Mass spectrometric identification of immunogenic SARS-CoV-2 epitopes and cognate TCRs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID-19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally if not more important than humoral responses in mediating recovery and immune protection. One major challenge in developing T cell-based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities. Our studies find that, in contrast to current convention, "immunodominant" SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined empirically by directly analyzing peptides eluted from the naturally processed peptide-major histocompatibility complex (MHC) and then validating immunogenicity by determining whether such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes derived from not only structural but also nonstructural genes in regions highly conserved among SARS-CoV-2 strains, including recently recognized variants. Finally, there are no reported T cell receptor-engineered T cell technology that can redirect T cell specificity to recognize and kill SARS-CoV-2 target cells. We report here several SARS-CoV-2 epitopes defined by mass spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity, and demonstrate engineered TCR-redirected killing.

Identification B and T-Cell epitopes and functional exposed amino acids of S protein as a potential vaccine candidate against SARS-CoV-2/COVID-19.

Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus that it disease spreads in over the world. Coronaviruses are single-stranded, positive-sense RNA viruses with a genome of approximately 30 KD, the largest genome among RNA viruses. Most people infected with the COVID-19 virus will experience mild to moderate respiratory illness and recover without requiring special treatment. Older people and those with underlying medical problems like cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop serious illness. At this time, there are no specific vaccines or treatments for COVID-19. So, there is an emergency need for vaccines and antiviral strategies. The spike protein is the major surface protein that it uses to bind to a receptor of another protein that acts as a doorway into a human cell. The putative antigenic epitopes may prove effective as novel vaccines for eradication and combating of COV19 infection. A combination of available bioinformatics tools are used to synthesis of such peptides that are important for the development of a vaccine. In conclusion, amino acids 250-800 were selected as effective B cell epitopes, T cell epitopes, and functional exposed amino acids in order to a recombinant vaccine against coronavirus.

Computational Identification and Characterization of a Promiscuous T-Cell Epitope on the Extracellular Protein 85B of Mycobacterium spp. for Peptide-Based Subunit Vaccine Design.

Tuberculosis (TB) is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using "Allele Frequency Database," we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB.

Isolation and partial characterization of an immunogenic antigen of Giardia lamblia.

Humoral and cellular immune responses play an important role during Giardia lamblia infection. Several Giardia proteins have been identified as immunogenic antigens based on their elicited humoral immune response. Poorly is known about Giardia antigens that stimulate a cellular immune response. The main purpose of this study was to isolate and partial characterize an immunogenic antigen (5G8) of G. lamblia. The 5G8 protein was isolated from G. lamblia trophozoite lysates by affinity chromatography using moAb 5G8-coupled CNBr-Sepharose. The isolated protein was analysed by electrospray tandem mass spectrometry (ESI-MS/MS), and by diverse bioinformatics tools (GiardiaDB, BLASTn, BLASTp and ExPASy). Additionally, several biochemical and immunological characteristics of the isolated protein were analysed. By ESI-MS/MS the amino acidic 5G8 sequence was deduced. The 5G8 antigen belongs to the VSP family proteins of G. lamblia. This protein is composed by one polypeptide chain (±71kDa). Using the algorithm SYFPHEITI, we identified candidate CD4+ T-cell epitopes from the 5G8 antigen, which can elicit cell-mediated immune responses. In this study, we have identified a G. lamblia protein that induces a strong immune response in infected mice. The biochemical and immunological characterization of the immunogenic 5G8 antigen may contribute to the rational design of a Giardia vaccine.

Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.

Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

Extraction of tissue antigens for functional assays.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined (1). This is particularly true in autoimmune diseases and cancer(2). Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes (1,3,4,5). To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays (6). Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

Multiple antigenic peptides based on H-2K(b)-restricted CTL epitopes from murine heparanase induce a potent antitumor immune response in vivo.

