1. Tracking Tropism: Epidemiology and Clinical Trials Jointly sponsored by Postgraduate Institute for Medicine and Clinical Care Options, LLC This program is supported by educational grants from Jürgen K. Rockstroh, MD Professor of Medicine, University of Bonn Bonn, Germany Mark A. Wainberg, PhD Director, McGill AIDS Centre, McGill University Montreal, Quebec, Canada

2. clinicaloptions.com/hiv HIV Entry and Tropism About These Slides Disclaimer The materials published on the Clinical Care Options Web site reflect the views of the authors of the CCO material, not those of Clinical Care Options, LLC, the CME providers, or the companies providing educational grants. The materials may discuss uses and dosages for therapeutic products that have not been approved by the United States Food and Drug Administration. A qualified healthcare professional should be consulted before using any therapeutic product discussed. Readers should verify all information and data before treating patients or using any therapies described in these materials.  These slides accompany CCO’s online Treatment Update on HIV Entry and Tropism: New Strategies for Effective Antiretroviral Therapy  The full program is available on the Clinical Care Options HIV Web site: clinicaloptions.com/hiventry  We are grateful to Jürgen K. Rockstroh, MD, Mark A. Wainberg, PhD, and all others who granted permission to publish these slides  Users are encouraged to use these slides in their own presentations, but we ask that content and attribution not be changed. Users are asked to honor this intent  These slides may not be published or posted online without permission from Clinical Care Options

3. clinicaloptions.com/hiv HIV Entry and Tropism Overview  Viral Tropism Defined  Prognostic Value of Viral Tropism Phenotype  Coreceptor Use in Different Patient Populations  Viral Tropism Assays  Summary

4. Viral Tropism Defined

5. clinicaloptions.com/hiv HIV Entry and Tropism Defining HIV Viral Tropism  Tropism = which coreceptor HIV uses to enter a CD4+ cell  Expression of CCR5 and/or CXCR4 on a cell determines whether it can be infected with R5 and/or X4 virus –R5 virus uses only the CCR5 coreceptor –X4 virus uses only the CXCR4 coreceptor  Dual-tropic virus can use either CCR5 or CXCR4 coreceptors  Mixed-tropic virus is a viral population with both R5 and X4 viruses

6. clinicaloptions.com/hiv HIV Entry and Tropism Viral Tropism Targets Esté JA, et al. Lancet. 2007;370:81-88. CXCR4CCR5CD4 Naive CD4+ cellsMemory CD4+ cellsMacrophages R5 X4D/M D/M or X4 occurs in up to half of pts as disease progresses Cause of most HIV transmissions Must be activated to memory phenotype to become target of R5

7. Prognostic Value of Viral Tropism Phenotype

8. clinicaloptions.com/hiv HIV Entry and Tropism History of Tropism Research Late 1980s: correlation found between infected cells’ tendency to cluster (syncytia) and HIV progression –NSI virus mostly in patients with asymptomatic HIV infection –SI virus mostly in patients with AIDS 1. Cocchi F, et al. Science. 1995;270:1811-1815. 2. Feng Y, et al. Science. 1996;272:872-877. 3. Dragic T, et al. Nature. 1996;381:667- 673. 4. Liu R, et al. Cell. 1996;86:367-377. 1990 2000 1985 December 1995: RANTES, MIP1α, MIP1ß identified as HIV suppressive factors in CD8+ cells [1] May 1996: fusin (CXCR4) identified as a coreceptor for T- tropic (X4) HIV [2] June 1996: CCR5 identified as a coreceptor for M-tropic (R5) HIV [3] August 1996: CCR5  32 identified as HIV resistance factor [4] 1995 Light microscope, KE Schneweis, Virology Bonn R5 (NSI)X4 (SI)

9. clinicaloptions.com/hiv HIV Entry and Tropism Tropism Confirmed as Marker of HIV Progression  R5 virus as correlate of less advanced HIV infection and X4 virus as correlate of more advanced infection confirmed in several studies  Cross-sectional British study of 563 antiretroviral-naive or antiretroviral-experienced patients found –112 (20%) had X4 or D/M virus –R5 HIV correlated with higher CD4+ cell count, higher natural killer cell count, lower HIV-1 RNA than X4 virus –Tropism not correlated with treatment experience, HIV-1 subtype, or resistance mutations Moyle GJ, et al. J Infect Dis. 2005;191:866-872.

