HIV-1 Antiretroviral Drug Resistance and Resistance Testing HIV and ARV resistance October 2009 Dr...

HIV-1 Antiretroviral HIV-1 Antiretroviral Drug Resistance and Drug Resistance and Resistance TestingResistance Testing

HIV and ARV resistance October 2009

Dr Michelle Gordon October 2009

Evolution of Viral MutationsEvolution of Viral Mutations

Mutations arise because HIV-1 RT makes Mutations arise because HIV-1 RT makes spontaneous errors (1 in 10spontaneous errors (1 in 1044))

HIV-1 genome is 10 000 (HIV-1 genome is 10 000 (101044)) bases long, bases long, therefore 1 error each time the genome is therefore 1 error each time the genome is replicatedreplicated

Production of virus = 10Production of virus = 1099 to 10 to 101010 virions per virions per day day quasispecies quasispecies

Every possible mutation present in Every possible mutation present in quasispecies before ARV therapyquasispecies before ARV therapy


How drug resistance arises. Richman, DD. Scientific American , July 1998

How drug resistance arisesHow drug resistance arises


Resistant mutants emerge as a result of Resistant mutants emerge as a result of genetic barrier (selective pressure of a regimen).genetic barrier (selective pressure of a regimen). residual replication (potency of a regimen).residual replication (potency of a regimen).

Drugs differ with respect to their genetic barrier and time it Drugs differ with respect to their genetic barrier and time it takes to develop resistance.takes to develop resistance.

1 nt change 1 nt change → aa change → aa change → → resistance = LOW genetic barrier resistance = LOW genetic barrier >1 nt change at 1>1 nt change at 1stst and 2nd codon position and 2nd codon position → aa change → aa change → resistance→ resistance = LOW = LOW

genetic barrier, but slightly longer to emergegenetic barrier, but slightly longer to emerge >1 mutation (accumulation) >1 mutation (accumulation) → → resistance = higher genetic barrier resistance = higher genetic barrier

Escape mutants will continue to replicate and develop additional Escape mutants will continue to replicate and develop additional (secondary/compensatory) mutations to:(secondary/compensatory) mutations to:

further increase resistance (decreased drug susceptibility)further increase resistance (decreased drug susceptibility) increase viral fitness (“compensatory mutations”)increase viral fitness (“compensatory mutations”)

Emergence of resistanceEmergence of resistance


Disappearance of Disappearance of resistanceresistance

When treatment interruptedWhen treatment interrupted

No drug selection of quasispeciesNo drug selection of quasispecies

Wild-type becomes dominantWild-type becomes dominant

Drug-resistant strains no longer Drug-resistant strains no longer detected by genotyping assaysdetected by genotyping assays

Still minority population that can re-Still minority population that can re-emerge if selection pressure re-emerge if selection pressure re-appliedapplied


Antiretroviral drugsAntiretroviral drugs

5 classes drugs5 classes drugs

PR Inhibitors (PIs) (9)PR Inhibitors (PIs) (9)

Nucleoside/nucleotide RT Inhibitors (NRTIs) Nucleoside/nucleotide RT Inhibitors (NRTIs) (8)(8)

Non-nucleoside RT Inhibitors (NNRTIs) (4)Non-nucleoside RT Inhibitors (NNRTIs) (4)

Fusion Inhibitors (2)Fusion Inhibitors (2)

Integrase Inhibitors (1)Integrase Inhibitors (1)

Current therapies imperfect because:Current therapies imperfect because:Regimen complexitiesRegimen complexitiesToxicitiesToxicitiesDrug resistanceDrug resistance


Relationship between drug Relationship between drug concentration, viral suppression and concentration, viral suppression and adverse effectsadverse effects

adequate drug concentrations are need in order to bring about desired pharmacological and virological effects.


• Targets HIV-1 PRTargets HIV-1 PR

• Attaches to the PR binding cleft that Attaches to the PR binding cleft that recognizes and cleaves precursor recognizes and cleaves precursor polyproteins. polyproteins.

Most PR resistance mutations alter the Most PR resistance mutations alter the structure of the substrate cleft structure of the substrate cleft

Causes resistance by reducing the binding affinity Causes resistance by reducing the binding affinity between the inhibitor and the mutant protease between the inhibitor and the mutant protease enzyme. enzyme.

Mutations also occur in the flaps Mutations also occur in the flaps • compensate for loss of fitnesscompensate for loss of fitness

PIs and ResistancePIs and Resistance

Dr Michelle Gordon August 2006

PI drug interactionsPI drug interactions

PIs are metabolized by CYP 3A4 and P450 (PIs are metabolized by CYP 3A4 and P450 (the body’s the body’s drug clearance mechanisms).drug clearance mechanisms).