Accumulating research suggests that heparanase may be a universal tumor-associated antigen (TAA). Several heparanase T-cell epitopes from humans and mice have already been identified. However, because of low immunogenicity, polypeptide vaccines usually have difficulty inducing effective antitumor immune responses in vivo. In this study, to increase the immunogenicity of polypeptide vaccines, we designed and synthesized two four-branch multiple antigenic peptides (MAP) on the basis of mouse heparanase (mHpa) T-cell epitopes (mHpa398 and mHpa519). The dendritic cells (DC) from mice bone marrow loaded with above MAP vaccines from heparanase were used to evaluate immune response against various tumor cell lines, compared with immune response to their corresponding linear peptides, ex vivo and in vivo. We further assessed IFN-γ release both in CD4(+) T-cell-depleted and nondepleted mice. The results showed that effectors generated from DCs, loaded with MAP-vaccinated mice splenocytes, induced a stronger immune response against target cells expressing both heparanase and H-2K(b) than did effectors generated from mice vaccinated with their corresponding linear peptides. Heparanase-specific CD8(+) T-cell responses induced by MAP and linear peptide vaccination required synergy of CD4(+) T cells. In addition, heparanse-derived MAP vaccines significantly inhibited the growth of B16 murine melanoma in C57BL/6 mice, while also increasing the survival rate of tumor-bearing mice. Our data suggest that MAP vaccines based on T-cell epitopes from heparanase are efficient immunogens for tumor immunotherapy.

Identification of HLA-A*0201/-A*2402-restricted CTL epitope-peptides derived from a novel cancer/testis antigen, MCAK, and induction of a specific antitumor immune response.

Cancer immunotherapy is a potential therapeutic strategy, in addition to surgical treatment, radiotherapy, and chemotherapy. Cancer-specific immunotherapy, such as the MAGE peptide vaccine, has been utilized clinically. How-ever, there are inherent limits to the effectiveness of vaccinotherapy using a single antigen because of the expression frequency of cancer-specific antigens on tumor cells. Thus, identification of a new cancer-specific antigen is needed. In this study, we examined the possibility of using cancer-specific immunotherapy based upon mitotic centromere-associated kinesin (MCAK) which was previously identified as a novel cancer/testis antigen. To evaluate the feasibility of developing cancer immunotherapy using MCAK peptides, we studied HLA-A*0201 and *2402 as targets for CTLs in the context of HLA class I molecules. By using a peptide with a sequence of AINPELLQL (amino acid positions 63-71 in MCAK, HLA-A*0201) and FFEIYNGKL (amino acid positions 401-409 in MCAK, HLA-A*2402), CTL responses could be induced from unseparated PBMCs by stimulation of freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and also by using interleukin-7 and keyhole limpet hemocyanin in primary culture. The induced CTLs could lyse HLA-A-*0201/*2402 colon and gastric cancer cells expressing MCAK, as well as the peptide-pulsed target cells, in an HLA class l, and CD8 restricted manner. The identification of the MCAK/HLA-A*0201 and *2402 peptides suggests the possibility of designing peptide-based immunotherapeutic approaches that might prove effective in treating patients with MCAK-positive cancer.

CD8+ T cell responses against TAP-inhibited cells are readily detected in the human population.

Target cell recognition by CTLs depends on the presentation of peptides by HLA class I molecules. Tumors and herpes viruses have adopted strategies to greatly hamper this peptide presentation at the important bottleneck, the peptide transporter TAP. Previously, we described the existence of a CD8(+) CTL subpopulation that selectively recognizes such TAP-deficient cells in mouse models. In this study, we show that the human counterpart of this CTL subset is readily detectable in healthy subjects. Autologous PBMC cultures were initiated with dendritic cells rendered TAP-impaired by gene transfer of the viral evasion molecule UL49.5. Strikingly, specific reactivity to B-LCLs expressing one of the other viral TAP-inhibitors (US6, ICP47, or BNLF2a) was already observed after three rounds of stimulation. These short-term T cell cultures and isolated CD8(+) CTL clones derived thereof did not recognize the normal B-LCL, indicating that the cognate peptide-epitopes emerge at the cell surface upon an inhibition in the MHC class I processing pathway. A diverse set of TCRs was used by the clones, and the cellular reactivity was TCR-dependent and HLA class I-restricted, implying the involvement of a broad antigenic peptide repertoire. Our data indicate that the human CD8(+) T cell pool comprises a diverse reactivity to target cells with impairments in the intracellular processing pathway, and these might be exploited for cancers that are associated with such defects and for infections with immune-evading herpes viruses.

Rapid identification of CD4+ T-cell epitopes using yeast displaying pathogen-derived peptide library.

