1. Virological predictors of clinical outcome Anna Maria Geretti Royal Free Hampstead NHS Trust & UCL Medical School London

2. Mortality risk with detectable viraemia during HAART Cumulative mortality Months after starting HAART 0 18 36 54 72 Lohse et al, Clin Infect Dis 2006 Cumulative mortality stratified by % VL ≥400 cps/ml recorded during 18 months after HAART initiation 100% 51%-75% 76%-99% 26%-50% 1%-25% 0% n=2046 patients who started HAART before 2002 Follow-up: 8898 patient-years after 18 months on HAART

3. Determinants of HAART outcomes Drug exposure Adherence PK Compartments Host-related factors Immune status Genetics Tolerability Virological factors Viral load Sequence variability Drug-resistance Drug potency IC 50 value Genetic barrier to resistance

4. First-line HAART in the UK  Drug-naïve cohort (n= 1175)  Started HAART in 1996-2006  82% achieved a VL<50 cps/ml  At median 3.5 months  IQR 2.4, 5.5 months Geretti et al, EHDRW 2008 HAART regimen started

5. Independent predictors of achieving a VL <50 HR95% CIP Year of starting HAART 1999-20010.610.46, 0.790.0002 2002-20030.780.67, 0.930.004 2004-20061-- AgePer 10 yrs older1.171.06, 1.280.001 Baseline VLPer 1 log  0.630.56, 0.70<0.0001 Baseline CD4Per 50 cells  0.970.94, 1.000.02 GSSPer 1 unit  1.501.19, 1.890.001 RegimenNNRTI1-- PI/r0.860.70, 1.050.13 PI0.390.22, 0.710.002 Triple NRTI0.510.35, 0.760.001 No effect of gender, risk group, ethnicity, or B vs non-B subtype Geretti et al, EHDRW 2008

6.  Impact of low-level viraemia  Impact of HIV-1 subtype  Impact of transmitted drug resistance Exploring the determinants of HAART failure

7.  Impact of low-level viraemia  Impact of HIV-1 subtype  Impact of transmitted drug resistance Exploring the determinants of HAART failure

8.  Investigate the long-term virological outcomes of a large cohort initially showing good responses to first-line HAART  Explore the occurrence and impact of low-level viraemia between 50 and 400 cps/ml Study objectives Geretti et al, Antiviral Therapy 2008

9. Study population  Drug-naïve cohort (n= 1386)  Started HAART in 1996-2005  Achieved a VL <50 cps/ml  In the following year: In follow-up On HAART No VL >400 cps/mL Geretti et al, Antiviral Therapy 2008 Note: 320 (23.1%) changed the initial regimen before achieving <50 cps/ml due to toxicity (allowed) HAART regimen at first VL <50 cps/ml

10. Virological status in the first year after achieving a VL <50 cps/m Low-level rebound = 50-400 cps/ml Geretti et al, Antiviral Therapy 2008

11. Virological status in the first year after achieving a VL <50 cps/m 269 patients Blips per person 1 84.8% 2 13.4% 3 1.5% 4 0.4% 85 patients Consecutive VL >50 2 in 54.1% 3 in 28.2% ≥4 in 17.6% Low-level rebound = 50-400 copies/ml Geretti et al, Antiviral Therapy 2008 Low-level rebound = 50-400 cps/ml

12. Predictors of low-level rebound  Multivariate model  Factors analysed: age, gender, risk group, ethnicity/country of birth, baseline CD4 and VL, CD4 at first VL <50 cps/ml, time from start of HAART to first VL <50 cps/ml, HAART regimen, year when first achieved a VL <50 cps/ml, HAART changes for toxicity prior to first VL <50 cps/ml OROR95% CIP 2 NRTIs + 1 NNRTI1.00-0.01 2 NRTIs + 1 PI/r1.391.00, 1.94 2 NRTIs + 1 PI1.480.96, 2.26 Triple NRTIs/Other1.731.23, 2.42 Geretti et al, Antiviral Therapy 2008

13. Virological outcomes  Follow-up started 1 year after 1 st VL <50 cps/m  Lasted for median 2.2 years (range 0.0–7.4)  Failure = VL >400 cps/ml  Failure rate = 86 patients (6.2%) Consistent undetectability 5.0% Transient low-level rebound 8.2% Persistent low-level rebound 14.1% Geretti et al, Antiviral Therapy 2008 Low-level rebound = 50-400 cps/ml

