D4 HIV Resistance Testing An Update Barnett

HIV Resistance Testing: An

UpdateBen J. Barnett, MD

University of Texas Health Science Center, Houston

May 26, 2010

Outline

Overview of HIV resistance Resistance testing

Types of tests available Indications for use Test limitations

Interpretation of resistance tests New drugs and new assays

Case

54 year old man from Houston, diagnosed HIV positive 12/09 Found by “universal screening” in HCHD

ER Presenting syndrome: pneumonia

PMHx GERD, psoriasis, depression

Social Hx HIV risk: MSM Not in relationship now, casual partners

in past

Initial Evaluation

Clinically appeared well except for wasting syndrome and oral thrush

Initial laboratory tests Blood counts, liver, kidney tests normal Hepatitis B and C negative CD4 = 16 (5%) HIV viral load 650,000 copies/mL (5.81

log)

Some Considerations in starting HAART

Patient adherence potential Comorbid conditions that complicate

ARV treatment Potential drug interactions Potential adverse effects Convenience Results of genotypic drug resistance

testing Do we need to be concerned about

resistance in this case?Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. DHHS. December 1, 2009; 1-161.

Initial Genotype

“Transmitted” HIV drug resistance

Definition The presence of a resistant strain of HIV in a

person who has never taken antiretroviral drugs

Transmission Drug resistant HIV can be transmitted to a

previously HIV negative person by any way HIV is usually transmitted

Also can occur by “super-infection” but this is rare

The only way to know if someone has been infected with a resistant strain is to do a resistance test on the newly diagnosed person Called “Baseline” resistance testing

Baseline Genotypes, Thomas Street clinic1999 2003-04

Sample size 44 40CD4 288 (6-904) 274 (9-727)Time since HIV diagnosis (mo.)

17 (0-144) 39 (1-236)

NRTI mutations

2 (4.5) 2 (5)

NNRTI mutants

1 (2.3) 2 (5)

PI 1 (2.3) 1 (2.5)Total mutants

4 (9.1) 5 (12.5)

Increasing Primary Drug Resistance: CDC Survey

1. Bennett D, et al. CROI 2002. Abstract 372. 2. Bennett D, et al. CROI 2005. Abstract 674.

Prevalence of Drug Resistance, %

1998[1] (n = 257)

1999[1

] (n = 239)

2000[1]

(n = 299)

2003-2004[2]

(n = 787)

Any drug 5.5 8.8 10.7 14.5

NRTI 5.1 7.1 7.7 7.1

NNRTI 0.4 2.1 1.7 8.4

PI 0 0.8 3.0 2.8

≥ 2 drug class

0 1.3 1.3 3.1

Transmitted Drug Resistance in

US: Newly Diagnosed 2007 CDC surveillance

for transmitted drug resistance (TDR) 10,496 with new HIV

Dx 2,480 with genotype

TDR detected in 16% of patients with new HIV diagnosis Most common: NNRTI 83% had single

mutation No demographic risks

factors identified

HIV TDR Surveillance Areas (2007)Seattle-King

County

HIV TDR (2007)

Per

cent

with

TD

R50% K103N

Kim D, et al. 17th CROI; San Francisco CA USA; February 16-19, 2010. Abst. 580.

What Is HIV Resistance?

The ability of HIV to multiply in the presence of suppressive levels of antiretroviral drugs

Does not make HIV more

pathogenic Perhaps less pathogenic?

How Does Resistance Occur?

Primary transmission of resistant virus (TDR)

Sub-suppressive levels of anti-viral drugs and then natural selection of mutant strains

Partial adherence to regimen

Sub-optimal dosing of drugs

Drug interactions

Incomplete absorption in intestinal tract

Hirsch. JAMA 1998;279:1984.

