Antiretroviral therapy and development of resistant HIV Louise Bruun Jørgensen Department of Virology Statens Serum Institut Presentation of the lab.

Virological diagnosis and monitoring of HIV-1 infektion Diagnostic Monitorering * HIV antibodies * HIV RNA (Viral load) (ELISA – WB) (PCR) * HIV antigen * CD4 counts (ELISA) (FACS) * HIV DNA * Resistance (PCR) (PCR-sequencing) Culture

MACS study: 209 pt inrolled in 1984 and 1985

MACS study: 209 pt inrolled in 1984 and 1985

Drugs approved for treatment of HIV Pommier, Nature Reviews, 2005

NRTI

NNRTI

Protease inhibitors

Protease inhibitors

Treatment of HIV patients

Recommendations for HIV treatment in Denmark HAART 3-4 antiviral drugs CD4 cell count < 200-300 cells/ul Acute infection Clinical Symptoms Pregnancy EFV + AZT + 3TC NEV + AZT + 3TC LOP/Rit + AZT + 3TC http://www.dsinfm.dk

Combination therapy: Drugs Clinical effect Virological effect HAART Highly Active Antiretroviral Therapy (3-4 drugs NRTI+NNRTI or PI)

Combination therapy: Drugs Clinical effect Virological effect HAART Måneder HAART HAART Highly Active Antiretroviral Therapy (3-4 drugs NRTI+NNRTI or PI)

Combination therapy: Drugs Clinical effect Virological effect HAART Måneder HAART Highly Active Antiretroviral Therapy (3-4 drugs NRTI+NNRTI or PI)

Combination therapy: Drugs Clinical effect Virological effect HAART Months HAART Highly Active Antiretroviral Therapy (3-4 drugs NRTI+NNRTI or PI) Compliance Site-effects Development of resistance

Compliance and Site-effects

Mutations in the Pro and RT gene associated with resistance Development of resistance Mutations in the Pro and RT gene associated with resistance NRTI: M41L, E44D, A62V, K65R, D67N, T69D/(S+xx), K70R, L74V, V75T/M/A/I, F77L, Y115F, F116Y, V118I, Q151M, M184V/I, L210W, T215Y/F, K219Q/E Primary: D30N, M46I/L, G48V, I50V, V82F/A/T/S, I84V, L90M Secondary: L10F/I/R/V, K20R/M, L24I/V, V32I, L33F, M36I, I47V, F53L, I54V/M/L, L63P, A71V/T, G73S/A, V77I, N88D/S NNRTI: A98G, L100I, K101E, K103N, V106A/I, V108I, V179D, Y181C/I, Y188L/C/H, G190A/S, P225H, M230L, P236L

Development of resistant HIV against antiretroviral drugs Viral load Fitness of the resistant virus Effectivity of the drug against wild type Time Speed of development of resistance

Methods for genotyping Commercial services: Virco Virologic etc. Commercial kits Abbott ViroSeq Visible Genetics In House: Home brewed asays

Genotypic resistance by direct sequencing EDTA-blood sample Extraction of HIV-RNA from plasma cDNA syntesis by MuLV reverse transcriptase Single PCR-amplification of 1,8 kb of the pol-gene Direct sequencing of the protease- and RT-gene Electroforesis (ABI 3100) Nucleotide sequence Translation to amino acidsequence Identification of mutations associated to resistance 90 10 54 84 82 71 63 20 32 46 24 48 50 33 36 73 47 77 30 88

Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: Department of Virology SSI EDTA-blood sample Extraction of HIV-RNA from plasma * Abbott ViroSeq 2 system Nucleotide sequence * Quality control by phylogenetic analysis * Identification of mutations associated to resistance * Interpretation of resistance * Report

Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: Department of Virology SSI EDTA-blood sample Extraction of HIV-RNA from plasma * Abbott ViroSeq 2 system Nucleotide sequence * Quality control and subtyping by phylogenetic analysis * Identification of mutations associated to resistance * Interpretation of resistance * Report

