Production of NY-ESO-1 peptide/DRB1*08:03 tetramers and ex vivo detection of CD4 T-cell responses in vaccinated cancer patients

We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121–138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1 125–134 and 124–134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122–135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122–135 on APC, but the peptide 122–135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123–135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring.     

Identification of two novel immunodominant UreB CD4 T cell epitopes in Helicobacter pylori infected subjects

An epitope-based vaccine is a promising option for treating Helicobacter pylori (H. pylori) infection. Epitope mapping is the first step in designing an epitope-based vaccine. A pivotal role of CD4⁺ T cells in protection against H. pylori has been accepted, but few Th epitopes have been identified. In this study, two novel UreB CD4⁺ T cell epitopes were identified using PBMCs obtained from two H. pylori infected subjects. We determined the restriction molecules by antibody blocking and used various Epstein–Barr virus-transformed B lymphocyte cell lines (BLCLs) with different HLA alleles as APCs to present peptides to CD4⁺ T cells. These epitopes were DRB1*1404-restricted UreB_373–385 and DRB1*0803-restricted UreB_438–452. The T cells specific to these epitopes not only recognized autologous DCs loaded with recombinant UreB but also those pulsed with H. pylori whole cell lysates, suggesting that these epitope peptides are naturally processed. These epitopes have important value for designing an effective H. pylori vaccine.     

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.     

A modified epitope identified for generation and monitoring of PSA-specific T cells in patients on early phases of PSA-based immunotherapeutic protocols

Efficacy of vaccination in cancer patients on immunotherapeutic protocols can be difficult to evaluate. The aim of this study was therefore to identify a single natural or modified epitope in prostate-specific antigen (PSA) with the ability to generate high levels of PSA-specific T cells to facilitate monitoring in patients after vaccination against prostate cancer. To the best of our knowledge, this study describes for the first time the peptide specificity of T cells stimulated by endogenously processed PSA antigen. The peptide specificity of HLA-A*0201-restricted CD8⁺ T cells against human and rhesus PSA was investigated both in vivo after DNA vaccination in HLA-A*0201-transgenic mice and in vitro after repetitive stimulation of human T cells with DNA-transfected human dendritic cells (DCs). One of seven native PSA peptides, psa53–61, was able to activate high levels of PSA-specific CD8⁺ T cells in HLA-A*0201-transgenic mice after PSA DNA vaccination. Psa53–61 was also the only peptide that induced human T cells to produce IFNγ after stimulation with PSA transfected DCs, however not in all donors. Therefore, plasmids encoding modified epitopes in predicted HLA-A*0201 sequences were constructed. One of these modified PSA plasmids consistently induced IFNγ producing CD8⁺ T cells to the corresponding modified peptide as well as to the corresponding native peptide, in all murine and human T cell cultures. This study demonstrates a novel concept of introducing a modified epitope within a self-tumor antigen, with the purpose of eliciting a reliable T cell response from the non-tolerized immune repertoire, to facilitate monitoring of vaccine efficacy in cancer patients on immunotherapeutic protocols. The purpose of such a modified epitope is thus not to induce therapeutically relevant T cells but rather to, in case of weak or divergent T cell responses to self antigens/peptides, help answer questions about efficacy of vaccine delivery and about the possibility to induce immune responses in the selected and often immunosuppressed cancer patients.     

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.     

Hepatitis B virus (HBV)-derived DRB1*0101-restricted CD4 T-cell epitopes help in the development of HBV-specific CD8+ T cells in vivo

We previously identified two HLA-DRB1*0101-restricted epitopes in hepatitis B virus (HBV) X protein (HBx) and in HBV envelope proteins (preS2). To evaluated their help in the development of CD8+ T-cell responses, mice transgenic for human class I and class II HLA molecules were immunized with HBV-T helper constructs. The preS2 epitope favored a well-balanced response with CD4+ and CD8+ T cells producing IFN-γ, IL-2 and TNF-α. The response was focused on CD8+ T cells with the HBx epitope. Fine characterization of helper activities may meet clinical needs in terms of enhancing the potency of preventive or therapeutic polyepitope vaccines.     

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.     