Viral Resistance is the Outcome of Viral Replication, Mutations and

Selection Pressure

Viral Resistance is the Outcome of Viral Replication, Mutations and

Selection Pressure

Original VirusOriginal VirusQuasispeciesQuasispecies

Selection PressureSelection Pressureexerted by Drugsexerted by Drugs

HIV

RN

A L

evel

New VirusNew VirusQuasispeciesQuasispecies

Resistant cloneResistant cloneResistantResistant clonesclones

Time

Resistance Testing

Methods to measure resistance

Genotype Direct sequencing of viral genes reverse

transcriptase and protease, less commonly integrase and envelope

Resistance to specific drugs is predicted based on known mutations

Requires some knowledge of which mutations affect which drugs

Phenotype Grow virus in culture with various amounts of

drugs added Direct measure of viral resistance Does not explore the underlying mutations, just

their affect on the ability of the drug to stop the virus

Genotype reporting Sequencing of various HIV genes and

comparing result to reference strain Example: M 184 V in reverse

transcriptase 184 refers to amino acid position 184 in

enzyme M (methionine) is the “wild-type” amino

acid V (valine) is the “mutant” amino acid

“Mixtures” are when both WT and mutant amino acids are detected M 184 M/V

Hirsch. JAMA 1998;279:1984.

Genotypic Assays

Relative Advantages

Wide Availability Results available

in days Less technically

demanding Less expensive FDA approved

kits

Relative Limitations Indirect measure

of susceptibility

Expert interpretation required

Unable to detect linkage of multiple mutations or the overall affect of many mutations

Example of Genotype Report

Phenotypic Assays

Direct measure of viral susceptibility to individual drugs, reported as “fold change”

Drug Concentration

Inh

ibit

ion

of

Vir

al

Re

plic

ati

on

(%

) 100

0

ICIC5050

50

ICIC5050

Fold Fold ResistanceResistance

Wild-type strain

Mutant strain

Reviewed in Wilson. AIDS Read 2000;10:469.

Phenotypic AssaysRelative Advantages Direct measure

of susceptibility Fold change

in IC50

May account for effects of multiple mutations at different sites

Relative Limitations Restricted availability Results may take 2-3

weeks Technically demanding Clinically significant

cut-off values notdefined for many drugs

More expensive

Hirsch. JAMA 1998;279:1984.

Example of Phenotype Report

General Limitations of Resistance Assays

Viral load must be at least 500 - 1000 copies

Mutations are detected only if mutant virus is at least 10-20% of virus population Viral “mixtures” or minor variants can be

missed by genotypes, and not accounted for by phenotypes.

Resistance tests are most accurate in assessing the current regimen If resistance has ever been detected, then

archived mutations exist If no drug pressure exists, “wild type” virus

will often overgrow the mutant strains

Discordance between GT and PT

Incomplete genotypic algorithms (rules) Novel mutations discovered Improper weighting of mutations in

algorithms (both over- and under-weighted) Non-B subtype resistance patterns

Mixtures are present (some wild type, some mutant)

Suppressive mutations or “re-sensitization” caused by specific mutations (e.g. 184V)

Discordance due to mixtures

Result: Phenotype reads “sensitive” because of presence of wild-type virus in the mixture But Genotype predicts “resistant” due to presence of mutant virus in the mixture

Incomplete Rules may lead to discordance

Result: Genotype predicts resistance based on rules-based algorithm, but Phenotype reads “sensitive” due to interactions of complex mutation patterns

PT GT NET

“Virtual” Phenotype

DHHS Guidelines 12/09 Recommended in acute HIV

infection In chronic infection, recommended

for all patients on entry into care, regardless of treatment plan If therapy deferred, consider retesting

on treatment initiation Recommended to assist in selecting

active drugs for patients with viral failure and HIV RNA > 1000 copies Consider for viral load > 500 copies

Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. DHHS. December 1, 2009; 1-161.

DHHS Guidelines 12/09

Should be performed when managing suboptimal viral load reduction

In the setting of viral failure, testing should be done while the patient is on therapy, or within 4 weeks of stopping

Should be done for all pregnant women prior to therapy, or for those entering pregnancy with a detectable HIV viral load on therapy

Which Resistance Test When? (DHHS)

Genotype preferred due to faster result, lower cost and enhances sensitivity for detecting “mixtures” In anti-retroviral naïve patients In patients with sub-optimal viral response on therapy Virologic failure on a first or second regimen

Phenotype “Addition of phenotypic testing to genotypic testing is

generally preferred for persons with known or suspected complex drug resistance mutation patterns, particularly to protease inhibitors”

Virtual Phenotype (not specifically stated in DHHS) As a substitute when actual phenotype not available

Interpretation of Resistance Tests

Genotype interpretation

Memorize common mutations Pattern recognition Most assays come with an “expert”

interpretation included, but be careful http://hivdb.stanford.edu is a reliable resource

Limited by complex interactions of multiple mutations, especially for protease inhibitors

Newer drugs come with “prediction rules”

Mutations in Reverse Transcriptase

Mutations in Reverse Transcriptase

NucleosideMutation Sites

Non-NucleosideMutation Sites

© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.

