Influence of flanking sequences on presentation efficiency of a CD8+ cytotoxic T-cell epitope delivered by parvovirus-like particles.
We have previously developed an antigen-delivery system based on hybrid recombinant porcine parvovirus-like particles (PPV-VLPs) formed by the self-assembly of the VP2 protein of PPV carrying a foreign epitope at its N terminus. In this study, different constructs were made containing a CD8(+) T-cell epitope of chicken ovalbumin (OVA) to analyse the influence of the sequence inserted into VP2 on the correct processing of VLPs by antigen-presenting cells. We analysed the presentation of the OVA epitope inserted without flanking sequences or with either different natural flanking sequences or with the natural flanking sequences of a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus nucleoprotein, and as a dimer with or without linker sequences. All constructs were studied in terms of level of expression, assembly of VLPs and ability to deliver the inserted epitope into the MHC I pathway. The presentation of the OVA epitope was considerably improved by insertion of short natural flanking sequences, which indicated the relevance of the flanking sequences on the processing of PPV-VLPs. Only PPV-VLPs carrying two copies of the OVA epitope linked by two glycines were able to be properly processed, suggesting that the introduction of flexible residues between the two consecutive OVA epitopes may be necessary for the correct presentation of these dimers by PPV-VLPs. These results provide information to improve the insertion of epitopes into PPV-VLPs to facilitate their processing and presentation by MHC class I molecules.
Inmunología y Genética Aplicada SA (INGENASA), C/Hnos García Noblejas 41, 28037 Madrid, Spain.
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Rueda
Unité de Biologie des Régulations Immunitaires, INSERM E 352, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris CEDEX 15, France.
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Morón
Inmunología y Genética Aplicada SA (INGENASA), C/Hnos García Noblejas 41, 28037 Madrid, Spain.
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Sarraseca
Unité de Biologie des Régulations Immunitaires, INSERM E 352, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris CEDEX 15, France.
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Leclerc
Inmunología y Genética Aplicada SA (INGENASA), C/Hnos García Noblejas 41, 28037 Madrid, Spain.
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Casal
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000821:virology*; D002645:Chickens; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010047:Ovalbumin / Q000276:immunology; D010321:Parvoviridae / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine
D000939:Epitopes; D011994:Recombinant Proteins; D010047:Ovalbumin