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Surveillance of IL-2 inducing CD4+ T cell epitopes in

Surveillance of IL-2 inducing CD4+ T cell epitopes in acute HIV-1 infection for the emergence of escape mutants. R. Brad Jones 1 , Feng Yun Yue 2 , Colin Kovacs 3 , Ruqaya Mohamed 2 , Kelly Macdonald 1,2 , Mario Ostrowski 1,2. 1 Department of Immunology, University of Toronto

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Surveillance of IL-2 inducing CD4+ T cell epitopes in

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  1. Surveillance of IL-2 inducing CD4+ T cell epitopes in acute HIV-1 infection for the emergence of escape mutants R. Brad Jones1, Feng Yun Yue2, Colin Kovacs3, Ruqaya Mohamed2, Kelly Macdonald1,2, Mario Ostrowski1,2 1 Department of Immunology, University of Toronto 2 Clinical Sciences Division, University of Toronto 3 Canadian Immunodeficiency Collaborative

  2. Introduction • CD4+ T cell responses are critical in control of other chronic viral • infections including: gamma herpesvirus (Cardin, 1996), • LCMV and vaccinia (Leist, 1989) • Strong HIV-specific CD4+ T cell proliferation is maintained only • in long-term nonprogressors (LTNP), (Pontesilli, 1999) • Vigorous HIV-1-specific IL-2 producing CD4+ T cell responses • are associated with control of viremia (Rosenberg, 1997) • Is this cause or effect? High level HIV-1 viremia suppresses viral • antigen specific CD4+ T cell proliferation (McNeil, 2001) • Prospective study suggests that IL-2 producing CD4+ T cell • response to gag does not have prognostic value for rate • of progression to AIDS (Miedema, 2006)

  3. Delineating Cause and Effect • HIV-1 is capable of rapidly acquiring mutations which confer • escape from selective pressure • We see this with gp120 mutations which escape antibody • responses, drug-resistance mutations, and certain CD8+ • T cell responses • If CD4+ T cells are capable of exerting immunological • pressure on HIV-1, we should see the emergence of CD4 • epitope escape mutations

  4. Subject: OM214 • Acute seroconverter - symptomatic: fever, rash HAART

  5. Methods Cloning: • Sample obtained from leukopheresis • After CD8+ depletion, cells were stimulated overnight with p55 • Enrichment for HIV-p55 specific CD4+ T cells achieved with IL-2 • secretion assay (Miltenyi) and MACS • Plated at limiting dilution with irradiated feeder cells • p55 specificity was screened by ELISPOT and confirmed by FACS Determining Eptiope Specificity of Clones: • Gross specificity determined by overlapping gag peptide pool • ELISPOT and confirmed by FACS • Minimal epitope determined using truncated peptides

  6. Results Gag p17: Gag p24: “HIVWASRELER” “FRDYVDRFYK” Gag p24: “REPRGSDIAGT”

  7. HLA Restriction • ELISPOTs repeated with core peptides in presence of either anti-DQ, • anti-DR, or isotype controls Peptide + clone + B cell line Clone + BCL Clone + BCL + anti-DR Clone + BCL + anti-DQ • Two clones from OM214 ‘MREPRGSDIAGT’ and ‘FRDYVDRFYK’ are DQ • restricted • Specifically ‘FRDYVDRFYK’ is restricted by DQB1*05011/DQA1*010101

  8. Epitope Responses in ex vivo PBMCs Month 2: Control “FRDYVDRFYK” Autologous p55 “REPRGSDIAGT” “HIVWASRELER” 0.025 0.021 0 0.053 0.015 IL-2 CD69 Month 12: Control “FRDYVDRFYK” Autologous p55 “REPRGSDIAGT” “HIVWASRELER” 0.005 0 0 0 0 IL-2 CD69

  9. Sequencing • Performed on circulating plasma viruses • Limiting dilution methodology with direct sequencing • from PCR product • Sequences screened for hypermutation • Phylogenetic trees constructed to ensure that patient’s • sequences cluster together

  10. Types ofMutations Observed Mutations in Core Epitope Extended Epitope/Processing Mutations

  11. Frequencies of Mutations Observed

  12. Frequencies of Mutations Observed

  13. Epitope Mutation Clone A2: FRDYVDRFYK FRDYVDQFYK 55.9 0 PBMCs: Control FRDYVDQFYK FRDYVDRFYK 0.006 0.025 0

  14. Flanking Mutations • Clone and express autologous p55 with and without • flanking mutations • Test ability to stimulate clones Flanking mutations(FM) wt 2ug/ml FM p55 2ug/ml SUMO-CAT 2.5ug/ml wt p55 10ug/ml p55 1ug/ml p55 10ug/ml p55 1ug/ml p55 Med Med SEB SEB Clone A2 ‘FRDYVDRFYKT’ Clone B2 ‘REPRGSDIAGT’ • Flanking mutations do not confer escape to A2 or B2

  15. Conclusions • Rapid progression occurred in OM214 despite early • induction of an IL-2 producing CD4+ T cell response - • including a potent MHR-directed response • Loss of IL-2 secreting CD4+ T-cell response accompanied • disease progression • An escape mutation in an IL-2 inducing CD4+ T cell • epitope within the MHR was confirmed • This mutation arose within 6 months of infection and was • maintained at a frequency of 10%, for at least 1 year • IL-2 producing CD4+ T cell responses are capable of • exerting immune pressure on HIV-1, resulting in escape • mutations • Generalized loss of IL-2 responses with time suggests that • immune dysfunction due to viremia is an important • mechanism for viral escape from immune pressure

  16. Acknowledgments Elizabeth Yue Mario Ostrowski Maple Leaf Clinic: Colin Kovacs Roberta Halpenny Macdonald Lab: Ruqaya Mohamed David Willer Kaul Lab Sample Donors

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