CTL Recognition of Gag Cleavage Sites Thomas Harrer Dept. for - - PowerPoint PPT Presentation

CTL Recognition of Gag Cleavage Sites

Thomas Harrer

• Dept. for Internal Medicine 3

University Hospital Erlangen

Protease cleavage site mutations Role

• f the

immune system ? Cote, JV 2001

TCR

CTL

HIV

HLA A 2 HLA CW 8 HLA CW 4 HLA A3 HLA B7 HLA B27

Gag Pol vif Env

Tat Rev Nef

vpr vpu

LTR LTR HLA A2,A3 B27,B8 Cw3,4 HLA A24,A32 B14,B51 Cw5,8

T E R Q A N F L G K I 13 6 2 - 2 2 - - 1 19 8

Prevalence of mutations in the NC/P1-Cleavage Site in 93 patients

Significant correlation to HLA alleles in red

Aa HLA Allele p-wert

• dds Ratio

n= T A32 0.054 13.82 3 T A28 0.05 4.44 11 E B40 0.049 10.13 6 R B61 0.043 89 2 K B64 0.041 21 2 K B41 0.041 21 2 I B13 0.036 5.46 17 T B40 0.037 3.7 6 K B35 0.024 4.35 13 Ins TNE A1 0.016 3.4 24 T B45 0.019 33.7 2 E A32 0.011 42 3 E B61 0.004 95 2 K Cw8 0.042 20.71 2 K Cw2 0.034 0.1 15 T Cw16 0.014 8 8

TERQANFLGKI – NC/p1 Cleavage Site Statistical Correlations of mutations to HLA-I in 93 patients

• C-terminal CS-mutations frequent in HIV without PI resistance (n=40)

436R (18%) in NC/P1 437L (8%) 449P (38%) 451N (10%) 453T (10%) 453L (13%)

• 436R significantly correlated with HLA-B13 (p<0,05)

(but could also be found in HLA B13 negative patients).

Analysis of CS mutations in Resina by Jens Verheyen

(Drug Resistance Workshop, Sitges 2008)

Influence

• f NC/P1-

mutations

• n recognition

by HLA B13-restricted CTL

The A to V mutation was observed in 29% of PI-treated patients but in none of the controls (only NRTI - therapy) Cote, JV 2001. The K to R – mutation correlated to HLA B13

RQAN / FLGKI

• -V-
• --R-

Influence o e of t the A e A/V - C

• Compensatory C

y Cleavage S e Site M Mutations

• n CTL Recognition
• n CTL Recognition

in 3 HLA B13 + Patients. n 3 HLA B13 + Patients.

20 40 60 80 100 120 140 160 1 2 3

SFU/100 000 PBMC

• -V------
• -A------

no peptide

RQANFLGKI epitope

25 50 75 100 125 150 10 µg/ml 1 µg/ml 100 ng/ml 10 ng/ml peptide concentration

SFU / 100 000 PBMC

RQANFLGKI RQVNFLGKI RQANFLGRI 2 4 6 8 10 12 14 16 18 20 10 µg/ml 1 µg/ml 100 ng/ml 10 ng/ml peptide concentration SFU/2*105 PBMC RQANFLGKI RQVNFLGKI RQANFLGRI

Patient #2

10 20 30 40 50 60 10 µg/ml 1 µg/ml 100 ng/ml 10 ng/ml peptide concentration SFU/1*105 PBMC RQANFLGKI RQVNFLGKI RQANFLGRI

Patient #3

50 100 150 200 250 10 µg/ml 1 µg/ml 100 ng/ml 10 ng/ml peptide concentration

SFU in RQANFLGKI-stimulated PBMC

RQANFLGKI RQVNFLGKI RQANFLGRI

Patient #SSY

Different patterns of recognition of CS-mutations

The TCR quality decisive for recognition of mutations

• Cleavage site mutations are correlated with several

HLA alleles suggesting an influence of CTL on emergence of cleavage site mutations.

• A431V and K436R in NC/P1 – CS can influence

recognition by HLA B13 – restricted CTL, dependend

• n the quality of the individual T-cell receptor.
• K436R seems to be selected in HLA B13+ patients,

but can emerge also in HLA B13 – negative patients

• More patients needed to assess an influence of CTL
• n A431V (so far only 1 of 4 patients HLA B13+).

Summary

• Sandra Müller

Bernd Spriewald

• Kathrin Eismann

Hauke Walter

• Silke Bergmann

Klaus Korn

• Pia Rauch
• Ellen Harrer

Jens Verheyen Rolf Kaiser

Kompetenznetzwerk HIV/AIDS DFG, Hector Stiftung, IZKF Erlangen

n HLA n mutations both + A32 x T 3 13 2 A28 x T 11 13 4 B40 x E 6 6 2 B61 x R 2 2 1 B61 split of B40 B64 x K 2 19 2 B41 x K 2 19 2 B13 X I 17 8 4 B40 x T 6 13 3 B35 x K 13 19 6 A1 x Insertion 24 5 4 B45 x T 2 13 2 A32 x E 3 6 2 B61 x E 2 6 2 B61 split of B40 Cw8 x K 2 19 2 Cw2 x K 15 19 0 negative correlation Cw16 x T 8 13 4

KEGHQMKDCTERQANFLGKIWPSYKGRPGNF KEGHQMKDCTERQANF A2 HQMKDCNERQAN A2 CTERQANFL B61 TERQANFL B*4002 RQANFLGKI B*13 FLGKIWPS A2 FLGKIWPSYK A*0201 KIWPSYKGR A*3101 Published CTL Epitopes in NC/P1 Cleavage Site

200 400 600 800 1000 1200 5 µg/ml 1 µg/ml 100 ng/ml 10 ng/ml peptide concentration

SFU i: RI9-K/R stimulated cells

RQANFLGKI RQVNFLGKI RQANFLGRI

Patient #R

100 200 300 400 500 600 700 800 900 1000 5 µg/ml 1 µg/ml 100 ng/ml 10 ng/ml peptide concentration

SFU in RQVNFLGKI-stimulated cells

RQANFLGKI RQVNFLGKI RQANFLGRI

Patient #R

100 200 300 400 500 600 700 800 900 10 µg/ml 1 µg/ml 100 ng/ml 10 ng/ml peptide concentration

SFU in 10E5 peptide PBMC

RQANFLGKI RQVNFLGKI RQANFLGRI

Patient #R

70 HIV genotypes from therapy-naïve patients Primary resistance in RT and Pol in 30 patients. All genotyped for HLA-A and HLA-B Analysis of CS mutations by Jens Verheyen (Köln)

Resina - Cohort