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

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

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

Introduction to HIV

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

Clinical course of HIV – no drugs

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

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

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

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

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

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

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

Lipodystrophia Associated with usage of protease inhibitors

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

Introduction to resistance

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

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

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=0.00001, r=–0.554 Paterson DL et al. Ann Intern Med 2000

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: 229-33)

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

Lamivudine mutation aminoacid 184 M > V R. Shafer, Clin Microbiol Rev 2002; 15: 247-277

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

Fitness Lamivudine Viral load Time M184V Schuurman R, et al. J Infect Dis 1995; 171: 1411-9

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

Sequencing +ddATP +ddCTP +ddGTP+ddTTP

Sequence

Mutations

Amino-acids Computer-software to detect mutations

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

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 © 2004. The International AIDS Society–USA Johnson VA, et al. Topics HIV Med. 2004. Updated on www.iasusa.org. 1. D’Aquila RT, Schapiro JM, Brun-Vezinet F, et al. Drug resistance mutations in HIV-1. Top HIV Med. 2002;10:11-15. ´

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

Retrogram M184V

Several systems exist… De Luca et al. Journal of infectious diseases 2003; 187: 1934-43

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

Epidemiological research

Transmission of resistance Drug resistant HIV can be transmitted to others USA: San Diego/ San Francisco 25% Little et al. NEJM 2002; 347: 385-94 Grant et al. JAMA 2002; 288: 181-8 Europe: until recently studies limited to single countries, different methodologies

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

32 Participating countries SPREAD 32 Participating countries www.spread-europe.org SPREAD is sponsored by the European Union (contract number QLK2-CT-2001-01344) Extended as EuropeHIVResistance WHO HIVResNet

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: 958-66

CATCH-study

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

Results 1996-2002 N=2208 Prevalence Resistance 10.4% 230/2208 NRTI SPREAD Results 1996-2002 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: 958-66.

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

Group M - viruses

Subtype B vs. non-B OR = 3.0 (95% CI 2.0-4.4; P<0.001)

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

Additional research Automatic subtyping tool De Oliveira, Deforche et al. Bioinformatics. 2005 Oct 1;21(19):3797-800 Subtype diversity and genetic barrier Van de Vijver, Wensing et al. J Acquir Immune Defic Syndr. 2006 Mar;41(3):352-60. Resistance in antiretroviral experienced patients CAPTURE

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