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 T-cell surface
HIV-Infection - Why R5-HIV at start of infection? - Why und when develops X4-HIV? - X4-HIV responsible for CD4+ loss?
HIV receptors Primary Co- Syncytia Replication in PBMC receptor receptors Slow fast CD4 CCR5 CXCR4 HIV-1 R5 + + - - + - HIV-1 X4 + - + + - + HIV-1 R5/X4 + + + + - + MIP-1 α, β Ligands MHC II SDF-1 Rantes
Questions Immunodeficiency ? ? X4 R5 CD4 + HIV HIV Therapy ? - Immunodeficiency result or cause coreceptor switch - Can therapy reverse coreceptor switch and/or restore the immune system - New infections with X4 HIV?
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
Summary (2008) 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 1013 of 114 samples sequences Western blot 477 all positive CCR5-Genotype 278 of 294 CCR+/CCR+: 238 CCR+/CCR-: 40 16x no DNA
HIV phenotyping
Generation of a CCR5-negative T-cell line MT-2- cells irradiated PBMC (CD8+ depleted) CCR5 - / - donor with 5000 rd HTLV-1 production CCR5 +/- CCR5 +/+ CCR5 -/- Co-culture, immortalization CCR5-negative T-Cell line IsnoR5 cultivation in RPMI-1640 / 10 % FKS / IL-2 Single cell cloning Cell clones: IsnoR5 clone 1, 2, 3
HIV phenotyping * * * HIV * * * * * AMD3100 AMD3100 * * * * * CXCR4 CCR5 CXCR4 IsnoR5 PBMC or T cell line
HIV phenotyping PBMC IsnoR5 or T cell line Tropism of HIV Isolate - - AMD3100 AMD3100 R5 - - + + X4 + - + - R5/X4 + - + + Y + + + + Y: alternative receptors
Phenotyping of HIV patient isolates 10 6 PBMC 10 5 IsnoR5 TCID 50 / ml 10 4 10 3 10 2 P-7 P-26 P-31 P-41-0 P-41-9 P-41-19 P-51 P-107 P-180 Neg. C. Coreceptor- prediction R5 R5 X4 X4 R5 X4 X4 R5 R5 (PSSM/ geno2pheno): Binninger-Schinzel et al. 2007
Replication of HIV-1 SF 2 (X4-trop) and SF162 (R5-trop) 10 5 10 5 PM-1 PM-1 10 4 10 4 10 3 10 3 10 2 10 2 SF 2 a HIV-1 p24 (pg / ml) SF 162 a SF 2 b SF 162 b SF 2 c SF 162 c 10 5 10 5 IsnoR5 cl 1 IsnoR5 cl 1 10 4 10 4 10 3 10 3 10 2 10 2 SF 2 a SF 162 a SF 2 b SF 162 b SF 2 c SF 162 c 10 1 10 1 0 5 10 15 20 0 5 10 15 20 D. p. i. D. p. i. Binninger-Schinzel et al. 2007
HIV growth in IsnoR5 cells only after corezeptor switch HIV-1 SF 162 (R5) CTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHC HIV-1 SF 162 (X4) ------------Y-------HT--R------K--- Binninger-Schinzel et al. 2007
PEI: Birgit Krause Albrecht Werner Britta Meyé Dorothea Binninger-Schinzel Gudrun Winskowsky Daniela Müller Uni-Klinikum Frankfurt: Reinhard Brodt Timo Wolf
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