Updates available at www.iasusa.org.

Mutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIs

Abacavir

Didanosine

Emtricitabine

Lamivudine

Stavudine

Tenofovir

Zidovudine



Mutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIs

Multi-nRTI Resistance: 69 Insertion Complex (affects all nRTIs currently approved by the US FDA)

Multi-nRTI Resistance: 151 Complex (affects all nRTIs currently approved by the US FDA except tenofovir)

Multi-nRTI Resistance: Thymidine Analogue-Associated Mutations (TAMs; affect all nRTIs currently approved by the US FDA)

Major nRTI mutations M184V

Resistance to lamivudine and emtricitabine Some resistance to didanosine and abacavir May restore some activity to zidovudine and

tenofovir K65R

Broad resistance to all nRTI except zidovudine

L74V Resistance to abacavir and didanosine

Thymidine analogue mutations (TAMS) Affect all nRTI to a variable degree

Even with nRTI resistance, most nRTI retain some activity and may help suppress virus with other agents



Mutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIs

Efavirenz

Etravirine

Nevirapine

Major NNRTI mutations K103N

Most common NNRTI mutation High level resistance to efavirenz and

nevirapine but not etravirine Y181C

High level resistance to nevirapine and intermediate efavirenz resistance

Some etravirine resistance, but provides a mutational foundation for development of higher levels of resistance

Broad cross resistance with nevirapine and efavirenz with low genetic barriers Single mutation can eliminate activity of EFV

or NVP No impact of NNRTI mutations on viral

replication ability, so continued use of NNRTI in the face of resistance adds nothing

Resistance Mutations in Protease

8282

8484

9090

4848

5454

3030



Mutations Selected by PIsMutations Selected by PIs

Atazanavir +/-ritonavir

Darunavir/ritonavir

Fosamprenavir/ritonavir

Indinavir/ritonavir

Lopinavir/ritonavir



Mutations Selected by PIs (cont)Mutations Selected by PIs (cont)

Nelfinavir

Saquinavir/ritonavir

Tipranavir/ritonavir

Major protease mutations

“Signature” mutations for non-boosted PI D30N: nelfinavir I50L: atazanavir I50V: fosamprenavir

Boosted PIs usually do not select for mutations if used as first PI with nucleosides Otherwise have broad general cross

resistance Gets very complicated very quickly

Newer protease “mutation score”

Daruavir is a newer PI with activity against resistant HIV, approved June 2006

POWER studies showed patients treated with darunavir and optimized background meds had VL < 50 copies/mL greater than for comparator PIs

Response to darunavir was found to be dependent on 11 PI mutations at baseline

Darunavir response by DRV score

0-2 mutations 3 mutations 4 mutations

50%

22%

10%Pati

en

ts (

%)

wit

h H

IV-1

R

NA

<

50

cop

ies/

mL

at

Week

24

Number of Darunavir mutations at baseline

A wrinkle: The common PI mutation at 82A may actually have a positive effect on viral response to darunavir

Pro

bab

ilit

y of

resp

on

se

Fold Change

Lower clinical cutoffResponse is significantly reduced

Upper clinical cutoffResponse is unlikely

“Zone of Intermediate Response”

Interpreting PhenotypesCutoffs differ for each drug

Newer drugs and assays

Etravirine “Second generation” NNRTI

Raltegravir Integrase inhibitor

Entry inhibitors Enfuvirtide Maraviroc

Etravirine

Second generation NNRTI FDA approved January 2008

May retain activity against HIV with NNRTI resistance from NVP or EVF K103N alone does not affect etravirine

Has a higher “genetic barrier” than other NNRTI, and therefore a “mutation score” has been developed similar to PIs



Mutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIs

Efavirenz

Etravirine

Nevirapine

TTCA0066-07332-29UN

Impact of BL Resistance in DUET: The Number of Baseline ETV RAMs Correlated with the Virologic Response (<50 copies/mL)

Number of ETV RAMs present at baseline

Placebo + OBT (n=414)