Ii-Key/HPV16 E7 hybrid peptide immunotherapy for HPV16+ cancers

Activation of antigen-specific CD4+ T cells is critical for vaccine design. We have advanced a novel technology for enhancing activation of antigen-specific CD4+ T helper cells whereby a fragment of the MHC class II-associated invariant chain (Ii-Key) is linked to an MHC class II epitope. An HLA-DR4-restricted HPV16 E7 epitope, HPV16 E7(8–22), was used to create a homologous series of Ii-Key/HPV16 E7 hybrids testing the influence of spacer length on in vivo enhancement of HPV16 E7(8–22)-specific CD4+ T lymphocyte responses. HLA-DR4-tg mice were immunized with Ii-Key/HPV16 E7(8–22) hybrids or the epitope-only peptide HPV16 E7(8–22). As measured by IFN-γ ELISPOT assay of splenocytes from immunized mice, one of the Ii-Key/HPV16 E7(8–22) hybrids enhanced epitope-specific CD4+ T cell activation 5-fold compared to the HPV16 E7(8–22) epitope-only peptide. We further demonstrated that enhanced CD4+ T cell activation augments the CTL activity of a H-2D^b-restricted HPV16 E7(49–57) epitope in HLA-DR4+ mice using an in vivo CTL assay. Binding assays indicated that the Ii-Key/HPV16 hybrid has increased affinity to HLA-DR4+ cells relative to the epitope-only peptide, which may explain its increased potency. In summary, Ii-Key hybrid modification of the HLA-DR4-restricted HPV16 E7(8–22) MHC class II epitope generates a potent immunotherapeutic peptide vaccine that may have potential for treating HPV16+ cancers in HLA-DR4+ patients.     

Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumour challenge in mice

Peptide based vaccines offer practical advantages, but unmodified peptides usually require an adjuvant or delivery vehicle to promote immunogenicity. When peptides containing ovalbumin (OVA) derived CD4 and CD8 T cell epitopes were conjugated to 0.05 μm nano-beads, they gave strong immune responses and inhibition of growth of tumour cells expressing the CD8 T cell epitope with MHC class I. These responses were inducible with both high (50 μg) and low (5 μg) peptide doses after a single immunisation. The helper CD4 T cell epitope was unnecessary for induction of CD8 T cell or tumour challenge responses. However, the CD4 T cell epitope contained a B cell epitope and triggered strong antibody responses. This simple approach offers a convenient experimental tool and a potentially useful clinical method for peptide immunisation.     

Ii-Key/MHC class II epitope hybrid peptide vaccines for HIV

The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Hybrid peptides linking an immunoregulatory segment of the Ii protein (Ii-Key peptide) through a polymethylene bridge to MHC class II epitopes of HIV gp160 or gag are potent vaccines to elicit CD4(+) T cell responses. More potent responses to two MHC class II epitopes, HIV gp160(843-852) or HIV gag(279-292), occurred in mice immunized with Ii-Key hybrid peptides than with epitope-only peptides, as measured in IL-4 and IFN-gamma ELISPOT assays of splenic lymphocytes stimulated in vitro by epitope-only peptides. Both the number of responding cells and cytokine output per cell were increased. The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Such antigenic peptide constructs create opportunities to enhance greatly Th1 or Th2 responses to MHC class II epitopes for therapeutic purposes.     

Characterization of immune response to novel HLA-A2-restricted epitopes from zinc transporter 8 in type 1 diabetes

ObjectiveZnT8-specific CD8+ T cells in human type 1 diabetes (T1D) have been reported recently, although the results from different laboratories are inconsistent. We aimed to characterize these ZnT8 specific CD8+ T cells and validate assays to screen peptide libraries.MethodsWe screened HLA-A2-restricted T cell candidate peptides of ZnT8 with different methods including computer algorithms, MHC-peptide binding and dissociation assays in T2 cell line, identification in HLA-A2 transgenic (Tg) mice and in vivo CTL assays. Then ELISpot assay was used to measure peptide-reactive T cell responses in 49 HLA-A2-restricted T1D patients.ResultsWe demonstrated that ZnT8_{107–116(115)}, ZnT8_110–118, and ZnT8_177–186 were novel HLA-A*0201-restricted CTL epitopes in T1D patients. ZnT8_{107–116(115)}, ZnT8_115–123, ZnT8_153–161, ZnT8_177–186 and ZnT8_291–300 represent potentially major biomarkers for T1D. T cell responses against these epitopes showed different distributions between recently diagnosed and long-standing patients. Furthermore, they displayed discriminating performance among different ethnicities. We also compared the performance of the epitope identification strategies used herein. The epitopes which exhibited strong immunogenicity in HLA-A2 Tg mice were also well recognized by T1D patients.ConclusionsThe differences in autoimmune T cell responses among T1D individuals may open new avenues toward T1D prediction and prevention. It also provides efficient strategies for immune intervention.     

Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumour challenge in mice

Peptide based vaccines offer practical advantages, but unmodified peptides usually require an adjuvant or delivery vehicle to promote immunogenicity. When peptides containing ovalbumin (OVA) derived CD4 and CD8 T cell epitopes were conjugated to 0.05 microm nano-beads, they gave strong immune responses and inhibition of growth of tumour cells expressing the CD8 T cell epitope with MHC class I. These responses were inducible with both high (50 microg) and low (5 microg) peptide doses after a single immunisation. The helper CD4 T cell epitope was unnecessary for induction of CD8 T cell or tumour challenge responses. However, the CD4 T cell epitope contained a B cell epitope and triggered strong antibody responses. This simple approach offers a convenient experimental tool and a potentially useful clinical method for peptide immunisation.     

Identification of HLA class II H5N1 hemagglutinin epitopes following subvirion influenza A (H5N1) vaccination

Prophylactic immunization against influenza infection requires CD4+ T-helper cell activity for optimal humoral and cellular immunity. Currently there is one FDA approved H5N1 subvirion vaccine available, although stockpiles of this vaccine are insufficient for broad population coverage and the vaccine has only demonstrated modest immunogenicity. Specific activation of CD4+ T-helper cells using class II H5N1 HA peptide vaccines may be a useful component in immunization strategy and design. Identification of HLA class II HA epitopes was undertaken in this report by obtaining PBMCs from volunteers previously immunized with an H5N1 inactivated subvirion vaccine, followed by direct ex vivo stimulation of CD4+ T cells against different sources of potential HA class II epitopes. In the 1st round of analysis, 35 donors were tested via IFN-γ ELISPOT using pools of overlapping HA peptides derived from the H5N1 A/Thailand/4(SP-528)/2004 virus, recombinant H5N1 (rHA) and inactivated H5N1 subvirion vaccine. In addition, a series of algorithm-predicted epitopes coupled with the Ii-Key moiety of the MHC class II-associated invariant chain for enhanced MHC class II charging were also included. Specific responses were observed for all 20 peptide pools, with 6–26% of vaccinated individuals responding to any given pool (donor response frequency) and a magnitude of response ranging from 3- to >10-fold above background levels. Responses were similarly observed with the majority of algorithm-predicted epitopes, with a donor response frequency of up to 29% and a magnitude of response ranging from 3–10-fold (11/24 peptides) to >10-fold above background (7/24 peptides). PBMCs from vaccine recipients that had detectable responses to H5N1 rHA following 1st round analysis were used in a 2nd round of testing to confirm the identity of specific peptides based on the results of the 1st screening. Sixteen individual HA peptides identified from the library elicited CD4+ T cell responses between 3- and >10-fold above background, with two peptides being recognized in 21% of recipients tested. Eight of the putative MHC class II epitopes recognized were found in regions showing partial to significant sequence homology with New Caledonia H1N1 influenza HA, while eight were unique to H5N1 HA. This is the first study to identify H5N1 HA epitope-specific T cells in vaccine recipients and offers hope for the design of a synthetic peptide vaccine to prime CD4+ T-helper cells. Such a vaccine could be used to provide at least some minimal level of H5N1 protection on its own and/or prime for a subsequent dose of a more traditional but supply-limited vaccine.     

