ARV Drug Resistance

Dr Pontiano Kaleebu

Pontiano Kaleebu MBchB PhD

MRC/UVRI Uganda Research Unit on AIDS

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

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

Selective Pressures of Therapy

Selection of resistant quasispecies

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

Vir

al lo

ad

Time

Drug-susceptible quasispeciesDrug-resistant quasispecies

Treatment begins (CD4 <200 cells/mm3)

100,000

900,000

< 400

Copies/mL

V. Failure

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

Patterns of mutation

• Degree of resistance by a mutation differs• E.g M184V mutation:

– <5 fold resistance to abacavir and didanosine >1000 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

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

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

Genotyping assays

Cross-check for contamination fromolder samples by phylogenetic analysis

Acquire consensus sequences And save them as text files

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

1.5

2

2.5

3

3.5

4

4.5

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96

Study week

Pla

sma

HIV

-1 R

NA

Lo

g

WT

M184V

M184V, T215T/YM41L/M, M184V, T215Y

M41L, M184V, L210L/W, T215Y

50 c/mL

400 c/mL

D67N/D, K70R/K, M184V

ABC=5.9, ZDV=4.1fold

ABC=6.2, ZDV=12.2 fold

ZDV/3TC/ABC: Example of Slow Stepwise Appearance of Mutations in Subjects With Virologic Failure

M41L, M184V, T215Y

28 weeks of M184V only5000 c/mL

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

Review of drug resistancein Uganda

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)

POTENTIAL IMPLICATIONS OF ART WITHOUT VIROLOGICAL MONITORING: FAILURE OF THERAPY

MONTHS

VL 1000

Viral load

CD4 count

Treatmentonset

Virologicalfailure

(>1000 c/ml)

Clinical failure(AIDS events)

INCREASING RESISTANCE

YEARS

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 <100 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 CBV+TDF

• Genotyping of those with VL >1000c/ml is underway

Evolution of resistance 24-48 weeks (n=7)

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

AZT + 3TC + TDF

Impact of viral subtype on resistance mutations

Mutation Subtype P-value

A(A1) (n=33) C (n=14) D (n=12)

M184V/I 24(73%) 9 (64%) 9 (75%) 0.8

K65R 5 (15%) 3 (21%) 1 (8%) 0.7

# TAMs01-34-6

10 (30%)17 (52%)6 (18%)

4 (29%)6 (43%)4 (29%)

5 (42%)5 (42%)2 (17%)

0.9

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;

Table 1: Prevalence of individual and class specific mutations

ABC (n=56)

NVP (n=31)

Individual Mutations* M41L 3 (5%) 2 (6%) D67NG 21 (38%) 6 (19%) K70R 24 (43%) 3 (10%) K103N 3 (5%) 7 (23%) Y181CI 0 6 (19%) M184V 49 (88%) 23 (74%) G190AS 1 (2%) 9 (29%) T215FY 11 (20%) 4 (13%) K219QEN 8 (14%) 1 (3%) Class mutations TAMs none 25 (45%) 22 (71%) 1-2 23 (41%) 7 (23%) 3+ 8 (14%) 2 (6%) NNRTI none 52 (93%) 9 (29%) 1+ 4 (7%) 22 (71%) Permutations of mutations None 6 (11%) 5 (16%) TAMs only 1 (2%) 0 M184V only 18 (32%) 2 (6%) NNRTI only 0 3 (10%) TAMs & M184V 27 (48%) 2 (6%) TAMs & NNRTI 0 0 M184V & NNRTI 1 (2%) 12 (39%) TAMs & M184V & NNRTI 3 (5%) 7 (23%)

* occurring with >5% prevalence

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 2007Note 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

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

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

The Country HIVDR PackageNational HIVDR strategy elements for

countries scaling up ARTA. Development of a national HIVDR

strategy working group, plan and budgetB. HIVDR prevention activitiesC. Regular evaluation of HIVDR "early

warning" indicators from all ART treatment sites

D. HIVDR transmission threshold surveys: geographic areas, populations, timing

E. Sentinel monitoring of HIVDR emerging in treated populations and related ART programme factors

F. HIVDR database developmentG. A designated HIVDR genotyping

laboratoryH. Preparation of national annual HIVDR

report and recommendations

23rd -24th January 2007, Kampala, Uganda

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.

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

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

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

QCMD 2007 ENVA7 HIV Drug Resistance Typing Proficiency Programme

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

Data on Early Warning Indicators for HIV Drug Resistance In Uganda

December 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

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

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

Composite ScoresSite %

First-lineT=100%

% Lost FU Yr 1T < 20%

% Retained First-line, Yr 1T > 70%

% Kept AppointmentT > 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

Composite Scores (ctd)Site % First-

lineT=100%

% Lost FU Yr 1T < 20%

% Retained First-line, Yr 1T > 70%

% Kept AppointmentT > 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

Composite Scores (ctd)Site % First-

lineT=100%

% Lost FU Yr 1T < 20%

% Retained First-line, Yr 1T > 70%

% Kept AppointmentT > 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

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

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