Anti-Gene IGF-I Vaccines in Cancer Gene Therapy: A Review of a Case of Glioblastoma.

OBJECTIVE: Vaccines for the deadliest brain tumor - glioblastoma (GBM) - are generally based on targeting growth factors or their receptors, often using antibodies. The vaccines described in the review were prepared to suppress the principal cancer growth factor - IGF-I, using anti-gene approaches either of antisense (AS) or of triple helix (TH) type. Our objective was to increase the median survival of patients treated with AS and TH cell vaccines. METHODOLOGY: The cells were transfected in vitro by both constructed IGF-I AS and IGF-I TH expression episomal vectors; part of these cells was co-cultured with plant phytochemicals, modulating IGF-I expression. Both AS and TH approaches completely suppressed IGF-I expression and induced MHC-1 / B7 immunogenicity related to the IGF-I receptor signal. RESULTS: This immunogenicity proved to be stronger in IGF-I TH than in IGF-I AS-prepared cell vaccines, especially in TH / phytochemical cells. The AS and TH vaccines generated an important TCD8+ and TCD8+CD11b- immune response in treated GBM patients and increased the median survival of patients up to 17-18 months, particularly using TH vaccines; in some cases, 2- and 3-year survival was reported. These clinical results were compared with those obtained in therapies targeting other growth factors. CONCLUSION: The anti-gene IGF-I vaccines continue to be applied in current GBM personalized medicine. Technical improvements in the preparation of AS and TH vaccines to increase MHC-1 and B7 immunogenicity have, in parallel, allowed to increase in the median survival of patients.

Optimized Anchor-Modified Peptides Targeting Mutated RAS Are Promising Candidates for Immunotherapy.

RAS mutations occur in approximately 20% of all cancers and given their clonality, key role as driver mutation, association with poor prognosis and undruggability, they represent attractive targets for immunotherapy. We have identified immunogenic peptides derived from codon 12 mutant RAS (G12A, G12C, G12D, G12R, G12S and G12V), which bind to HLA-A*02:01 and HLA-A*03:01 and elicit strong peptide-specific CD8+ T cell responses, indicating that there is an effective CD8+ T-cell repertoire against these mutant RAS-derived peptides that can be mobilized. Alterations in anchor residues of these peptides enhanced their binding affinity to HLA-A*02:01 molecules and allowed generation of CD8+ T cells that responded to target cells pulsed with the anchor-modified and also with the original peptide. Cytotoxic T cells generated against these peptides specifically lysed tumor cells expressing mutant RAS. Vaccination of transgenic humanized HLA-A2/DR1 mice with a long peptide encompassing an anchor-modified 9-mer G12V epitope generated CD8+ T cells reactive to the original 9-mer and to a HLA-A*02:01-positive human cancer cell line harboring the G12V mutation. Our data provide strong evidence that mutant RAS can be targeted by immunotherapy.

Presence of T cells directed against CD20-derived peptides in healthy individuals and lymphoma patients.

Preclinical and clinical studies have suggested that cancer treatment with antitumor antibodies induces a specific adaptive T cell response. A central role in this process has been attributed to CD4+ T cells, but the relevant T cell epitopes, mostly derived from non-mutated self-antigens, are largely unknown. In this study, we have characterized human CD20-derived epitopes restricted by HLA-DR1, HLA-DR3, HLA-DR4, and HLA-DR7, and investigated whether T cell responses directed against CD20-derived peptides can be elicited in human HLA-DR-transgenic mice and human samples. Based on in vitro binding assays to recombinant human MHC II molecules and on in vivo immunization assays in H-2 KO/HLA-A2+-DR1+ transgenic mice, we have identified 21 MHC II-restricted long peptides derived from intracellular, membrane, or extracellular domains of the human non-mutated CD20 protein that trigger in vitro IFN-γ production by PBMCs and splenocytes from healthy individuals and by PBMCs from follicular lymphoma patients. These CD20-derived MHC II-restricted peptides could serve as a therapeutic tool for improving and/or monitoring anti-CD20 T cell activity in patients treated with rituximab or other anti-CD20 antibodies.