ETV + OBT (n=406)

0 2 3 41

7/28 3/18

25

17

25

41

6/2413/32

25

58

17/6837/64

38

59/15773/121

60

64/147

121/161

75

44

Pati

en

ts w

ith

con

firm

ed

vi

ral

load

<

50 H

IV-1

RN

A c

op

ies/

mL

(%

)

0

20

40

60

80

Vingerhoets J, et al. 11th EACS 2007. Abstract P7.3/05

7/28 3/186/2413/3217/6837/6459/15773/12164/147

121/161

3 or more ETV associated mutations give a reduced response to ETV

TTCA0100-08173-8UN

aMedian (Q1–Q3) FC for all isolates was 3.0 (1.1–9.3);bV179F was never present as single INTELENCE RAM (always with Y181C)

Median Q1–Q3 n

Y181I 1.5 42.0 23.2–129.7 34 12.5 High 3Y181V 0.9 10.4 3.9–60.6 28 17.4 High 3K101P 2.6 22.3 5.6–42.9 65 6.2 High 2.5L100I 8.4 6.7 2.7–17 264 1.8 Medium 2.5Y181C 32.0 4.4 2.1–11.6 552 3.9 Medium 2.5M230L 1.1 4.3 2.7–10.5 20 3.4 High 2.5E138A 2.5 2.9 1.4–10.6 44 2.0 Medium 1.5V106I 4.4 2.6 1.4–5.2 63 NA Low 1.5G190S 3.7 0.8 0.6–1.7 32 0.2 Low 1.5V179Fb 0.7 – – 0 0.1 Medium 1.5V90I 6.8 2.0 0.8–3.6 97 1.5 Low 1

V179D 2.1 1.7 1.0–4.7 33 2.6 Low 1K101E 9.9 1.5 0.8–2.5 24 1.7 Low 1K101H 2.2 1.1 0.6–2.8 8 1.3 Low 1A98G 9.5 1.0 0.5–1.9 127 2.5 Low 1V179T 0.6 0.9 0.7–1.2 2 0.8 Low 1G190A 23.3 0.8 0.5–1.5 226 0.8 Low 1

Effect on FC in linear model

Weight factor

INTELENCE FC in the subset of HIV-1 clinical isolates with 1 INTELENCE RAM

(n=1,619), regardless of the presence of other NRTI or

NNRTI RAMsa

Prevalence (%) in the panel of

4,248 HIV-1 clinical isolatesMutation

INTELENCE FC in

a single SDM

Updated List of INTELENCE RAMs: Weight Factors for 2008 INTELENCE RAMs

Vingerhoets J, et al. IHDRW 2008; Abstract 24Mutation not in table is scored 0; RAMs, resistance-associated mutations; FC, fold change

Raltegravir

First HIV “integrase” inhibitor FDA approved October 2007

Should be very little transmitted resistance as it is the first of a new class of agents

Genotype assays for integrase are now available, but clinical significance undefined

Phenotypic assay for raltegravir resistance also now available



Mutations in the Integrase Gene Associated Mutations in the Integrase Gene Associated With Resistance to Integrase InhibitorsWith Resistance to Integrase InhibitorsMutations in the Integrase Gene Associated Mutations in the Integrase Gene Associated With Resistance to Integrase InhibitorsWith Resistance to Integrase Inhibitors

Raltegravir

Raltegravir resistance Raltegravir failure is associated with

integrase mutations in at least 3 genetic pathways defined by at least 2 mutations Major mutation at 148, 155, or 143 One or more additional minor mutations

Most common and highest degree of resistance is Q148H plus G140S

Continued raltegravir in the presence of viral failure and resistance is not recommended

HIV-1 Entry Inhibitors

Virus core

Enfuvirtide

TNX-355Maraviroc

Enfuvirtide First HIV “fusion” inhibitor

FDA approved March 2003 Patients who have not taken it before

generally have virus susceptible to it Mutations in “envelope” gene region

gp41 have been seen in viruses exposed to ENF, but these mutations do not easily predict loss of anti-viral effect

Phenotypic assay is available but its clinical utility is not well defined



Mutations in the Envelope Gene Associated Mutations in the Envelope Gene Associated With Resistance to Entry InhibitorsWith Resistance to Entry InhibitorsMutations in the Envelope Gene Associated Mutations in the Envelope Gene Associated With Resistance to Entry InhibitorsWith Resistance to Entry Inhibitors

