Emerging patterns of drug resistance and viral tropism in cART-naïve and failing patients infected with HIV-1 subtype C

Thumbi Ndung’u, BVM, PhDAssociate Professor

Director, HIV Pathogenesis ProgrammeDoris Duke Medical Research InstituteNelson R. Mandela School of Medicine

University of KwaZulu-Natal

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

H

C

F1

F2

K

D

B

J

G

A1

A2

N-group

O-groupCPZ ANT

CPZ GABCPZ US

M-group

5%

HIV-1 Phylogeny

47.2%

27.0%

12.3%

Phenotypic Classification of HIV-1

• Slow/low versus rapid/high

• Syntitium-inducing (SI) versus NSI

• Slow/Low = NSI (Early, slow progression)

• Rapid/High = SI (Late, rapid progression)

CD4 CD4 CD4

CCR5

CCR5

CXCR4

CXCR4

M-tropic Dual tropic T-tropicVirus Variants

HIV-1 coreceptor usage and viral tropism

Target Cell Types

Macrophage Primary T cell

T-cell line

>25 years of HIV/AIDS>25 years of HIV/AIDS

> 33

For every 2 people put on treatment, 5 others are infected

Treatment begins

Selection of resistant quasispecies

Incomplete suppression•Inadequate potency•Inadequate drug levels•Inadequate adherence•Pre-existing resistance

Selection of Resistant strains

Time

Vira

l loa

d

Drug-susceptible quasispecies

Drug-resistant quasispecies

Study rationale

Background:• Relatively limited information on coreceptor usage by

HIV-1 subtype C isolates, particularly in children. However, most studies suggest very rare CXCR4 usage

• Some reports suggest increasing X4 usage (in adults) eg. Johnston et. al. (n=28), 50% using X4 among ART experienced viremic patients

• Previously used methods may be biased because they involved first generating viral isolates by co-culture

Study rationale

• ART may boost T-cell immune responses which have been shown to preferentially suppress X4 viruses. Thus partially effective therapy may select against X4 viruses (Deeks et al, JID 2004; Harouse et al, PNAS 2003)

• ART reduces CCR5 expression on T cells (due to reduction in T cell activation) potentially selecting for X4 viruses (Brumme et al, JID 2005; Anderson et al, AIDS 1998)

• Suboptimal drug metabolism (such as AZT) in the cellular reservoirs for X4 viruses has been suggested and could lead to selection for X4 viruses (Boucher et al, AIDS 1992)

Aims

Specific Aims:1) To determine the prevalence of major drug

mutations in ART-naïve and failing children and adults

2) Determine overall prevalence of X4 tropism among children and adults initiating and failing HAART

3) Compare prevalence of X4-utilizing viruses between ART-naïve and ART-experienced subjects with detectable viremia

4) Explore factors associated with viral tropism in HIV-1C infection

HIV-1 Genotyping Assay

plasma

Blood cells

centrifugation

RNA

cDNA

DNA

RT-PCR

PCR

Dye terminatorsPCR

A T G C

ATAGACCAG : consensus sequence I Q QATCGACCTG : patient sequence I Q *L

TT C

T C GT C G A

Software analysis

CMV pA

Env

5’LTR gagpol env

vif

vpr

vpu

tatrev

Luc

3’LTR

+

Trofile assay summary- for coreceptor usage

pcDNA-env

0.2µfilter

0.2µfilter

Luciferase assay

CCR5 cells CXCR4 cells293T cells

Table 1: Children Demographic and Clinical Characteristics

NOTE. Data are no. (%) of children unless otherwise indicated. For cases where the data is incomplete, the n value is indicated. Statistical tests: a Mann-Whitney U test and b Fisher’s exact test (for WHO stage analysis, stages I, II and III were grouped together).