Identification of novel HLA-A11-restricted T-cell epitopes in the Ebola virus nucleoprotein.

The Ebola virus (EBOV) is a very contagious virus that is highly fatal in humans and animals. The largest epidemic was in West Africa in 2014, in which over 11,000 people died. However, to date, there are no licensed vaccines against it. Studies show that CD4+ and CD8+ T-cell responses, especially cytotoxic T-lymphocyte (CTL) responses, play key roles in protecting individuals from EBOV infection. Since HLA-restricted epitope vaccines are likely to be effective and safe immunization strategies for infectious diseases, the present study screened for CTL epitopes in the EBOV-nucleoprotein that are restricted by HLA-A11 (a common allele in Chinese people). Predictive computer analysis of the amino-acid sequence of EBOV-nucleoprotein identified ten putative HLA-A11-restricted epitopes. ELISPOT assay of immunized HLA-A11/DR1 transgenic mice showed that five (GR-9, VR-9, EK-9, PK-9, and RK-9) induced effective CTL responses. Additional epitope analyses will aid the design of epitope vaccines against EBOV.

The ADAMTS131239-1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope.

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239-1253-loaded HLA-DR tetramers.

Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells.

Research on human immunology has been hindered by the lack of optimal small animal models, given that the protective immune responses of human and non-human species show significant differences. However, due to ethical constraints[1] and the high cost of clinical trials, it is urgent to improve the current animal models that can mimic faithfully human physiology, particularly the human immune system (HIS). HIS mice had been generated recently by engrafting human hematopoietic stem cells (hHSCs) or human peripheral mononuclear cells (hPBMCs) into highly immuno-deficient mice such as NSG, NOG or NRG mice. However, a major experimental drawback for studies using these models is the rapid onset of Graft-versus-Host Disease (GvHD). In the present study, we overcome this limitation by generating new immuno-deficient mice named "HUMAMICE" (HLA-A2+/+/DR1+/+/H-2-ß2m-/-/IAß-/-/Rag2-/-/IL2rγ-/-/Perf-/- mice), which expressed human HLA molecules instead of mouse MHC molecules (H-2), and whose immuno-deficient status was reversed by transferring functional HLA-matched PBMCs thus producing mice with an immuno-competent status with a functional human immune system. We showed that in this HLA-matched context, the hPBMC-transfer led to high lymphocytes engraftment rates without GvHD over three months in this novel mouse model. Furthermore, to evaluate the utility of the hPBMC-HUMAMICE, we immunized them with commercial vaccine of Hepatitis B virus (HBsAg, Hepvac@) which resulted in robust and reproducible production of high levels of HBsAg-specific antibodies, implying that both transferred T and B lymphocytes were functional in HUMAMICE. These responses are comparable to those observed in human clinical trials with this identical vaccine. In conclusion, these findings indicated that the HLA-matched-hPBMC-HUMAMICE represents a promising model for dissecting human immune responses in various human diseases, including infectious diseases, cancers and tumors, and to facilitate the development of novel vaccines and cellular therapies.

Generation of human MHC (HLA-A11/DR1) transgenic mice for vaccine evaluation.

The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-)) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-) mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and ß2m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2D(b) were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans.

Characterization of effector components from the humoral and cellular immune response stimulated by melanoma cells exhibiting modified IGF-1 expression.

Modified melanoma B16 cells inhibited in their IGF-1 expression (B16MOD), on the contrary to the IGF-1 fully expressed parental wild-type (B16WT) counterpart, were shown to stimulate humoral as well as cellular immune responses. Among humoral components, the neutralizing and complement-fixing antibodies of IgM and essentially IgG2 (a+b) isotypes exhibited in vitro and in vivo effects upon tumour growth, while the IgG1 antibody isotype promoted enhanced tumour proliferation. As for the cellular immunity, it was found that the T CD8(+) lymphocyte subpopulation remained the main potent and long lasting immune active effector regulating tumour growth.

Development of a humanized HLA-A2.1/DP4 transgenic mouse model and the use of this model to map HLA-DP4-restricted epitopes of HBV envelope protein.

