A preclinical study on the efficacy and safety of a new vaccine against Coxsackievirus B1 reveals no risk for accelerated diabetes development in mouse models.

AIMS/HYPOTHESIS: Enterovirus infections have been implicated in the aetiology of autoimmune type 1 diabetes. A vaccine could be used to test the causal relationship between enterovirus infections and diabetes development. However, the development of a vaccine against a virus suspected to induce an autoimmune disease is challenging, since the vaccine itself might trigger autoimmunity. Another challenge is to select the enterovirus serotypes to target with a vaccine. Here we aimed to evaluate the function and autoimmune safety of a novel non-adjuvanted prototype vaccine to Coxsackievirus serotype B1 (CVB1), a member of the enterovirus genus. METHODS: A formalin-inactivated CVB1 vaccine was developed and tested for its immunogenicity and safety in BALB/c and NOD mice. Prediabetic NOD mice were vaccinated, infected with CVB1 or mock-treated to compare the effect on diabetes development. RESULTS: Vaccinated mice produced high titres of CVB1-neutralising antibodies without signs of vaccine-related side effects. Vaccinated mice challenged with CVB1 had significantly reduced levels of replicating virus in their blood and the pancreas. Prediabetic NOD mice demonstrated an accelerated onset of diabetes upon CVB1 infection whereas no accelerated disease manifestation or increased production of insulin autoantibodies was observed in vaccinated mice. CONCLUSIONS/INTERPRETATION: We conclude that the prototype vaccine is safe and confers protection from infection without accelerating diabetes development in mice. These results encourage the development of a multivalent enterovirus vaccine for human use, which could be used to determine whether enterovirus infections trigger beta cell autoimmunity and type 1 diabetes in humans.

HLA-DR and HLA-DP restricted epitopes from human cytomegalovirus glycoprotein B recognized by CD4+ T-cell clones from chronically infected individuals.

PURPOSE: Helper CD4(+) T cells presumably play a major role in controlling cytomegalovirus (CMV) by providing help to specific B and CD8(+) cytotoxic T cells, as well as through cytotoxicity-mediated mechanisms. Since CMV glycoprotein B (gB) is a major candidate for a subunit vaccine against CMV, we searched for gB-epitopes presented by human leukocyte antigen (HLA)-class II molecules. METHODS: Dendritic cells obtained from CMV-seropositive donors were loaded with a recombinant gB and co-cultured with autologous CD4(+) T cells. Microcultures that specifically recognized gB were cloned by limiting dilution using autologous Epstein-Barr virus (EBV)-immortalized B cells pulsed with gB as antigen-presenting cells. To pinpoint precisely the region encoding the natural epitope recognized by a given CD4(+) clone, we assessed the recognition of recombinant Escherichia coli expressing gB-overlapping polypeptides after their processing by autologous EBV-B cells. RESULTS: We isolated several gB-specific CD4(+) T-cell clones directed against peptides gB(190-204), gB(396-410), gB(22-36) and gB(598-617) presented by HLA-DR7, HLA-DP10 and HLA-DP2. While their precise role in controlling CMV infection remains to be established, gB-specific CD4(+) T cells are likely to act by directly targeting infected HLA-class II cells in vivo, as suggested by their recognition of EBV-B cells infected by the Towne CMV strain. CONCLUSIONS: The characterization of such gB-epitopes presented by HLA-class II should help to understand the contribution of CD4(+) T-cell responses to CMV and may be of importance both in designing a vaccine against CMV infection and in immunomonitoring of subjects immunized with recombinant gB or with vectors encoding gB.

A MAGE-3 peptide presented by HLA-DR1 to CD4+ T cells that were isolated from a melanoma patient vaccinated with a MAGE-3 protein.

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific Ags, which have been used in therapeutic vaccination trials of cancer patients. MAGE-3 is expressed in 74% of metastatic melanoma and in 50% of carcinomas of esophagus, head and neck, bladder, and lung. We report here the identification of a new MAGE-3 peptide, which is recognized by three different CD4(+) T cell clones isolated from a melanoma patient vaccinated with a MAGE-3 protein. These clones, which express different TCRs, recognize an HLA-DR1 peptide ACYEFLWGPRALVETS, which corresponds to the MAGE-3(267-282) and the MAGE-12(267-282) protein sequences. One of the T cell clones, which expresses LFA-1 at a high level, lysed tumor cells expressing DR1 and MAGE-3. Another of these DR1-restricted CD4(+) clones recognized not only the MAGE-3/12 peptide but also homologous peptides encoded by genes MAGE-1, 2, 4, 6, 10, and 11.

CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A.

Mild/moderate hemophilia A patients carrying certain mutations in the C1 domain of factor VIII (FVIII) have a higher risk of inhibitor occurrence. To analyze the mechanisms responsible for inhibitor development in such patients, we characterized FVIII-specific CD4(+) T-cell clones derived from a mild hemophilia A patient carrying an Arg2150His substitution in the C1 domain and who presented with a high titer inhibitor toward normal but not self-FVIII. All T-cell clones recognized synthetic peptides encompassing Arg2150. The peptides were presented to the T-cell clones by DRB1*0401/DRB4*01 or DRB1*1501/DRB5*01. Interestingly, the latter haplotype was previously reported as being associated with an increased incidence of inhibitor formation. Peptide I2144-T2161 also bound to other DR molecules such as DRB1*0101 and DRB1*0701, indicating that the peptide binds to major histocompatibility complex (MHC) class II molecules expressed in more than 60% of the population. None of the T-cell clones recognized recombinant FVIII carrying the substitution Arg2150His, even when FVIII was presented by an FVIII-specific B-cell line. The mutation likely alters T-cell recognition of the mutated peptide associated to MHC molecules, because the mutated peptide bound to immunopurified DR molecules nearly as effectively as the native peptide. These observations demonstrate that T cells of this patient with mutation Arg2150His distinguish between self- and wild-type FVIII and provide a plausible mechanism for the frequent occurrence of an inhibitor in patients carrying this substitution. A similar phenomenon may occur with other mutations associated to an increased incidence of inhibitor formation.

Tumor-specific shared antigenic peptides recognized by human T cells.

The first tumor-specific shared antigens and the cancer-germline genes that code for these antigens were identified with antitumor cytolytic T lymphocytes obtained from cancer patients. A few HLA class I-restricted antigenic peptides were identified by this 'direct approach'. A large set of additional cancer-germline genes have now been identified by purely genetic approaches or by screening tumor cDNA expression libraries with the serum of cancer patients. As a result, a vast number of sequences are known that can code for tumor-specific shared antigens, but most of the encoded antigenic peptides have not yet been identified. We review here recent 'reverse immunology' approaches for the identification of new antigenic peptides. They are based on in vitro stimulation of naive T cells with dendritic cells that have either been loaded with a cancer-germline protein or that have been transduced with viruses carrying cancer-germline coding sequences. These approaches have led to the identification of many new antigenic peptides presented by class I or class II molecules. We also describe some aspects of the processing and presentation of these antigenic peptides.