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.     

Highly conserved pattern of recognition of influenza A wild-type and variant CD8+ CTL epitopes in HLA-A2+ humans and transgenic HLA-A2+/H2 class I-deficient mice

As an in vivo model for studying human MHC (HLA) class I-restricted CTL responses to viral infection, we established a series of HLA Tg mice expressing HLA-A2, -B7 or -B27 human/mouse hybrid genes on a background deficient for H2 class I (Tg HLA(hyb)/H2 class I DKO). To determine whether CTL recognition of influenza A (flu) infection in Tg HLA-A2(hyb)/H2 DKO mice is similar to HLA-A2+ humans, we compared the HLA-A2-restricted Tg mouse and human CD8+ T-cell responses to an immunodominant flu epitope (wild-type [WT] M1 58-66), as well as a variant of this peptide (var. M1 58-66). Similar to HLA-A2+ humans, our results show WT M1 58-66 is likely the dominant CTL epitope recognized in infected Tg HLA-A2(hyb)/H2 DKO mice. Var. M1 58-66 was also recognized by WT peptide-reactive T cells from both HLA-A2+ humans and Tg mice, although slightly less efficiently than WT M1 58-66 in both cases. Reduced variant recognition was shown to be associated with reduced peptide/A2 binding, as well as a more limited repertoire of utilized TCR Vbeta chains. The similar pattern of recognition and cross reaction observed here for the WT and variant M1 58-66 epitopes with HLA-A2 by human and Tg HLA mouse CTLs indicates that A2-dependent events of Ag processing, presentation and recognition are well-conserved between species. These findings demonstrate that this Tg HLA-A2/H2 DKO model will aid identification and development of epitopes as vaccines for numerous viral and tumor antigens for the HLA-A2 supertype.     

Peptide-pulsed dendritic cells induce the hepatitis C viral epitope-specific responses of naïve human T cells

Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8⁺) and class II-restricted (CD4⁺) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells.     

Prediction and identification of novel IBV S1 protein derived CTL epitopes in chicken

Infectious bronchitis virus (IBV) is a major pathogen common in the poultry industry. Broad cytotoxic T lymphocyte (CTL) response against IBV is one of the crucial factors that help to control viral replication. Spike glycoproteins on the surface of the IBV virion harbor major T cell epitopes. In this study, based on the peptide-binding motifs of chicken MHC I molecules for the BF2*4, BF2*12, BF2*15, and BF2*19 haplotypes, potential CTL epitopes were predicted using S1 proteins from different IBV strains. Twenty-one peptides were predicted to be potential CTL epitopes; they were manually synthesized and the CTL responses to them tested in vitro. Spleen lymphocytes were collected from specific-pathogen free (SPF) chicken that had been immunized with the S1 protein expression plasmid, pV-S1, and were stimulated by the synthesized peptides. IFN-γ secretion and CD8⁺ T cell proliferation in chickens were tested by ELISpot array and flow cytometry, respectively. Four epitopes (P8_SRIQTATDP, P9_SRNATGSQP, P18_GAYAVVNV, and P19_SRIQTATQP) were identified to stimulate CD8⁺ T cell proliferation and IFN-γ secretion, indicating their efficacy as CTL epitopes in chicken. Poly-CTL-epitope DNA vaccine (pV-S1T) was constructed by inserting nucleotide sequences encoding the P8, P9, P18, and P19 CTL epitopes into the pVAX1 vector. Chickens were vaccinated with either pV-S1, pV-S1T, or pVAX1 and the protection efficacy was analyzed, revealing that ninety percent of chickens immunized with pV-S1T were protected after challenge with 10⁶ ELD₅₀ of IBV, demonstrating that these novel CTL epitopes were effective against IBV challenge. This study provides a new method to screen virus CTL epitopes in chicken and to develop poly-CTL-epitope DNA vaccines.     

