15th Conference on Retroviruses and Opportunistic Infections Boston, Massachusetts - February 3-6, 2008

Conf Retrovir Opportunistic Infect 2008 Feb 3-6;15: (abstract no. 24)

E Lazaro¹, P Stamegna¹, D Heckerman², B Walker^1,3, and Sylvie Le Gall ^1,3
1Massachusetts Gen Hosp, Harvard Med Sch, Boston, US; ²Microsoft Res, Seattle, WA, US; and ³Howard Hughes Med Inst, Chevy Chase, MD, US

BACKGROUND: Accumulating evidence shows that some immunodominant cytotoxic leukocyte (CTL) responses are not protective whereas subdominant ones may contribute to viral control. Thus it may be necessary to alter natural dominance in order to elicit protective CTL responses from vaccines. Epitopes arise from the degradation of proteins in the antigen processing pathway. We previously showed that antigen processing contributes to immunodominance by modulating the production of epitopes. We also identified portable flanking sequences altering the production and antigenicity of epitopes. Whether the stability of optimal epitopes varies, contributes to epitope hierarchy, or could be altered to modulate epitope presentation has never been studied.

METHODS: We combine novel assays of peptide degradation with computational analysis of HIV sequences and killing assays. Optimal HIV epitopes were incubated with peripheral blood mononuclear cell (PBMC) extracts for 30 minutes or less and the amount of peptides was quantified by high-performance liquid chromatography (HPLC) profile analysis, where the surface of the peak corresponding to one peptide is proportional to the amount of epitope. We performed a computational analysis of stable and unstable peptides to identify motifs involved in epitope stability. Finally we introduced these motifs in epitopes and measured the stability and antigenicity of mutant sequences.

RESULTS: The analysis of 130 HIV experimentally defined epitopes demonstrates a great heterogeneity in epitope stability with 0 to 90% of the original peptide remaining after a 10-minute incubation with PBMC extracts. For HLA alleles for which stable epitopes were identified, stability largely correlates with epitope hierarchy. Stable epitopes are mainly located in Gag. The computational analysis of sequences identified several motifs associated with stability. Introducing these motifs into unstable epitopes increased their stability by 8-fold whereas mutating these sequences in stable epitopes decreased stability by as much as 10-fold. The antigenicity of mutated epitopes introduced into target cells used in killing assays was altered accordingly.

CONCLUSIONS: This is a first demonstration of a new factor involved in HIV epitope hierarchy, namely, the critical role of epitope intracellular stability. These data also show that epitope presentation can be modulated through alteration of intracellular epitope stability. We propose that changes in epitope sequences at non-essential positions may offer novel way to optimize epitope presentation from vaccine vectors.

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2008-02-03
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