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

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

Jan 11, 2023 340 likes •528 views

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

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Correlations between transmitted HIV-1 drug resistance mutations and the HLA of therapy- naive HIV-patients

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Background: Immune Pressure

immune escape mutants

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Background: Drug Pressure

Genotypic resistance testing:

Identification of drug resistance mutations Adjustment of therapy

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Research Problem

including drug resistance mutations

10% of therapy-naive patients already hold drug resistance mutations!!!

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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

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Results of Statistical Analysis

Confirm statistically obtained results by means of an

biological assay: ELISPOT

Samples of each indicated group

Drug resistance mutation HLA class I type P value

L33F HLA-A* 01 & B* 35 0.0125 M46I/L HLA-A* 03 & B* 35 0.0109 V75I HLA-A* 11 0.0323 K103R HLA-B* 44 0.0018 V118I HLA-B* 07 & B* 40 0.0344 L210F HLA-B* 44 0.0074

protease

Reverse transcriptase

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Principle: ELISPOT

viral peptide interferon-γ PBMC EDTA blood

  • 2. Recruitment of Patients
  • 1. Design of Peptides
  • 3. ELISPOT assay

2 weeks incubation restimulation

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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

KEALLDTGADDTVLEEMSLPGRWKPKMIGGI

20 30 40 50 B*35 (potential) A*01

Peptide Sequence

Pol07-20 EALLDTGADDTVLEE Pol07-21 DTGADDTVLEEMNLP Pol07-22 DDTVLEEMNLPGRWK Pol07-23

LEEMNLPGRWKPKMI

required motifs: HLA-A* 01: . . [DE] . . . . . [Y] HLA-B* 3501: . [PAV] . . . . . . . HLA-B* 3501: . [PAVYRD] . . . . . . [YFMLI]

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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

TKIEELRQHLLRWGLTTPDKK

200 210 220 B*44

Peptide Sequence

Negative control

  • L210

EELRQHLLRW L210W EELRQHLWRW L210F EELRQHLFRW Positive control PHA

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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

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

  • 3. Detail ELISPOT: assay

coat plate with antibody incubate overnight Cells with or without (control) peptide add biotinylated antibody add streptavidin enzyme add substrate (AEC) to develop spots

Previous step: 2 weeks incubation

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

ELISPOT Result: L210F

negative control L210 L210W L210F positive control

ID 663 2,5 30 24 316,5 100 200 300 400 negative control L210F L210W L210 positive control SFU / 100000 cells

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Comparison to a „L210F“ patient from Erlangen

ID 663 2,5 30 24 316,5 100 200 300 400 negative control L210F L210W L210 positive control SFU / 100000 cells

Patient ID 16, Erlangen 48,5 94,5 524,5 359 100 200 300 400 500 600 negative control L210F L210W L210 SFU / 100000 cells

L210F:

immune escape in background of HLA-B* 44

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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

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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

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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
  • n T-cell activity
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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

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AREVIR - Meeting 10./11.04.2008 Finja Schweitzer

Thank you for your attention!