Potent enhancement of cellular and humoral immune responses against recombinant hepatitis B antigens using AS02A adjuvant in healthy adults

Recombinant subunit protein vaccines generally elicit good humoral immune responses, weak helper T cell responses and no cytotoxic T cell responses. Certain adjuvants are known to enhance humoral and cellular immune responses. This study evaluated the humoral, CD4+ T helper and CTL responses induced by the recombinant SL* protein adjuvanted with AS02A in comparison with non-adjuvanted SL* in PBS in two groups of 15 healthy adult volunteers. The AS02A adjuvant contains monophosphoryl lipid A (MPL), QS21 and an oil in water emulsion. The adjuvanted vaccine induced fast and vigorous humoral and helper T cell responses of the Th1 type. Using a pool of overlapping 20mer peptides a cytotoxic response was detected in 6 out of 14 HLA-A2-positive (+) and HLA-A2-negative (-) recipients of the adjuvanted vaccine. All HLA-A2-positive subjects in the adjuvanted group and up to 30% of the subjects in the SL* PBS group displayed a CTL response against selected HLA-A2-restricted CD8+ T cell epitopes. The non-adjuvanted vaccine induced a very weak antibody response and no helper T cell responses. Local and general reactions were more frequently reported by AS02A recipients than in the non-adjuvanted group but the safety profile was considered acceptable. AS02A can be considered as a useful adjuvant that strongly enhances the cellular and humoral responses of subunit protein vaccines.     

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8 T cells

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8⁺ T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8⁺ T cells recognized D^d-restricted 8mer peptides, NY-ESO-1_81-88. MAGE-A4-specific CD8⁺ T cells recognized D^d-restricted 9mer peptides, MAGE-A4_265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8⁺ T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.     

Novel CD8+ T cell-based vaccine stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic HLA-A2 mice

The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-γ antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (α1 and α2 domains of HLA-A2 and α3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency.     

CD8 T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein

Cytotoxic CD8⁺ T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8⁺ T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ⁺CD8⁺ T cells and CD107a/b⁺CD8⁺ T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8⁺ T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8⁺ T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8⁺ T cells represented a memory subset with CD45RB^high and CD62L^low. Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8⁺ T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8⁺ T cells, which are important in the development of vaccine against SARS-CoV infection.     

DNA vaccination as a tool to identify subdominant CD8 T cell epitopes

DNA vaccination is highly efficient at inducing CD8⁺ T cell responses in animal models. Here we investigated whether DNA vaccine technology could be exploited to identify subdominant cytotoxic T lymphocytes (CTL) epitopes. Previous studies have shown that the Sendai virus HN protein does not induce a CD8⁺ T cell response in C57BL/6 mice. Thus, we vaccinated C57BL/6 mice with a DNA vaccine encoding Sendai virus hemagglutinin neuraminidase (HN) protein. The data show that this strategy elicited a potent D^b-restricted CD8⁺ CTL response against at least one subdominant HN-derived epitope. These CTL were able to lyse Sendai virus-infected target cells, demonstrating that the epitope was appropriately processed and present at sufficient levels for T cell recognition. However, these cells did not confer protection against lethal challenge with Sendai virus. These data demonstrate the capacity of DNA vaccine to raise CTL responses to subdominant epitopes, but show that such responses may be limited in their efficacy against non-persistent viruses.     

Induction of HLA-A2-restricted CTL responses by a tubular structure carrying human melanoma epitopes

Epitope-based vaccination strategies designed to induce strong tumor-specific CD8(+) T cell responses are being widely considered for cancer immunotherapy. Here, two recombinant tubular structures, NS1-Mela 1 and NS1-Mela 2, carrying, respectively two HLA-A2 epitopes derived from human melanoma antigens were constructed and their capability to induce CTL responses in vivo were studied in HLA-A2 transgenic mice. Strong CTL responses specific for GnT-V/NA 17-A and gp100 (154-162) epitopes were generated in HLA-A2 transgenic mice immunized by the construct NS1-Mela l carrying these two epitopes. The second construct NS1-Mela 2 carrying both Tyrosinase (369-377Da) and Melan-A/Mart-1 (27-35) epitopes induced a weak Tyrosinase-specific CTL response in mice but failed to induce specific CTL responses against the Melan-A/Mart-1 (27-35) epitope in the tested mice. Thus, recombinant tubular structures containing multiple tumoral epitopes may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.     