10. clinicaloptions.com/hiv HIV Entry and Tropism Association Between Tropism and BL CD4+ Cell Count Confirmed  Cross-sectional Canadian study of 979 patients beginning triple therapy –Strong association between presence of D/M or X4 virus and baseline CD4+ cell count –Proportion of D/M and X4 virus ranging from 50% at CD4+ cell count < 25 cells/mm 3 –D/M or X4 virus progressively more likely in each lower CD4+ cell stratum BL CD4+ cell count, cells/mm 3 R5 virus, % D/M or X4 virus, % > 500937 350-499919 200-349919 100-1997228 50-997426 25-496931 < 254654 Brumme ZL, et al. J Infect Dis. 2005;192:466-474.

11. clinicaloptions.com/hiv HIV Entry and Tropism 0.6 0.4 0.5 BL Tropism Correlated With Disease Progression Daar ES, et al. ICAAC 2003. Abstract 1722c. Daar ES, et al. Clin Infect Dis. 2007;45:643-649.  Kaplan-Meier curves showing progression to AIDS for patients with R5 or D/M virus –BL tropism measured in 126 children and adolescents –D/M virus at BL associated with lower BL CD4+ cell count and higher VL –BL D/M virus associated with 3.8-fold higher risk of progression to AIDS Conclusion: coreceptor tropism independently influences natural history of HIV disease. R5 virus D/M virus 012345678 0.0 0.1 0.2 0.3 0.7 0.8 0.9 1.0 Proportion AIDS free Time, years

12. clinicaloptions.com/hiv HIV Entry and Tropism CCR5 wild type   CCR5  32 2 normal copies 1 copy of  32 2 copies of  32 wt/wt wt/  32  32/  32 Standard disease progression Delayed disease progression “Resistant” to HIV infection NormalHeterozygousHomozygous CCR5 Wild Type and CCR5  32 Liu R, et al. Cell. 1996;86:367-367. Samson M, et al. Nature. 1996;382:722-725. Dean M, et al. Science. 1996;273:1856-1862. Huang Y, et al. Nat Med.1996;2:1240-1243. Michael NL, et al. Nat Med. 1997;3:1160-1162. Eugen-Olsen J, et al. AIDS. 1997;11:305-310.

13. clinicaloptions.com/hiv HIV Entry and Tropism Patients Homozygous or Heterozygous for CCR5  32  Homozygous –~ 1% of white population [1] –Lack CCR5 molecules on CD4+ cell surface [2,3] –Resistant to R5 viruses –Susceptible to infection with X4 virus –Relatively normal immune function  Heterozygous –10% to 15% of white population [1] –Fewer CCR5 molecules on CD4+ cell surface [4] –Normal immune function [2] 1. McNicholl JM, et al. Emerg Infect Dis. 1997;3:261-271. 2. Liu R, et al. Cell. 1996;86:367-367. 3. Samson M, et al. Nature. 1996;382:722-725. 4. Wu L, et al. J Exp Med. 1997;185:1681-1691.

