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STATENS SERUM INSTITUT Antiretroviral therapy and development of resistant HIV Louise Bruun Jørgensen Department of Virology Statens Serum Institut.

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Presentation on theme: "STATENS SERUM INSTITUT Antiretroviral therapy and development of resistant HIV Louise Bruun Jørgensen Department of Virology Statens Serum Institut."— Presentation transcript:

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2 STATENS SERUM INSTITUT Antiretroviral therapy and development of resistant HIV Louise Bruun Jørgensen Department of Virology Statens Serum Institut

3 STATENS SERUM INSTITUT

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

5 STATENS SERUM INSTITUT MACS study: 209 pt inrolled in 1984 and 1985

6 STATENS SERUM INSTITUT MACS study: 209 pt inrolled in 1984 and 1985

7 STATENS SERUM INSTITUT

8

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

10 STATENS SERUM INSTITUT NRTI

11 STATENS SERUM INSTITUT NNRTI

12 STATENS SERUM INSTITUT Protease inhibitors

13 STATENS SERUM INSTITUT Protease inhibitors

14 STATENS SERUM INSTITUT Treatment of HIV patients

15 STATENS SERUM INSTITUT Recommendations for HIV treatment in Denmark HAART 3-4 antiviral drugs 1.EFV + AZT + 3TC 2.NEV + AZT + 3TC 3.LOP/Rit + AZT + 3TC http://www.dsinfm.dk CD4 cell count < 200-300 cells/ul Acute infection Clinical Symptoms Pregnancy

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

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

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

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

20 STATENS SERUM INSTITUT Compliance and Site-effects

21 STATENS SERUM INSTITUT Mutations in the Pro and RT gene associated with resistance 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 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 NNRTI: A98G, L100I, K101E, K103N, V106A/I, V108I, V179D, Y181C/I, Y188L/C/H, G190A/S, P225H, M230L, P236L Development of resistance

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

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

24 STATENS SERUM INSTITUT 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 by direct sequencing

25 STATENS SERUM INSTITUT 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

26 STATENS SERUM INSTITUT

27 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

28 STATENS SERUM INSTITUT Quality control by phylogenetic analysis Subtyping eg. subtype D

29 STATENS SERUM INSTITUT 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

30 STATENS SERUM INSTITUT Identification of mutationer associated to resistance

31 STATENS SERUM INSTITUT 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

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

33 STATENS SERUM INSTITUT Interpretation of genotypic resistance Dept. of Virology, SSI

34 STATENS SERUM INSTITUT 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

35 STATENS SERUM INSTITUT Report Dept. of Virology, SSI

36 STATENS SERUM INSTITUT New phenotypic assay: Recombinant Virus Assay (RVA) Recombinant virus MTT-assay (IC-50)

37 STATENS SERUM INSTITUT Cross-resistance Susceptible GenotypicWT (K103) Phenotypic Fold = 1 J.Virology 2001, 75(11)

38 STATENS SERUM INSTITUT Cross-resistance Susceptible K103NGenotypicWT (K103) Resistant Phenotypic Fold = 1Fold = 57-437X J.Virology 2001, 75(11)

39 STATENS SERUM INSTITUT 0 100 200 300 400 500 600 31-01-9315-06-9428-10-9511-03-9724-07-9806-12-99 CD4 count 0 50000 100000 150000 200000 250000 300000 350000 Viral load AZT 3TC SAQ ddC D4T CRX NOR ddI NEV NEL EFA ABC HYD M184V M41L D67N K70R L210W T215Y K219E G190A Y181C L10I M46I L63P A71V V77I I84V L90M

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

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

42 STATENS SERUM INSTITUT Detection limit of Genotypic resistance assay

43 STATENS SERUM INSTITUT 0 1000 2000 3000 4000 5000 6000 17.03.9716.04.9728.07.9724.09.9729.12.97 01.04.98 02.07.9808.09.9807.12.98 01.03.99 07.06.99 07.09.9901.11.99 07.02.00 Copies/ml Protease RT L63P V77I L74V M184V New ultra sensitive assay for genotypic resistance

44 STATENS SERUM INSTITUT 0 1000 2000 3000 4000 5000 6000 17.03.9716.04.9728.07.9724.09.9729.12.97 01.04.98 02.07.9808.09.9807.12.98 01.03.99 07.06.99 07.09.9901.11.99 07.02.00 Copies/ml Protease RT L63P V77I L74V M184V Improved extraction Nested / double PCR => 20 copies pr ml New ultra sensitive assay for genotypic resistance

45 STATENS SERUM INSTITUT 0 1000 2000 3000 4000 5000 6000 17.03.9716.04.9728.07.9724.09.9729.12.97 01.04.98 02.07.9808.09.9807.12.98 01.03.99 07.06.99 07.09.9901.11.99 07.02.00 Copies/ml Protease RT L63P V77I L74V M184V L74V M184V L63P V77I M36I L63P V77I N88D L90M* L33F M36I L63P V77I I84V N88D L90M* L33F M36I L63P V77I I84V N88D L33F M36I L63P V77I I84V N88D L33F M36I L63P V77I I84V N88D L90M* M36I L63P V77I I84V L90M* L33F M36I L63P V77I I84V N88D L90M* K20T L24I D30N* M36I L63P V77I N88D L90M* Improved extraction Nested / double PCR => 20 copies pr ml New ultra sensitive assay for genotypic resistance

