HIV Resistance Basics

Michael J. Harbour, MDClinical Assistant Professor

Stanford University School of MedicineStanford Positive Care Clinic

19 Drugs – How to Pick the Best Regimen?

3

The AIDS pandemicAdults and children living with HIV/AIDS, end 2003

• 5 Million new infections in 2003• 3 Million deaths due to HIV/AIDS in 2003• 40 Million living with HIV/AIDS; 50% Female

North AmericaNorth America790,000-1.2M790,000-1.2M

CaribbeanCaribbean350-590,000350-590,000

Latin AmericaLatin America1.3-1.9 M1.3-1.9 M

North AfricaNorth Africa& Middle East& Middle East470-730K470-730K

Sub-Saharan AfricaSub-Saharan Africa25-28.2 M25-28.2 M

East Asia East Asia & Pacific& Pacific

700k-1.3M700k-1.3M

S & SE AsiaS & SE Asia4.6-8.2M4.6-8.2M

AustraliaAustralia& New Zealand& New Zealand

12-18K12-18K

Western EuropeWestern Europe520-680K520-680K

Eastern EuropeEastern Europe& Central Asia& Central Asia

1,2-1.8M1,2-1.8M

700-1K

34-54K

30-40k

120-180k

610-1.1M

3-3.4M

45-80K

43-67k

180-280K

150-270K

2002-03 increase

Source: USAIDS

Leading Causes of Death 1987-2000:Leading Causes of Death 1987-2000:Persons 25-44 Years of AgePersons 25-44 Years of Age

0

5

10

15

20

25

30

35

40

87 88 89 90 91 92 93 94 95 96 97 98 99 '00

CDC: Preliminary Mortality data for 2000.

Unintentional injury

Cancer

Heart disease

Suicide

HIV infectionHomicide

Chronic liver diseaseStrokeDiabetes

Dea

ths/

100,

000

Po

pu

lati

on

Year

6

High-risk sex in HIV+ adults with known drug-resistant HIV

• SCOPE (Study of the Consequences Of the Protease inhibitor Era) cohort in San Francisco

• 168 patients on treatment but viremic and with genotypically proven drug-resistant HIV

Factor Proportion engaging in unprotected sex

OddsRatio p

Age <35 years 60% 8.8 <0.01

Sildenafil use 37% 5.4 <0.01

Depression 23% 1.9 <0.01

Chin-Hong PV, et al. 11th CROI, San Francisco 2004, #845

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Seroconversion for MSM in the VaxGen trial

• 4510 MSM participating in the VaxGen 004 Phase III study1

1. Ackers M, et al. 11th CROI, San Francisco 2004, #857; 2. Shepherd B, et al. ibid, #284

Adjusted hazard ratio

Seroconversion less likely:

Aged 41-50 (vs age <41) 0.58

Aged 50+ 0.45*

Seroconversion more likely:

>10 sexual partners 2.1

Use of amphetamines 1.9

Sex with an HIV+ partner 1.9

Use of poppers 1.7

Unprotected anal sex 1.4

All p<0.0001 except *p<0.01

• AIDSVAX had no influence on pretreatment HIV RNA set-point in those who seroconverted2

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HIV superinfection

• 1 of 32 (3.1%) newly infected subjects from the MACS6

– CD4+ progressed to <200 cells/mm3 2.4 years postinfection

• Implications:– Counseling of HIV-infected partners

– Concern regarding vaccine strategies

1. Altfeld M, et al. Nature 2002;420:434; 2. Jost S, et al. NEJM 2002;347:731; 3. Koelsch K, et al. AIDS 2003;17:F11; 4. Ramos A, et al. J Virol 2002;76:7444; 5. Smith D, et al. 11th CROI, San Francisco 2004, #21; 6. Gottlieb G, et al. ibid, #454

Mean change in HIV RNA and CD4+ after superinfection

ΔRNA(log10 c/mL)

ΔCD4+ (cells/mm3)

p=0.05 vs controls without superinfection

+1.6 log10 c/mL

–132 cells/mm3

• Superinfection recently described in the literature1−4

• 3 of 78 (4.1%) patients in the first 6 to 20 months of infection in San Diego and Los Angeles5

