The absence of a detectable immune response during transmissible spongiform encephalopathies is likely due to the fact that the essential component of infectious agents, the prion protein (PrP), is a self Ag expressed on the surface of many cells of the host. To overcome self-tolerance to PrP, we used 30-mer PrP peptides previously shown to be immunogenic in Prnp−/− mice, together with CFA or CpG-oligodeoxynucleotides (CpG) in IFA. Generation of anti-PrP T and B cell responses was analyzed in the spleen, lymph nodes, and serum of immunized C57BL/6 wild-type mice. Immunization with PrP peptides emulsified in CFA did not trigger an immune response to PrP. When CpG were used, vaccination with peptides P143–172 and P158–187 generated IFN-γ-secreting splenic T cells, and only P158–187 significantly stimulated IL-4-secreting T cells. Both peptides induced few Ab-producing B cells, and low and variable serum Ab titers. In contrast, immunization with peptide P98–127 did not induce significant levels of T cell responses but elicited specific peptide Abs. T cell epitope mapping, performed using 15-mer peptides covering PrP segment 142–182, revealed that an immunogenic motif lies between positions 156 and 172. These results demonstrate that T and B cell repertoires against PrP can be stimulated in C57BL/6 when adjuvant of the innate immunity such as CpG, but not CFA, is added to PrP peptides, and that the pattern of immune responses varies according to the epitope.

Recent reports suggest that immunity against the prion protein (PrP) retards transmissible spongiform encephalopathies progression in infected mice. A major obstacle to the development of vaccines comes from the fact that PrP is poorly immunogenic, as it is seen as self by the host immune system. Additional questions concern the immune mechanisms involved in protection and the risk of eliciting adverse reactions in the central nervous system of treated patients. Peptide-based vaccines offer an attractive strategy to overcome these difficulties. We have undertaken the identification of the immunogenic regions of PrP, which trigger helper T cells (Th) associated with antibody production. Our results identify two main regions, one between the structured and flexible portion of PrP (98-127) and a second between alpha 1 and alpha 2 helix (143-187). Peptides (30-mer) corresponding to these regions elicit class II-restricted Th cells and antibody production against native PrP and could therefore be of potential interest for a peptide-based vaccination.

Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnpo/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnpo/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnpo/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnpo/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnpo/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.

Prion diseases are presumed to be caused by the accumulation in the brain of a pathological protein called prion protein (PrP) scrapie which results from the transconformation of cellular PrP, a ubiquitous glycoprotein expressed in all mammals. Since all isoforms of PrP are perceived as self by the host immune system, a major problem in designing efficient immunoprophylaxis or immunotherapy is to overcome tolerance. The present study was aimed at investigating whether bone-marrow-derived dendritic cells (DCs) loaded with peptides previously shown to be immunogenic in PrP-deficient mice, can overcome tolerance in PrP-proficient wild-type mice and protect them against scrapie. Results show that, in such mice, peptide-loaded DCs elicit both lymphokine release by T cells and antibody secretion against native cellular PrP. Repeated recalls with peptide-loaded DCs reduces the attack rate of 139A scrapie inoculated intraperitoneally and retards disease duration by 40 days. Most interestingly, survival time in individual mice appears to be correlated with the level of circulating antibody against native cellular PrP. INTRODUCTIONPrion diseases, also called transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders which progress slowly, induce severe neurological loss and are transmissible. The agent is most presumably the scrapie prion protein (PrP) or PrPSc, a misfolded protein, rich in beta-sheets, resulting from the transconformation of cellular PrP (PrPc). PrPc is a ubiquitous, membrane-anchored protein, which has been remarkably conserved through evolution, but whose function in health is still enigmatic. The presence of PrPSc in tissues of diseased individuals confirms the diagnosis TSE (Prusiner et al., 1998;Wisniewski et al., 1998).Prions do not spontaneously evoke antibody (Ab) or T-cell responses in diseased subjects, most likely because PrPSc, like PrPc, is not perceived as foreign or 'dangerous' (Matzinger, 1998) by the host immune system. In recent years, however, following encouraging immunotherapy results in Alzheimer's disease patients (Schenk et al., 1999), several teams have attempted to passively or actively generate adaptive immune responses against prions in wild-type (wt) mice and have shown that passive Ab treatment (Sigurdsson et al., 2003;White et al., 2003), constitutive transgenic secretion of anti-PrP IgM (Heppner et al., 2001) or active immunization using whole PrP (Sigurdsson et al., 2002), PrP peptides (Schwarz et al., 2003), PrP fragments (Bade et al., 2006) or DNA constructs encoding Prnp gene sequences (Fernandez-Borges et al., 2006) had beneficial effects on disease evolution.In order to identify major histocompatibility (MHC) class II-restricted T-cell epitopes in C57BL6 (B6)-H-2 b mice, we previously screened a bank of overlapping peptides of mouse PrP and identified two main epitopes. One is contained in peptide PrP , the other is shared by peptides PrP 143-172 and PrP . Immunization of mice made genetically deficient for PrP (Prnp 2/2 ) with PrP 97-1...