Quality control by phylogenetic analysis Subtyping eg. subtype D

Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: Department of Virology SSI EDTA-blood sample Extraction of HIV-RNA from plasma * Abbott ViroSeq 2 system Nucleotide sequence * Quality control by phylogenetic analysis * Identification of mutations associated to resistance * Interpretation of resistance * Report

Identification of mutationer associated to resistance

Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: Department of Virology SSI EDTA-blood sample Extraction of HIV-RNA from plasma * Abbott ViroSeq 2 system Nucleotide sequence * Quality control by phylogenetic analysis * Identification of mutations associated to resistance * Interpretation of resistance * Report

Interpretation of genotypic resistance Tables www.iasusa.org Rulebased algoritms www.hivdb.stanford.edu/hiv/ Virtual phenotypes www.virco.be Neural networks

Interpretation of genotypic resistance Dept. of Virology, SSI

Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: Department of Virology SSI EDTA-blood sample Extraction of HIV-RNA from plasma * Abbott ViroSeq 2 system Nucleotide sequence * Quality control by phylogenetic analysis * Identification of mutations associated to resistance * Interpretation of resistance * Report

Report Dept. of Virology, SSI

Recombinant Virus Assay (RVA) New phenotypic assay: Recombinant Virus Assay (RVA) Recombinant virus MTT-assay (IC-50)

Cross-resistance Susceptible Genotypic WT (K103) Fold = 1 Phenotypic J.Virology 2001, 75(11) Susceptible Genotypic WT (K103) Fold = 1 Phenotypic

Cross-resistance Susceptible Resistant Genotypic WT (K103) K103N J.Virology 2001, 75(11) Susceptible Resistant Genotypic WT (K103) K103N Fold = 1 Fold = 57-437X Phenotypic

M184V M41L D67N K70R L210W T215Y K219E L74I Y181C L10I M46I L63P L90M 0.1 1.0 10.0 100.0 1000.0 NEV ABC AZT 3TC D4T CRX NOR SAQ M184V M41L D67N K70R L210W T215Y K219E G190A Y181C L10I M46I L63P A71V V77I I84V L90M 0.1 1.0 10.0 100.0 1000.0 NEV ABC D4T AZT 3TC NEL CRX NOR SAQ 600 350000 300000 500 250000 400 200000 CD4 count Viral load 300 150000 200 100000 100 50000 31-01-93 15-06-94 28-10-95 11-03-97 24-07-98 06-12-99 AZT 3TC SAQ ddC D4T CRX NOR ddI NEV NEL EFA ABC HYD

(x 1000 kopier/ml) TI-1 start TI-2 start M=mutant W=wildtype R=revertant (x 1000 kopier/ml) W184,M215 W184,W215 M184,M215 W184,R215 W184,M215 RNA/DNA M184,R215 TI-1 start M184,M215 TI-2 start M184,M215 (jul 2002) W184,M215 W184,W215 RNA/DNA Resumed treatment AZT 3TC d4T ABC ddI Nev Ind Nel Rit

Fylogeni af Pol-genet T215Y T215C/S *M184V Treatment interuption 1 120194 Fylogeni af Pol-genet 281191 300502D Treatment interuption 2 61 150502D 150502 150296 97 300502 T215Y T215C/S *M184V 56 250597* 201097* 170698* 070499* 121200 030701* 180702* 170102* 29 201099 Treatment interuption 1 201099D ASE7253 AUG037

Detection limit of Genotypic resistance assay

New ultra sensitive assay for genotypic resistance Copies/ml 6000 5000 4000 3000 2000 1000 17.03.97 16.04.97 28.07.97 24.09.97 29.12.97 01.04.98 02.07.98 08.09.98 07.12.98 07.06.99 07.09.99 01.11.99 01.03.99 07.02.00 L63P V77I Protease RT L74V M184V

New ultra sensitive assay for genotypic resistance Copies/ml 6000 Improved extraction Nested / double PCR => 20 copies pr ml 5000 4000 3000 2000 1000 17.03.97 16.04.97 28.07.97 24.09.97 29.12.97 01.04.98 02.07.98 08.09.98 07.12.98 07.06.99 07.09.99 01.11.99 01.03.99 07.02.00 L63P V77I Protease RT L74V M184V