Identification of HLA-DR4-restricted T-cell epitope on MPT51 protein,a major secreted protein derived from Mycobacterium tuberculosis using MPT51 overlapping peptides screening and DNA vaccination

We identified a novel HLA-DR4-restricted CD4+ T-cell epitope on a secreted antigen of Mycobacterium tuberculosis, MPT51, in 004149-MM HLA-DR4-transgenic mice which express HLA-DRB1*0401, but not murine MHC class II molecules. The mice were immunized with plasmid DNA encoding MPT51 using gene gun and interferon (IFN)-γ production from the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, only one peptide, p191–210, stimulated the splenocytes to produce IFN-γ. Further analysis using flow cytometry and computer-assisted algorithm, ProPred, narrowed down the region of CD4+ T-cell epitope to p191–202. The CD4+ T-cell epitope would be feasible for vaccine design against tuberculosis as well as for analysis of MPT51-specific T-cells in M. tuberculosis infection.     

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.     

Towards an immunosense vaccine to prevent toxoplasmosis: protective Toxoplasma gondii epitopes restricted by HLA-A*0201

The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8⁺ T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4⁺ epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8⁺ T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.     

H5N1 strain-specific Hemagglutinin CD4+ T cell epitopes restricted by HLA DR4

CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain-specific HLA class II restricted epitopes is essential for monitoring cellular strain-specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA_441–460 and H5HA_57–76 were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA_57–76 epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA_57–76 response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA_57–76 immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide.     

Effectiveness of a novel immunogenic nanoparticle platform for Toxoplasma peptide vaccine in HLA transgenic mice

We created and produced a novel self-assembling nanoparticle platform for delivery of peptide epitopes that induces CD8⁺ and CD4⁺T cells that are protective against Toxoplasma gondii infection. These self-assembling polypeptide nanoparticles (SAPNs) are composed of linear peptide (LP) monomers which contain two coiled-coil oligomerization domains, the dense granule 7 (GRA7_20–28 LPQFATAAT) peptide and a universal CD4⁺T cell epitope (derived from PADRE). Purified LPs assemble into nanoparticles with icosahedral symmetry, similar to the capsids of small viruses. These particles were evaluated for their efficacy in eliciting IFN-γ by splenocytes of HLA-B*0702 transgenic mice and for their ability to protect against subsequent T. gondii challenge. This work demonstrates the feasibility of using this platform approach with a CD8⁺ epitope that binds HLA-B7 and tests the biological activity of potentially protective peptides restricted by human major histocompatibility complex (HLA) class I molecules in HLA transgenic mice.     

Identification and characterization of HIV-1-specific CD8+ T cell epitopes presented by HLA-A*2601

Since HLA-A*26 is one of the most common alleles in Asia, where approximately 20% of people have this allele, identification of HIV-1-specific epitopes presented by HLA-A*26 is necessary for studies on the immunopathogenesis of AIDS and vaccine development in Asia. As presented herein, we used the reverse immunogenetics approach to identify HIV-1 epitopes presented by HLA-A*2601, one of the major HLA-A*26 subtypes. We selected 24 HLA-A*2601-binding peptides out of 110 HIV-1 peptides by using a HLA-A*2601 stabilization assay. The ability of these HLA-A*2601-binding peptides to induce peptide-specific CD8(+) T cells was tested by stimulating PBMCs from HIV-1-infected individuals having HLA-A*2601 with these peptides. Four HLA-A*2601-binding peptides induced peptide-specific CD8 T cells. Analysis using HIV-1 recombinant vaccinia-infected C1R-A*2601 cells indicated that these four peptides were HIV-1 epitopes endogenously presented by HLA-A*2601. Two epitope-specific CD8(+) T cells were predominantly detected in HIV-1 infected individuals, suggesting that these epitopes may be useful for vaccine development.     

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS “HLA Peptide Binding Predictions” program, we have identified and further characterized novel H-2^d-restricted CD8+ epitopes within the WHV core (peptides C#12–21, C#18–32, C#19–27, C#61–69) and surface antigens (peptides preS2#10–18, preS2#27–35, S#76–84, S#133–140 and S#257–265), respectively. These peptides bind to H-2^d with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-γ producing T cells. More importantly, WHV core peptides C#19–27 and C#61–69 and WHV surface peptides S#133–140 and S#257–265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18–32, S#76–84, and S#257–265 resulted in significant production of IFN-γ. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection.