HIV-1-specific CTL and Drug Resistance Aim of Studies - - PowerPoint PPT Presentation

HIV-1-specific CTL and Drug Resistance

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Aim of Studies – Interaction CTL/Resistance

Analysis of the influence of the immune system on

sequence variation and drug resistance mutations in HIV-1 PR/RT

Cohort of 198 chronically infected HIV-1 patients

Do mutations occur more frequently/infrequently in patients with

certain HLA types?

Does CTL recognition influnce the development of resistance mutations

Cooperation with Roland Kaiser’s group - analysis of

patients from the RESINA study (n=73 therapy naive patients)

Is the maintenance of transmitted drug resistance mutations in

treatment-naïve patients influenced by their HLA alleles?

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Methods

• serological or/and genotypic HLA-typing was performed –

HLA A, HLA B and HLA Cw were determined

• HIV-1 PR and RT sequences are determined
• univariate correlations were evaluated by Fisher’s exact test
• results were verified in biological assays:

γ-interferon ELISPOT analyses restriction analyses standard Cr51 release assays

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Interaction between CTL response and Development of Drug Resistance - Erlangen

Strong influence of CTL on protease polymorphisms and drug

resistance mutations

resistance mutations/polymorphisms correlating with HLA- types acted as escape mutations in most patients (JVi 2007, Mueller et al.) Of special interest: newly identified HLA B62 epitope KF9

(KMIGGIGGF aa 45 to 53 in PR)

• 70% of the B62+ patients showed strong recognition of the

epitope

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Interaction between CTL and Development of Drug Resistance - Erlangen

Major drug resistance mutations are located within

the B62 epitope KMIGGIGGF (KF9)

KF9 (HxB2) KMIGGIGGF KF9-I (M46I)

• I-------

( I DV,ATV,FPV,LPV,NFV) KF9-V1 (I47A)

• -A------

( LPV) KF9-V3 (I47V)

• -V------

( LPV,DRV,FPV,TPV) KF9-V4 (G48V)

• --V-----

( SQV,ATV) KF9-V5 (I50V)

• ----V---

( DRV,FPV,LPV)

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ELISPOT analyses

100 200 300 400 500 600

n.P. KMIGGIGGF

• I-------
• -A------
• -V------
• --V-----
• ---V----

SFU/100.000 cells Patient 1

200 400 600 800 1000 1200 1400

Patient 4 n.P. KMIGGIGGF

• I-------
• -A------
• -V------
• --V-----
• ---V----

SFU/100.000 cells KF9-V5 KF9-V4 KF9-V3 KF9-V1 KF9-I KF9 n.P. KMIGGIGGF

• I-------
• -A------
• -V------
• --V-----
• ---V----

100 200 300 400 500 600

SFU/100 000 cells Patient 8

• strong recognition of KF9
• none or very week recognition of KF9-V4 (48V)
• strong recognition of KF9-V3 (47V)

and KF9-V5 (50V)

• recognition of KF9-V1 (47A) and KF9-I (46I)

varies in the patients

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Results KF9

• KF9 is a frequently targeted CTL epitope in HLA B6 2 -positive patients
• The drug m utations 4 6 I , 4 8 V, 4 7 A strongly influence recognition of

the KF9 epitope by B6 2 -restricted CTL

• Only 6 / 3 7 B6 2 + patients show ed m utations w ithin KF9 , including 2 7

patients on PI treatm ent

• Reason: strong fitness im pairm ent by m utations in KF9 ?

=> in cooperation with Bernhard Thiele and Martin Däumer 454 sequencing will be carried out for the most interesting patients Please send us blood sam ples from patients show ing 4 6 I , 4 7 A or 4 8 V

50% 100% no therapy/"neutral HLA alleles" pre-selecting HLA alleles therapy + Pre-selecting HLA Allele

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Interaction between CTL and Maintenance of transmitted Drug Resistance – Köln (RESINA)

Maintenance of Drug resistance mutations can be assigned to specific HLA alleles

Drug resistance associated mutation HLA class I type P value Risk Ratio L33F (PR) HLA-A*01 0.0440 9.1667 M46I/L (PR) HLA-A*03 0.0181 10.6 V75I (RT) HLA-A*11 0.0251 >100 K103R (RT) HLA-B*44 0.00046 23.0769 V118I (RT) HLA-B*07 0.0146 2.95 L210F (RT) HLA-B*44 0.0366 6.9231

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Interaction between CTL and Maintenance of transmitted Drug Resistance – Köln (RESINA)

B* 44

Sequence-wt: GQHRTKIEELRQHLLRWGFTTPD L210F: EELRQHLFRW L210W: EELRQHLWRW AZT mutation

Pat 20 HLA A24, A24, B44, B13, Cw5, Cw6

Known HLA B44 epitope 210F is an escape mutation

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Results RESINA cohort

Correlations between HLA class I alleles und the

maintenance of drug resistance mutations in treatment-naïve patients

peptides comprising these aa positions were

recognized by CD8+ T cells

210F acts as escape mutation in B44+ patients

selection for this mutation may occur in B44+ patients

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Thom as Harrer

Kathrin Eism ann Silke Bergm ann Pia Rauch Ellen Harrer Hauke W alter Bernd Spriew ald Thom as‘ Group in Erlangen

Thanks to…

Martin Däum er and the Laboratory Bernhard Thiele, Kaiserslautern All Patients taking part in these studies

Rolf Kaiser

Finja Schw eizer and collegues RESI NA DFG,I ZKF, Hector Stiftung HI V Kom petenznetz

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Thank you for your attention

… and please don’t forget to send us blood sam ples from patients show ing 4 6 I , 4 7 A or 4 8 V ;-)

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Interaction between CTL and Maintenance of transmitted Drug Resistance – Köln (RESINA)

Epitope mapping

• sequence comparison with known peptide binding motifs or epitope prediction

tools (ELF, SYFPEITHI)

• ELISPOT analyses with overlapping peptides

cells are stimulated with peptides including the aa position which displayed

a correlation to a HLA allele and tested for cross recognition of the neighboring peptides

50 100 150 200

n.P. AYFSVPLDKDFRKYT_68 VPLDKDFRKYTAFTI_69 KDFRKYTAFTIPSIN_70 SFU/100 000 cells Pol69

VPLDKDFRKYT VPLDKDFRK (B*0703 .[P].......) VPLDKDFRK (B*0705 .[P].......)

http://www.hiv.lanl.gov/cgi-bin/ELF/epitope_analyzer.cgi

Statistic: correlation between V118I and HLA B7

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Summary (both studies)

The HLA-system plays a role in m aintenance of transm itted drug resistance m utations in treatm ent-naïve patients and influences the developm ent of resistance m utations in chronically infected patients

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Restriction Analyses, Standard Cr51 Release Assays

Patient 4 A2, A3, B57, B62, Cw3, Cw6

100 200 300 400 500 600 +Cw6 +B57,Cw6 +Cw3 +B62 +A3 +A2 +KF9 no peptide SFU

Identical results were obtained using CD8 + cells stimulated with the peptides Including the Mutations at position 46, 47 and 50

HLA B62 can present all variants apart from peptides including 48V

Patient 9 A2, A3, B27, B62, Cw3, Cw2

KF9 Clone + B62 B cell control 10 20 30 40 50 60 % specific cell lysis

CTL specific killing of antigen presenting cells