Ritonovir (RTV) is one of the most potent CYP 3A4 Ritonovir (RTV) is one of the most potent CYP 3A4 inhibitors known.inhibitors known.

Most PIs are co-administered with sub-therapeutic Most PIs are co-administered with sub-therapeutic doses of RTV doses of RTV thereby increasing the plasma levels of thereby increasing the plasma levels of the PI.the PI.

Boosted PI levels Boosted PI levels can overcome small reductions in can overcome small reductions in susceptibility conferred by early PI mutations, thus susceptibility conferred by early PI mutations, thus prolonging viral suppression. prolonging viral suppression.

NNRTI’s (NVP and EFV) and TB drugs are inducers of NNRTI’s (NVP and EFV) and TB drugs are inducers of CYP 450 and affect PI concentrations.CYP 450 and affect PI concentrations.


NRTIsNRTIs

NRTIs are ddNTPsNRTIs are ddNTPs

Phosphorylated NRTIs Phosphorylated NRTIs compete with natural compete with natural dNTPs for dNTPs for incorporation into the incorporation into the newly synthesized DNA newly synthesized DNA chains - cause chain chains - cause chain termination termination

Nucleosides require 3 Nucleosides require 3 phosphates; nucleotides phosphates; nucleotides require 2 phosphatesrequire 2 phosphates


Chain termination with NRTIsChain termination with NRTIs

If AZT was the last nt added, then the RT enzyme cannot add new nucleotides


PyrophosphorylationPyrophosphorylation•After incorporation of a nucleoside, two released phosphates may After incorporation of a nucleoside, two released phosphates may attack the link, causing the nucloside to be released again. attack the link, causing the nucloside to be released again.

NRTIs and ResistanceNRTIs and Resistance


The non-nucleoside RT inhibitors (NNRTIs) bind to a The non-nucleoside RT inhibitors (NNRTIs) bind to a hydrophobic pocket (called the NNRTI-binding hydrophobic pocket (called the NNRTI-binding pocket)pocket)

NNRTIs inhibit replication by displacing the enzyme NNRTIs inhibit replication by displacing the enzyme active site relative to the polymerase binding siteactive site relative to the polymerase binding site

A single mutation in the NNRTI-binding pocket A single mutation in the NNRTI-binding pocket may result in high-level resistance to one or more may result in high-level resistance to one or more NNRTIs (low genetic barrier). NNRTIs (low genetic barrier).

NNRTIs and NNRTIs and ResistanceResistance


Fusion InhibitorsFusion Inhibitors

• During HIV-1 infection, the virus’s gp120 binds to both a During HIV-1 infection, the virus’s gp120 binds to both a CD4 and chemokine receptor (CCR5 or CXCR4) on the CD4 and chemokine receptor (CCR5 or CXCR4) on the target cellstarget cells

• causes a conformational change in gp120causes a conformational change in gp120

• promotes the fusion of the viral and cellular membranes promotes the fusion of the viral and cellular membranes


This class of drugs interferes with the binding, fusion This class of drugs interferes with the binding, fusion and entry of an HIV virion to a human (host) cell.and entry of an HIV virion to a human (host) cell.

Enfuvirtide (Fuseon)Enfuvirtide (Fuseon)•Binds to gp41 transmembrane protein and blocks fusion Binds to gp41 transmembrane protein and blocks fusion of hiv-1 to the host cell.of hiv-1 to the host cell.

Maraviroc (Selzentry; Celsentri )Maraviroc (Selzentry; Celsentri )•Binds to CCR5, preventing an interaction with gp120. Binds to CCR5, preventing an interaction with gp120.

Fusion InhibitorsFusion Inhibitors


Integrase InhibitorsIntegrase Inhibitors

Integrase is one of three viral enzymes necessary Integrase is one of three viral enzymes necessary for HIV replication that integrates or blends HIV for HIV replication that integrates or blends HIV genetic material into the DNA of human CD4 cells. genetic material into the DNA of human CD4 cells.

This makes it possible for the infected cell to This makes it possible for the infected cell to make new copies of HIV make new copies of HIV

Isentress (raltegravir) Isentress (raltegravir) By interfering with integrase, the integrase By interfering with integrase, the integrase

inhibitors prevent HIV genetic material from inhibitors prevent HIV genetic material from integrating into the CD4 cell, thus stopping HIV integrating into the CD4 cell, thus stopping HIV replication. replication.