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Evolving high-risk groups

San Francisco4

• Rising unprotected anal sex in Asian MSM

1. Millett G. 11th CROI, San Francisco 2004, #83; 2. Hightow LB, et al. ibid, #84; 3. Fitzpatrick L, et al. ibid, #85LB; 4. Troung HM, et al. ibid, #844

Screening and Tracing Active Transmission (“STAT”)2,3

• Cases of HIV among 18−30 year old men (n=998) in North Carolina attending college rose from 4% (late 2001) to 15% (2003)

• More likely to be African-American, have acute/recent infection, have sex with men and women, use ecstasy, travel outside of NC

• 73% of HIV+ felt they were at low risk for HIV acquisition

Men on the Down Low (“DL”)1

• Heterosexually identified black men who have sex with men but do not tell their female partners

– Don’t identify with gay subculture

– Usually unaware or non-disclosing of their HIV status

GENESEQ™ AND PHENOSENSE™ COMPLEMENTARY TECHNOLOGIES

GeneSeq™

Patient virus

PR-RT DNA

RT-PCR

Protein Sequence

Sequencing

Resistance Mutations

Selection

Prediction of DrugSusceptibility

Interpretation

PhenoSense™ HIV

Patient virus

PR-RT DNA

RT-PCR

Resistance Test Vector

Vector Assembly

Recombinant Virus

Transfection

Measure of Drug Susceptibility

Infection

?

PhenoSense HIV

Allows determination of drug susceptibility against patient’s virus compared to wild-type virus

Measurement of single drug, not the combination

Can be performed on samples with viral loads 500 copies/mL

Results based on direct measurement --not inference from genotype (Virtual Phenotype)

Able to detect majority and (depending on percentage) some minority species

Genotype Test

High degree of accuracy and reproducibility

Results are specific to HIV RNA sequence

Detects many minority populations of drug-resistant virus

Can be performed on samples with viral loads 500 copies/mL

Software analyzes every position of the nucleotide sequence, therefore may detect mutations other tests may miss

Reports mixtures

Reports all mutations (including polymorphisms)

Reports subtype

WHEN DOES GENOTYPING HELP?

When mixtures of viral strains are present

* Homogeneous population with low level resistance

* Mixed population with highly-resistant minor species

* Genotype is less sensitive than phenotype for some mutations, e.g. G190S

When reversion mutations are present

* Drug-sensitive variants with increased ability to convert to resistance mutations compared with wild type virus (e.g. T215C)

When the clinical cutoff is unknown

* Known resistance mutations associated with clinical failure, e.g. T69D

ADVANTAGES OF ACCESSING BOTH GENOTYPIC AND PHENOTYPIC RESISTANCE DATA

Combining genotypic data with phenotypic data provides acomprehensive picture of the resistance profile of the patient’svirus

Allows clinician maximum efficiency in antiretroviralmanagement

Enhances ability to preserve future treatment options

Facilitates individualization of antiretroviral therapymanagement for optimal clinical outcomes

Phenotypic and genotypic results can be provided from a single patient sample

Combination phenotype/genotype test report form

Page 1 provides all info necessary for clinical interpretation

Page 2 provides more detailed assay data

PHENOSENSE GTTM TEST REPORT

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A. Fold-change bar graph

B. Phenotypic cutoff indication

C. Resistance mutations by drug class

D. Side-by-side phenotype/genotype interpretations

E. Net assessment of susceptibility (based on proprietary algorithm; helpful in cases of discordance)

F. References for detailed comments found on Page 2

G. HIV-1 subtype info

PhenoSense GT Test Report

A

D

E

B

C

G

F

Glossary of Phenotype Terminology

Fold change: The change in susceptibility above or below wild type reference strain

Cut point (Cut off): The fold change value above which drug susceptibility declines. There are different means of assessing a cut point

Hypersusceptibility: Increased drug susceptibility compared to wild type

HIV drug resistance cutoffs Clinical cutoffs:

– based on outcome data from clinical trials involving patients

Biologic cutoffs:– based on natural variability of wild-type

viruses from patients

Reproducibility cutoffs:– based on assay variability with

repeated testing of patient samples

Clin

ical

Rel

evan

ce

Highest

Moderate

Patient’s virus is sensitive to the drug

%

Patient:Control:

Nelfinavir

% I

nh

ibit

ion

IC50(patient)IC50(control)

Fold Change =

FC=1

PhenoSense Inhibition Curves

Patient’s virus is highly resistant to the drug

%

Patient:Control:

Nelfinavir

% I

nh

ibit

ion FC=200

High-Level Drug Resistance

Hypersusceptibility

Patient:Control:

Structure and Function in Biology:

What’s the practical difference between a genotype and a phenotype?

