SLIDE 1
HIV-1 Envelope glycoprotein
CD4 CCR5/CXCR4 gp120 gp41
T-cell surface HIV-1
HIV HIV-
- 1
1 Envelope Envelope glycoprotein glycoprotein
Evolution of HIV geno-and phenotypes during anti-retroviral therapy - - PowerPoint PPT Presentation
Evolution of HIV geno-and phenotypes during anti-retroviral therapy - - PowerPoint PPT Presentation
Evolution of HIV geno-and phenotypes during anti-retroviral therapy Barbara Schnierle Paul-Ehrlich-Institut HIV-1 Envelope glycoprotein HIV-1 gp41 gp120 HIV- HIV -1 1 Envelope Envelope glycoprotein glycoprotein CD4 CCR5/CXCR4
SLIDE 2
SLIDE 3
HIV-Infection
- Why R5-HIV at start of infection?
- Why und when develops X4-HIV?
- X4-HIV responsible for CD4+ loss?
SLIDE 4
CD4 CCR5 CXCR4 HIV-1 R5 + +
- +
- HIV-1 X4
+
- +
+
- +
HIV-1 R5/X4 + + + +
- +
Ligands MHC II MIP-1α, β SDF-1 Rantes Primary receptor Co- receptors Syncytia Replication in PBMC Slow fast
HIV receptors
SLIDE 5
CD4+
X4 HIV R5 HIV Immunodeficiency
Therapy ? ? ?
- Immunodeficiency result or cause coreceptor switch
- Can therapy reverse coreceptor switch and/or
restore the immune system
- New infections with X4 HIV?
Questions
SLIDE 6
Study design
- clinical documentation of study participants
- Establishment of a data base for documentation and analysis of
clinical and experimental patient data
- Serological examinations of patients (WB)
- Virus isolation
- Sequencing of the C2V5-region of the outer envelope
glycoprotein
- Determination of patients CCR5-genotype
- Phenotypic determination of coreceptor usage
SLIDE 7
Amount comments
Patients (total) 291 Samples 801 133 x 1 sample per patient 86 x 2-3 samples per patient 75 x 4-13 samples per patient Virus isolations 576 (86 % pos.) C2V5 sequences 2581 of 256 samples Protease sequences 1013 of 114 samples Western blot 477 all positive CCR5-Genotype 278 of 294 CCR+/CCR+: 238 CCR+/CCR-: 40 16x no DNA
Summary (2008)
SLIDE 8
HIV phenotyping
SLIDE 9
Generation of a CCR5-negative T-cell line
PBMC (CD8+ depleted) CCR5-/- donor MT-2- cells irradiated with 5000 rd HTLV-1 production
Co-culture, immortalization
CCR5-negative T-Cell line IsnoR5 Single cell cloning cultivation in RPMI-1640 / 10 % FKS / IL-2 Cell clones: IsnoR5 clone 1, 2, 3
CCR5+/+ CCR5 +/- CCR5-/-
SLIDE 10
* * * * * * *
PBMC or T cell line IsnoR5
CXCR4 CXCR4 CCR5
* * * * * *
HIV phenotyping HIV
AMD3100 AMD3100
SLIDE 11
R5
- +
+ X4 +
- +
- R5/X4
+
- +
+ Y + + + +
IsnoR5 Tropism of HIV Isolate
AMD3100
Y: alternative receptors
HIV phenotyping
PBMC
- r T cell line
- AMD3100
SLIDE 12
TCID50 / ml
102 103 104 105 106
P-7 P-26 P-31 P-41-0 P-41-9 P-41-19 P-51 P-107 P-180 Neg. C.
PBMC IsnoR5
Phenotyping of HIV patient isolates
Binninger-Schinzel et al. 2007
Coreceptor- prediction (PSSM/ geno2pheno): R5 R5 R5 R5 X4 X4 X4 X4 R5
SLIDE 13
Replication of HIV-1 SF 2 (X4-trop) and SF162 (R5-trop)
Binninger-Schinzel et al. 2007 102 103 104 105
SF 2 a SF 2 b SF 2 c
PM-1 HIV-1 p24 (pg / ml)
5 10 15 20
SF 2 a SF 2 b SF 2 c
IsnoR5 cl 1
- D. p. i.
101 102 103 104 105 105 102 103 104 105
SF 162 a SF 162 b SF 162 c
PM-1
5 10 15 20
SF 162 a SF 162 b SF 162 c
IsnoR5 cl 1
- D. p. i.
101 102 103 104
SLIDE 14
HIV-1 SF 162 (R5) HIV-1 SF 162 (X4)
CTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHC
- -----------Y-------HT--R------K---
Binninger-Schinzel et al. 2007
HIV growth in IsnoR5 cells only after corezeptor switch
SLIDE 15