Identification of CD4+ T-cell epitopes is a critical step in studying and modulating the immune responses to tumors, infectious agents, and autoantigens. Here we report a facile, accurate, and high-throughput method for CD4+ T-cell epitope identification using yeast displaying pathogen-derived peptide library. A library of DNA fragments that encode all the possible peptides with 10-20 amino acids from the antigens (single antigenic proteins or pathogenic organisms) are fused to the gene encoding the restriction single-chain MHC class II molecule in a yeast display vector. The resultant library of recombinant yeast cells are analyzed by FACS to identify those containing peptides with high affinity towards the restriction MHC molecule, which are subsequently screened for their ability to induce antigen-specific T-cell activation. DNA sequence analysis of selected positive clones results in direct identification of the antigenic peptides. We show that this method can be used to rapidly pinpoint the HA(306-322) epitope from the haemagglutinin protein and the entire influenza virus X31/A/Aichi/68 genome, respectively.

Identification of breast cancer peptide epitopes presented by HLA-A*0201.

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.

Computational prediction and identification of dengue virus-specific CD4(+) T-cell epitopes.

In this study, we tried to identify dengue virus-specific CD4(+) T-cell epitopes, which can induce PBMC (peripheral blood mononuclear cells) isolated from DF convalescent patients (dengue virus type 1 infection) to secrete IFN-gamma. PBMC of DF convalescent patients were stimulated in vitro with dengue virus-derived peptides, which were prepared based on the prediction of dengue virus-specific CD4(+) T-cell epitopes by using RANKpep online software. Subsequently, the frequency of IFN-gamma producing T cells and percentage of IFN-gamma(+) CD4(+) T cells were measured by using ELISPOT assay and ICS assay (intracellular cytokine straining), respectively. The positive response of PBMC by ELISPOT showed that the numbers of SFC (spots forming cells) ranged from 50 to 310 SFC/1x10(6) PBMC. The positive response of PBMC by ICS assay showed that the percentage of IFN-gamma(+) CD4(+) T cells ranged from 0.03 to 0.27%. As a result, C(45-57) (KLVMAFIAFLRFL), E(396-408) (SSIGKMFEATARG), NS3(23-35) (YRILQRGLLGRSQ), and NS3(141-155) (NREGKIVGLYGNGVV) were identified as dengue virus-specific CD4(+) T-cell epitopes.

[Identification of a naturally presented MAGE-A3 epitope on the surface of HLE cell line by mass spectrometry].

OBJECTIVE: To identify a naturally presented HLA-A2-restricted epitope of MAGE-A3 antigen, FLWGPRALV (MAGE-A3(271 - 279)), on the surface of a human hepatocellular carcinoma (HCC) cell line HLE. METHODS: Synthetic peptide FLWGPRALV, served as positive control target, was analyzed by HPLC and HPLC-ESI-TOF-MSMS, in order to determine its HPLC elution time, mass-spectrometric characteristics and the lowest detection limitation by the two approaches. 3 x 10(9) HLE cells were collected, peptides naturally presented by major histocompatibility complex (MHC) molecules on the cell surface were isolated by mild acid elution, and concentrated by lyophilization, then the mixtures of peptides were fractioned by HPLC. The ingredient ranged from 2 min before the elution time determined by the synthetic peptide to 2 min after that was collected, concentrated by lyophilization, and analyzed by HPLC-ESI-TOF-MSMS, to identify the existence of the MAGE-A3(271 - 279) peptide. RESULTS: The HPLC-ESI-TOF-MSMS detection provided an evidence for the existence of a doubly charged ion of (m/z)(2) 529.9, which was further analyzed by collision induced dissociation. The doubly charged ion was ultimately identified as the MAGE-A3(271 - 279) peptide, its amino sequence was FLWGPRALV and its molecular weight was 1058.4 Da. CONCLUSIONS: MAGE-A3(271 - 279) epitope could be naturally presented by HLA-A2 molecules to the surface of HCC cell line and MAGE-A3(271 - 279) peptide may have potential immunotherapeutic value in HCC patients.

Identification of a highly immunogenic HLA-A*01-binding T cell epitope of WT1.

PURPOSE: The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. EXPERIMENTAL DESIGN: Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC. RESULTS: One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer. CONCLUSION: In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.

Leishmania chagasi T-cell antigens identified through a double library screen.

Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines.

Screening and identification of severe acute respiratory syndrome-associated coronavirus-specific CTL epitopes.