46 STATENS SERUM INSTITUT Therapy Development of Drug resistance

47 STATENS SERUM INSTITUT Therapy Development of Drug resistance Transmission of resistance

48 STATENS SERUM INSTITUT 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 Total MDR: Primary HIV: 1995-1998 8% 4% 1999-2000 23%10% RTI:PI: Seroconv. 6% 1% IDVU 0% 0% New diagn.13% 3% Military10%10% USA

49 STATENS SERUM INSTITUT Reversion of genotypic resistance AZT 215 ACC(T) WT A. DeRonde J.Virol.2001 012År

50 STATENS SERUM INSTITUT Reversion of genotypic resistance AZT 215 TAC(Y)ACC(T) WT A. DeRonde J.Virol.2001 012År STOP of therapy

51 STATENS SERUM INSTITUT Reversion of genotypic resistance AZT 215 TCC(S) GAC(D) TAC(Y)ACC(T)AAC(N) WT A. DeRonde J.Virol.2001 012År STOP of therapy

52 STATENS SERUM INSTITUT Reversion of genotypic resistance AZT 215 TCC(S) GAC(D)AGC(S) TAC(Y)ACC(T)AAC(N)GAC(D) WT A. DeRonde J.Virol.2001 012År STOP of therapy

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

54 STATENS SERUM INSTITUT HIV-1 subtypes Based on the variation of the genome HIV can be divided in to different subtypes D C F E O B G A H

55 STATENS SERUM INSTITUT 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.

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

57 STATENS SERUM INSTITUT Epidemilogy

58 STATENS SERUM INSTITUT 2000: 2,0% (2/104) 41L, 210S, 215S B41 % 0/2 215S B 2001: 4,8 % (7/147) 90M, 63P, 71V, 77I B39 % 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 B37 % 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 B34 % 4/7 67N, 219E 84V, 10V,33F,36I,63P AE 69D AE 215N B 215S B 103N A 181C AE 2004: 0,7 % (1/137) 190A AD(34 %) 0/1 Total prv. RT PI Subtype % non-B Prv. Non-B Prevalence of drug resistance mutations in newly diagnosed HIV-1 patients in Denmark 2000-2004

59 STATENS SERUM INSTITUT Transmission of HIV

60 STATENS SERUM INSTITUT 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

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

62 STATENS SERUM INSTITUT 94 100 78 (Daughter) (Mother) (Stepfather) POL 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

63 STATENS SERUM INSTITUT 100 68 Stepfather Mother Daughter Gag 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

64 STATENS SERUM INSTITUT GAG Daughter!

65 STATENS SERUM INSTITUT Dublication GAG

66 STATENS SERUM INSTITUT 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.

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

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

69 STATENS SERUM INSTITUT Transmission 1991 1994 2001

70 STATENS SERUM INSTITUT 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.

71 STATENS SERUM INSTITUT Transmission 1991 1994 2001 9/2-2000 14/7-1999 14/1-2002 Analysed

72 STATENS SERUM INSTITUT New mutation against NRTI?

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

74 STATENS SERUM INSTITUT Jun 2000 Jan 2002 Jan 2003 Mar 2005 Mutation D67G_Y

75 STATENS SERUM INSTITUT

76 RegalInst algoritme

77 STATENS SERUM INSTITUT juli 2000januar 2002januar 2003marts 2005 D67NNNGG D67GY GY T69NN K70RRR R L74V V V118I I M184V V K219QQQ Q K103NKN V108I I Y181CCC C M46IMI V82AAA A L90MMM M L10III I K20R R M36III I F53L L I54VV V L63PPP P A71V V V77I I NRTINNRTIPI 1998ddi+abcnvpidv Apr. 2003(abc+3tc+azt)+tf rtv+amp Jun. 2003(abc+3tc+azt)+tf rtv+saq Mar. 2005(abc+3tc+azt)+tf rtv+saq HIV drug mutations 2000-2005

78 STATENS SERUM INSTITUT Treatment history NRTINNRTIPIVLGenotypic Resistance 1990-1998AZT+ddI 1995-dec.1996AZT+3TC Dec.1996-1998d4T+3TC IDV 1998DDI+ABCNVPNFV 1998DDI+ABCNVPIDV juli 2000DDI+ABCNVPIDV54100No insert: D67GY Jan. 2002DDI+ABCNVPIDV56300No insert: D67GY Jan. 2003DDI+ABCNVPIDV40000Plus insert: D67GY Apr. 2003(Abc+3TC+AZT)+TF RTV+Amp Jun. 2003(Abc+3TC+AZT)+TF RTV+SAQ marts 2005(Abc+3TC+AZT)+TF RTV+SAQ16300Plus insert: D67GY

79 STATENS SERUM INSTITUT 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


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