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 Toronto2 Clinical Sciences Division, University of Toronto

3 Canadian Immunodeficiency Collaborative

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)

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

Subject: OM214

Acute seroconverter - symptomatic: fever, rash

0

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100000

150000

200000

250000

300000

350000

400000

450000

0 2 4 6 8 10 12 14 16

Month Post Infection

Vir

al Load

0

50

100

150

200

250

300

350

400

450

500

CD

4 C

ount

Viral Load

CD4

HAART

Methods

Cloning:

Sample obtained from leukopheresis

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

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

ResultsGag p17:

“HIVWASRELER”

Gag p24:

“FRDYVDRFYK”

Gag p24:

“REPRGSDIAGT”

Fine mapping of peptide-specific Elispot responses of cloneA1

0 10000 20000 30000 40000 50000 60000WASREL ERFAUN

HIVWA SREL ERFA UN

WASREL ER

VWASREL ER

IVWA SREL E

HIVWA SREL

HIVWA SREL ER

YKLKHIVWA SREL ER

SFC/ mi llion

Finemapping of peptide-specific Elispot responses of clone A2

0 10 000 0 20 000 0

YVDRFYKTL

DYVDRF YKTL R

FRDYVDRFYKT LRA E

YVDRFYKT

F RDYVDRF Y

DYVDRFYKT

FRDYVDRFYKT

PKEPF RDYVDRFYKT

SFC/million

Fine mapping of peptide- specific E lispot responses of clone B2

RGSDIAGTT

PRGSDIAGT

PRGSDIAGTT

REPRGSDIAGTTSTL

EPRGSDIA

REPRGSDI

REPRGSDIA

REPRGSDIAGT

PGQMREPRGSDIAGT

SFC/ million

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

IL-2

Control

100 101 102 103 104100

101

102

103

104G1ÉFL2-H, FL4-H subset

FL1-H: CD69-FITC

FL

2-H

: IL

-2-P

E

0.025

100 101 102 103 104100

101

102

103

104

G5ÉFL4-H, FL2-H subset

FL1-H: CD69-FITC

FL

2-H

: IL

-2-P

E

0.015

“FRDYVDRFYK” “REPRGSDIAGT” “HIVWASRELER”

CD69

Epitope Responses in ex vivo PBMCs

100 101 102 103 104100

101

102

103

104

P55ÉFL2-H, FL4-H subset

FL1-H: CD69-FITC

FL

2-H

: IL

-2-P

E

0.053

Autologous p55

100 101 102 103 104100

101

102

103

104

G21ÉFL2-H, FL4-H subset

FL1-H: CD69-FITC

FL

2-H

: IL

-2-P

E

0

100 101 102 103 104100

101

102

103

104june-G5ÉFL3-H, SSC-H subset

FL4-H: CD69-APC

FL

1-H

: IL

-2-F

ITC

0

100 101 102 103 104100

101

102

103

104

june-G1ÉFL3-H, SSC-H subset

FL4-H: CD69-APC

FL

1-H

: IL

-2-F

ITC

0

100 101 102 103 104100

101

102

103

104June-g7ÉFL3-H, SSC-H subset

FL4-H: CD69-APC

FL

1-H

: IL

-2-F

ITC

4.96e-3

100 101 102 103 104100

101

102

103

104June-p55ÉFL3-H, SSC-H subset

FL4-H: CD69-APC

FL

1-H

: IL

-2-F

ITC

0

100 101 102 103 104100

101

102

103

104

june-dmsoÉFL3-H, FL1-H subset

FL4-H: CD69-APC

FL

1-H

: IL

-2-F

ITC

0

“FRDYVDRFYK” “REPRGSDIAGT” “HIVWASRELER” Autologous p55Control

IL-2

CD69

Month 2:

Month 12:

0 0.025 0.021 0.015 0.053

00 0.005 0 0

Sequencing

Performed on circulating plasma viruses

Limiting dilution methodology with direct sequencing from PCR product

Phylogenetic trees constructed to ensure that patient’s sequences cluster together

Sequences screened for hypermutation

Types of Mutations Observed

Mutations in Core Epitope

Extended Epitope/Processing Mutations

Frequencies of Mutations Observed

Month 2 Month 5 Month 12

TSILDIRQGPKEPFRDYVDRFYK 4/10 0/8 0/10

ASILDIRQGPKEPFRDYVDQFYK 0/10 1/8 1/10

ASILDIRQGPKEPFRDYVDRFYK 6/10 7/8 9/10

Month 2 Month 5 Month 12

MREPRGSDIAGT 5/10 0/8 0/10

MREPGGSDIAGT 1/10 0/8 0/10

IREPRGSDIAGT 4/10 8/8 10/10

Mon Month 2 Month 12

RLRPGGKKKYRLKHIVWASRELERFAVNPGLLESAS 10/10 3/10

QLRPGGKKKYRLKHIVWASRELERFAVNPGLLESAS 0/10 3/10

RLRPGGQKKYRLKHIVWASRELERFAVNPGLLESAS 0/10 2/10

RLRPGGKKKYRLKHIVWASRELERFAVDPGLLESAS 0/10 1/10

RLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSE 0/10 1/10

Frequencies of Mutations Observed

Epitope Mutation

FRDYVDRFYK FRDYVDQFYK

100 101 102 103 104100

101

102

103

104OM214 clone A2+G5ÉFL3-H, FL2-H subset

FL3-H: CD4-Percp

FL

2-H

: IL

-2-P

E

55.9

100 101 102 103 104100

101

102

103

104OM214 clone A2+G21ÉFL3-H, FL2-H subset

FL3-H: CD4-Percp

FL

2-H

: IL

-2-P

E

0

100 101 102 103 104100

101

102

103

104G21ÉFL2-H, FL4-H subset

FL1-H: CD69-FITC

FL

2-H

: IL

-2-P

E

0

100 101 102 103 104100

101

102

103

104G1ÉFL2-H, FL4-H subset

FL1-H: CD69-FITC

FL

2-H

: IL

-2-P

E

0.025

100 101 102 103 104100

101

102

103

104SUMO-CATÉFL2-H, FL4-H subset

FL1-H: CD69-FITC

FL2

-H: I

L-2

-PE

6.4e-3

Clone A2:

PBMCs:Control FRDYVDRFYK FRDYVDQFYK

0.025 0

55.9 0

0.006

Flanking Mutations

Clone and express autologous p55 with and without flanking mutations

Test ability to stimulate clones

Med10ug/ml p55

1ug/ml p55 SEB

wt Flanking mutations(FM)

10ug/ml p55

1ug/ml p55 Med

2ug/mlFM p55

2ug/mlSUMO-CAT

2.5ug/mlwt p55 SEB

Clone A2‘FRDYVDRFYKT’

Clone B2‘REPRGSDIAGT’

Flanking mutations do not confer escape to A2 or B2

Conclusions

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

Rapid progression occurred in OM214 despite early induction of an IL-2 producing CD4+ T cell response - including a potent MHR-directed response

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

Acknowledgments

Elizabeth YueMario Ostrowski

Maple Leaf Clinic:Colin Kovacs

Roberta Halpenny

Macdonald Lab:Ruqaya Mohamed

David Willer

Kaul Lab

Sample Donors