Severe acute respiratory syndrome (SARS) is a highly contagious and life-threatening disease that emerged in China in November 2002. A novel SARS-associated coronavirus was identified as its principal etiologic agent; however, the immunopathogenesis of SARS and the role of special CTLs in virus clearance are still largely uncharacterized. In this study, potential HLA-A*0201-restricted spike (S) and nucleocapsid protein-derived peptides were selected from an online database and screened for potential CTL epitopes by in vitro refolding and T2 cell-stabilization assays. The antigenicity of nine peptides which could refold with HLA-A*0201 molecules was assessed with an IFN-gamma ELISPOT assay to determine the capacity to stimulate CTLs from PBMCs of HLA-A2(+) SARS-recovered donors. A novel HLA-A*0201-restricted decameric epitope P15 (S411-420, KLPDDFMGCV) derived from the S protein was identified and found to localize within the angiotensin-converting enzyme 2 receptor-binding region of the S1 domain. P15 could significantly enhance the expression of HLA-A*0201 molecules on the T2 cell surface, stimulate IFN-gamma-producing CTLs from the PBMCs of former SARS patients, and induce specific CTLs from P15-immunized HLA-A2.1 transgenic mice in vivo. Furthermore, significant P15-specific CTLs were induced from HLA-A2.1-transgenic mice immunized by a DNA vaccine encoding the S protein; suggesting that P15 was a naturally processed epitope. Thus, P15 may be a novel SARS-associated coronavirus-specific CTL epitope and a potential target for characterization of virus control mechanisms and evaluation of candidate SARS vaccines.

Identification of a novel HIV type 1 CRF01_AE cytotoxic T lymphocyte (CTL) epitope restricted by an HLA-Cw0602 allele and a novel HLA-A0206/peptide restriction.

This report describes specific T cell responses to HIV-1 CRF01_AE Env and A Gag peptides in 20 HIV-1 CRF01_AE-infected Thai individuals using an interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISpot) assay. Twenty-six potentially novel HLA class I-restricted CD8+ T cell epitopes were identified in 14/20 subjects. Fine mapping analysis using the chromium release cytotoxic T lymphocyte (CTL) assay revealed a novel HLA-Cw0602 restricted epitope of HIV-1 CRF01_AE Env (NAKTIIVHL) and a previously identified HIV-1 A Gag epitope (ATLEEMMTA) with a novel HLA-A0206 restriction.

Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus.

This paper describes mapping of antigenic and host-protective epitopes of infectious bronchitis virus proteins by assessing the ability of defined peptide regions within the S1, S2 and N proteins to elicit humoral, cell-mediated and protective immune responses. Peptides corresponding to six regions in the S1 (Sp1-Sp6), one in the S2 (Sp7) and four in the N protein (Np1-Np4) were synthesized and coupled to either diphtheria toxoid (dt) or biotin (bt). Bt-peptides were used to assess if selected regions were antigenic and contained B- or T-cell epitopes and dt-peptides if regions induced an antibody response and protection against virulent challenge. All S1 and S2 peptides were antigenic, being recognised by IBV immune sera and also induced an antibody response following inoculation into chicks. Three S1-and one S2-bt peptides also induced a delayed type hypersensitivity response indicating the presence of T-cell epitopes. The S2 peptide Sp7 (amino acid position 566-584) previously identified as an immundominant region, was the most antigenic of all peptides used in this study. Two S1 (Sp4 and Sp6) and one S2 peptide (Sp7), protected kidney tissue against virulent challenge. From four N peptides located in the amino-terminal part of the N protein, only one, Np2 (amino acid position 72-86), was antigenic and also induced a delayed type hypersensitivity response. None of the N peptides induced protection against virulent challenge. The results suggest that the S1 glycoprotein carries additional antigenic regions to those previously identified and that two regions located in the S1 and one in the S2 at amino acid positions 294-316 (Sp4), 532-537 (Sp6) and 566-584 (Sp7) may have a role in protection.

[Generation of HER2 specific, HLA-A24 restricted CTLs derived from a healthy donor].

Cancer vaccine therapy is one of the latest treatment modalities for advanced cancer. In this study, we have newly identified two HER2 peptide epitopes restricted by HLA-A24, which are the most common alleles in Japanese. We have generated mature DCs from PBMCs in the HLA-A24+ healthy donor. Mature DCs were co-incubated with HER2 peptide, and then autologous PBMCs were co-incubated with antigen-loaded DCs. In this way, we have generated HER2 reactive and HLA-A24-restricted CTL lines. The CTL's specificity was evaluated with ELISPOT analysis and cytotoxic assay. In the two CTL lines, the specificity of TISI loaded with HER2/neu peptide used for each CTL induction was recognized, and that specific cytotoxicity was also found against cancer cell lines expressing HLA-A24 and HER2. In conclusion, we have newly identified two HER2 peptide epitopes restricted by HLA-A24 and confirmed that these epitopes will be new targets for cancer vaccine therapy.