14. Predictors of virological failure  Multivariate model  Factors analysed: As in previous analysis + VL status in the first year after achieving a VL <50 cps/m PredictorsRR95% CIP Virological status in 1 st yr after achieving <50 cps/ml Consistent <501.00–0.02 Transient rebound1.410.86, 2.34 Persistent rebound2.181.15, 4.10 GenderMale1.00–0.02 Female1.791.12, 2.85 HAART regimen2 NRTIs + 1 NNRTI1.00–0.09 2 NRTIs + 1 PI/r1.881.02, 3.46 2 NRTIs + 1PI1.230.66, 2.31 3 NRTIs/Other1.871.04, 3.36 Geretti et al, Antiviral Therapy 2008

15. Effect of VL on the detection of resistance (multivariate analysis) VLN% RAMsRR (95% CI) <300449 600.94 (0.87-1.01) 300-1000552 720.99 (0.94-1.04) 1000-30001120 761 3000-100001312 771.01 (0.97-1.05) 10000-300001326 670.91 (0.87-0.95) 30000-1000001438 600.84 (0.80-0.88) ≥1000001682 490.70 (0.66-0.74) RAMs: resistance-associated mutations RR: Relative risk Geretti et al, IHDRW 2009

16. Number of mutations detected by VL strata* *in patients with ≥1 mutation N= 270 399 850 1014 888 858 809 Geretti et al, IHDRW 2009

17.  Impact of low-level viraemia  Impact of HIV-1 subtype  Impact of transmitted drug resistance Exploring the determinants of HAART failure

18. Study population  Drug-naïve cohort (n=2116)  Started HAART in 1996-2006 Most commonly 2 NRTIs + 1 NNRTI  ≥12 months of follow-up  Excluded patients with TDR Outcomes measured:  Time to VL suppression <50 cps/ml  For those who achieved 1000 cps/ml  Median follow-up of 39 months (IQR 23, 67) Geretti et al, Clin Infect Dis 2009 Subtype  Ethnicity and risk group strongly associated with subtype (P<0.001)

19. Responses to first-line HAART by subtype N = 64 1381 255 37 51 118 N = 13 238 51 7 7 19 1906/2116 (90%) achieved VL suppression <50 cps/ml 335/1906 (18%) rebounded >1000 cps/ml Geretti et al, Clin Infect Dis 2009

20. VL suppression 0.00 0 0.20 0.40 0.60 0.80 1.00 36912 Probability of achieving <50 cps/ml Analysis time (months) Log-rank p<0.0005 ABCABC Number at risk A6623952 B1550785332218169 C27210412317 Median time to VL suppression Subtype A 2.6 months Subtype C 2.8 months Subtype B 3.1 months Multivariate analysis* Time to VL suppression shorter for A (HR 1.35 [1.04,1.74] P=0.02) and C (HR 1.16 [1.01,1.33] P=0.04) vs B *adjusted for age, centre, HAART regimen, calendar year, baseline CD4 and VL Geretti et al, Clin Infect Dis 2009

21. VL rebound *adjusted for age, centre, HAART regimen, calendar yr, baseline CD4 and VL, and time to VL suppression Number at risk A644934221715 B13811167830630454317 C255197116744830 0.00 0 0.20 0.40 0.60 0.80 1.00 12243648 Probability of remaining <50 cps/ml Analysis time (months) Log-rank p=0.09 ABCABC 60 Multivariate analysis* Time to VL rebound shorter for C vs B (HR 1.40 [1.00, 1.95] P=0.05) 143 rebounds: virological failure 192 rebounds: non-adherence or treatment discontinuation Time to virological failure similar for C vs B Geretti et al, Clin Infect Dis 2009

22. CD4 recovery over time Geretti et al, Clin Infect Dis 2009 100 0 200 300 400 500 61218 CD4 count (cells/mm 3 ) Time since ART initiation (months) ABCABC Number in analysis A66505742382820 B155012531174970796668552 C2722252011671219363 302436

23.  Impact of low-level viraemia  Impact of HIV-1 subtype  Impact of transmitted drug resistance Exploring the determinants of HAART failure