14. clinicaloptions.com/hiv HIV Entry and Tropism Rheumatoid arthritis  32/wt: milder course Kidney transplantation  32/  32: improved transplant survival Disseminated sclerosis  32/wt: delayed onset, less relapse HIV-1  32/  32: resistance to infection  32/wt: delayed disease progression Hepatitis CData controversial Atopic asthmaData controversial Multiple sclerosis  32/  32: predisposition? Sarcoidosis  32/wt: increased disease manifestation rate and activity Lupus erythematosus  32/wt: increased disease severity West Nile virus  32/  32: increased susceptibility to disease Ahlenstiel G, et al. J Antimicrob Chemother. 2004;53:895-898. CCR5  32 Potential Impact on Other Diseases

15. clinicaloptions.com/hiv HIV Entry and Tropism 4% D/M Virus R5 Virus  Phase IIb study  At screening, higher proportion with X4 or D/M virus than in British and Canadian studies  Significantly lower median BL CD4+ cell count in patients with D/M vs R5 virus  CD4+ cell count not significantly lower in patients with only X4 virus than those with R5 virus at screening Wilkin TJ, et al. Clin Infect Dis. 2007;44:591-595. R5-only virus 50% D/M virus 46% X4-only virus Tropism at Screening Median BL CD4+ Cell Count (cells/mm 3 ) 0 40 80 120 160 200 103 170 P <.001 ACTG 5211: BL Tropism and CD4+ Cell Count in Treatment-Experienced Pts

16. clinicaloptions.com/hiv HIV Entry and Tropism  D/M vs R5 virus associated with greater and more rapid CD4+ cell count decline at 12 months before HAART initiation  After HAART initiation, no differences in response to HAART observed Waters LJ, et al. ICAAC 2006. Abstract H-1667. 50 100 Month 12Month 24 0 150 200 250 300 185 182 247 292 Mean CD4+ Cell Count Increase (cells/mm 3 ) 25 50 Month 6Month 12 0 75 100 71 75 72 68 Patients Achieving HIV-1 RNA < 50 copies/mL (%) Month 24 73 78 R5D/M Treatment Response in Naive Patients Same Despite Tropism Differences

17. Coreceptor Use in Different Patient Populations

18. clinicaloptions.com/hiv HIV Entry and Tropism 1. Brumme ZL, et al. J Infect Dis. 2005;192:466-474. 2. Moyle GJ, et al. J Infect Dis. 2005;191:866-872. 3. Demarest J, et al. ICAAC 2004. Abstract H-1136. 4. Coakley E, et al. International Workshop on Targeting HIV Entry 2006. Abstract 8. 82% 81% 88% 85% HOMER cohort [1] (N = 979) Chelsea and Westminster cohort [2] (N = 402) Demarest et al. [3] (N = 299) MERIT cohort [4] (N = 1428) 18% 19% 12% 15% < 1% R5D/MX4 HIV Tropism in Antiretroviral-Naive Populations  R5-only virus in 80% to 90% of patients, with D/M or X4 virus in remainder

19. clinicaloptions.com/hiv HIV Entry and Tropism 50% 59% 56% TORO 1 and 2 ENF trials [1] (N = 612) ACTG A5211 [2] (N = 391) SCOPE cohort [3] (N = 186) 48% 46% 39.5% 41% 4% 0.5% 1. Melby J, et al. J Infect Dis.2006;194:238-246. 2. Wilkin TJ, et al. Clin Infect Dis. 2007;44:591-595. 3. Hunt PW, et al. J Infect Dis. 2006;194:926-930. 4. Coakley E, et al. International Workshop on Targeting HIV Entry 2006. Abstract 8. MOTIVATE 1 and 2 MVC trials [4] (N = 2560) 2% 3% HIV Tropism in Antiretroviral- Experienced Populations  R5-only virus in 50% to 60% of patients, with D/M or X4 virus in remainder R5D/MX4

20. clinicaloptions.com/hiv HIV Entry and Tropism Kuhmann SE, et al. J Viral Entry. 2005;1:4-16. Moore JP, et al. AIDS Res Hum Retroviruses. 2004;20:111-126. R5 InfectionR5 + X4 Infection Time Since Seroconversion HIV-1 RNA Level Current assays’ X4 detection limit WeeksYears HIV Tropism and Disease Progression R5 virus X4 virus

21. clinicaloptions.com/hiv HIV Entry and Tropism Possible Outcomes in Patients With D/M Viruses Treated With CCR5 Antagonist Initial Virus Pool R5/X4 CCR5 Antagonist