Why may they not tell you the same thing?

Genotype Depicts Structural Changes

Translation

Processing and Folding

HIV RNA

Linear sequence of amino acids

Genotype Depicts Structural Changes

Translation

Processing and Folding

HIV RNA

Linear sequence of amino acids

Genotype sees this…And needs an algorithmTo predict this

Phenotype Assesses Functional Aspects

HIV RNA

PhenoSense tests the ability of each drug to

interfere with the FUNCTION

of the viral enzymes that are the actual targets of the drugs.

RTV

Interpreting Resistance Test Reports

How We Identify a Mutation

How do we identify a resistance mutation?

“M” is the “wild type” amino acid

“184” is the codon position

“V” is the mutant amino acid

M 184 V

20 Amino Acid Symbols

A = alanine C = cysteine D = Aspartate E = glutamate F = phenylalanine G = glycine H = histidine I = Isoleucine K = lysineL L = leucine

M = methionine N = asparagine P = proline Q = glutamine R = arginine S = serine T = threonine V = valine W = tryptophan Y = tyrosine

What Are TAMs? Thymidine Analog (Resistance) Mutations

Previously known as ZDV resistance mutations

Selected by ZDV and/or d4T– 41L– 67N– 70R– 210W– 215Y/F– 219Q/E

Other ZDV-selected mutations include– 44D/A, 118I, 207D/E, 208Y

TAMs Confer Cross-resistance to NRTIs

ZDV resistance mutations now recognized as multinucleoside resistance mutations – Cross-resistance with d4T, ddI, ddC, 3TC

Presence of 2 TAMs + 184V significantly reduces potency of ABC

Presence of 3 TAMs including 41L + 210W significantly reduces activity of TDF

Gentotype and Phenotype Disconcordance

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What do we mean by discordance?

Discordance refers to disagreement between the results of the phenotypic measurement of susceptibility and the genotypic interpretation of susceptibility based on mutational patterns

Discordance Review

Observed differences between results from phenotypic/genotypic resistance testing is more common than generally thought

Discordance occurs because the interpretation of the results may be different and the tests “see” different aspects of the virus

Genotypic and phenotypic tests provide complimentary information that gives the most complete picture of resistance

In many cases, interpretation of these results can be facilitated by learning several patterns and rules

PT-Resistant, GT-Susceptible 25% PT-S, GT-R, mixtures* absent 41% PT-S, GT-R, mixtures* present 34%

Types of Pheno/Geno Discordance

Parkin et al, JAIDS 2002; 31: 128-136

*Mixtures = patient sample has mixture of drug resistant and drug sensitive virus, usually observed during transitionbetween completely drug resistant and completely drug sensitive virus, such as shortly after interrupting therapy or during the brief period when drug resistant virus first emerges

Major explanations of discordance

Incomplete genotypic algorithms (rules)– Novel mutations – Improper weighting of mutations in algorithms (both over-

and under-weighted)– Non-B subtype resistance patterns

Immaturity of interpretive algorithms

Mixtures present

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

Case Study (1):35 yr. old Gay Hispanic Male

HIV+ since 1996 Presents for care November, 2000

Past Medical History

Treated with IDV/AZT/3TC for 2 mos Stopped HAART due to side effects Last CD4=230 HIV RNA=unknown No Prior OI’s Pancreatitis 1999 No Prior Surgery NKDA No current Medications

Social History

Alcohol Abuse (6-12 beers daily) No drug use No tobacco Living with male partner for 2 yrs who is also

active alcoholic Works as shipping clerk at the Gap

Review of Systems

33 lb weight loss over 6 mos Chills, sweats Diarrhea Vomiting on occasion Allergic rhinitis Visual changes Depressive Symptoms

Physical Exam

Wt=68.6 kg BP=122/84 T=98.3 Pulse=74 Resp=18

Nervous Appearing Smelled of ETOH No swollen nodes No thrush Chest Clear No HSM Skin clear Normal genitals Guiac Neg

