ARV Drug Resistance

1
ARV Drug Resistance

• Dr Pontiano Kaleebu
• Pontiano Kaleebu MBchB PhD
• MRC/UVRI Uganda Research Unit on AIDS

2
Summary of presentation

• Mechanisms of ARV resistance
• How do we look for resistance
• Clinical implications
• Some review of resistance in Uganda (Including
  Dart)
• National HIVDR Drug Resistance Prevention,
  Monitoring and Surveillance Plan

3
Mechanisms of Drug Resistance and Diversity

• HIV-1 genetic variability is generated by the
  lack of proof-reading ability of reverse
  transcription
• Rapid turnover of HIV in vivo
• Host selective immune pressures
• Recombination events during replication
• Of the 10 billion new viruses produced, 1/1000
  (10 million) viruses per day will have one new
  "error" (mutation) some of which make the virus
  resistant to ART drugs

4
Selective Pressures of Therapy
Copies/mL
Treatment begins (CD4 lt200 cells/mm3)
900,000
Drug-susceptible quasispecies Drug-resistant
quasispecies
Selection of resistant quasispecies
100,000

• Incomplete suppression
• Inadequate potency
• Inadequate drug levels
• Inadequate adherence
• Pre-existing resistance

Viral load
V. Failure
lt 400
Time
5
Other factors

• Genetic barrier
• Some drugs like lamivudine and NNRTIs have a low
  genetic barrier in that they only require a
  single mutation to cause resistance.
• On the other hand drugs like abacavir and
  indinavir require at least three mutations before
  significant loss of activity
• Half life
• Long half life e.g Tenofovir

6
Patterns of mutation

• Degree of resistance by a mutation differs
• E.g M184V mutation
• lt5 fold resistance to abacavir and didanosine
  gt1000 fold resistance to lamuvidine
• K103R leads to 20-30 fold resistance to NNRTI
• Cross-resistance 151M arises primarily with DDI
  but leads to resistance to most NRTIs

7
Patterns of mutation

• K65R causes tenofovir resistance but increases
  sensitivity to AZT
• NVP Y181C can suppress effect of AZT 215 mutation
• Mutation patterns observed in combination
  treatment have become complex and interpretation
  needs experience

8
Measurement of HIV Drug Resistance

• Genotypic assays Commercial e.g ViroSeq Kit
  (Abbot Diagonostics), TrueGene Kit In house
• Phenotypic assays Virco Laboratories (Belgium,
  USA)
• Virtual phenotype
• Use of genotype results to predict phenotypic
  susceptibility based originally on database of
  paired genotype and phenotype data or, more
  recently, through scores derived from linear
  regression analysis

9
Genotyping assays
10
Acquire consensus sequences And save them as
text files
Cross-check for contamination from older samples
by phylogenetic analysis
11
Phenotypic assays

• Culture and sensitivity
• In vitro determination of drug susceptibility
  compare the concentration at which virus
  replication is inhibited by 50 (IC50) compare
  with a reference strain
• Cost about US 300

12
ZDV/3TC/ABC Example of Slow Stepwise Appearance
of Mutations in Subjects With Virologic Failure
M184V
D67N/D, K70R/K, M184V
M184V, T215T/Y
M41L/M, M184V, T215Y
M41L, M184V, T215Y
WT
M41L, M184V, L210L/W, T215Y
28 weeks of M184V only
5000 c/mL
ABC6.2, ZDV12.2 fold
400 c/mL
ABC5.9, ZDV4.1fold
50 c/mL
13
Should we worry about drug resistance

• USA 1999 an estimated 87 of patients with
  detectable viremia receiving treatment with ARVs
  had evidence of genotypic mutations associated
  with HIV resistance to at least one drug (70
  for NRTI, 31 for NNRTI and 42 for protease
  inhibitors)
• In recently infected individuals resistance
  prevalence ranges between 10-25 in some
  communities in Europe and USA

14
Review of drug resistancein Uganda
15
Some information on resistance in Uganda

• Drug naïve
• Becker-Pergola et al. AIDS Res Hum Retro 2000
• Weidle PJ et ak JAIDS 2001
• Richard N et al. ARHR 2004
• Gale C et al. ARHR 2006
• NO resistant mutations but appreciable
  polymorphisms-minor mutations that could have
  relevance in resistance development
• Transmitted resistance
• Ndembi N et al ARHR In press (No resistance
  mutations)

16
POTENTIAL IMPLICATIONS OF ART WITHOUT VIROLOGICAL
MONITORING FAILURE OF THERAPY
17
Dart virology studies

• 300 patients on Combivir Tenofovir
• 100 in each of 3 clinical sites in Uganda (2) and
  Zimbabwe (1)
• 50 with baseline CD4 lt100 cells/mm3, 50 CD4
  (100-199)
• Plasma HIV-1 RNA assayed on stored specimens at
  0, 4, 12, 24 and 48 weeks after initiation of
  CBVTDF
• Genotyping of those with VL gt1000c/ml is underway

