positive CD8 positive cells in splenocytes (2- to 3-fold increase). Moreover, ex vivo restimulation with E7 CTL epitope peptide dramatically increased the number of pentamer-positive CD8 positive cells in splenocytes obtained from mice immunized with PPC-treated, peptide-loaded DCs (6- to 7-fold increase) when compared to splenocytes from mice immunized with PPC-nontreated, peptide-loaded DCs. Experiments using pCA and pCoA are in progress. We conclude that 1) ex vivo treatment with the natural PPC complex significantly potentiates the in vivo efficacy of DC-based HPV vaccines and 2) tumor antigen-specific CD8+ T cells are significantly expanded ex vivo by restimulating splenocytes from mice primed in vivo with PPC-treated, peptide-loaded BMDCs. In addition, our preliminary studies show that PPC, pCA and pCoA significantly increase the expression levels of CD8α, XCR1 and/or CLEC9A on murine BMDCs that have been demonstrated to be essential for the induction of CTL responses. Our approaches will provide innovative strategies for the design of more effective DC-based vaccines and adaptive T-cell therapies for the treatment of patients with cancer and other immune disorders. Citation Format: Soichi Haraguchi, Tina Pastoor, William Guy Bradley, Akiko Tanaka. Improving the efficacy of dendritic cell-based human papillomavirus vaccines in mice using a natural lignin-carbohydrate complex and/or synthetic lignin-like polyphenol polymers. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B29.

Potential Contribution of a Novel Tax Epitope–Specific CD4+ T Cells to Graft-versus-Tax Effect in Adult T Cell Leukemia Patients after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for adult T cell leukemia/lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). We previously reported that Tax-specific CD8(+) cytotoxic T lymphocyte (CTL) contributed to graft-versus-ATL effects in ATL patients after allo-HSCT. However, the role of HTLV-1-specific CD4(+) T cells in the effects remains unclear. In this study, we showed that Tax-specific CD4(+) as well as CD8(+) T cell responses were induced in some ATL patients following allo-HSCT. To further analyze HTLV-1-specific CD4(+) T cell responses, we identified a novel HLA-DRB1*0101-restricted epitope, Tax155-167, recognized by HTLV-1-specific CD4(+) Th1-like cells, a major population of HTLV-1-specific CD4(+) T cell line, which was established from an ATL patient at 180 d after allo-HSCT from an unrelated seronegative donor by in vitro stimulation with HTLV-1-infected cells from the same patient. Costimulation of PBMCs with both the identified epitope (Tax155-167) and known CTL epitope peptides markedly enhanced the expansion of Tax-specific CD8(+) T cells in PBMCs compared with stimulation with CTL epitope peptide alone in all three HLA-DRB1*0101(+) patients post-allo-HSCT tested. In addition, direct detection using newly generated HLA-DRB1*0101/Tax155-167 tetramers revealed that Tax155-167-specific CD4(+) T cells were present in all HTLV-1-infected individuals tested, regardless of HSCT. These results suggest that Tax155-167 may be the dominant epitope recognized by HTLV-1-specific CD4(+) T cells in HLA-DRB1*0101(+)-infected individuals and that Tax-specific CD4(+) T cells may augment the graft-versus-Tax effects via efficient induction of Tax-specific CD8(+) T cell responses.

Identification of an H2-Kb or H2-Db restricted and glypican-3-derived cytotoxic T-lymphocyte epitope peptide