Characteristics HAART-Failures (n=41) HAART-Naïve (n=40) P value

Age, median years (IQR) 7.9 (4.8-10.4) 0.9 (0.5-2.8) <0.0001a

Black Race 41 (100.0) 39 (97.5) 0.49b

Male Gender 24 (58.5) 18 (45.0) 0.27b

Nadir CD4%, median (IQR) 9.0 (3.1-13.5) (n=33) 14.0 (7.5-22.0) (n=37) 0.008a

Current CD4%, median (IQR) 18.0 (9.0-24.0) 14.0 (7.5-22.0) (n=37) 0.47a

Current CD8%, median (IQR) 51.0 (40.5-58.0) 48.0 (35.5-56.5) (n=37) 0.38a

Current CD3%, median (IQR) 72.0 (67.0-77.0) 66.0 (56.0-77.5) (n=37) 0.18a

Current plasma HIV-1 viral load,

median log 10 copies/ml (IQR)4.9 (4.4-5.4) 5.9 (5.6-6.8) <0.0001a

Current WHO Stage: (n=40)

I 1 (2.5) 0 (0.0)

0.003bII 15 (37.5) 1 (2.5)

III 18 (45.0) 20 (50.0)

IV 6 (15.0) 19 (47.5)

NOTE. Data are no. (%) of children unless otherwise indicated. For cases where the data is incomplete, the n value is indicated. Prior treatment indicated with underlined drug/s changed ● d4T, 3TC, ritonavir (n=1); * unknown; ○ d4T, 3TC, EFV (n=1) and AZT, 3TC, NVP (n=1); d4T, 3TC, kaletra; d4T, 3TC, EFV.Statistical tests: a Mann-Whitney U test and b Fisher’s exact test

Table 1: Patient Demographic and Clinical Characteristics Cont. Characteristics HAART-Failures (n=41) HAART-Naïve (n=40) P value

Current Drug regimen:

D4T, 3TC, EFV 25 (61.0)

D4T, 3TC, LPV/r ● 6 (14.6)

D4T, DDI, EFV * 1 (2.4)

AZT, 3TC, NVP 3 (7.3)

AZT, 3TC, EFV ○ 3 (7.3)

AZT, DDI, EFV 1 (2.4)

AZT, DDI, LPV/r 1 (2.4)

D4T, ABC, LPV/r * 1 (2.4)

Duration of HAART prior to study

recruitment, median months (IQR) 28.6 (19.7-37.5) (n=38)

History of single-dose NVP for PMTCT 10 (26.3) (n=38) 18 (47.4) (n=38) 0.09b

Frequency of drug resistance mutations and levels of resistance in HAART-failing children to the NRTIs (a) and NNRTIs (b)

58.5% had TAMs39% had ≥3 TAMs

• d4T/3TC/EFV (n=25)

– 3 patients have no DRMs (VLs are 617; 79,400; 228,000)

– 20 NRTI DRM

– 2 NNRTI DRM

(one patient had a PI DRM)

• d4T/3TC/kaletra (n=5)

– 3 patients have no DRMs (VLs are 143,000; 198,000; 4,410,000)

– 1 patient has 1 NRTI DRM (M184V) only

– 1 patient has 1 NRTI (M184V) and 1 NNRTI DRM (Y181C)

Average no. of major mutation in patients failing standard first line treatment (n=30)

How many major mutations compromise the standard second line treatment?

d4T/3TC/EFV (n=25) → AZT/ddI/Kaletra• 3 patients susceptible to all drugs – no change needed• All patients susceptible to kaletra• 3 patients susceptible to 3 drugs in standard second line tx.

AZT Resistance ddI ResistanceSusceptible (n=2) High-Level (n=2)

Low level (n=5)

Potential low-level (n=2)

Low-level (n=1)

Intermediate (n=2)

Intermediate (n=8)

Potential low-level (n=2)

Low-level (n=3)

Intermediate (n=2)

High-level (n=1)

High-Level (n=4)Intermediate (n=2)

High-Level (n=2)

• Overall, 13 of 25 (52%) patients will have some degree of resistance (low to high) to two of the three drugs in their new regimen (excluding potential low-level resistance)

d4T/3TC/kaletra (n=5) → AZT/ddI/(NVP/EFV)

• 4 of 5 patients are susceptible to all second line drugs

• 1 patient had intermediate resistance to EFV (3.7 yrs old)(Y181C)

Note: Overall better if not changed

• All still susceptible to PIs and d4T with 3 patients still susceptible to 3TC

[2 high-level resistance to 3TC (M184V)]

90.6%

9.4%

HAART-Naïve

R5-tropic

D/M-tropic

45.7%

11.4%

42.9%

HAART-Failures

R5-tropic

X4-tropic

D/M-tropic

p<0.0001

45.7%

11.4%

42.9%

HAART-Failures

R5-tropic

X4-tropic

D/M-tropic

Comparison of coreceptor usage in HAART-failing and HAART-naïve children

Evaluation of Several Genotypic Tools for the Prediction of CXCR4-usage

a A total of 52 pure subtype C isolates with both phenotypic and genotypic data were included in this analysis. bA false positive rate of 10% was used. c A combination of the first four genotypic tools were used where the majority prediction was considered as the final genotype prediction (n=47).

