1. Drug resistant HIV, an introduction and overview of epidemiological studies
David van de Vijver PharmD PhD
Eijkman-Winkler Institute
Dept. of virology
University Medical Centre Utrecht

2. Introduction In 1996, several novel anti-HIV drugs became available
Dramatic reduction mortality among HIV-infected patients
Resistance to anti-HIV drugs develops rapidly
Objective
Principles of HIV drug resistance
Epidemiological studies

3. Contents General introduction to HIV Drug resistance
Natural course, epidemiology, and treatment
Drug resistance
Principles, genotyping, fitness, drug resistance interpretation systems
Epidemiological research
Transmisison of resistance across Europe

4. Introduction to HIV

5. HIV Human immunodeficiency virus (HIV)
Primarily infects vital components of the human immune system
CD4-cells which is vital for proper functioning of immune system
Immune system is slowly being demolished
Patients will eventually develop AIDS without treatment

6. Clinical course of HIV – no drugs

8. Treatment of HIV Currently four classes of antiretrovirals available
NRTI
NNRTI
Protease inhibitors
Fusion-inhibitor
Future
CCR5-inhibitors
Treatment with antiretroviral drugs is expensive

9. NRTI (nucleoside reverse transcriptase inhibitors)
Lamivudine, zidovudine (AZT) and others
A, C, T and G are building blocks of DNA
NRTI’s nucleoside analogues of A,C,T or G
Chain prolongation of DNA inhibited

10. NNRTI (non-nucleoside reverse transcriptase inhibitors)
Efavirenz, Nevirapine
Binds to RT

11. Protease inhibitors
Lopinavir, amprenavir and others
Inhibits the enzyme protease which results in immature non-infectious viruses
Boosted protease inhibitors

12. Boosted protease inhibitors
Liver and kidney are main organs responsible for metabolism of drugs
Protease inhibitors are metabolized by group of enzymes named CYP450 in liver
Ritonavir inhibits CYP450 already at small dosage
Boosting is combination of protease inhibitor with ritonavir
Metabolism inhibited –> drugs levels and half-life increase

13. Fusion-inhibitor
Enfuvirtide or T-20
Peptide, only by injection
Binds to the envelope, prevents HIV from entering cell
€ 6000 per month
Only for treatment of drug-resistant HIV

14. Side effects Antiretroviral drugs have many serious side effects
Some side effects:
Nausea, diarrhea
Diabetes, increase in cholesterol > increased risk for heart disease
Central nervous system: vivid dreams

15. Lipodystrophia
Associated with usage of protease inhibitors

16. Treatment of HIV
Long-term data show that a single inhibitor is not successful (Lopinavir/r ?)
Combination of at least two different classes of antiretrovirals
HAART (Highly Active AntiRetroviral Treatment)
Treatment should be started as late as possible
Always: opportunistic infection or CD4<200
CD4 between 200 and 350
Measure of success of treatment is viral load
<50 copies/ml

17. Introduction to resistance

18. Mechanism of resistance
Large daily production of HIV (~109/day)
HIV reverse transcriptase makes a lot of errors
Large genetic variation in HIV
Cloud of genetically different viruses
Quasi-species

19. Mechanism HAART Wild-type Not complete inhibition of HIV Resistant
Fastest replicating virsuses
Wild-type
Not complete inhibition of HIV
Resistant
Replication in absence of drugs

20. Proportion with virologic failure (%)
Adherence
100 80 60 40 20
Proportion with virologic failure (%)
>95 90–95 80–90 70–80 <70
Medication taken (%)
p= , r=–0.554
Paterson DL et al. Ann Intern Med 2000

21. Adherence Patients have to comply strictly to dosing regimen
>95% of prescribed dosages (side effects!)
Heart disease
Statines after heart attack
>80% to prevent new heart attack (Heart 2002; 88: )

22. Mutations
Drug resistance is caused by the emergence of mutations in HIV-genome
Genetic barrier
Number of mutations required to overcome drug selective pressure
Differs per drug
High for boosted protease inhibitors
Low for the NNRTI´s and the NRTI lamivudine

23. Lamivudine mutation aminoacid 184 M > V
R. Shafer, Clin Microbiol Rev 2002; 15:

24. Fitness
Resistance associated mutations do not replicate as fast as wild-type virus
Resistant virus less fit
What are the consequences of decreased fitness?