Mutations associated with resistance to Mutations associated with resistance to PIsPIs


http://hivdb.stanford.edu/http://hivdb.stanford.edu/

Mutations associated with resistance toMutations associated with resistance to NRTIsNRTIs



Mutations associated with resistance to NNRTIsMutations associated with resistance to NNRTIs



Mutations associated with resistance to Fusion InhibitorsMutations associated with resistance to Fusion Inhibitors



Mutations associated with resistance to Integrase InhibitorsMutations associated with resistance to Integrase Inhibitors



How do you measure drug How do you measure drug resistance?resistance?

Genotyping:Genotyping:

Indirect assay: Detects drug resistance mutations Indirect assay: Detects drug resistance mutations that are present in the relevant virus genes.that are present in the relevant virus genes.

Phenotyping:Phenotyping:

Direct assay: Measures the ability of the virus to Direct assay: Measures the ability of the virus to grow in various concentrations of antiretroviral grow in various concentrations of antiretroviral drugs.drugs.


Resistance TestingResistance Testing

1.1. Genotypic TestingGenotypic TestingMore commonly used in clinical settingsMore commonly used in clinical settingswider availability, lower cost and quicker turnaround wider availability, lower cost and quicker turnaround (1-2 weeks)(1-2 weeks)

PI and RTISPI and RTIS HIV-1 GenotypR PLUS (Specialty Laboratories)HIV-1 GenotypR PLUS (Specialty Laboratories) TRUGENE HIV-1 genotyping testTRUGENE HIV-1 genotyping test (Bayer) (Bayer) VircoGEN II (Virco)VircoGEN II (Virco) Viroseq HIV genotyping systemViroseq HIV genotyping system (Abbott Diagnostics) (Abbott Diagnostics) GeneSeq (ViroLogic)GeneSeq (ViroLogic) HIV-1 Mutation Analysis (Focus Technologies)HIV-1 Mutation Analysis (Focus Technologies) HIV ViroTYPE (Rheumatology Diagnostoc Laboratory)HIV ViroTYPE (Rheumatology Diagnostoc Laboratory) GenoSure (LabCorp and Virco)GenoSure (LabCorp and Virco)

Fusion InhibitorsFusion Inhibitors TRUGENE HIV-1 Envelope (gp41) Genotyping Assay (Bayer)TRUGENE HIV-1 Envelope (gp41) Genotyping Assay (Bayer) HIV GenoSure Fusion (LabCorp)HIV GenoSure Fusion (LabCorp)


Genotyping: AdvantagesGenotyping: Advantages

Identification of all nucleotides, amino acid differences, deletions & insertions

Genotyping has the ability to detect resistant virus that constitutes only a small proportion (about 20%) of the viral population.

This can provide “predictive” early warning of developing resistance before full resistance develops

Faster and less expensive than phenotype assay


Genotyping: DisadvantagesGenotyping: Disadvantages

Reports may be difficult to interpret unless clinician is very experienced

Labs use different software programs to predict resistance - need a consensus on which mutations are important

There is a lot of variation in the quality of the “product” from different laboratories especially in the ability to detect minority species in the population

Current limitation of use is that viral load needs to 1000 copies/ml - need greater sensitivity


Phenotyping: AdvantagesPhenotyping: Advantages

Provides resistance information on each drug regardless of the presence of multiple mutations

Interpretation may be more intuitive than for genotype assay

Very useful in patients with complex drug history and complicated mutation profile

Very useful for deciphering cross-resistance

May be more useful than genotyping for new drugs until appropriate mutations are established by clinical data


Phenotyping: DisadvantagesPhenotyping: Disadvantages

If drug resistant population is minor the phenotypic effect may not be detected

Current limitation of use is that viral load needs to 1000 copies/ml - need greater sensitivity

Very expensive and time-consuming

The relevance of small changes in drug sensitivity not yet fully determined - drugs to which patient is actually still sensitive may be unnecessarily eliminated


There is continued effort to develop cheaper in-house assays for resistance genotyping.

However, there needs to be some mechanism of standardization between laboratories.

Also, because all in-house methods are nested reactions (with an increased chance of contamination) there needs to be the same level of adherence to quality control.