New ultra sensitive assay for genotypic resistance Copies/ml 6000 Improved extraction Nested / double PCR => 20 copies pr ml 5000 4000 3000 2000 1000 17.03.97 16.04.97 28.07.97 24.09.97 29.12.97 01.04.98 02.07.98 08.09.98 07.12.98 01.03.99 07.06.99 07.09.99 01.11.99 07.02.00 L63P V77I L74V M184V L63P V77I M36I N88D L90M* L33F I84V K20T L24I D30N* Protease RT L74V M184V

Development of Drug resistance Therapy

Development of Drug resistance Therapy Transmission of resistance

Transmission of resistant HIV-1 strains RTI: PI: Spain 3% 1% Italy 11% 2% Belgium 13% 4% Luxembourg 12% 0% Germany 16% 3% France 17% 2% Europe RTI: PI: Seroconv. 6% 1% IDVU 0% 0% New diagn. 13% 3% Military 10% 10% USA Total MDR: Primary HIV: 1995-1998 8% 4% 1999-2000 23% 10%

Reversion of genotypic resistance AZT 215 ACC(T) WT 1 2 År A. DeRonde J.Virol.2001

Reversion of genotypic resistance AZT 215 ACC(T) TAC(Y) WT 1 2 År STOP of therapy A. DeRonde J.Virol.2001

Reversion of genotypic resistance AZT 215 GAC(D) ACC(T) TAC(Y) AAC(N) WT TCC(S) 1 2 År STOP of therapy A. DeRonde J.Virol.2001

Reversion of genotypic resistance AZT 215 GAC(D) AGC(S) TAC(Y) ACC(T) AAC(N) GAC(D) WT TCC(S) 1 2 År STOP of therapy A. DeRonde J.Virol.2001

Prevalence of drug resistance mutations in newly diagnosed HIV-1 patients in Denmark 2000-2004 Total prv. RT PI 2000: 2,0% (2/104) 41L, 210S, 215S 215S 2001: 4,8% (7/147) 90M, 63P, 71V, 77I 215E 215L (x2) 215S 82F, 10I, 63P 98G, 215S 2002: 5,0% (7/140) 103N, 215Y 103N, 188C 41L, 215D 90M, 10I, 63P, 71T, 77I 215D (x2) 67N 2003: 5,5% (7/127) 67N, 219Q 67N, 219E 84V, 10V,33F,36I,63P 69D 215N 103N 181C 2004: 0,7% (1/137) 190A

HIV-1 subtypes Based on the variation of the genome HIV can be divided in to different subtypes

HIV-1 subtypes 9 identified subtypes (A-K) and 16 CRF (circulating recombinant forms) In the industrialised world subtype B is most prevalent. However, globally subtype C is the most prevalent subtype.

Prevalence of subtype non-B in newly diagnosed HIV-1 patients in Denmark Global subtype distribution Andre D A C B Ref.: J.Clin. Virol. 29 (2004)

Epidemilogy

Prevalence of drug resistance mutations in newly diagnosed HIV-1 patients in Denmark 2000-2004 Total prv. RT PI Subtype % non-B Prv. Non-B 2000: 2,0% (2/104) 41L, 210S, 215S B 41 % 0/2 215S B 2001: 4,8% (7/147) 90M, 63P, 71V, 77I B 39 % 0/7 215E B 215L (x2) B+B 215S B 215S 82F, 10I, 63P B 98G, 215S B 2002: 5,0% (7/140) 103N, 215Y B 37 % 4/7 103N, 188C AG 41L, 215D 90M, 10I, 63P, 71T, 77I B 215D (x2) B+D 215S AE 67N A 2003: 5,5% (7/127) 67N, 219Q B 34 % 4/7 67N, 219E 84V, 10V,33F,36I,63P AE 69D AE 215N B 103N A 181C AE 2004: 0,7% (1/137) 190A AD (34 %) 0/1

Transmission of HIV

Case African/danish family African mother - HIV pos in 2004 African daugter – HIV pos in 2005 Probably infected at birth Arrived as teenager in DK Has no HIV symptoms Danish stepfather – HIV positiv 2003 Infected by african wife

Questions: Can we confirm the mother has infected the daughter? Is it possible to identify af transmission chain after 17 years?