Genotypic ToolPrediction of CXCR4-usagea

Sensitivity (%) Specificity (%) PPV (%) NPV (%)

11/25 charge rule 30.0 96.9 83.0 74.0

Net V3 charge rule 65.0 78.1 59.0 82.0

C-PSSMsinsi 75.0 87.5 75.0 88.0

Geno2pheno[coreceptor]b 60.0 87.5 70.0 82.0

Combined Rulesc 63.2 100.0 100.0 85.0

C4.5 25.0 100.0 100.0 73.0

C4.5 positions 8-12 25.0 100.0 100.0 73.0

PART 30.0 100.0 100.0 75.0

SVMwetcat 40.0 96.9 86.0 77.0

Patient Characteristic

HAART-Experienced Patients failing

Treatment (n=45)

HAART-Naïve Patients (n=45)

p-value

Age, median years (Q1-Q3)

36 (24-51)

36 (20-78)

0.65

Gender: Female 28 (65%) 27 (60%)  

Black race 45 (100%) 45 (100%)  

CD4 count, median cells/mm3 (Q1-Q3) Current Nadir

174 (9-718)57 (3-197)

123 (8-660) 0.0360.0004

Vial load, median copies/ml

6, 653 (225-220,010)

44,042(1,702-1,167,759)

0.001

WHO stage at visit I-III IV

32 (71 %)13 (29 %)

9 (20 %)36 (80%)

Adult patient information

Patterns of drugresistance

• What is the outcome of patients failing if started on the standard second line of treatment without having genotypic data?

• d4T/3TC/ (EFV/NVP) (n=16) (Note: 2 on NVP)– No major PI mutations

– 1.75 NRTI DRM

– 1.69 NNRTI DRM

Average no. of major mutation in patients failing standard first line treatment (n=16)

How many compromise the standard second line treatment?

d4T/3TC/ (EFV/NVP) (n=16) → AZT/ddI/LPV/r• All patients susceptible to kaletra (LPV/r)• 6 patients susceptible to all 3 drugs in standard second line tx.

AZT Resistance ddI Resistance

Susceptible (n=4)Potential low-level (n=3)

High-Level (n=1)

Potential low-level (n=2)Susceptible(n=1)

Low-level (n=1)

Low-level (n=2) Low-level (n=2)

Intermediate (n=2) Low-level (n=2)

• 4 of 16 (25%) patients will have some degree of resistance (low to intermediate) to two of the three drugs in their new regimen (excluding potential low-level resistance).

• 6 of 16 (37.5%) will have some degree of resistance (low to high) to one of the three drugs in their new regimen (excluding potential low-level resistance).

High levels of CXCR4 viruses in patients failing therapy- limited salvage options

Method % of sequences correctly predicted

% of R5 sequences correctly predicted

% of X4/D/M sequences correctly predicted

11/25 78 90 55Overall net V3 charge 75 71 81C-PSSM 81 85 72Geno2Pheno 84 86 82Combined algorithm* 87 90 80

*In the combined algorithm, concordant results from at least 3 of 4 methods (i.e. the amino acids at positions 11 and/or 25, the overall net V3 charge, C-PSSM prediction and Geno2Pheno prediction) were used.

V3 loop-based methods for coreceptor usage prediction

Conclusions• Virologic failure is mainly due to DRMs• High levels of TAMs is source of concern- suggests

subpotimal adherence and need for intensive monitoring

• Higher levels of CXCR4 using viruses among HAART experienced patients- need to explore CCR5 antagonists as part of first-line/early treatment

• Collectively, these data highlight the need for intensified adherence counselling and better HAART monitoring to maximize benefits.

AcknowledgementsUKZN• Taryn Green• Ashika Singh• Mohendran Archary• Michelle Gordon• Raziya Bobat• Hoosen Coovadia

McCord Hospital• Henry Sunpath• Richard Murphy

Monogram Biosciences• Jacqueline Reeves• Yolanda Lie• Elizabeth Anton

Harvard University• Daniel Kuritzkes• Bruce Walker

Funding• IMPAACT Network, NIH• Harvard University CFAR• South African DST/NRF• Hasso Plattner Foundation