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

3. 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

4. MACS study: 209 pt inrolled in 1984 and 1985

5. MACS study: 209 pt inrolled in 1984 and 1985

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

9. NRTI

10. NNRTI

11. Protease inhibitors

12. Protease inhibitors

13. Treatment of HIV patients

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

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

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

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

18. 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

19. Compliance and Site-effects

20. 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

21. 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

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

23. 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

24. 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. 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

27. Quality control by phylogenetic analysis
Subtyping
eg. subtype D

28. 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

29. Identification of mutationer associated to resistance

30. 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

31. Interpretation of genotypic resistance
Tables
Rulebased algoritms
Virtual phenotypes
Neural networks

32. Interpretation of genotypic resistance
Dept. of Virology, SSI

33. 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

34. Report
Dept. of Virology, SSI

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

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

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

38. 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
AZT
3TC
SAQ
ddC
D4T
CRX
NOR
ddI
NEV
NEL
EFA
ABC
HYD

39. (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

40. 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

41. Detection limit of Genotypic resistance assay

42. New ultra sensitive assay for genotypic resistance
Copies/ml
6000
5000
4000
3000
2000
1000
L63P
V77I
Protease RT
L74V
M184V

43. New ultra sensitive assay for genotypic resistance
Copies/ml
6000
Improved extraction
Nested / double PCR
=> 20 copies pr ml
5000
4000
3000
2000
1000
L63P
V77I
Protease RT
L74V
M184V

44. New ultra sensitive assay for genotypic resistance
Copies/ml
6000
Improved extraction
Nested / double PCR
=> 20 copies pr ml
5000
4000
3000
2000
1000
L63P
V77I
L74V
M184V
L63P
V77I
M36I
N88D
L90M*
L33F
I84V
K20T
L24I
D30N*
Protease RT
L74V
M184V

45. Development of
Drug resistance
Therapy

46. Development of
Drug resistance
Therapy
Transmission
of resistance

47. 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:
% 4%
% 10%

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

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

50. 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

51. 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

52. 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) M, 63P, 71V, 77I
215E
215L (x2)
215S F, 10I, 63P
98G, 215S
2002: 5,0% (7/140) 103N, 215Y
103N, 188C
41L, 215D M, 10I, 63P, 71T, 77I
215D (x2)
67N
2003: 5,5% (7/127) 67N, 219Q
67N, 219E V, 10V,33F,36I,63P
69D
215N
103N
181C
2004: 0,7% (1/137) 190A

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

54. 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.

55. 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)

56. Epidemilogy

57. 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 % /2
215S B
2001: 4,8% (7/147) M, 63P, 71V, 77I B 39 % /7
215E B
215L (x2) B+B
215S B
215S F, 10I, 63P B
98G, 215S B
2002: 5,0% (7/140) 103N, 215Y B 37 % /7
103N, 188C AG
41L, 215D M, 10I, 63P, 71T, 77I B
215D (x2) B+D
215S AE
67N A
2003: 5,5% (7/127) 67N, 219Q B 34 % /7
67N, 219E V, 10V,33F,36I,63P AE
69D AE
215N B
103N A
181C AE
2004: 0,7% (1/137) 190A AD (34 %) /1

58. Transmission of HIV

59. 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

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

61. 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

62. 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

63. GAG
Daughter!

64. GAG
Dublication

65. 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.

66. 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.

67. 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. ?

68. Transmission
1991
1994
2001

69. 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.

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

71. New mutation against NRTI?

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

73. Mutation D67G_Y
Jun 2000
Jan 2002
Jan 2003
Mar 2005

75. RegalInst algoritme

76. 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
NRTI
NNRTI PI
1998
ddi+abc
nvp
idv
Apr. 2003
(abc+3tc+azt)+tf
rtv+amp
Jun. 2003
rtv+saq
Mar. 2005

77. Treatment history NRTI NNRTI PI VL Genotypic Resistance 1990-1998
AZT+ddI
1995-dec.1996
AZT+3TC
Dec
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

78. 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