Glypican-3 (GPC3) is overexpressed in human hepatocellular carcinoma (HCC) but not expressed in normal tissues except for placenta and fetal liver and therefore is an ideal target for cancer immunotherapy. In this study, we identified an H2-Kb or H2-Db restricted and murine GPC3 (mGPC3)-derived cytotoxic T-lymphocyte (CTL) epitope peptide in C57BL/6 (B6) mice, which can be used in the design of preclinical studies of various therapies with GPC3-target immunotherapy in vivo. First, 11 types of 9- to 10-mer peptides predicted to bind with H2-Kb or H2-Db were selected from the mGPC3 amino acid sequence based on the binding score as calculated by the BIMAS software. We evaluated the peptide-binding affinity and confirmed that all peptides were able to bind to H2-Kb or H2-Db by in vitro cellular binding assay. Subsequently, a mixed peptide vaccine and single peptide vaccine were given to B6 mice to evaluate immunogenic potential of the 11 selected peptides. Using the splenocytes from peptide-vaccinated mice, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays showed that mGPC3-1127–136 (AMFKNNYPSL) peptide was the most efficient for inducing CTLs among the 11 peptides. Next, we demonstrated that the mGPC3-1 peptide-specific CTL line could recognize mGPC3-expressing cancer cells, suggesting that mGPC3-1 peptide was an endogenously presented peptide. In conclusion, we identified mGPC3-1 as an H2-Kb or H2-Db restricted, mGPC3-derived CTL epitope peptide.

Establishment of a stable T lymphoma cell line transduced with HLA-A^|^lowast;24:02-restricted WT1-specific TCR genes and its application to antigen-specific immunomonitoring

Wilms' tumor gene 1 (WT1) has been proposed as an attractive target for cancer immunotherapy. A natural 9-mer peptide (CYTWNQMNL), which bound to human leukocyte antigen (HLA)-A*24:02, was identified from among WT1-specific cytotoxic T lymphocyte (CTL) epitopes. This natural WT1 CTL epitope peptide was further modified (CMTWNQMNL) to enhance its binding affinity to HLA-A*24:02. This modified WT1 CTL epitope peptide was superior to the natural peptide for inducing HLA-A*24:02-restricted WT1-specific CTLs. Here we induced several WT1 CTLs that reacted with both modified and natural WT1 tetramers from peripheral blood mononuclear cells. Then, T-cell receptor (TCR) genes were isolated from these WT1 CTLs to determine their Vα and Vβ usage. These TCR genes were transduced into human T lymphoma cells to establish a stable cell line, SK37, which expressed a WT1-specific TCR. We confirmed that SK37 cells reacted with both modified and natural WT1 tetramers, which indicated that SK37 cells could be a useful tool for WT1 tetramer reagent quality assurance. One the basis of these findings, we propose that this WT1 tetramer, which was quality-assured using established SK37 cells, will contribute to reliable immunomonitoring of tumor-specific CTL responses of cancer patients who receive WT1-targeted cancer vaccine therapy or TCR-gene therapy.

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.

A Novel DNA/Peptide Combined Vaccine Induces PSCA-specific Cytotoxic T-lymphocyte Responses and Suppresses Tumor Growth in Experimental Prostate Cancer

To develop a completely novel DNA peptide-combined vaccine and determine whether it can efficiently improve tumor-specific cytotoxic T lymphocyte (CTL) responses and inhibit tumor progression in experimental prostate cancer models. The DNA/peptide combined vaccine was prepared by the self-assembly of a cationic peptide ([K]18P9) containing 18 lysines and a CTL epitope peptide, prostate stem cell antigen (PSCA (14-22)) (HLA-A2 restricted) with a recombinant plasmid encoding human full-length PSCA gene (pcDNA3.1(+)-PSCA) through electrostatic interactions. The formation of a DNA/peptide complex was examined by DNA retardation assay, DNase I protection assay, and transmission electron microscopy. The efficacy of vaccination using this complex was demonstrated in terms of the PSCA-specific CTL activity and antitumor immunity to PSCA(+) tumors in a murine model. This form of DNA/peptide complex could efficiently transfer the plasmid encoding full-length PSCA gene into mammalian cells and induced potent CTLs cytotoxicity against a human prostate carcinoma cell line established from the left supraclavicular lymph node metastasis from a 50-year-old man with prostate carcinoma in 1977. Expressing PSCA compared with pcDNA3.1(+)-PSCA, [K]18P9 peptide, or pcDNA3.1(+). Moreover, the vaccination of mice with this complex induced a potent antitumor immunity to prostate carcinomas in a xenograft tumor model in nude mice. This study suggests that a specific antitumor immune response can be induced by this DNA/peptide combined vaccine, which represents a new strategy for use in the immunotherapy of prostate cancer.

