Correlations between transmitted HIV-1 drug resistance mutations and - PowerPoint PPT Presentation

Correlations between transmitted HIV-1 drug resistance mutations and the HLA of therapy- naive HIV-patients AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Background: Immune Pressure immune escape mutants AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Background: Drug Pressure Genotypic resistance testing: � Identification of drug resistance mutations � Adjustment of therapy AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Research Problem including drug resistance mutations 10% of therapy-naive patients already hold drug resistance mutations!!! AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Hypothesis Drug Resistance Mutation = Escape Variants Previous Statistical Analysis: � Correlations of HLA-A & -B and drug resistance mutations AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Results of Statistical Analysis Drug resistance mutation HLA class I type P value protease L33F HLA-A* 01 & B* 35 0.0125 M46I/L HLA-A* 03 & B* 35 0.0109 V75I HLA-A* 11 0.0323 transcriptase K103R HLA-B* 44 0.0018 Reverse V118I HLA-B* 07 & B* 40 0.0344 L210F HLA-B* 44 0.0074 � Confirm statistically obtained results by means of an biological assay: ELISPOT � Samples of each indicated group AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Principle: ELISPOT 2. Recruitment of Patients EDTA blood 3. ELISPOT assay 2 weeks incubation restimulation PBMC viral peptide interferon- γ 1. Design of Peptides AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

1. Design of Peptides: WT • Search databases for (potential) HLA restricted CTL epitopes for 6 amino acid positions • Design overlapping WT peptides (length: 15AA) in order to confirm epitopes eg. 33 A*01 Peptide Sequence B*35 (potential) KEALLDTGADDTV L EEMSLPGRWKPKMIGGI Pol07-20 EALLDTGADDTV L EE 20 30 40 50 Pol07-21 DTGADDTV L EEMNLP Pol07-22 DDTV L EEMNLPGRWK required motifs: HLA-A* 01: . . [DE] . . . . . [Y] L EEMNLPGRWKPKMI Pol07-23 HLA-B* 3501: . [PAV] . . . . . . . HLA-B* 3501: . [PAVYRD] . . . . . . [YFMLI] AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

1. Design of Peptide: Mutant • Design of mutant peptide (length: 10AA) according to patients amino acid substitutions e.g. described epitope at amino acid position 210 Peptide Sequence Negative control - B*44 TKIEELRQHL L RWGLTTPDKK L210 EELRQHL L RW 210 220 200 L210W EELRQHL W RW L210F EELRQHL F RW Positive control PHA AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

2. Recruitment of Patients • 75 patients in statistical analysis � 19 with corresponding HLA types and drug resistance mutation • Problems, recruiting patients: � patients come from different clinics � blood needs to be processed within 48h � cells cannot be frozen and stored before ELISPOT (5 samples lost) � cells cannot be cultured longer than 24h before ELISPOT AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

3. Detail ELISPOT: assay Previous step: 2 weeks incubation coat plate add streptavidin with antibody enzyme incubate overnight add substrate Cells with or without (AEC) to (control) peptide develop spots add biotinylated antibody AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

ELISPOT Result: L210F negative control ID 663 L210 positive control 316,5 L210 24 L210W 30 L210W L210F 2,5 negative control 0 0 100 200 300 400 L210F SFU / 100000 cells positive control AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Comparison to a „L210F“ patient from Erlangen ID 663 positive control 316,5 L210 24 L210W 30 L210F 2,5 Patient ID 16, Erlangen negative control 0 0 100 200 300 400 SFU / 100000 cells L210 359 L210W 524,5 L210F 94,5 � L210F: immune escape in negative control 48,5 background of HLA-B* 44 0 100 200 300 400 500 600 SFU / 100000 cells AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Result of a clinical follow up of therapy-experienced patients holding a L210F Therapy-experienced (20): • 8 patients show mutations of TAM pathways � 68.8% TAM I � 31.2% TAM II � Seems to push the drug resistance development towards the TAM-I pathway AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

ELISPOT Results: remaining amino acid positions • 3/5 sharing only HLA-A* 01 recognized peptide covering amino acid 33 in PR • 6/10 HLA-A* 03: 46 PR • 4/7 HLA-A* 11: 75 RT � Indications of epitopes within investigated regions AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Next steps • Further studies to confirm the presence of these epitopes • Investigte the influence of mutations within epitopes on T-cell activity AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Thanks to.. My colleagues at the Institute of Virology, Cologne Martin Däumer and the Laboratory Dr.Thiele, Kaiserslautern Sandra Mueller and Thomas Harrer, University of Erlangen The clinicians especially at the University Hospital Düsseldorf AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Thank you for your attention! AREVIR - Meeting 10./11.04.2008 Finja Schweitzer