A new homozygous humanized transgenic mouse strain, HLA-A2.1(+/+)HLA-DP4(+/+) hCD4(+/+)mCD4(-/-)IAß(-/-)ß2m(-/-) (HLA-A2/DP4), was obtained by crossing the previously characterized HLA-A2(+/+)ß2m(-/-) (A2) mouse and our previously created HLA-DP4(+/+) hCD4(+/+)mCD4(-/-)IAß(-/-) (DP4) mouse. We confirmed that the transgenes (HLA-A2, HLA-DP4, hCD4) inherited from the parental A2 and DP4 mice are functional in the HLA-A2/DP4 mice. After immunizing HLA-A2/DP4 mice with a hepatitis B DNA vaccine, hepatitis B virus-specific antibodies, HLA-A2-restricted and HLA-DP4-restricted responses were observed to be similar to those in naturally infected humans. Therefore, the present study demonstrated that HLA-A2/DP4 transgenic mice can faithfully mimic human cellular responses. Furthermore, we reported four new HLA-DP4-restricted epitopes derived from HBsAg that were identified in both vaccinated HLA-A2/DP4 mice and HLA-DP4-positive human individuals. The HLA-A2/DP4 mouse model is a promising preclinical animal model carrying alleles present to more than a quarter of the human population. This model should facilitate the identification of novel HLA-A2- and HLA-DP4-restricted epitopes and vaccine development as well as the characterization of HLA-DP4-restricted responses against infection in humans.

A CCR4 antagonist combined with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self antigens.

Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.

Characterization of the specific CD4+ T cell response against the F protein during chronic hepatitis C virus infection.

BACKGROUND: The hepatitis C virus (HCV) Alternate Reading Frame Protein (ARFP or F protein) presents a double-frame shift product of the HCV core gene. We and others have previously reported that the specific antibodies against the F protein could be raised in the sera of HCV chronically infected patients. However, the specific CD4(+) T cell responses against the F protein during HCV infection and the pathological implications remained unclear. In the current study, we screened the MHC class II-presenting epitopes of the F protein through HLA-transgenic mouse models and eventually validated the specific CD4(+) T cell responses in HCV chronically infected patients. METHODOLOGY: DNA vaccination in HLA-DR1 and-DP4 transgenic mouse models, proliferation assay to test the F protein specific T cell response, genotyping of Chronic HCV patients and testing the F-peptide stimulated T cell response in the peripheral blood mononuclear cell (PBMC) by in vitro expansion and interferon (IFN)- γ intracellular staining. PRINCIPAL FINDINGS: At least three peptides within HCV F protein were identified as HLA-DR or HLA-DP4 presenting epitopes by the proliferation assays in mouse models. Further study with human PBMCs evidenced the specific CD4(+) T cell responses against HCV F protein as well in patients chronically infected with HCV. CONCLUSION: The current study provided the evidence for the first time that HCV F protein could elicit specific CD4(+) T cell response, which may provide an insight into the immunopathogenesis during HCV chronic infection.

An H5N1 M2e-based multiple antigenic peptide vaccine confers heterosubtypic protection from lethal infection with pandemic 2009 H1N1 virus.

BACKGROUND: A 2009 global influenza pandemic caused by a novel swine-origin H1N1 influenza A virus has posted an increasing threat of a potential pandemic by the highly pathogenic avian influenza (HPAI) H5N1 virus, driving us to develop an influenza vaccine which confers cross-protection against both H5N1 and H1N1 viruses. Previously, we have shown that a tetra-branched multiple antigenic peptide (MAP) vaccine based on the extracellular domain of M2 protein (M2e) from H5N1 virus (H5N1-M2e-MAP) induced strong immune responses and cross-protection against different clades of HPAI H5N1 viruses. In this report, we investigated whether such M2e-MAP presenting the H5N1-M2e consensus sequence can afford heterosubtypic protection from lethal challenge with the pandemic 2009 H1N1 virus. RESULTS: Our results demonstrated that H5N1-M2e-MAP plus Freund's or aluminum adjuvant induced strong cross-reactive IgG antibody responses against M2e of the pandemic H1N1 virus which contains one amino acid variation with M2e of H5N1 at position 13. These cross-reactive antibodies may maintain for 6 months and bounced back quickly to the previous high level after the 2nd boost administered 2 weeks before virus challenge. H5N1-M2e-MAP could afford heterosubtypic protection against lethal challenge with pandemic H1N1 virus, showing significant decrease of viral replications and obvious alleviation of histopathological damages in the challenged mouse lungs. 100% and 80% of the H5N1-M2e-MAP-vaccinated mice with Freund's and aluminum adjuvant, respectively, survived the lethal challenge with pandemic H1N1 virus. CONCLUSIONS: Our results suggest that H5N1-M2e-MAP has a great potential to prevent the threat from re-emergence of pandemic H1N1 influenza and possible novel influenza pandemic due to the reassortment of HPAI H5N1 virus with the 2009 swine-origin H1N1 influenza virus.

