TCR clonotypes responding to an immunodominant HLA-B*27 restricted epitope are relatively stable over extended periods but

do not correlate with viral escape in HIV-1 infected Long-term Non-progressors (LTNP)

David van Bockel

Immunovirology and Pathogenesis Program (IPP)Centre for Immunology (CFI)

National Centre in HIV Epidemiology and Clinical Research (NCHECR)

University of New South Wales (UNSW), Sydney, Australia

• Vaccination studies— Current viral vector (prime/boost) delivery systems are immunogenic— Neutralizing antibodies not generated— T-cell responses do not prevent or limit disease

• Long-term non-progressors (LTNP)— Ideal candidates for potential vaccine determinants

— Maintain effective response to chronic HIV infection— Symptom free for a minimum of 8 years— CD4+ T-cell count > 500 cells/uL in the absence of therapy

—HLA-B*2705 allele associated with delayed progression (n = 19)— Immunodominant target— p24 Gag epitope, KRWIILGLNK (KK10)

Definitive correlates of protective immunity

Schneidewind et al., J. Virol., April 2009, vol. 83, no. 8, p. 3993 - 3997

Early (L268M)Early (L268M)Late (R264K/G)Late (R264K/G)

Late (S173A)Late (S173A)

Parameters Wild-type (n = 12)

Escape(n = 7)

P-value

Median follow up (years) 16 18 0.18

Median follow up at censored time (years) 16 13 0.25

Surrogate markers

Viral load censored median (copies/ml) 3,495 21,000 0.01 CD4+ T cell count censored (cells/uL) 557 360 0.06 Viral load time weighted (copies/ml) 441 44,401 0.01 CD4+ T-cells time weighted (cell/uL) -10 -35.5 0.48

Host factors

CCR5 (wt/h/Δ32) 10/2/0 3/4/0 0.13

CCR2 (wt/h/64I) 8/4/0 7/0/0 0.25

SDF1 (wt/h/3’A) 6/5/1 6/5/1 >0.65

Virus

gp41 (escape) 2 1 >0.99

p17 (escape) 2 0 0.51

nef (escape/deletion) 0 0 -

Project design• Hypothesis:

– T-cell receptor (TCR) repertoire breadth correlates with viral control– Response to HIV infection will be more variable over time compared to

latent cytomegalovirus (CMV) infection

• Aim:– Using the KK10 immune escape event, create a definitive model to

highlight the qualitative effect of HIV-specific CTL in vivo

• Method: – TCR repertoire (variable β-chain CDR3) clonal sequence analysis – Longitudinal observation (n = 6; median = 7.2 years, range = 4.4 – 9.4)– Tetramer isolated CD8+ T-cells direct ex vivo

• HIV: HLA-B*2705/KRWII(L/M)GLNK (KK10)• CMV: HLA-A*0201/NLVPMVATV (NV9)

TCRβ CDR3 sequence data collection

n = number of TRBV CDR3 sequences collected per sampleDS = standardised measure of TCR repertoire diversity

Standardised measurement of clonal expansion

BV 7-2 CASSFPSNQPQHF

BV 7-2 CASSSFGTGGYF

BV 4-3 CASSPGQGNTIYF

BV 7-2 CASSFPSNQPQHF

BV 4-3 CASSPGQGNTIYF BV 7-2 CASSSFGTGGYF

DS = 0.93

DS = 0.11

35%

33%

31%

97%

2%1%

1.

2.

Tetramer labelled CDR3β clonotype freq. Simpson’sDiversity Index (Ds)

Standardised measurement of TCR repertoire diversity does not segregate by patients with immune escape

p > 0.50 p > 0.90

Standardised measurement of clonal evolution

KK10

NV9

Specificity Clonotype (years) Morisita-HornIndex(CMH)

CMH = 0.98

CMH = 0.28

KK10 NV9

ESCAPE

Elevated clonal evolution (turn-over) is observed in CMV (NV9)-specific CD8+ T-cells

Summary of TCR repertoire analysis• No correlation between standardized TCR repertoire diversity

– Immune escape– Clinical features

• Clonotype evolution was opposite to proposed hypothesis:HIV (++ immune activation) = limited turn-overCMV (latent infection) = significant turn-over

• HIV-specific clonotype longevity – Observed up to 9.4 years– Irrespective of viral escape– Limited to dominant clonotypes

Functional avidities for HIV and CMV viral epitopes do not change with time

KK10 NV9

Increased IL-7R (CD127) and Bcl-2 expression is associated with dominant (Vβ+) clonotypes

CD45RO CD28 CD27 CD127 Bcl2 Perforin

KK10

NV9

Conclusions• Viral escape at R264 within the KK10 epitope is the

only determinant of disease progression• Diversity of HIV KK10-specific CD8+ T-cell TCR

repertoire:– Not associated with viral escape– Static, despite chronic HIV-infection– Clonal dominance maintained for several years

• Functional avidity remained unchanged• Clonal dominance associated with increased

capacity for CD8+ T-cell survival

Acknowledgments1. Tony KelleherPalanee AmmaranondJohn ZaundersMee-ling MunierKaz SuzukiCiara McGinleyNabila SeddikiKate McGhieJulie YeungMaria PiperiasLeakhena LeasMichelle Bailey

University of New South Wales (UNSW), Sydney, AustraliaCentre for Immunology, St Vincent’s Hospital (SVH)1

National Centre in HIV Epidemiology and Clinical Research (NCHECR)2

Centre for Vascular Research3

National Health and Medical Research Council4

National Institutes of Health (NIH), Bethesda, USAVaccine Research Centre (Human Immunology Group)5

Queen Mary College, University of LondonNational Mycobacterium Reference Laboratory

4. Dora Lush Biomedical Research Scholarship

5. David A. PriceTedi AsherDanny DouekJason BrenchleyBrenna HillDavid Ambrozak

LTNP patientsSVH clinicians

2. David CooperJan GeurinMelanie

MiddletonLinda Gelgor

3. Vanessa Venturi

Miles Davenport

Hui-Yee Chin