POL (Mother) 94 100 (Stepfather) 78 (Daughter) Metode: Neighbour joining tree, 100 bootstrap replikations 1200 bp All HIV-1 subtype A POL sequences from Los Alamos included Subtype A sequences from SERO project Bootstrapvalues over 50% is shown

Gag 100 68 Stepfather Mother Daughter Metode: Neighbour joining tree, 100 bootstrap replikations 370 bp All HIV-1 subtype A gag sequences from Los Alamos included Bootstrapvalues over 50% is shown

GAG Daughter!

GAG Dublication

Epidemiological Study of HIV-1 in Greenland using Phylogenetic Analyses TV Madsen, N Obel, N Lohse, J Gerstoft, AB Petersen, C Nielsen & LB Jørgensen 90 samples from Greenland HIV-1 positive patients with high viral load, and as early in the infection as possible, was chosen for the study. HIV-1 sequences from Danish patients are also included in order to compare the genetic composition of the two populations. Sequences were obtained from selected regions (gag: p17-region; pol: protease and 250 aa of RT; env: V3-region) using in-house PCR and sequencing methods, and ViroSeqTM HIV Genotyping System (Abbott). In order to study the genetic variation over time and to identify local outbreaks and chains of infection in the Greenland HIV-1 population the phylogeny was correlated with epidemiological data from the Danish HIV Cohort.

Figure 1. Phylogeny on pol-region (NJ, 100 bootstrap replicates) Figure 1. Phylogeny on pol-region (NJ, 100 bootstrap replicates). Isolates from Greenland have been marked in green.

Figure 1. Phylogeny on pol-region (NJ, 100 bootstrap replicates) Figure 1. Phylogeny on pol-region (NJ, 100 bootstrap replicates). Isolates from Greenland have been marked in green. ?

Transmission 1991 1994 2001

Three patients: GR41: 31 year old female HTX infected in Greenland first positive test is from 27/9-94. Analysed sample from 14/7-99. GR57: 51 year old male HTX infected in Greenland first positive test 1/1-91. † 6/3-01. Analysed sample from 9/2-00. GR75: 52 year old female HTX infected in Greenland first positive test is from 1/8-01. Analysed sample from 14/-02.

Transmission Analysed 1991 1994 2001 9/2-2000 14/7-1999 14/1-2002

New mutation against NRTI?

History Heavily treated patient with non-compliant behaviour Four samples sent for HIV-1 genotypic resistance

Mutation D67G_Y Jun 2000 Jan 2002 Jan 2003 Mar 2005

RegalInst algoritme

HIV drug mutations 2000-2005 juli 2000 januar 2002 januar 2003 marts 2005 D67N N G D67GY   GY T69N K70R R L74V V V118I I M184V K219Q Q K103N KN V108I Y181C C M46I MI V82A A L90M M L10I K20R M36I F53L L I54V L63P P A71V V77I HIV drug mutations 2000-2005   NRTI NNRTI PI 1998 ddi+abc nvp idv Apr. 2003 (abc+3tc+azt)+tf rtv+amp Jun. 2003 rtv+saq Mar. 2005

Treatment history NRTI NNRTI PI VL Genotypic Resistance 1990-1998 AZT+ddI   1995-dec.1996 AZT+3TC Dec.1996-1998 d4T+3TC IDV 1998 DDI+ABC NVP NFV juli 2000 54100 No insert: D67GY Jan. 2002 56300 Jan. 2003 40000 Plus insert: D67GY Apr. 2003 (Abc+3TC+AZT)+TF RTV+Amp Jun. 2003 RTV+SAQ marts 2005 16300

Virus BL-3 laboratory, Department of Virology, SSI Deptartment of infectious diseases Rigshospitalet Hvidovre Hospital Skejby Sygehus Odense Universitets Hospital Ålborg Sygehus Sygehus Øresund Support ABI and Abbott