Data

CBC Normal CD4=169 HIV RNA=230,000 ALT=90 AST=120 Amylase=96 RPR Negative Hep A non immune Hep B immune Hep C Negative Normal UA Testosterone=355 (400-1080) CXR Normal

HIV Genotype

PR Mutations– L63P

RT Mutations– None

Initial Treatment

HIV therapy held until ETOH abuse was under better control

AA meetings encouraged, but pt did not connect Short course of Antabuse PCP Prophylaxis begun Testosterone Replacement Paxil 20 mg/d Referred for counseling

Follow-up Care

Tcell count=200 HIV RNA=58,000 six weeks later Patient able to substantially reduce ETOH intake Started on Sustiva 600 qhs and Combivir bid on

March 2, 2001

Patient Complaints

Called office complaining of nausea and vomiting Feels as if he has a hangover in the morning Headache Just “can’t function” Feels like “shit” Nightmares Partner complains of somniloquy

Should Sustiva Be Withheld in Certain Patients?

Patients with Depression or Anxiety? Patients with Active Substance use? Patients with Schitzophrenia or other severe

mental illness?

How I Treated Patient

Telephone Reassurance Follow-up Office Visit to check compliance Ativan 0.5-1.0mg qhs Compazine 10 mg q 8 hrs prn

Case Study 2

32 Year old White maleDx: HIV + 1990Transferred Care: 6/1/00 on CBV BID only

HIV PCR= 1,000 Tcell=138 (23%)PMH: No hx OI’s

Depression (severe)HIV Risk Factor: “Prolific” Sex (mostly hetero, some

homo)SH: Works for technology start up company

Case 2 Contd

HIV Med History– AZT monotherapy followed by AZT/ddC dual

therapy– SQV/RTV dual therapy in late 1990’s– Transferred on Combivir and Septra

– Genotype Performed 7/00– PR Mutations: None– RT Mutations: D67N, T69N, K70R, M184V, T215,

K219Q

– What would you have done in 7/2000?

Case 2 Contd

7/00 Meds Changed to:– Viramune 200 BID– ddI 400 QD – d4T 40 BID

– LAB DATA: HIV PCR = BDL T cell = 398 (25%)

– Regimen Maintained until 9/02 when HIV PCR = 2900 T cell = 387 (22%)

Case 2 Contd

Gentotype Done 7/02– PR—None– RT—D67N, T69N, K103N, V106A, V118I, T215F,

K219Q, New Medication Started 9/02

– Viread 300 QD– Abacavir 300 BID– ddIEC 250 QD

– Lab Data HIV PCR = 1500 T cell = 358 (20%)

Case 2 Contd Genotype Done 3/03

PR: NoneRT: D67N, T69N, K70R, K103N, V118I, T215F, K219Q (M184V is

now undetected)

Labs on 6/03– HIV PCR = 460– T cell = 339 (19%)

Phenotype Done 8/03

What would you do?

RC is reported as a percentage that compares the ability of the patient’s virus to replicate with that of the average wild-type virus

RC value of wild-type virus is set at 100%

Replication Capacity (RC)

RC is a measure of viral fitness

Case 2 Contd

New Regimen Started 10/1/03 Kaletra 3 BID T-20 90 s.c. BID Maintain ddI EC 250 QD, TDF 300 QD, ABC 300 BID

Labs After Initiation of Treatment

10/03 1/04 5/04 12/04 HIV PCR = BDL HIV PCR = BDL HIV PCR = BDL HIV = BDL T cell = 368 (21%) T cell = 429 (25%) T cell = 398 (24%) T cell = 542

(30%)

87

88

89

90

Simplified Model for Patients Initiating Enfuvirtide Treatment*

Factor Odds ratio 95% CI P-value

Disease stage

BL CD4+ count (>100 cells/mm3) 2.4 (1.6, 3.5) <.0001

BL plasma HIV-1 RNA (<100K) 1.8 (1.2, 2.6) <.0022

Treatment history

No. of prior ARVs (10) 1.8 (1.2, 2.6) 0.0058

Activity of background regimen

2 active ARVs in background 2.8 (2.0, 4.0) <.0001

* HIV RNA<400 copies/ml at Week 24.

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Case 3--Patient D.L.