Evaluation of antitumor immunity efficacy of epitope-based vaccine with B16 cell line coexpressing HLA-A2/H-2kb and CTL multiepitope in HLA transgenic mice

Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. HLA-A2-transgenic mouse is a useful tool for measuring the CTL responses in vitro. However, tumor vaccine development is required to address the variables that are not easily evaluated by in vitro assays. With the objective of extending the usage of A2-tansgenic mouse in vaccine efficacy assay, here, we established a B16 tumor cell line coexpressing HLA-A*0201/H-2Kb chimeric gene and a polyepitope construct based on the use of a mammalian expression vector pIRES. The value as a tool for evaluating the antitumor efficacy in vitro as well as in experimental tumor challenge model in vivo has been tested. We found that priming with the polyepitope construct and boosting with the mixture of peptide in A2-transgenic mice resulted in: (1) CTL responses not only against the peptide-sensitized T2 and SW480 cell lines but also the non-sensitized reconstructed B16 cell line; (2) expression of HLA-A*0201/H-2Kb chimeric gene and polyepitopes by B16 led to its rejection by immunized A2-transgenic mice. These data established that the reconstructed B16 cell line stably expressed and efficiently presented the HCC-derived CTL epitopes, making B16 based melanoma suitable for the evaluation of the antitumor efficacy of immune responses to these epitopes. Collectively, these data indicate that the use of this method allows for directly testing of HLA-A2 restricted epitope immunogenicity in the A2-transgenic mice.     

Silencing an immunodominant epitope of hepatitis B surface antigen reveals an alternative repertoire of CD8 T cell epitopes of this viral antigen

Immunodominance hierarchies operating in immune responses to viral antigens limit the diversity of the elicited T cell responses. The L^d/S_28–39-restricted CD8 T cell response to the hepatitis B surface antigen (HBsAg or S) prevents copriming of D^d- and K^b-restricted CD8 T cell responses. We exchanged L to V at position S₃₉ of HBsAg to construct mutant S_L39V. Comparable levels of wild-type S and mutant S_L39V were produced by transiently transfected cells, and mice immunized with the pCI/S and pCI/S_L39V DNA vaccines showed comparable serum antibody responses to HBsAg. The pCI/S but not pCI/S_L39V DNA vaccination induced L^d/S_28–39-specific CD8 T cell responses. However, the pCI/S_L39V DNA vaccine efficiently primed CD8 T cell responses to the subdominant D^d- and K^b-restricted epitopes, confirming the immunosuppressive phenotype of the L^d/S_28–39-specific CD8 T cell response. A single point mutation within the HBsAg can hence completely silence a ‘dominant’ CD8 T cell response thereby facilitating priming of a multispecific repertoire of suppressed, ‘subdominant’ epitopes. The data have practical implications for understanding HBV-specific CD8 T cell responses and for the design of novel vaccination strategies.     

DNA/Ad5 vaccination with SIV epitopes induced epitope-specific CD4⁺ T cells, but few subdominant epitope-specific CD8⁺ T cells

The goals of a T cell-based vaccine for HIV are to reduce viral peak and setpoint and prevent transmission. While it has been relatively straightforward to induce CD8⁺ T cell responses against immunodominant T cell epitopes, it has been more difficult to broaden the vaccine-induced CD8⁺ T cell response against subdominant T cell epitopes. Additionally, vaccine regimens to induce CD4⁺ T cell responses have been studied only in limited settings. In this study, we sought to elicit CD8⁺ T cells against subdominant epitopes and CD4⁺ T cells using various novel and well-established vaccine strategies. We vaccinated three Mamu-A*01⁺ animals with five Mamu-A*01-restricted subdominant SIV-specific CD8⁺ T cell epitopes. All three vaccinated animals made high frequency responses against the Mamu-A*01-restricted Env TL9 epitope with one animal making a low frequency CD8⁺ T cell response against the Pol LV10 epitope. We also induced SIV-specific CD4⁺ T cells against several MHC class II DRBw*606-restricted epitopes. Electroporated DNA with pIL-12 followed by a rAd5 boost was the most immunogenic vaccine strategy. We induced responses against all three Mamu-DRB*w606-restricted CD4 epitopes in the vaccine after the DNA prime. Ad5 vaccination further boosted these responses. Although we successfully elicited several robust epitope-specific CD4⁺ T cell responses, vaccination with subdominant MHC class I epitopes elicited few detectable CD8⁺ T cell responses. Broadening the CD8⁺ T cell response against subdominant MHC class I epitopes was, therefore, more difficult than we initially anticipated.     

