Peptide-loaded chimeric influenza virosomes for efficient in vivo induction of cytotoxic T cells

Virus-specific CD8+ T cells are thought to play an important role in resolving acute hepatitis C virus (HCV) infection as viral clearance has been associated with a strong and sustained CD8+ T cell response. During the chronic state of HCV infection virus-specific T cells have a low frequency and a reduced responsiveness. Based on this, a therapeutic vaccine increasing the frequency of specific T cells is a promising alternative for the treatment of chronic HCV infection. We improved an existing vaccine platform based on immunopotentiating reconstituted influenza virosomes (IRIVs) for efficient delivery of peptide epitopes to the MHC class I antigen presentation pathway. IRIVs are proteoliposomes composed of phospholipids and influenza surface glycoproteins. Due to their fusogenic activity, IRIVs are able to deliver encapsulated macromolecules, e.g. peptides to immunocompetent cells. We developed a novel method based on chimeric virosomes [chimeric immunopotentiating reconstituted influenza virosomes (CIRIVs)] combining the high peptide-encapsulation capacity of liposomes and the fusion activity of virosomes. This new approach resulted in a 30-fold increase of the amount of incorporated soluble peptide compared with current preparation methods. To study the immunogenicity of chimeric virosomes HLA-A2.1 transgenic mice were immunized with CIRIVs containing the HCV Core132 peptide. Core132-CIRIVs efficiently induced specific cytotoxic and IFNγ-producing T cells already with low peptide doses. Vaccine formulations, which include combinations of different HCV-derived CTL epitopes could be used to induce not only a strong but also a multi-specific CTL response, making them potential candidates for therapeutic and maybe prophylactic T cell vaccines in human

A Randomized Placebo-Controlled Phase Ia Malaria Vaccine Trial of Two Virosome-Formulated Synthetic Peptides in Healthy Adult Volunteers

BACKGROUND AND OBJECTIVES: Influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination. METHODOLOGY/PRINCIPAL FINDINGS: The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18-45 years. Five groups of 8 subjects received virosomal formulations containing 10 microg or 50 microg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1(st), 7 after the 2(nd) and 5 after the 3(rd) vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1(st) injection, 10 after the 2(nd) and 6 after the 3(rd). Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 microg) or the synthetic antigen vaccines (PEV301 and PEV302) used for immunization was found. In terms of immunogenicity, both PEV301 and PEV302 elicited already after two injections a synthetic peptide-specific antibody response in all volunteers immunized with the appropriate dose. In the case of PEV301 the 50 microg antigen dose was associated with a higher mean antibody titer and seroconversion rate than the 10 microg dose. In contrast, for PEV302 mean titer and seroconversion rate were higher with the lower dose. Combined delivery of PEV301 and PEV302 did not interfere with the development of an antibody response to either of the two antigens. No relevant antibody responses against the two malaria antigens were observed in the control group receiving unmodified virosomes. CONCLUSIONS: The present study demonstrates that three immunizations with the virosomal malaria vaccine components PEV301 or/and PEV302 (containing 10 microg or 50 microg of antigen) are safe and well tolerated. At appropriate antigen doses seroconversion rates of 100% were achieved. Two injections may be sufficient for eliciting an appropriate immune response, at least in individuals with pre-existing anti-malarial immunity. These results justify further development of a final multi-stage virosomal vaccine formulation incorporating additional malaria antigens. TRIAL REGISTRATION: ClinicalTrials.gov NCT00400101

Molecular Mimicry of Human Cytochrome P450 by Hepatitis C Virus at the Level of Cytotoxic T Cell Recognition

Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8+ cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A*0201–restricted HCV-derived epitope, i.e., HCV core 178–187, which shows sequence homology with human CYP2A6 and CYP2A7 8–17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYP-derived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8+ CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity

Small-Animal PET Imaging of Amyloid-Beta Plaques with [11C]PiB and Its Multi-Modal Validation in an APP/PS1 Mouse Model of Alzheimer's Disease

In vivo imaging and quantification of amyloid-β plaque (Aβ) burden in small-animal models of Alzheimer's disease (AD) is a valuable tool for translational research such as developing specific imaging markers and monitoring new therapy approaches. Methodological constraints such as image resolution of positron emission tomography (PET) and lack of suitable AD models have limited the feasibility of PET in mice. In this study, we evaluated a feasible protocol for PET imaging of Aβ in mouse brain with [11C]PiB and specific activities commonly used in human studies. In vivo mouse brain MRI for anatomical reference was acquired with a clinical 1.5 T system. A recently characterized APP/PS1 mouse was employed to measure Aβ at different disease stages in homozygous and hemizygous animals. We performed multi-modal cross-validations for the PET results with ex vivo and in vitro methodologies, including regional brain biodistribution, multi-label digital autoradiography, protein quantification with ELISA, fluorescence microscopy, semi-automated histological quantification and radioligand binding assays. Specific [11C]PiB uptake in individual brain regions with Aβ deposition was demonstrated and validated in all animals of the study cohort including homozygous AD animals as young as nine months. Corresponding to the extent of Aβ pathology, old homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous (23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was shown to be suitable as an intracerebral reference region. PET results were cross-validated and consistent with all applied ex vivo and in vitro methodologies. The results confirm that the experimental setup for non-invasive [11C]PiB imaging of Aβ in the APP/PS1 mice provides a feasible, reproducible and robust protocol for small-animal Aβ imaging. It allows longitudinal imaging studies with follow-up periods of approximately one and a half years and provides a foundation for translational Alzheimer neuroimaging in transgenic mice

Peptide-loaded chimeric influenza virosomes for efficient in vivo induction of cytotoxic T cells

ISSN:0953-8178ISSN:1460-237