In prion diseases, PrPc, a widely expressed protein, is transformed into a pathogenic form called PrPSc, which is in itself infectious. Antibodies directed against PrPc have been shown to inhibit PrPc to PrPSc conversion in vitro and protect in vivo from disease. Other effectors with potential to eliminate PrPSc-producing cells are cytotoxic T cells directed against PrP-derived peptides but their ability to protect or to induce deleterious autoimmune reactions is not known. The natural tolerance to PrPc makes difficult to raise efficient adaptive responses. To break tolerance, adenovirus (Ad) encoding human PrP (hPrP) or control Ad were administered to wild-type mice by direct injection or by transfer of Ad-transduced dendritic cells (DCs). Control Ad-transduced DCs from Tg650 mice overexpressing hPrP were also used for immunization. DC-mediated but not direct administration of AdhPrP elicited antibodies that bound to murine native PrPc. Frequencies of PrP-specific IFNγ-secreting T cells were low and in vivo lytic activity only targeted cells strongly expressing hPrP. Immunohistochemical analysis revealed that CD3+ T cell infiltration was similar in the brain of vaccinated and unvaccinated 139A-infected mice suggesting the absence of autoimmune reactions. Early splenic PrPSc replication was strongly inhibited ten weeks post infection and mean survival time prolonged from 209 days in untreated 139A-infected mice to 246 days in mice vaccinated with DCs expressing the hPrP. The efficacy appeared to be associated with antibody but not with cytotoxic cell-mediated PrP-specific responses.

BackgroundActive immunization against Aβ was reported to have a therapeutic effect in murine models of Alzheimer’s disease. Clinical Aβ vaccination trial AN1792 was interrupted due to the development in 6 % of the patients of meningoencephalitis likely involving pro-inflammatory CD4+ T cells. However, the potential implication of auto-aggressive anti-Aβ CD8+ T cells has been poorly investigated.MethodsPotential MHC-I-restricted Aβ-derived epitopes were first analyzed for their capacity to recruit functional CD8+ T cell responses in mouse models. Their impact on migration of CD8+ T cells into the brain parenchyma and potential induction of meningoencephalitis and/or neuronal damage was investigated upon vaccination in the APPPS1 mouse model of AD.ResultsWe identified one nonamer peptide, Aβ33-41, which was naturally processed and presented in association with H-2-Db molecule on neurons and CD11b+ microglia. Upon optimization of anchor residues for enhanced binding to H-2-Db, immunization with the modified Aβ33-41NP peptide elicited Aβ-specific IFNγ-secreting CD8+ T cells, which are cytotoxic towards Aβ-expressing targets. Whereas T cell infiltration in the brain of APPPS1 mice is dominated by CD3+CD8− T cells and increases with disease evolution between 4 and 7 months of age, a predominance of CD3+CD8+ over CD3+CD8− cells was observed in 6- to 7-month-old APPPS1 but not in WT animals, only after vaccination with Aβ33-41NP. The number of CD11b+ mononuclear phagocytes, which significantly increases with age in the brain of APPPS1 mice, was reduced following immunization with Aβ33-41NP. Despite peripheral activation of Aβ-specific CD8+ cytotoxic effectors and enhanced infiltration of CD8+ T cells in the brain of Aβ33-41NP-immunized APPPS1 mice, no clinical signs of severe autoimmune neuroinflammation were observed.ConclusionsAltogether, these results suggest that Aβ-specific CD8+ T cells are not major contributors to meningoencephalitis in response to Aβ vaccination.

a b s t r a c tPrion diseases are caused by the transconformation of the host cellular prion protein PrP c into an infectious neurotoxic isoform called PrP Sc . While vaccine-induced PrP-specific CD4 + T cells and antibodies partially protect scrapie-infected mice from disease, the potential autoreactivity of CD8 + cytotoxic T lymphocytes (CTLs) received little attention. Beneficial or pathogenic influence of PrP c -specific CTL was evaluated by stimulating a CD8 + T-cell-only response against PrP in scrapie-infected C57BL/6 mice. To circumvent immune tolerance to PrP, five PrP-derived nonamer peptides identified using prediction algorithms were anchored-optimized to improve binding affinity for H-2D b and immunogenicity (NP-peptides). All of the NP-peptides elicited a significant number of IFNc secreting CD8 + T cells that better recognized the NP-peptides than the natives; three of them induced T cells that were lytic in vivo for NP-peptide-loaded target cells. Peptides 168 and 192 were naturally processed and presented by the 1C11 neuronal cell line. Minigenes encoding immunogenic NP-peptides inserted into adenovirus (rAds) vectors enhanced the specific CD8 + T-cell responses. Immunization with rAd encoding 168NP before scrapie inoculation significantly prolonged the survival of infected mice. This effect was attributable to a significant lengthening of the symptomatic phase and was associated with enhanced CD3 + T cell recruitment to the CNS. However, immunization with Ad168NP in scrapie-incubating mice induced IFNc-secreting CD8 + T cells that were not cytolytic in vivo and did not influence disease progression nor infiltrated the brain. In conclusion, the data suggest that vaccine-induced PrP-specific CD8 + T cells interact with prions into the CNS during the clinical phase of the disease.

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