Liposomes and nanotechnology in drug development: focus on oncotargets

Nanotechnology is the development of an engineered device at the atomic, molecular, and macromolecular level in the nanometer range. Advances in nanotechnology have proven beneficial in therapeutic fields such as drug-delivery and gene/protein delivery. Antigen delivery systems are important for inducing and modifying immune responses. In cellular immunity, cytotoxic T lymphocytes (CTLs) are important in the host defense against tumors. Key to the development of CTL-inducible vaccines is the ability to deliver antigens to antigen-presenting cells efficiently and to induce the subsequent activation of T cell-mediated immunity without adjuvants, as they can induce excessive inflammation leading to systemic febrile disease. Since expression and cloning methods for tumor-associated antigens have been reported, cancer vaccines that induce effective cell immunity may be promising therapeutic candidates, but Th2 cells are undesirable for use in cancer immunotherapy. Peptide vaccines have immunological and economic advantages as cancer vaccines because CTL epitope peptides from tumor-associated antigens have high antigen-specificity. However, cancer vaccines have had limited effectiveness in clinical responses due to the ability of cancer cells to “escape” from cancer immunity and a low efficiency of antigen-specific CTL induction due to immunogenic-free synthetic peptides. In contrast, carbohydrate-decorated particles such as carbohydrate-coated liposomes with encapsulated antigens might be more suitable as antigen delivery vehicles to antigen-presenting cells. Oligomannose-coated liposomes (OML) can eliminate established tumors in mouse cancer models. In addition, OMLs with an encased antigen can induce antigen-specific CTLs from peripheral blood mononuclear cells obtained from patients. Feasibility studies of OML-based vaccines have revealed their potential for clinical use as vaccines for diseases where CTLs act as effector cells. Furthermore, use of the hepatitis B core particle, in which tumor-antigen epitopes are set, has consistently been shown to induce strong CTL responses without the use of an adjuvant. Thus, nanoparticles may provide a new prophylactic strategy for infectious disease and therapeutic approaches for cancer via the induction of T-cell immunity.

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.

Crystal Structure of Swine Major Histocompatibility Complex Class I SLA-1*0401 and Identification of 2009 Pandemic Swine-Origin Influenza A H1N1 Virus Cytotoxic T Lymphocyte Epitope Peptides

The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1 0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIV(NW9); NSDTVGWSW) or Ebola virus (Ebola(AY9); ATAAATEAY) were determined in this study. The overall peptide-SLA-1 0401 structures resemble, as expected, the general conformations of other structure-solved peptide major histocompatibility complexes (pMHC). The major distinction of SLA-1 0401 is that Arg(156) has a "one-ballot veto" function in peptide binding, due to its flexible side chain. S-OIV(NW9) and Ebola(AY9) bind SLA-1 0401 with similar conformations but employ different water molecules to stabilize their binding. The side chain of P7 residues in both peptides is exposed, indicating that the epitopes are "featured" peptides presented by this SLA. Further analyses showed that SLA-1 0401 and human leukocyte antigen (HLA) class I HLA-A 0101 can present the same peptides, but in different conformations, demonstrating cross-species epitope presentation. CTL epitope peptides derived from 2009 pandemic S-OIV were screened and evaluated by the in vitro refolding method. Three peptides were identified as potential cross-species influenza virus (IV) CTL epitopes. The binding motif of SLA-1 0401 was proposed, and thermostabilities of key peptide-SLA-1 0401 complexes were analyzed by circular dichroism spectra. Our results not only provide the structural basis of peptide presentation by SLA I but also identify some IV CTL epitope peptides. These results will benefit both vaccine development and swine organ-based xenotransplantation.

