Surveillance of IL2 inducing CD4 T cell epitopes in

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
  DQB105011/DQA1010101

8
Epitope Responses in ex vivo PBMCs
Month 2
Control
FRDYVDRFYK
REPRGSDIAGT
Autologous p55
HIVWASRELER
0.025
0
0.021
0.015
0.053
IL-2
CD69
Month 12
Control
FRDYVDRFYK
REPRGSDIAGT
Autologous p55
HIVWASRELER
0.005
0
0
0
0
IL-2
CD69
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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
  patients
• sequences cluster together

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Types of Mutations Observed
Mutations in Core Epitope
Extended Epitope/Processing Mutations
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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
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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

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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