22. clinicaloptions.com/hiv HIV Entry and Tropism Possible Outcomes in Patients With D/M Viruses Treated With CCR5 Antagonist Initial Virus Pool R5/X4 CCR5 Antagonist Viral Load R5 X4 Hypothetical Virologic Responses to CCR5 Antagonist CCR5 Antagonist

23. clinicaloptions.com/hiv HIV Entry and Tropism Possible Outcomes in Patients With D/M Viruses Treated With CCR5 Antagonist Initial Virus Pool R5/X4 CCR5 Antagonist Viral Load R5 X4 Viral Load R5 X4 Hypothetical Virologic Responses to CCR5 Antagonist CCR5 Antagonist

24. clinicaloptions.com/hiv HIV Entry and Tropism Possible Outcomes in Patients With D/M Viruses Treated With CCR5 Antagonist Initial Virus Pool R5/X4 CCR5 Antagonist Viral Load R5 X4 Viral Load R5 X4 Viral Load R5 X4 Hypothetical Virologic Responses to CCR5 Antagonist CCR5 Antagonist

25. Viral Tropism Assays

26. clinicaloptions.com/hiv HIV Entry and Tropism Predictive Value of Genotype  Theoretically may be effective in predicting viral tropism  Amino acid sequence of V3 of gp120, as well as of V1-V2, determines coreceptor use  Different methods of using genotype to predict phenotype –SensiTrop assay reported to detect X4 virus representing 1% of viral population –Virco phenotypic/genotypic platform successfully sequenced 151/161 isolates (94%) [1] 1. Rondelez E, et al. European HIV Drug Resistance Workshop 2007. Abstract 58.

27. clinicaloptions.com/hiv HIV Entry and Tropism Algorithm, %SensitivitySpecificity 11/25 rule30.593.4 Neural network method44.487.5 WebPSSM SI/NSI33.895.3 WebPSSM X4/R524.596.9 SVM genomaniac21.889.6 SVM geno2pheno44.790.6 Low A, et al. HIV Resistance Workshop 2007. Abstract 149. Genotyping Algorithms  6 genotyping algorithms found inadequate in predicting X4 and D/M tropism compared with phenotypic coreceptor tropism assay –Relatively high specificity for predicting X4 virus but uniformly low sensitivity

28. clinicaloptions.com/hiv HIV Entry and Tropism Utility of Genotyping Algorithms  High specificity means algorithm can confirm X4 virus with ~ 90% accuracy if sample includes X4 virus  Some experts believe a highly specific algorithm can be used to screen a viral sample –If X4 tropism confirmed, CCR5 antagonist should not be prescribed –If X4 virus not confirmed, phenotypic test should be ordered  Other experts believe even 90% specificity is not enough to make this approach useful

29. clinicaloptions.com/hiv HIV Entry and Tropism Phenotypic Tropism Assays  1 CLIA-validated phenotypic assay, Trofile, commercially available  Other phenotypic assays in development –XTrack and PhenX-R highly sensitive in early studies, detecting R5 or X4 HIV representing only 1% of viral population [1] –Phenoscript-Env assay detected X4 virus representing < 2% of viral population [2] 1. Hamy F, et al. European HIV Drug Resistance Workshop 2007. Abstract 60. 2. Roulet V, et al. European HIV Drug Resistance Workshop 2007. Abstract 59.

30. clinicaloptions.com/hiv HIV Entry and Tropism CD4+ CCR5+ Infection CD4+ CXCR4+ ++ HIV signaling enzyme HIV Env expression vector* Inserted into cell line so that HIV can replicate Viral pseudotypes produced *Amplified from patient’s blood sample. How Phenotypic Tropism Assay Works Whitcomb J, et al. Antimicrob Agents Chemother. 2007;51:566-575.