25. Fitness Lamivudine Viral load Time M184V
Schuurman R, et al. J Infect Dis 1995; 171:

26. Lab measurement of resistance
Genotyping
Assess nucleotide sequence of reverse transcriptase and protease region
Find presence of drug resistance associated mutations

27. Sequencing
+ddATP +ddCTP
+ddGTP+ddTTP

28. Sequence

29. Mutations

30. Amino-acids
Computer-software to detect mutations

31. VIRADAPT: HIV RNA <200/mL
Randomized study
Open study
32.3
31.3 40
30.4
29.2 30
30.5
Patients (%) 20 10
Control
Genotypic
14.0
14.0
12.5 3 6 9 12
Time (mo)
Durant J, et al. Lancet 1999;353:2195-9

32. Genotyping © 2004. The International AIDS Society–USA
Zidovudine M L 41 D R N K 67 70 W
210 YF QE T
215
219 E 44 I V
118
Stavudine M L 41 D R N K 67 70 W
210 YF QE T
215
219 E 44 I V
118 65
Didanosine K R 65 L V 74
Zalcitabine K R 65 D T 69 L V 74 M
184
Abacavir K R 65 L V 74 Y F
115 M
184
Lamivudine E D 44 I V
118 M VI
184 K R 65
Emtricitabine K R 65 M VI
184
Tenofovir K R 65
© The International AIDS Society–USA
Johnson VA, et al. Topics HIV Med Updated on
1. D’Aquila RT, Schapiro JM, Brun-Vezinet F, et al. Drug resistance mutations in HIV-1. Top HIV Med. 2002;10:11-15.

33. Genotyping Interpretation is difficult
Cross-resistance
Fitness
Interaction between mutations
Re-sensitization due to particular mutations
E.g. M184V leads to hyper-susceptibility to zidovudine
Several drug resistance interpretation systems have been developed
Retrogram

34. Retrogram
M184V

35. Several systems exist…
De Luca et al. Journal of infectious diseases 2003; 187:

36. ViroLab Funded by EU (6th framework) Coordinated by Peter Sloot
Build a virtual laboratory
Drug resistance interpretation system at its core

37. Epidemiological research

38. Transmission of resistance
Drug resistant HIV can be transmitted to others
USA: San Diego/ San Francisco 25%
Little et al. NEJM 2002; 347:
Grant et al. JAMA 2002; 288: 181-8
Europe: until recently studies limited to single countries, different methodologies

39. What is SPREAD?
Prevalence transmission of drug resistant HIV in newly diagnosed patients across Europe
Representative sampling of 4000 patients in two rounds
Quality control of participating labs
Same methods in all 31 participating countries
Coordination: dept. of virology, UMC Utrecht, the Netherlands
Funding by European Union

40. 32 Participating countries
SPREAD
32 Participating countries
SPREAD is sponsored by the European Union
(contract number QLK2-CT )
Extended as EuropeHIVResistance
WHO HIVResNet

41. CATCH study CATCH Aim SPREAD
First large scale study on transmission of resistance in Europe
On behalf of SPREAD – program
Aim
To determine the prevalence of drug resistance in antiretroviral naïve patients in Europe from 1996 to 2002
Wensing, Van de Vijver et al. Journal of infectious diseases 2005; 192:

42. CATCH-study

43. Population n=2208, 19 countries
Age (mean) 36  10 years
Male 73%
Subtype B 70%
HIV-RNA (mean) 4.82  0.86 log cp/ml
CD4 (median) 408 (1-1764) cells/mm3

44. Results 1996-2002 N=2208 Prevalence Resistance 10.4% 230/2208 NRTI
SPREAD
Results
N=2208
Prevalence
Resistance
10.4%
230/2208
NRTI
7.6%
165/2177
NNRTI
2.9%
64/2190
Protease
1.7%
36/2178
≥2 classes
2.0%
45/2207
Wensing, van de Vijver Journal of infectious diseases 2005; 192:

45. Phylogenetic trees HIV is genetically very diverse
Identification of different groups
99% of HIV
Branch length is genetic distance ≈ number of different nucleotides

46. Group M - viruses

47. Subtype B vs. non-B
OR = 3.0 (95% CI ; P<0.001)

48. Implications
As soon as patient is diagnosed, and patient is likely to be infected with a subtype B virus
genotyping

49. Additional research Automatic subtyping tool
De Oliveira, Deforche et al. Bioinformatics Oct 1;21(19):
Subtype diversity and genetic barrier
Van de Vijver, Wensing et al. J Acquir Immune Defic Syndr Mar;41(3):
Resistance in antiretroviral experienced patients
CAPTURE

50. Conclusions Antiretroviral drugs available for treatment of HIV
Serious side effects
Drug resistance occurs frequently
Good adherence is key
Drug resistance interpretation tools have variable prediction
Drug resistance frequently limits efficacy
10% transmission of resistance