Identification of a new MAGE-A10 antigenic peptide presented by HLA-A*0201 on tumor cells

MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues with the exception of testis and placenta. Among MAGE-A antigens, MAGE-A10 is extensively expressed in various histological types of tumors, representing an attractive target for tumor immunotherapy. Cytotoxic T lymphocytes (CTLs) play a key role in anti-tumor immune responses, so the identification of CTL epitopes derived from MAGE-A10 would contribute a lot to the design of epitope-based vaccines for tumor patients. In this study, we predicted HLA-A*0201-restricted CTL epitope peptides of MAGE-A10, followed by peptide/HLA-A*0201 binding affinity and complex stability assays, and induced peptide-specific CTL immune responses. Of the selected three peptides (designated P1, P2 and P3), P1 (MAGE-A10310-318, SLLKFLAKV) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. And, the induced CTLs could lyse MAGE-A10-expressing tumor cells in a HLA-A*0201-restricted fashion but not MAGE-A10-negative tumor cells. Our results demonstrate that the peptide MAGE-A10310-318 is a HLA-A*0201-restricted CTL epitope of MAGE-A10 and could serve as a target for therapeutic antitumoral vaccination.

A CTL-based liposomal vaccine capable of inducing protection against heterosubtypic influenza viruses in HLA-A*0201 transgenic mice

The current vaccination strategy against influenza is to induce the production of antibodies directed against surface antigens of viruses. However, the frequent changes in the surface antigens of influenza viruses allow the viruses to avoid antibody-mediated immunity. On the other hand, it is known that cytotoxic T-lymphocyte (CTL) populations directed against internal antigens of influenza A virus are broadly cross-reactive to influenza virus subtypes. In the present study, liposomal conjugates with CTL epitope peptides derived from highly conserved internal antigens of influenza viruses were evaluated for their ability to protect against infection with influenza viruses. Liposomal conjugates with peptide M1 58-66, an HLA-A*0201-binding CTL epitope present within the amino-acid sequence of the M1 coding region, successfully induced antigen-specific CD8 + T-cells and CTLs in HLA-A*0201-transgenic mice. Moreover, after nasal infection with either the H1N1 or H3N2 virus, viral replication in the lung was significantly inhibited in the immunized mice. These protective activities lasted at least 6 months after the immunization. Thus, these results suggest that liposome-coupled CTL epitope peptides derived from highly conserved internal antigens of influenza viruses might be applicable to the development of vaccines that induce protection against infection with heterosubtypic influenza viruses.

Highly Efficient Antiviral CD8+T-Cell Induction by Peptides Coupled to the Surfaces of Liposomes

In previous studies, we have demonstrated that liposomes with differential lipid components display differential adjuvant effects when antigens (Ags) are chemically coupled to their surfaces. When ovalbumin was coupled to liposomes made by using unsaturated fatty acids, it was found to be presented not only to CD4(+) T cells but also to CD8(+) T cells and induced cytotoxic T lymphocytes (CTLs) which effectively eradicated the tumor from mice. In this study, we coupled liposomes to immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus (LCMV) and evaluated its potency as an antiviral vaccine. The intramuscular immunization of mice with the peptide-liposome conjugates along with CpG resulted in the efficient induction of antiviral CD8(+) T-cell responses which conferred complete protection against not only LCMV Armstrong but also a highly virulent mutant strain, clone 13, that establishes persistent infections in immunocompetent mice. The intranasal vaccination induced mucosal immunity effective enough to protect mice from the virus challenge via the same route. Complete protection was achieved in mice even when the Ag dose was reduced to as low as 280 ng of liposomal peptide. This form of vaccination with a single CTL epitope induced Ag-specific memory CD8(+) T cells in the absence of CD4(+) T-cell help, which could be shown by the complete protection of CD4-knockout mice in 10 weeks as well as by the analysis of recall responses. Thus, surface-linked liposomal peptide might have a potential advantage for the induction of antiviral immunity.