Identification of a dominant CD8+ CTL epitope in the SARS-associated coronavirus 2 spike protein

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, has been spreading throughout the world. To date, there are still no approved human vaccines for this disease. To develop an effective vaccine, the establishment of animal models for evaluating post-vaccination immune responses is necessary. In this study, we have identified a CTL epitope in the SARS-CoV-2 spike (S) protein that could be used to measure the cellular immune response against this protein. Potential predicted CTL epitopes of the SARS-CoV-2 S protein were investigated by immunizing BALB/c mice with a recombinant of the receptor-binding domain (RBD) of the S protein. Then, CD8⁺ T cells specific for S-RBD were detected by stimulating with potential epitope peptides and then measuring the interferon-gamma production. Truncation of this peptide revealed that S-RBD-specific CD8⁺ T cells recognized a H2-D^d-restricted S_526–533 peptide. In conclusion, this animal model is suitable for evaluating the immunogenicity of SARS-CoV-2 vaccines.     

A self-adjuvanting lipopeptide-based vaccine candidate for the treatment of hepatitis C virus infection

Effective CD8(+) T cell responses have been induced using totally synthetic self-adjuvanting lipopeptides containing the dipalmitoyl-S-glyceryl cysteine lipid moiety, which is a ligand for Toll-like receptor 2 (TLR2) on dendritic cells (DC). In this study, we evaluated the use of lipopeptide vaccine candidates containing HLA-A2-restricted epitopes for DC-based immunotherapy of HCV infection. Lipopeptides were able to induce specific CD8(+) T cell responses in HLA-A2 transgenic mice and consistently activated human monocyte-derived DC from both healthy individuals and HCV infected patients. Lipopeptide-pulsed human DC were also found to secrete the pro-inflammatory cytokine IL-12p70 and were able to activate antigen-specific IFN-gamma production by autologous CD8(+) T cells obtained from a hepatitis C patient. These results show that DC from HCV patients can be matured and antigen loaded with TLR2-targeting lipopeptides for effective presentation of CD8(+) T cell epitopes; the use of autologous lipopeptide-pulsed DC or direct lipopeptide vaccination may be successful approaches for the priming or boosting of anti-HCV CD8(+) T cell responses to aid in the clearance of the virus in chronically infected individuals.     

In silico analysis of MHC-I restricted epitopes of Chikungunya virus proteins: Implication in understanding anti-CHIKV CD8+ T cell response and advancement of epitope based immunotherapy for CHIKV infection

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus, responsible for acute febrile infection. The high morbidity and socio-economic loss associated with the recent CHIKV epidemics worldwide have raised a great public health concern and emphasize the need to study the immunological basis of CHIKV infection to control the disease. MHC-I restricted CD8⁺ T cell response represent one of the major anti-viral immune responses. Accordingly, it is essential to have a detailed understanding towards CHIKV specific MHC-I restricted immunogenic epitopes for anti-viral CD8⁺ CTL immunogenicity. In the present study, a computational approach was used to predict the conserved MHC-I epitopes for mouse haplotypes (H2-Db and H2-Dd) and some alleles of the major HLA-I supertypes (HLA-A2, -A3, -A24, -B7, -B15) of all CHIKV proteins. Further, an in-depth computational analysis was carried out to validate the selected epitopes for their nature of conservation in different global CHIKV isolates to assess their binding affinities to the appropriate site of respective MHC-I molecules and to predict anti-CHIKV CD8⁺ CTL immunogenicity. Our analyses resulted in fifteen highly conserved epitopes for H2-Db and H2-Dd and fifty epitopes for different HLA-I supertypes. Out of these, the MHC-I epitopes VLLPNVHTL and MTPERVTRL were found to have highest predictable CTL immunogenicities and least binding energies for H2-Db and H2-Dd, whereas, for HLA-I, the epitope FLTLFVNTL was with the highest population coverage, CTL immunogenicity and least binding energy. Hence, our study has identified MHC-I restricted epitopes that may help in the advancement of MHC-I restricted epitope based anti-CHIKV immune responses against this infection and this will be useful towards the development of epitope based anti-CHIKV immunotherapy in the future. However, further experimental investigations for cross validation and evaluation are warranted to establish the ability of epitopes to induce CD8⁺ T cell mediated immune responses.     

