1 / 16

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

zachery-cruz
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related