• 60 year old Latino man • HIV + since 1984

– HSV and Candida Esophagitis– Bacterial Pneumonia– Wasting

• PMH– Hypertension Gout– Hypogonadism Erectile Dysfunction– Anemia Fatigue/Malaise– GERD Hypothyroidism

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Case 3--Patient D.L.

• Current Non-HIV Medications– Verapamil, Dapsone, Prilosec, – Androgel, Diflucan, Procrit

• Antiviral History– AZT, 3TC, ABC– Combivir– Nevarapine– Fortovase and Norvir 600/300 BID– Kaletra (refuses to take again)

• HAART Regimen as of 11/02– EFV, TDF, ddI EC 250, 3TC

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Patient D.L. Resistance Testing

• 2/03 Genotype Obtained on EFV, TDF, 3TC, ddI – PI: L63P– NRTI: M41L, L74V, V118I, M184V, G190S,

T215Y

• 5/03 HIV PCR = >100k Tcell= 63Treatment Plan– Sustiva Stopped– SQV (HGC)/ritonavir 1000/100 bid

• d/c after 1 month due to pill count

• Patient Maintained on TDF, FTC, and ddI– HIV PCR= 29,000 T cell = 83

95

Patient D.L. Resistance Testing

• Phenotype– NRTI:

• Resistant to ABC, 3TC, (ddI fold change = 2.1)

• Sensitive to d4T, TDF, AZT

– PI: Pansensitive– NNRTI: resistance EFV, NVP– Replication Capacity = 21%

96

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Patient D.L.

• New HAART regimen started 10/03– Reyataz/ritonavir 300/100 QD– Maintained TDF, FTC, and DDI EC 200 (as per

weight)

• Prilosec discontined and replaced with Pepcid 20 mg qhs

• Verapamil discontinued and replaced with lisinopril;

• EKG normal

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Patient D.L.

• New Labs on HAART 11/03 one month later– HIV PCR = BDL– T cell = 105

• Current Labs 7/04– HIV PCR = BDL– T cell = 140

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Case 4: A.W.-- 47 Year old White Man

• HIV+ diagnosed mid 1980’s• Presented to Stanford University in 5/97• Reports previous AZT, ddI, SQV and NLF• Current medications at transfer are d4T,

3TC, IDV 1000 TID and NVP

A.W.

PMH– Gonorrhea and chlamydia– Pubic lice– Hepatitis B

Social History– Lives alone– Drinks alcohol daily (2 beers or 2 glasses wine)– No tobacco or drugs

Family History– Mom with depression– Dad healthy– Sibling with Alcohol abuse

A.W.

Initial labs 5/97– CD4 = 320 %CD4 =23 H/S = 0.3– HIV RNA = BDL– Cholesterol = 285

A.W.

Initial labs 5/97– CD4 = 320 %CD4 =23 H/S = 0.3– HIV RNA = BDL– Cholesterol = 285

Returns one month later 6/97– CD4 = 240 – HIV RNA = 230

Next labs 9/97 (Swears to Adherence)– CD4 = 250– HIV RNA 180

A.W. Next Labs 2/98

– CD4 = 290– HIV RNA = 13,000

Genotype Performed

PI Mutations:

L10I, N37S, D60E, L63P

A71T, I72E, G73A, V77I

L90M, I93L

RT mutations:– M16V, K20R, T39A, M41I,

K49R, V60I, D67N, T69D, R83K, V90I, K103N, V118I I135T, S162D, E169D M184V, G196E, I202V Q207E, L210W, R211K T215Y, A272P

A.W.

With known K103N, patient is taken off of NVP and maintains IDV 1000 TID, 3TC, and d4T

Next labs 7/98– CD4 = 220– HIV RNA = 5100

A.W.

Patient is noncompliant with appointments and returns only yearly for several visits

4/99 CD4 = 230 HIV RNA = 1100 2/00 CD4 = 260 HIV RNA = 1300 12/01 CD4 = 355 HIV RNA = 1300

A Phenosense is Obtained

A.W.

New Regimen Started 6/02– LPV/r 3 BID– TDF 300 QD– ddI EC 400mg QD

Follow-up Labs 7/02CD4 = 317 HIV RNA = BDL

7/30/02 Pt develops severe HZV Right V-1, treated

8/02 Videx EC dose is reduced to 250 mg/day

Viral load continues BDL to present T cell is 423 as of 12/04