Enfuvirtide

Maraviroc

0.001 0.01 0.1 1.0 10.0

Normalized IC50 (g/mL)

ENF Naive

ENF Failure

0.001 0.01 0.1 1.0 10.0

Enfuvirtide PT Susceptibility During TORO 1 & 2

Biological Cutoff

Maraviroc

First HIV “CCR5 inhibitor” FDA approved in August 2007

Only effective against strains that use the CCR5 co-receptor for cell entry Little effect on viruses which use CXCR4

co-receptor, or which use both Activity of maraviroc is tested by co-

receptor “tropism” test, which is not a resistance test

HIV tropism defined R5 HIV: uses only CCR5 co-receptor X4 HIV: uses only CXCR4 co-receptor Dual-tropic HIV: uses either co-

receptor Mixed tropism: some R5 and some X4

virus present in the same sample

Maraviroc binds to the co-receptor, which is a HUMAN protein that cannot “mutate” in a given individual patient

Maraviroc resistance?? Emergence of X4 Virus

Switch of virus tropism from R5 to X4: unusual

Outgrowth of Dual/Mixed or X4 virus from pre-existing minority population present at levels too low to be detected by current tropism assays

Virus remains R5, with true resistance Mutations in HIV gp120 that allow HIV to bind

to CCR5 even though maraviroc is also bound Most mutations are in the V3 loop, but no

consensus yet on the specific mutations required

DHHS on New Classes The clinical utility of testing for integrase

and fusion inhibitor resistance is limited Lack of availability of second-line drugs in

class In patients failing integrase or fusion

inhibitor treatment, resistance testing may be helpful to determine whether to include these drugs in subsequent regimens, or in preparation for newer drugs in these classes

In naïve patients, there is no general recommendation to test integrase, but this may become more common in the future As yet, no documented cases of integrase TDR

Management of a treatment experienced patient

The goal of treatment for patients with prior drug exposure and resistance is to re-establish maximal viral suppression (<50 copies/mL)

Treatment history and past and current resistance tests should be used to identify fully active agents for the new regimen This is complex, and expert advice is

criticalPanel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. DHHS. December 1, 2009; 1-161.

Management of a treatment experienced patient

Adding at least two (preferably three) fully active agents to an optimized background ARV regimen can provide significant activity

For some highly treatment experienced patients, maximal viral suppression is not possible. In this case, ARV therapy should be continued with regimens designed to minimize toxicity, preserve CD4 cell counts and avoid clinical progression Expert advice is again essential

Case Follow up

Patient was started on anti-retrovirals BEFORE results of GT were returned Baseline VL was 650,000 Initial regimen was 2 nRTI and boosted-

PI

Resistance tests repeated Genotype, virtual phenotype, tropsim test

DATE Viral Load CD4 Meds

1/14/10 650,000 16 Naïve

2/5/10 3,230 2nRTI, PI/r

2/22/10 595,000 175 Same

Resistance tests for Case

Case follow up

Regimen was then changed to Tenofovir/emtricitabine Darunavir/r 600/100 mg BID

(resistance dose) Added raltegravir Added etravirineDATE Viral Load CD4 Meds

1/14/10 650,000 16 Naïve

2/5/10 3,230 2nRTI, PI/r

2/22/10 595,000 175 Same

4/14/10 1,150 193 Above, one month

Conclusions Resistance can occur in patients new to

ARV Resistance testing can be used to optimize

an antiretroviral regimen Must use in context of treatment history and

results of all prior resistance tests Goal for all HIV infected patients is HIV RNA <

50 Factors other than resistance may

cause regimen failure Resistance testing is reliable and cost-

effective but must be interpreted in context and may require expert advice

More information IAS-USA mutation cards and notes

http://www.iasusa.org Update of the Drug Resistance

Mutations in HIV-1: December 2009 Topics HIV Med. 17(5):138-145.

Stanford resistance database http://hivdb.stanford.edu

DHHS guidelines for HIV http://www.aidsinfo.nih.gov/guidelines/

D4 HIV Resistance Testing An Update Barnett

Health & Medicine

Transcript of D4 HIV Resistance Testing An Update Barnett