HIV-1 Drug Resistance Testing:A Practical Discussion

Lee T. Bacheler, PhDVP Clinical Virology

VircoLab, Inc

Durham, NC

Topics for today

• National Guidelines for use of resistance testing

• Types of resistance testing– genotype

– conventional phenotype

– virtualPhenotypeTM-LM

• A tour of the vircoTYPE HIV-1 report• Some Illustrative examples

DHHS Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents - October 10, 2006

Similar recommendations from IAS-USA

Genotypicsequence

MeasuredFold Change

(FC)

CalculatedFold Change

(FC)

All Resistance Tests begin by Amplifying the Patient’s Virus Population

Genotype Test

• Determine the genetic composition of patient’s virus

• Compare the genetic composition of patient’s virus to the genetic composition of wild type virus= identify mutations

• Use established algorithms to determine which mutations are associated with drug resistance

Test Result

Analysis of Sequencing Results

Sample Genotype Report

Conventional Phenotype Test

• ‘Grow’ patient’s virus in the presence of drug

• Determine the amount drug required to suppress 50% viral growth = IC50

• Compare the IC50 of the patient’s virus to the IC50 of a wild type virus = Fold Change

• “Clinical” or “Biological” cutoff to define drug resistance

• Commercially Available Tests:

– Antivirogram (Virco)

– PhenoSense (Monogram)

– Phenoscript (Viralliance)

The Antivirogram® Assay Step by Step

PATIENT

PLASMA(> 200 µl)

total RNA cDNA gag//PRO/RT GENES(amplicon)

extraction

RT 2x PCR

Creation of deleted molecular clone

WT molecular clone (HXB2)

gag, PRO, RT

Delete gag, PRO & RT genes

Deleted molecular clone

CD4+ T-cells (MT4)

Infectious RECOMBINANT

virusSusceptibility assay(2-3 complete viral replication cycles)

gag (p7/p1-p1/p6); PRO(1-99); RT (1-400)

Gene transfer (nucleofection)

ANTIVIROGRAM®: susceptibility assay

Res.

Res.

WT

WT

3TC

0.0064 0.032 40.80.16 20 100 µM0

AZT

Drug Concentration (µM)Drug Concentration (µM)

100100

5050

00lowlow ICIC5050 ICIC5050

Wild-type isolate

highhigh

9090Patient isolate

<<

10x more

Fold Change in IC50

IC50 100 µM

IC50 10 µM =10

% In

hib

itio

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IV R

ep

licat

ion

% In

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IV R

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Sample Conventional Phenotype Report Antivirogram (Virco)

HIV-1 Resistance Testing Technologies

Phenotyping Assay

• Direct measure of the ability of the virus to grow in the presence of Antiretroviral Drugs

• Quantitative

• Simple interpretation based on (quantitative) fold-change and cut-off values (Biological or Clinical)

• Less sensitive than genotype for the early detection of mutants

• Costly and long TAT

Genotyping Assay

• Indirect measure of the virus’ susceptibility to Antiretroviral Drugs based on sequence (mutations) of relevant parts of the viral genome

• Qualitative

• Requires sophisticated analysis & interpretation of sequence information

• Early detection of mutants

• Affordable and rapid TAT

RESISTANCE

• The ability of HIV to replicate in the presence of Antiretroviral Drugs• Caused by changes in relevant parts of the virus genome (mutations)

Predicting the Drug Susceptibility Phenotype from the Viral Genotype

virco®NET

5-60 minutes

•For most US customers, genotyping is done locally (eg LabCorps)•The resulting nucleic acid sequence is sent to Belgium for vircoTYPE analysis

virco®TYPE HIV-1 report

Re

pea

t fo

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ch

dru

g

GENOTYPE(nucleotide sequence)

Compare to reference sequence

Identify all mutations in sample virus

Identify all mutations and mutation pairs in sample virus that contribute to resistance

to a given drug according to LM

For each mutation or mutation pair in the sample virus that contributes to resistance, retrieve the Resistance Weight Factor

Add all Resistance Weight Factors to obtain a predicted Fold Change

Every 2 months

G/P correlative database

Resistance Weight Factors(drug specific)

LM

Virco’s correlative database

Genotypes Phenotypes

Multiple Linear Regression Analysis

Determine which mutations contribute to resistance for each ARV, and by how much (Resistance Weight Factor)

[Note: RWF’s are re-calculated after every database update (2-monthly)]

Obtaining Resistance Weight Factors

Virco’s Databases*

Clinical Outcomes Database(>17,000 patients on therapy)

• Routine clinical testing• Clinical trials• Research collaborations

Genotypicdata >285,000

Phenotypicdata >83,000

VirtualPhenotype™-LM engine

>50,000 matched G/P samples

Predicted fold change

values in IC50

Nucleotide sequence (…AAGTCTCCGCATGCATA…)