24. Detection of TDR 0.001 0.01 0.1 1 10 100 Detected by ultrasensitive methods Mutation Frequency Detected by routine methods Natural background 6-13% of naïve patients in Europe & N America 1-9 1. Masquelier JAIDS 2005; 2. Wensing JID 2005; 3. Booth JAC 2007; 4. Geretti COID 2007; 5. SPREAD AIDS 2008; 6. Vercauteren AIDS RHR 2008; 7. Peuchant AIDS 2008; 8. Bannister JAIDS 2008; 9. Weinstock JID 2004; 10. Peuchant AIDS 2008; 11. Metzner AIDS 2005; 12. Johnson PLoS ONE 2007; 13. Johnson PLOS Med 2008; 14. Siemen JID 2009; 15. Geretti JAIDS 2009; 16. Goodman IHDRW 2009 Rates doubled 10-16

25. The clinical significance of low-frequency TDR  Different methods, interpretative cut-offs, populations, HAART regimens Metzner, Antivir Ther 2007 Peuchant, AIDS 2008 Geretti, JAIDS 2009 Johnson, PLOS Med 2008 Siemens, JID 2009 Goodman IHDRW 2009 Impact on virological responses No Yes

26. The FIRST study MutationsMethodP BulkUDS NNRTI 6.6%15.1%<0.001 NRTI 6.2%14.0%<0.001 PI 2.3% 4.7% 0.03 Any13.6%28.3%<0.001 N=258 HR for failure in patients with NNRTI resistance  Bulk : 12.4 [3.41-45.1]  UDS: 2.50 [1.17-5.36] Siemen et al, JID 2009 USD = Ultra deep sequencing

27. Impact of NNRTI TDR on responses to first-line NNRTI-based HAART  Case control study  Patients with virological failure (n=18) vs those who achieved and maintained VL suppression <50 for ≥24 weeks (n=75)  Pre-HAART sample tested by sensitive PCR  Targets: K65R, K103N, G190A, Y181C, M184V  Interpretative cut-off of 0.5-0.9% Geretti et al, JAIDS 2009

28. NNRTI TDR reduces responses to first-line NNRTI-based HAART Resistance at baseline  7/18 cases vs 0/75 controls  2 K103N HIGH 1 G190A HIGH 4 K103N LOW Odds of virological failure  Bulk resistance p=0.006  4 K103N LOW p=0.001  Combined p <0.0001 Geretti et al, JAIDS 2009 HIGH= detected also by bulk genotyping; LOW= detected only by PCR (>0.9%)

29. K103N >2% or >2000 cps/ml predicts failure of first-line NNRTI-based HAART Goodman et al, IHDRW 2009 Fit plot for VF (>400 c/mL) 01234 Log copies of RNA with K103N mutation Clinical virology VF (>400 c/mL) -0.5 0 0.5 VF Non-VF Fit99% confidence band 10 3.3 = 1995 c/mL 02.5512.515 Percent of RNA with K103N mutation Clinical virology VF (>400 c/mL) -0.5 0 0.5 VF Non-VF Fit99% confidence band 1 7.510 GS-01-934 (n=487)  TDF/FTC/EFV  ZDV/3TC/EFV  K103N LOW in 16/476 (3.4%) patients  6/16 (38%) of patients with K103N LOW had VF VF = virological failure

30. Conclusions-1  Low risk of virological failure once suppression is achieved and maintained for 1 year  Consistent suppression <50 the optimal target  NNRTI-based HAART least likely to result in low-level viraemia and virological failure  Women at increased risk of virological failure  Problems with long-term adherence  Cultural and social-economic factors  Resistance testing at low VL yields clinically useful information

31. Conclusions-2  Predominantly NNRTI-based HAART achieves excellent outcomes regardless of the infecting subtype  Subtype C patients show an increased risk of rebound possibly related to non-adherence

32. Conclusions-3  Low-frequency K103N mutants as prevalent as bulk-detectable resistant variants  Significantly associated with virological failure  Patients infected with single NNRTI mutants  ? Impact on second generation NNRTIs Etravirine Nevirapine Efavirenz

33. Thank you a.geretti@medsch.ucl.ac.uk