18
Evolution of resistance 24-48 weeks (n7)
AZT 3TC TDF
Patient Mutations at week 24 Additional mutations by week 48
A 184V 67N,70R,215F
B 41L,67N,70R,184V,215Y 210W
C 184V 41L,67N,210W,215Y
D 67N,70R,184V 41L,215Y,219Q
E 67N,70R,215F 184V,219E
F 67N,70R,184V,215N 41L,215Y
G 41L,67N,181I,184V,215Y 70R
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Impact of viral subtype on resistance mutations
Mutation Subtype Subtype Subtype P-value
Mutation A(A1) (n33) C (n14) D (n12) P-value
M184V/I 24 (73) 9 (64) 9 (75) 0.8
K65R 5 (15) 3 (21) 1 (8) 0.7
TAMs 0 1-3 4-6 10 (30) 17 (52) 6 (18) 4 (29) 6 (43) 4 (29) 5 (42) 5 (42) 2 (17) 0.9

20
Differences in the dynamics of viral rebound and
evolution of resistance between CBV/NVP and
CBV/ABC (NORA sub study of DART Trial) uncovered
in the absence of viral load monitoring in
real-time.

• Nicaise Ndembi1, Deenan Pillay2, Ruth Goodall3,
  Adele McCormick4, Andy Burke3, Fred Lyagoba1,
  Paula Munderi1, Pauline Katundu5, Stefano
  Tugume5, Pontiano Kaleebu1 on behalf of the DART
  Virology and Trial Teams

1 Med Res Council/Uganda Virus Res Inst Prgm on
AIDS, Entebbe, Uganda 2 UCL/Health Protection
Agency, London, UK 3 Med Res Council Clin Trials
Unit, London, UK 4 UCL, London, UK and 5 Joint
Clin Res Ctr, Kampala, Uganda
21
Table 1 Prevalence of individual and class
specific mutations
occurring with gt5 prevalence
22
On treatment in clinics (JCRC and UNAIDS HIV
drug access initiative clinics)

• Weidle PJ et al. JAIDS 2001
• Weidle PJ et al. Lancet 2002
• Weidle P.J et al. AIDS 2003
• Richard N et al. ARHRetro 2004
• Oyugi JH et al. AIDS 2007
• Note late 1990s some were on dual NRTI therapy,
  most paying and price high
• Summary findings Resistance detected in those
  with virological failure, mutations were similar
  to subtype B phenotypic resistance corresponded
  to genotypic resistance treatment interruptions
  lead to resistance

23
Resistance under PMTCT(HIVNET 006 012)

• Jackson JB et al. AIDS 2000 Eshleman SH et al.
  AIDS 2001 Eshleman SH et al. JAIDS 2004
  Eshleman SH et al. ARHR 2004 Eshleman SH et al.
  JID 2005
• Summary In women K103N NVP mutation 6-8 weeks
  after delivery and fades by 12-24 months!!
  Minor population missed
• In infants Y181C
• What will happen when these women and infants
  start HAART will NVP containing regimens be
  effective

24
Is it possible to prevent HIV Drug Resistance?

• No, NOT ENTIRELY
• Some degree of HIV drug resistance (HIVDR)
  is inevitable
• high rate of mutation
• treatment is life long

25
The Country HIVDR PackageNational HIVDR
strategy elements for countries scaling up ART

1. Development of a national HIVDR strategy working
   group, plan and budget
2. HIVDR prevention activities
3. Regular evaluation of HIVDR "early warning"
   indicators from all ART treatment sites
4. HIVDR transmission threshold surveys geographic
   areas, populations, timing
5. Sentinel monitoring of HIVDR emerging in treated
   populations and related ART programme factors
6. HIVDR database development
7. A designated HIVDR genotyping laboratory
8. Preparation of national annual HIVDR report and
   recommendations

26
23rd -24th January 2007, Kampala, Uganda
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Goal of Plan

• To support ART program practices and country
  planning in order to minimize the unnecessary
  emergence of HIV drug resistance, and to restrict
  the extent to which emerging resistance
  jeopardizes the effectiveness of the limited ART
  regimens available, within the context of the
  national HIV prevention and treatment plan.

28
Specific Objectives and key activities

• Develop an support capacity for HIVDR prevention,
  monitoring and surveillance
• Develop a list of EWI that will be regularly
  evaluated
• Support and coordinate surveillance of HIVDR
  transmission in different geographical regions
• Support and coordinate the monitoring of HIVDR
  arising in paediatric and adult populations
  starting and continuing treatment

29
Objectives continue

• Accredit and support local laboratories to
  support HIVDR activities
• Develop and maintain a data management system
• Develop and maintain a system to disseminate
  program findings and results for evidence based
  HIV drug resistance containment strategies (
  translate into policy