31. clinicaloptions.com/hiv HIV Entry and Tropism Reliability of Phenotypic Coreceptor Tropism Assay  Used in clinical trials of CCR5 antagonists  Has analyzed > 23,000 viral samples in clinical trials and successfully called 94%  Can detect X4 virus with 100% accuracy when X4 makes up ≥ 10% of viral population  Detects X4 virus 83% of time when it makes up ≥ 5% of viral population  Success rate of viral amplification drops at HIV-1 RNA < 1000 copies/mL  Enhanced luciferase activity with potential modifications to assay [1] –Possible to detect X4 variants representing 0.1% of viral population –Earlier detection of X4 variants in longitudinal samples 1. Reeves, JD, et al. ICAAC 2007. Abstract H-1026.

32. clinicaloptions.com/hiv HIV Entry and Tropism Predictive Value of Tropism Testing 1. Gulick R, et al. IAS 2007. Abstract TUAB102. 2. Mayer H, et al. IAC 2006. Abstract THLB0215.  Negative predictive value of tropism testing confirmed in clinical trials –Identify patients not likely to benefit from CCR5-antagonist therapy  ACTG 5211: VCV + OBR vs placebo + OBR in treatment- experienced patients with only R5 virus [1] –12/118 had only R5 virus at screening but D/M virus at study entry –D/M or X4 virus present in 35% of VCV-treated patients vs 11% of placebo-treated patients who experienced VF  Study A4001029: MVC + OBR vs placebo + OBR in treatment- experienced patients with D/M virus [2] –Similar HIV-1 RNA reductions in all treatment arms, suggesting no virologic benefit from CCR5 antagonist in this population

33. clinicaloptions.com/hiv HIV Entry and Tropism Limitations of Phenotypic Tropism Assay  Logistics, cost, turnaround time (14-16 days)  ~ 83% accurate when a minority HIV variant makes up ≥ 5% of viral population  In trial of antiretroviral-experienced patients, 10% of samples rated as D/M-tropic at study entry, after rating them R5-tropic at screening a few weeks before [1]  Differentiation between dual-tropic and mixed-tropic virus not possible 1. Gulick R, et al. IAC 2006. Abstract THLB0217.

34. clinicaloptions.com/hiv HIV Entry and Tropism FDA Recommendations for Use of Maraviroc Selzentry [package insert]. Pfizer. New York, New York. August 2007.  Tropism testing and treatment history should guide use of MVC  MVC indicated for patients with detectable R5 virus only, evidence of viral replication, and resistance to multiple antiretroviral agents  Use of MVC not recommended in patients with D/M or X4 HIV-1, as efficacy not demonstrated in a phase II study in this patient group  Safety and efficacy of MVC not established in treatment- naive adult patients or pediatric patients

35. Summary

36. clinicaloptions.com/hiv HIV Entry and Tropism Summary  HIV must use either coreceptor CCR5 or CXCR4 to attach to cell –Infection established by R5 virus –X4 virus may emerge during disease progression  R5 virus in 80% to 90% of antiretroviral-naive patients  D/M or X4 virus in 40% to 50% of antiretroviral-experienced patients  Emergence of X4 associated with disease progression  Tropism test needed to determine coreceptor usage in each patient’s viral population –Genotypic algorithms too insensitive for clinical use –Only 1 phenotypic tropism test CLIA-validated for clinical use  CCR5 antagonists not recommended in patients with detectable X4 virus

37. clinicaloptions.com/hiv HIV Entry and Tropism For a detailed overview of this topic, visit clinicaloptions.com/HIVentry  Free, online CME credit for educational modules written by our expert faculty: Eric S. Daar, MD; Robert W. Doms, MD, PhD; Anton L. Pozniak, MD, FRCP; Jürgen K. Rockstroh, MD –Understanding HIV Entry and Targets for Therapy –Tracking Tropism: Epidemiology and Clinical Tools –Recent Clinical Studies of HIV Entry Inhibitors –Entry Inhibitors in Clinical Practice: Strategies to Achieve Treatment Goals  Downloadable slides are also available covering each of these important topics!