title: Designing the epitope flanking regions for optimal generation of CTL epitopes.
abstract: The flanking amino acids that surround epitopes are critical for effective antigen processing and maintenance of epitope integrity. In the present study, the frequency and characteristics of each amino acid that flanked the peptides generated from the proteasomal degradation of three different subtypes of HIV-1 Gag-p24 were determined. Synthetic flanking regions were designed based on the highest and the lowest frequencies of amino acid with the ideal characteristics at positions upstream and downstream of the proteasomal cleavage site. Peptides were synthesized that contained known CD8+ CTL-epitopes from HIV-1 Gag, CMV pp65, and vaccinia proteins HRP-2, and C16, flanked by amino acid sequences specifically designed to either generate or inhibit the known CD8+ CTL-epitopes. As predicted, the known CD8+ CTL-epitopes were effectively generated from the peptides with synthetic flanking regions specifically designed to promote epitope generation in a proteasome-dependent manner. The majority of the proteasome-generated epitopes were cleaved immediately after the C-terminal amino acid of the specific CTL-epitope. The synthetic peptide sequences containing known CD8+ CTL-epitopes with the flanking regions that promote epitope generation were effectively processed and presented to epitope specific CD8+ T-cells resulting in the production of IFN-γ. These results highlight the importance of flanking regions in promoting efficient antigen processing and presentation. This concept can have important implications in vaccine design and development strategies.
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockledge, MD 20852, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Steers
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockledge, MD 20852, USA.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Currier
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockledge, MD 20852, USA.
authors.forename: Ousman
authors.initials: O
authors.lastname: Jobe
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockledge, MD 20852, USA.
authors.forename: Sodsai
authors.initials: S
authors.lastname: Tovanabutra
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockledge, MD 20852, USA.
authors.forename: Silvia
authors.initials: S
authors.lastname: Ratto-Kim
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Marovich
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Jerome H
authors.initials: JH
authors.lastname: Kim
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Nelson L
authors.initials: NL
authors.lastname: Michael
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Carl R
authors.initials: CR
authors.lastname: Alving
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. Electronic address: mrao@hivresearch.org.
authors.forename: Mangala
authors.initials: M
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D016655:HIV Core Protein p24 / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D046988:Proteasome Endopeptidase Complex / Q000276:immunology
chemical_list: D018984:Epitopes, T-Lymphocyte; D016655:HIV Core Protein p24; D010455:Peptides; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D007371:Interferon-gamma; D046988:Proteasome Endopeptidase Complex
keywords: CTL-epitope; Gag-p24; HIV-1; Immunogen design; Proteasome; Synthetic flanking region