30
Some achievements so far

• Consensus workshop Jan 2007
• HIVDR working group created
• HIVDR transmitted Threshold survey (In press
  ARHR)
• Early Warning Indicators (Pilot completed)
• UVRI National reference laboratory final stages
  of WHO Accreditation
• Some equipments and reagents obtained from The
  Global Fund

31
QCMD 2007 ENVA7 HIV Drug Resistance Typing
Proficiency Programme
32
Next activities

• Sentinel HIVDR monitoring at selected treatment
  sites
• Repeat Threshold transmitted resistance in
  Kampala and later Mbarara
• Collaborate with other partners such as
  PharmAccess

33
Data on Early Warning Indicators for HIV Drug
Resistance In UgandaDecember 2007

• Dr Wilford Kirungi, Dr Elizabeth Madraa, Dr Norah
  Namuwenge, Dr Frank Lule, Dr Beatrice Crahay,
  Miss Marion Acieng, Dr Pontiano Kaleebu
• and
• The National HIVDR Technical Working group

34
WHO Recommended HIVDR EWI

• The HIVDR TWG prioritised 6 HIVDR EWI and set
  thresholds for each
• Indicator 1 Prescribing practices
• Indicator 2 Defaulter rates during the first 12
  months of ART
• Indicator 3 Retention on first-line during the
  first 12 months of ART
• Indicator 4 Appointment keeping over a 12 months
  period
• Indicator 5 Pill count adherence
• Indicator 6 Continuity of ARV drug supply in
  facilities

35
Methods

• Sample of 41 ART sites during Nov Dec 2007
• Sampled from all regions, various partners,
  different levels and modes of ART service
  delivery that had had ART established for at
  least 1 year
• Trained field workers and constituted 6 teams of
  2
• 5 teams comprised of persons with medical
  training and clinical experience of ART 28
  sites
• One team of 2 data managers with IT background
  and experience in EMRS - 13 sites

36
Composite Scores
Site First-line T100 Lost FU Yr 1 T lt 20 Retained First-line, Yr 1 T gt 70 Kept Appointment T gt 80 Quarters with continuous ARV supply
NI Hosp 100 0 65 71 1
N2 Hosp 100 2 81 100 3
N3 RRH 99.4 0 83 ND 2
N4 Hosp 100 0 73 97 4
N5 Hosp 94 2 79 ND 0
N6 RRH 100 79 18 ND 1
N7 Hosp 100 0 52 97 0
N8 RRH 97.0 0 69 63 0
N9 Hosp 100 52.9 24 85 4
C1 HC 100 0 94 ND 1
C2 HC 100 0 85 22 0
C3 Hosp 100 2 93 77 3
C4 Hosp 100 ND ND ND 0
37
Composite Scores (ctd)
Site First-line T100 Lost FU Yr 1 T lt 20 Retained First-line, Yr 1 T gt 70 Kept Appointment T gt 80 Quarters with no continuous ARV supply
C5 CCE 100 0 87 ND ND
C6 Comm 78 2 63 ND 3
C7 HC 100 5 36 36 4
C8 RRH 99 0 83 54 4
C9 Hosp 98 1.6 81 ND 0
C10 Hosp 97 0 55 43 0
C11 HC 100 0 80 61 3
C12 Hosp 98 0 80 ND 0
E1 Hosp 78 0 50 55 2
E2 RRH 100 8.5 74 40 0
E3 HC 100 0 96 64 1
E4 Hosp 100 0 79 66 1
E5 NGO 100 0 91 ND 0
E6 NGO 99 0 93 ND 0
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Composite Scores (ctd)
Site First-line T100 Lost FU Yr 1 T lt 20 Retained First-line, Yr 1 T gt 70 Kept Appointment T gt 80 Quarters with no continuous ARV supply
E7 CE 99 ND ND ND 0
E8 CE 100 0 81 ND 4
W1 RRH 100 4 95 74 0
W2 Hosp 100 0 76 31 3
W3 NGO OR 100 24 76 38 4
W4 Hosp 100 5 76 29 0
W5 Hosp 100 24 62 74 0
W6 Hosp 100 10 83 59 3
W7 HC 100 0 97 53 2
W8 RRH 100 2.0 78 61 0
W9 Hosp 97 5 90 44 0
W10 H 100 0 72 66 0
W11 Hosp 100 0 90 ND 0
W12 HC 100 7.7 73 47 1
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Conclusions

• Resistance develops in those who do not suppress
  virus
• We need to study more how resistance develops in
  absence of virological monitoring
• A national ART prevention and monitoring plan is
  operational
• A small study has not demonstrated transmitted
  resistant viruses
• A significant proportion of patients in ART
  treatment centers start on appropriate ART
  regimens
• Drug stock-out afflict many centers and poses a
  danger for poor adherence and resistance
  development

40
Acknowledgements

• Dart team MRV-UVRI JCRC IDI University of
  Zimbabwe, MRC-CTU UCL, Imperial College
• National HIVDR working group
• WHO
• MRC
• Dart virology supported by Roche, GSK, Abbot,
  Gilead, Boehringer Ingelheim