Toll like receptor agonists augment HPV 11 E7-specific T cell responses by modulating monocyte-derived dendritic cells

Impaired local cellular immunity is one of the mechanisms responsible for condyloma acuminatum (CA) recurrence. The activation of dendritic cells (DCs) is important in vaccine development. We investigated the effect of different toll like receptor (TLR) agonists including LPS (TLR4 agonist), polyinosinic acid-polycytidylic acid (PIC, TLR3 agonist), CpG oligonucleotide (TLR9 agonist), and imiquimod (TLR7 agonist) on human monocyte-derived dendritic cells (mdDCs) loading of human papillomavirus (HPV) type 11 E7 epitope. As a result, we found that mdDCs loading HLA-A*0201-restricted HPV 11 E7 CTL epitope peptide could respond to the TLR agonists, especially LPS and PIC. This was characterized by an enhanced expression of CD40, CD80, CD86, CD83 and HLA-DR, and a high level of IL-12 production. TLR agonists, especially PIC, enhanced the ability of E7-loaded mdDCs to induce IFN-gamma-secretion CD4(+) naïve T cells. Moreover, E7-loaded mdDCs exposed to TLR agonists augmented autologous T cell responses including effector cytokines production and specific cytotoxic T lymphocyte (CTL) responses. In addition, the inhibitory effect of IL-10 on mdDCs maturation could be partially restored by LPS, PIC or imiquimod. Taken together, these results demonstrate that TLR agonists promoted the maturation of E7-loaded mdDCs and their ability to induce T help type 1 polarization and augment E7-specific T cell responses. These data also indicated that TLR3/4 agonists might be effective adjuvants of mdDC-based vaccines against CA.

Induction of specific and flavivirus—Cross-reactive CTLs by immunization with a single dengue virus-derived CTL epitope peptide

Specificities of cytotoxic T lymphocyte (CTL) effector cells induced in BALB/c mouse by immunization with the single modified CTL epitope peptide derived from NS3 of dengue virus types 1 and 3, or that of dengue virus types 2 and 4 were examined. The effector cells included CTLs specific for the epitope peptide used for immunization and those cross-reactive to epitope peptides of other flaviviruses. A CTL clone, 2F7, was established from the effector cells. The clone 2F7 was specific for the epitope peptide used for immunization. Recognition by the effector cells was remarkably impaired by amino acid substitutions at positions 3, 5, and 6 of the epitope peptides. These results indicate that immunization with a single CTL epitope peptide of dengue viruses induces serotype-specific CTLs as well as CTLs cross-reactive to the other flaviviruses, and that the a.a. residues at positions 3, 5, and 6 are critical for cross-reaction.

Immunization with dendritic cells loaded with α-galactosylceramide at priming phase, but not at boosting phase, enhances cytotoxic T lymphocyte activity against infection by intracellular bacteria

We evaluated the effect of immunization with dendritic cells (DCs) pulsed with alpha-galactosylceramide (alphaGalCer) and listeriolysin O (LLO) 91-99 peptide, a dominant cytotoxic T lymphocyte (CTL) epitope of Listeria monocytogenes by observing the responses of specific CD8(+) T cells and in vivo CTL activity. DCs were pulsed with various combinations of alphaGalCer and LLO91-99 peptide and administered to BALB/c mice. Immunization with DCs pulsed with alphaGalCer and LLO91-99 at priming phase and with DCs pulsed with LLO91-99 alone at boosting phase induced stronger in vivo CTL activity, reduced the bacterial load in spleens of Listeria-challenged mice and augmented CD62L(+) CD8(+) central memory T cells compared with other immunization protocols. The blockade of interferon-gamma (IFN-gamma) at boosting phase reversed the induction of CD8(+) central memory T cells and reduced the bacterial load in spleens of Listeria-challenged mice immunized with DCs pulsed with alphaGalCer and LLO91-99 at both phases, suggesting that alphaGalCer at boosting phase has deleterious effects through IFN-gamma production. These results indicate that immunization with DCs pulsed with CTL epitope peptide together with alphaGalCer at priming phase, but not at boosting phase, is feasible for eliciting a specific CTL activity and protective immunity against infection of intracellular bacteria.