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-gamma, IL-2 and TNF-alpha. 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.

Hepatitis B virus as a gene delivery vector activating foreign antigenic T cell response that abrogates viral expression in mouse models.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is characterized by functionally impaired T cell responses. To ensure active immunotherapy, the immune response must be switched from exhausted T cells to functional effectors that can attain the liver and cure the viral infection. We thus designed a recombinant HBV (rHBV) containing a modified viral core gene that specifically delivers a foreign antigenic polyepitope to the liver. This recombinant virus could only be self-maintained in hepatocytes already infected by HBV through capsid complementation. A strong foreign epitope-specific T cell response was first primed in the periphery by way of DNA immunization in human leukocyte antigen (HLA)-A2/DR1 transgenic mice. After the hydrodynamic (hyd.) injection of rHBV, expression of the foreign antigenic polyepitope in hepatocytes attracted/reactivated a vigorous T cell response in situ. Most liver-infiltrating CD8(+) T cells proved to be functional effectors. Following DNA priming and hyd. injection, the rHBV-based expression of hepatitis B surface antigen (HBsAg) in mouse liver was almost completely inhibited without causing major liver injury. Studies in HBsAg/HLA-A2/DR1 transgenic mice further validated our approach. CONCLUSION: For the first time, HBV was used as a gene delivery vector, which strongly triggered functional T cell response and subsequently controlled the viral expression in the liver of surrogate mouse models for HBV infection. It might represent an innovative and promising strategy of active immunotherapy during HBV persistent infection. This concept could even be more generally extended to other chronic viral diseases.

Role of class I human leukocyte antigen molecules in early steps of echovirus infection of rhabdomyosarcoma cells.

Several echoviruses use decay accelerating factor (DAF) as a cell surface receptor. However, most of them require additional cell surface coreceptors. We investigated the respective roles of DAF and class I human leukocyte antigen (HLA) molecules in the early steps of the echovirus 11 (EV11) lifecycle in rhabdomyosarcoma (RD) cells. EV11 infection was inhibited at an early stage by anti-beta2-microglobulin (beta2m) and anti-HLA monoclonal antibodies and by a soluble monochain HLA class I molecule. Expression of class I HLA molecules restored the early steps of the EV11 lifecycle, but its expression was not sufficient for EV11 replication and particle production. Expression of HLA class I molecules was associated with leukocyte cell line permissiveness to EV11 infection. In conclusion, HLA class I molecules are involved in the early steps of EV11 infection of RD cells and appear to participate in a complex interplay of surface molecules acting as coreceptors, including DAF.

A vector-based minigene vaccine approach results in strong induction of T-cell responses specific of hepatitis C virus.

Multiepitope-based vaccines against hepatitis C virus (HCV) were designed in the form of three minigenes encompassing four domains of the NS3, NS4 and NS5B proteins that contain multiple class I/II restricted epitopes. The polyEp-WT minigene encodes all four domains in fusion, the polyEp-C minigene encodes the same fusion but optimised for mammalian translation and the polyEp-E3 minigene has an additional endoplasmic reticulum targeting sequence. Whereas the minigenes vectorised by DNA were poorly immunogenic, adenovirus vectorisation induced strong and broader IFNgamma-ELISpot and CTL responses in HLA-A2 transgenic mice. In addition, polyEp-WT and polyEp-E3 responses were found cross-reactive in a recombinant Listeria-NS3-based surrogate challenge. This study illustrates the potency of vectorised minigenes in the field of HCV vaccine development.