In-house genotyping assaysIn-house genotyping assays

Genotyping kitsGenotyping kits• Already optimizedAlready optimized• Standardized Standardized • Built-in QC procedures Built-in QC procedures • Thus, prevent possible sample Thus, prevent possible sample

to sample contaminationto sample contamination• Very expensive Very expensive • Insensitive to presence of Insensitive to presence of

minor variants minor variants • Interpretation requires prior Interpretation requires prior

knowledge of genetic knowledge of genetic determinants of resistance determinants of resistance

In-house assaysIn-house assays• Most require optimizationMost require optimization• Not standardized btw labsNot standardized btw labs• Not usually have built-in Not usually have built-in

QCQC• Increased risk of Increased risk of

contaminationcontamination• Much cheaperMuch cheaper• More sensitive, because a More sensitive, because a

nested reactionnested reaction• Interpretation the sameInterpretation the same

Kits vs In-house assaysKits vs In-house assays

1.8kb1.8kb

Load samples onto ABI 3100Load samples onto ABI 3100

AA BB

CCFF

GG HH

proteaseprotease RTRT

plasmaplasma dried blood spotdried blood spot

Genotyping using the Viroseq Genotyping using the Viroseq KitKit


Sequence AnalysisSequence Analysis


Interpretation of Genotypic Resistance Interpretation of Genotypic Resistance TestsTests

• Interpretation complicated by complex patterns of Interpretation complicated by complex patterns of mutationsmutations

2 basic approaches:2 basic approaches:

i)i) Rules-based algorithms: Rules-based algorithms:

Developed by experts;Developed by experts;

Based on published data on phenotypic impact and Based on published data on phenotypic impact and clinical significance of drug resistance mutationsclinical significance of drug resistance mutations

• TruGene (Bayer)TruGene (Bayer)

• Viroseq (Celera)Viroseq (Celera)

• HIVdb (Stanford)HIVdb (Stanford)

• ANRS (French)ANRS (French)

• Rega (Belgium)Rega (Belgium)


ii) Machine-learning algorithmsii) Machine-learning algorithms

Contain rules discovered by a computer program by Contain rules discovered by a computer program by analyzing data linking genotype to phenotype or clinical analyzing data linking genotype to phenotype or clinical outcome.outcome.

Learn from a training data set and test performance using a Learn from a training data set and test performance using a test data settest data set

• Decision treesDecision trees

• Categorical analysis and regression trees (CART)Categorical analysis and regression trees (CART)

• Neural networksNeural networks

• Virtual phenotypeVirtual phenotype

Interpretation of Genotypic Resistance Interpretation of Genotypic Resistance TestsTests


http://hivdb.stanford.edu/http://hivdb.stanford.edu/Dr Michelle Gordon October 2009

Limitations of Drug Resistance Limitations of Drug Resistance TestingTesting

i) Relationship between drug resistance and clinical failure i) Relationship between drug resistance and clinical failure is complexis complex

Non-adherenceNon-adherence Sub-optimal regimensSub-optimal regimens PharmacokineticsPharmacokinetics Host factorsHost factors Many drug resistant variants are less fitMany drug resistant variants are less fit

ii) Complex quasispeciesii) Complex quasispecies

iii) Cross-resistanceiii) Cross-resistance

iv) Potentially miss minor populationsiv) Potentially miss minor populations


AdheranceAdherance

New DrugsNew Drugs

Salvage TherapySalvage Therapy

IntensificationIntensification

Overcoming ResistanceOvercoming Resistance


ConclusionConclusion

Resistance-conferring mutations occur continuously, in Resistance-conferring mutations occur continuously, in absence and presence of drug therapy.absence and presence of drug therapy.

Occur more rapidly when viral load is high: more Occur more rapidly when viral load is high: more replication, more mutations.replication, more mutations.

Mutant strains become dominant under drug selection Mutant strains become dominant under drug selection pressure.pressure.

Resistance testing gives information on which drugs no Resistance testing gives information on which drugs no longer potent in regimen. longer potent in regimen.

Resistance testing limited because rResistance testing limited because relationship between elationship between drug resistance and clinical failure is complex; may miss drug resistance and clinical failure is complex; may miss minor populations; cross-resistance.minor populations; cross-resistance.

Understanding resistance is important for optimal patient Understanding resistance is important for optimal patient management!management!


ReferencesReferences

http://hivdb.stanford.edu http://www.hivresistanceweb.com http://hiv.medscape.com/Home/Topics/AIDS/AIDS.html http://www.hivatis.org http://hiv-lanl.gov/seq-db.html


http://hivdb.stanford.edu/

http://www.hivresistanceweb.com/

http://hiv.medscape.com/Home/Topics/AIDS/AIDS.html

http://www.hivatis.org/

http://hiv-lanl.gov/seq-db.html

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