* Status December 06

FC Assessment: Example of TipranavirPI Mutation Analysis

10F & 47V 22V & 84V 34Q & 84V 36L 46L & 82T 53L 54V 74S

10F & 82A 24I & 82A 35D & 36I 36L & 58E 47V & 54M 54A & 55R 54V & 70E 76V

10F & 84V 24I & 82T 35D & 54A 36L & 83D 47V & 54V 54A & 84V 54V & 74P 82A & 84V

10V 30N 35D & 54V 36L & 95F 47V & 83D 54L 54V & 84V 82C

10V & 33F 30N & 74S 35D & 58E 38W 47V & 84V 54L & 82A 58E & 84V 82L

10V & 88D 33F 35D & 73T 41K 48A & 71V 54M 60E 82T

13V & 69K 33F & 82A 35G & 71V 41T 48M & 53L 54M & 74S 60F 84V

13V & 71V 33I & 36I 35N & 84V 43T 48V 54M & 82A 69K 85V

13V & 82S 33M 36I & 47V 43T & 82T 48V & 54V 54S 71V & 73T 88D

13V & 84V 33V 36I & 54T 46L & 53L 50L 54S & 82T 71V & 95F 90M

20R 33V & 84V 36I & 84V 46L & 71V 50V 54V 74P & 82A

10F, 13wt/V, 32I, 33F, 41K, 46I, 58E, 63P, 71wt/V, 73S, 77I, 84V, 89V, 90M, 93L

FC assessment: Example of TipranavirMutation Analysis: Defining Fold-Change

Mutations

33F41K84V90M

10F & 84V13V & 71V13V & 84V58E & 84V

no mut

10F, 13wt/V, 32I, 33F, 41K, 46I, 58E, 63P, 71wt/V, 73S, 77I, 84V, 89V, 90M, 93L

RWF(adjusted for mixtures)

0.217-0.0410.1490.061

-0.1740.0160.0430.079

-0.099

FC = 10 0.252 = 1.8

Log(FC) = 0.252

RWF*

0.217-0.0410.1490.061

-0.1740.0640.0870.079

-0.099

*Resistance Weight Factor: Weight and Direction for mutations which Impact TPV

virco®TYPE HIV-1 v4.1.00Powered byVirtualPhenotype™-LM

How to Read and Use the Report

Page 1Summary Report

Page 2Detailed Report

Page 3Definitions and

Disclaimers

virco®TYPE HIV-1 V4.1.00

1. Resistance-associated mutations2. VirtualPhenotype™-LM predicted Fold Change in IC50

3. Cut-Offs (BCO or CCO)4. Resistance Analysis5. Reference to Additional Clinical Notes

BCO: Biological Cut OffsCCO: Clinical Cut Offs

Page 1: Summary Report (1)

1

1

1

2 3 4 5

Page 1: Summary Report (2)

• Mutations– Mutations that are known to be associated with

resistance / treatment response and are listed on the report

– Mixtures• 4 amino acids: all residues are printed

– e.g. 41 wt/L = mixture of Wild Type (Methionine) and Leucine at position 41 in reverse Transcriptase

– 5 or more amino acids: printed as ‘X’

– e.g. 215X = more than 4 residues detected at position 215 in RT

CCO1

(low)CCO2

(high)FC value Resistance Analysis

BCO

Page 1: Summary Report (3)

• Cut-Off values– 2 Clinical Cut-Offs (CCO1 and CCO2) if available– 1 Biological Cut-Off (BCO), if CCO not available

• BCO printed in italics

Terminology Used in the Resistance Analysis

Page 2: Detailed Report (1)

ResistanceAnalysis

Details

AdditionalClinical

Notes

Key

1. Resistance-associated mutations2. Graphical representation of the Resistance Continuum3. Predicted Fold change in IC50, and 95% confidence limits4. Cut-Off values

1

1

1

2 3 4

Page 2: Detailed Report (2)

Page 2: Detailed Report (3)

CCO1 CCO2

Patient Virus FC

BCO

Page 2: Detailed Report (1)

ResistanceAnalysis

Details

AdditionalClinical

Notes

Key

Case Condition Drug Text

215 Revertants

T215A/C/D/E/G/H/I/L/N/S/V detected AND T215F and/or T215Y not detected

AZT

d4T

Mutations T215A,C,D,E,G,H,I,L,N,S or V in the RT gene can arise as revertants of resistance associated mutations T215Y or F, and may lead to reduced response to AZT and d4T treatment

NNRTI Cross-

resistance

Predicted Resistance to one NNRTI (EFV or NVP) AND susceptibility to the other

EFV

NVP

Cross resistance among EFV and NVP resistant viruses is extensive. Resistance to one of these drugs usually leads to reduced response to all currently approved NNRTIs

Additional Clinical Notes (1)

Page 3: Definitions and Disclaimers (1)

A Resistance Test Result for a Naïve Patient

A Resistance Report for a Patient With a Mixed Virus Population

In virtualPhenotype – LM 4.1, the contribution of mutations in a mixture is divided across the number of amino acids in the mixture Most accurate prediction of the measured phenotype

A Resistance Report for a Patient With a Mixed Virus Population

Standing Definition on pg 3

• In virtualPhenotype – LM 4.2, only the contribution of the mutation with the highest level of resistance in a mixture will be taken into account “Worst Case Scenerio”

A Resistance Report for a Patient With Extensive Drug Resistance: What should we do next?

What about other classes of Antiretroviral Drugs?

• Fusion inhibitors (enfuvirtide, T-20)– Resistance develops rapidly if virus replicates presence of T-2O– Resistance testing generally not utilized in patient management

• Integrase Inhibitors ( – Virologic failure associated with emergence of resistance mutations– Cross resistance between different inhibitors in the class is likely to be

important– No currently available commercial assays

• Tropism (maraviroc)– Not a resistance test per se– Used to Identify patients without detectable CXCR4 tropic virus who

could be candidates for use of a CCR5 antagonist– Commercially available assays

• Trofile™ ( Monogram)• SensiTrop™ HIV Co-Receptor Tropism Assay ( Pathway

Diagnostics, available through Quest, Mayo Reference Labs)

Summary:

• Use of HIV-1 drug resistance testing is recommended in a variety of settings

• Three types of resistance tests: genotype, phenotype, and virtual phenotype

• Tour of the vircoTYPE report• Examples

– Transmitted drug resistance

– Mixed virus populations

– Extensive resistance to approved drugs