Mapping T and B cell epitopes in sperm protein 17 to support the development of an ovarian cancer vaccine

Ovarian cancer (OC) is the seventh most common cancer in women worldwide, and the leading cause of death from gynaecological malignancy. Immunotherapeutic strategies including cancer vaccines are considered less toxic and more specific than current treatments. Sperm surface protein (Sp17) is a protein aberrantly expressed in primary as well as in metastatic lesions in >83% of ovarian cancer patients. Vaccines based on the Sp17 protein are immunogenic and protective in animal models. To map the immunogenic regions and support the development of human Sp17 peptide based vaccines, we used 6 overlapping peptides of the human Sp17 sequence adjuvanted with CpG to immunise humanised HLA-A2.1 transgenic C57BL/6 mice, and assessed immunogenicity by ELISPOT and ELISA. No CD8 T cells were found to be induced to a comprehensive panel of 10 HLA-A2.1 or H-2K^b binding predicted epitopes. However, one of the 6 peptides, hSp17_111–142, induced high levels of antibodies and IFN-γ producing T cells (but not IL-17 or IL-4) both in C57BL/6 and in C57BL/6-HLA-A2.1 transgenic mice. C57BL/6 mice immunised with CpG adjuvanted hSp17_111–142 significantly prolonged the life-span of the mice bearing the ovarian carcinoma ID8 cell line. We further mapped the immuno-dominant B and T cell epitope regions within hSp17_111–142 using ELISPOT and competition ELISA. Herein, we report the identification of a single immuno-dominant B cell (134–142 aa) epitope and 2 T helper 1 (Th1) cell epitopes (111–124 aa and 124–138 aa). These result together support further exploration of hSp17_111–142 peptide formulations as vaccines against ovarian cancer.     

Chlamydia pneumoniae genome sequence analysis and identification of HLA-A2-restricted CD8+ T cell epitopes recognized by infection-primed T cells

In the present study, we performed in silico analysis of Chlamydia pneumoniae genome sequence to identify human HLA-A2-restricted T cell epitopes. Thirty-one Chlamydia-specific protein antigens were selected and peptides were derived thereof using an HLA-A2 epitope predictive algorithm. Firstly, we tested binding of 55 selected 9mer peptides to HLA-A2 in vitro. Next, infection of HLA-A2 transgenic mice with C. pneumoniae elementary bodies and assessment of effector CD8+ T cells allowed us to identify which of the epitopes binding to HLA-A2 in vitro were recognized by C. pneumoniae infection-primed CD8+ T cells. Finally, we could confirm that CD8+ T cells in association with HLA-A2 recognized the most reactive peptides when the corresponding full-length genes were used to DNA-immunize HLA-A2 transgenic mice. By using this approach, a novel HLA-A2-restricted epitope in the outer membrane protein A (OmpA) of C. pneumoniae was identified, which proved to mediate specific lysis of peptide-loaded target cells.     

Influenza virus CTL epitopes,remarkably conserved and remarkably variable

Virus-specific cytotoxic T lymphocytes (CTL) contribute to the control of virus infections including those caused by influenza viruses. Especially under circumstances when antibodies induced by previous infection or vaccination fail to recognize and neutralize the virus adequately, CTL are important and contribute to protective immunity. During epidemic outbreaks caused by antigenic drift variants and during pandemic outbreaks of influenza, humoral immunity against influenza viruses is inadequate. Under these circumstances, pre-existing CTL directed to the relatively conserved internal proteins of the virus may provide cross-protective immunity. Indeed, most of the known human influenza virus CTL epitopes are conserved. However, during the evolution of influenza A/H3N2 viruses, the most important cause of seasonal influenza outbreaks, variation in CTL epitopes has been observed. The observed amino acid substitutions affected recognition by virus-specific CTL and the human virus-specific CTL response in vitro. Examples of variable epitopes and their HLA restrictions are: NP_383–391/HLA-B*2705, NP_380–388/HLA-B*0801, NP_418–426/HLA-B*3501, NP_251–259/HLA-B*4002, NP_103–111/HLA-B*1503. In some cases amino acid substitutions occurred at anchor residues and in other cases at T cell receptor contact residues. It is of special interest that the R384G substitution in the NP_383–391 epitope was detrimental to virus fitness and was only tolerated in the presence of multiple functionally compensating co-mutations. In contrast, other epitopes, like the HLA-A*0201 restricted epitope from the matrix protein, M1_58–66, are highly conserved despite their immunodominant nature and the high prevalence of HLA-A*0201 in the population. A mutational analysis of this epitope indicated that it is under functional constraints. Also in influenza A viruses of other subtypes, including H5N1, the M1_58–66 is highly conserved.