![Page 1: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/1.jpg)
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
![Page 2: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/2.jpg)
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)
![Page 3: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/3.jpg)
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
![Page 4: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/4.jpg)
Subject: OM214
Acute seroconverter - symptomatic: fever, rash
0
50000
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
![Page 5: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/5.jpg)
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
![Page 6: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/6.jpg)
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
![Page 7: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/7.jpg)
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
![Page 8: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/8.jpg)
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
![Page 9: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/9.jpg)
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
![Page 10: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/10.jpg)
Types of Mutations Observed
Mutations in Core Epitope
Extended Epitope/Processing Mutations
![Page 11: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/11.jpg)
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
![Page 12: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/12.jpg)
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
![Page 13: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/13.jpg)
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
![Page 14: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/14.jpg)
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
![Page 15: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/15.jpg)
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
![Page 16: Surveillance of IL-2 inducing CD4+ T cell epitopes in](https://reader036.vdocuments.us/reader036/viewer/2022062309/568135ec550346895d9d5d91/html5/thumbnails/16.jpg)
Acknowledgments
Elizabeth YueMario Ostrowski
Maple Leaf Clinic:Colin Kovacs
Roberta Halpenny
Macdonald Lab:Ruqaya Mohamed
David Willer
Kaul Lab
Sample Donors