title: Fragmentation of SIV-gag vaccine induces broader T cell responses.
abstract: BACKGROUND
  High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition.
  
  
  RESULTS
  three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an in vitro human priming system. The breadth of the CD8 T cell response, defined by the number of distinct epitopes, was assessed by IFN-γ-ELISPOT and memory phenotype and cytokine production evaluated by flow cytometry. We observed an increase of two- to six-fold in the number of epitopes recognised in the ubiquitin-fused fragments compared to the ubiquitin-fused full-length gag. In contrast, although proteasomal targeting was achieved, there was a marked reduction in the number of epitopes recognised in the ubiquitin-fused full-length gag compared to the full-length unmodified gene, but there were no differences in the number of epitope responses induced by non-ubiquitinated full-length gag and the ubiquitin-fused mini genes. Fragmentation and ubiquitination did not affect T cell memory differentiation and polyfunctionality, though most responses were directed against the Ad5 vector.
  
  
  CONCLUSIONS
  Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response against SIV-mac239 gag. Thus gene fragmentation of HIV vaccines may maximise responses.
authors.affiliation: Department of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom.
authors.forename: Adel
authors.initials: A
authors.lastname: Benlahrech
authors.forename: Andrea
authors.initials: A
authors.lastname: Meiser
authors.forename: Shanthi
authors.initials: S
authors.lastname: Herath
authors.forename: Timos
authors.initials: T
authors.lastname: Papagatsias
authors.forename: Takis
authors.initials: T
authors.lastname: Athanasopoulos
authors.forename: Fucheng
authors.initials: F
authors.lastname: Li
authors.forename: Steve
authors.initials: S
authors.lastname: Self
authors.forename: Veronique
authors.initials: V
authors.lastname: Bachy
authors.forename: Catherine
authors.initials: C
authors.lastname: Hervouet
authors.forename: Karen
authors.initials: K
authors.lastname: Logan
authors.forename: Linda
authors.initials: L
authors.lastname: Klavinskis
authors.forename: George
authors.initials: G
authors.lastname: Dickson
authors.forename: Steven
authors.initials: S
authors.lastname: Patterson
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D015683:Gene Products, gag / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D015658:HIV Infections / Q000517:prevention & control; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D059748:Proteolysis; D020871:RNA Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D015302:Simian Immunodeficiency Virus / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D014161:Transduction, Genetic; D054875:Ubiquitination; D014765:Viral Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D016207:Cytokines; D015683:Gene Products, gag; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014765:Viral Vaccines