Stimulation of anti-melanoma immune effectors via modified tumour cells exhibiting inhibited IGF-I and low CD9.

Modified melanoma cells (B16-F0.MOD) characterized by inhibited IGF-I, CD9 low but not their wild-type counterparts (B16-F0.WT), IGF-I positive, CD9 high, were shown to be immunogenic for syngeneic hosts. C57BL/6 syngeneic recipients vaccinated with B16-F0.MOD cells developed immune effectors that were observed at the humoral as well as cellular levels. These immune effectors were shown to be capable of controlling in vitro tumour growth and in vivo tumour progression.

B subunit of Shiga toxin-based vaccines synergize with alpha-galactosylceramide to break tolerance against self antigen and elicit antiviral immunity.

The nontoxic B subunit of Shiga toxin (STxB) targets in vivo Ag to dendritic cells that preferentially express the glycolipid Gb(3) receptor. After administration of STxB chemically coupled to OVA (STxB-OVA) or E7, a polypeptide derived from HPV, in mice, we showed that the addition of alpha-galactosylceramide (alpha-GalCer) resulted in a dramatic improvement of the STxB Ag delivery system, as reflected by the more powerful and longer lasting CD8(+) T cell response observed even at very low dose of immunogen (50 ng). This synergy was not found with other adjuvants (CpG, poly(I:C), IFN-alpha) also known to promote dendritic cell maturation. With respect to the possible mechanism explaining this synergy, mice immunized with alpha-GalCer presented in vivo the OVA(257-264)/K(b) complex more significantly and for longer period than mice vaccinated with STxB alone or mixed with other adjuvants. To test whether this vaccine could break tolerance against self Ag, OVA transgenic mice were immunized with STxB-OVA alone or mixed with alpha-GalCer. Although no CTL induction was observed after immunization of OVA transgenic mice with STxB-OVA, tetramer assay clearly detected specific anti-OVA CD8(+) T cells in 8 of 11 mice immunized with STxB-OVA combined with alpha-GalCer. In addition, vaccination with STxB-OVA and alpha-GalCer conferred strong protection against a challenge with vaccinia virus encoding OVA with virus titers in the ovaries reduced by 5 log compared with nonimmunized mice. STxB combined with alpha-GalCer therefore appears as a promising vaccine strategy to more successfully establish protective CD8(+) T cell memory against intracellular pathogens and tumors.

The Th1 immune response against HIV-1 Gag p24-derived peptides in mice expressing HLA-A02.01 and HLA-DR1.

Using HLA-DR1-transgenic H-2 class II knockout mice, we identified two new HLA-DR1-restricted HIV-1 Gag p24-derived epitopes (Gag(321-340 )and Gag(331-350)) and confirmed the immunogenicity of seven that have been previously described. The human relevance was confirmed for the two new ones (Gag(321-340 )and Gag(331-350)) assaying peripheral blood mononuclear cells from HLA-DR1(+) HIV-1-infected long-term asymptomatic subjects and showing that Gag(331-350) could prime CD4(+) T cells from two HLA-DR1(+) HIV-1 seronegative donors in vitro. Seven of these epitopes, structurally conserved among HIV-1 clade B isolates, were selected for a comparative evaluation of their Th1 helper potential by immunizing HLA-A02.01/HLA-DR1-transgenic, H-2 class I/class II knockout mice with recombinant mouse invariant chain constructs in which each helper epitope was inserted in association with two reporter HIV-1-derived HLA-A02.01-restricted CD8(+) T cell epitopes. A T helper effect was demonstrated in all cases, and was particularly strong with epitopes Gag(301-320),Gag(321-340 )and Gag(271-290), which should, therefore, be considered in the design of new vaccines.

Hepatitis B virus splice-generated protein induces T-cell responses in HLA-transgenic mice and hepatitis B virus-infected patients.

Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.