1. XVII International AIDS Conference 3-8 August 2008, Mexico City, Mexico Abstract Session on: "Molecular Epidemiology and Diversity of HIV" 04 August 2008, Salle SR05 Trends in Molecular Epidemiology and Global Surveillance of HIV-1 Diversity Dr Saladin Osmanov Coordinator WHO-UNAIDS HIV Vaccine Initiative World Health Organization Geneva, Switzerland

2. WHO-UNAIDS HIV Vaccine Initiative 2 27 years of the AIDS pandemic and 25 years from the discovery of HIV June 1981: Official registration of the first AIDS cases in USA November 1983 – May 1984 Identification of the human immunodeficiency virus (HIV) as a causative agent of AIDS October-November 1987 The first HIV vaccine trial started

3. Global estimates for adults and children, 2007 People living with HIV 33 million [30 – 36 mln] New HIV infections 2.7 million [1.6 – 3.9 mln] Deaths due to AIDS 2.0 million [1.8 – 2.3 mln

4. Total: 33 million (30 – 36 million) Western & Central Europe 730 000 [580 000 – 1.0 million] Middle East & North Africa 380 000 [280 000 – 510 000] Sub-Saharan Africa 22.0 million [20.5 – 23.6 million] Eastern Europe & Central Asia 1.5 million [1.1 – 1.9 million] South & South-East Asia 4.2 million [3.5 – 5.3 million] Oceania 74 000 [66 000 – 93 000] North America 1.2 million [760 000 – 2.0 million] Latin America 1.7 million [1.5 – 2.1 million] East Asia 740 000 [480 000 – 1.1 million] Caribbean 230 000 [210 000 – 270 000] Adults and children estimated to be living with HIV, 2007

5. Estimated number of adults and children newly infected with HIV, 2007 Western & Central Europe 27 000 [3200 – 69 000] Middle East & North Africa 40 000 [18 000 – 69 000] Sub-Saharan Africa 1.9 million [1.3 – 2.4 million] Eastern Europe & Central Asia 110 000 [53 000 – 200 000] South & South-East Asia 330 000 [76 000 – 680 000] Oceania 13 000 [12 000 – 15 000] North America 54 000 [7600 – 130 000] Latin America 110 000 [37 000 – 200 000] East Asia 51 000 [27 000 – 86 000] Caribbean 20 000 [14 000 – 27 000] Total: 2.7 million (1.6 – 3.9 million) in 2007 or 7,400 new HIV infections every day

6. Over 7400 new HIV infections a day in 2007 More than 96% are in low and middle income countries ~ 1000 are in children under 15 years of age ~ 6300 are in adults aged 15 years and older of whom: - ~ 50% are among women - ~ 45% are among young people (15-24)

7. J A G C B D F1 K H HIV-1 Group M HIV-1 Group O HIV-1 Group N HIV-2 SIVcpz SIVsm SIVagm SIVmnd SIVsun SIVlhoest SIVsyk

8. WHO-UNAIDS HIV Vaccine Initiative 8 The current classification and nomenclature of HIV HIV Types: HIV-1, HIV-2 Groups: M, N, O Subtypes: A-D, F-H, J, K Intersubtype recombinants: Curculating Recombinant Forms: - CRF01_AE, CRF02_AG, etc. - Unique Recombinant Forms

9. WHO-UNAIDS HIV Vaccine Initiative 9 0.05 C F D K B A1 A2 CRF01_AE G J H S, SE Asia (C IN ) ET (C”) S America (C BR ) F1 F2 BR W Africa E Africa CD ZA CM-TD KR SE Asia-CN (B’) W Africa (A3) E Africa FSU (IDU-A) SE Asia CF NG ES CU Genetic variability within HIV-1 Subtypes and CRFs

10. CRF01_AE LTR gag pol vif vpr tat vpu rev tatnef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env CRF12_BF CRF02_AG LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif tat vpu rev tat nef LTR vpr env CRF13_cpx CRF03_AB LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env CRF14_BG CRF04_cpx CRF05_DF LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env CRF15_01B LTR gag pol tat vpu tatnef rev LTR v i f vpr env CRF16_A2D LTR gag pol tat vpu tatnef rev LTR v if vpr env CRF06_cpx LTR gag pol vif vpr tat vpu rev tat nef LTRenv CRF07_BC LTR gag pol vif tat vpu rev tat nef env vpr LTR CRF18_cpx LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev CRF19_cpx LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tatnef LTR env CRF08_BC CRF10_CD CRF11_cpx LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR nef vif vpr vputat rev gag pol env rev CRF20_BG LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev LTR CRF23_BG CRF24_BG A2ABCDEFGHJK01U MOSAIC STRUCTURES OF HIV-1 CIRCULATING RECOMBINANT FORMS (CRFs) CRF01_AE LTR gag pol vif vpr tat vpu rev tatnef LTR env LTR gag pol vif vpr tat vpu rev tatnef LTR gag pol vif vpr tat vpu rev tatnef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env CRF12_BF CRF02_AG LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif tat vpu rev tat nef LTR vpr env LTR gag pol vif tat vpu rev tat nef LTR gag pol vif tat vpu rev tat nef LTR vpr env CRF13_cpx CRF03_AB LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env CRF14_BG CRF04_cpx CRF05_DF LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTR env CRF15_01B LTR gag pol tat vpu tatnef rev LTR v i f vpr env LTR gag pol tat vpu tatnef rev LTR v i f vpr env CRF16_A2D LTR gag pol tat vpu tatnef rev LTR v if vpr env LTR gag pol tat vpu tatnef rev LTR v if vpr env CRF06_cpx LTR gag pol vif vpr tat vpu rev tat nef LTRenv LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTRenv CRF07_BC LTR gag pol vif tat vpu rev tat nef env vpr LTR gag pol vif tat vpu rev tat nef env LTR gag pol vif tat vpu rev tat nef LTR gag pol vif tat vpu rev tat nef env vpr LTR CRF18_cpx LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev CRF19_cpx LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tatnef LTR env LTR gag pol vif vpr tat vpu rev tatnef LTR gag pol vif vpr tat vpu rev tatnef LTR env CRF08_BC CRF10_CD CRF11_cpx LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTR gag pol vif vpr tat vpu rev tat nef LTR env LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev CRF20_BG LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev LTR nef vif vpr vputat rev gag pol env rev LTR CRF23_BG CRF24_BG A2ABCDEFGHJK01U

11. WHO-UNAIDS HIV Vaccine Initiative 11 Global proportion of HIV-1 infections caused by different HIV-1 subtypes and CRFs by 2005 C A B CRF02_AG CRF01_AE D Other subtypes Other recombinants

12. WHO-UNAIDS HIV Vaccine Initiative 12 ABC D F G01 020607/08111219 33 Subtypes/CRF Geographic distribution of HIV-1 subtypes and CRFs B B B B 12_BF C,F G F A C C C A B A A 01_AE, B A,D,C 02_AG, G,A,06_cpx A, G,C,D,F,H,J 05_DF,11_cpx, 13_cpx,18_cpx 07_BC,08_BC, B, 01_AE C 33_01B

13. WHO-UNAIDS HIV Vaccine Initiative 13 Importance of Molecular Epidemiology Studies and the Global Surveillance for the Development of HIV vaccines - Tracking the dynamics and spread of genetic subtypes of HIV-1 and CRFs on a global, regional and population basis. - Generation of key data to provide rationale for matching the locally prevalent HIV strains with vaccine candidates. -Provision of critical information for improved diagnostic and treatment strategies

14. WHO-UNAIDS HIV Vaccine Initiative 14 Importance of Molecular Epidemiology Studies However: - Molecular epidemiology of HIV-1 is characterized by increasingly complex patterns with a clear shift from pure subtypes to CRFs. - The present HIV-1 classification is becoming more and more complicated and it does not directly reflect/predict biology and immunology of the virus. - Relevance of genetic subtypes and CRFs of HIV-1 for transmission, pathogenesis and vaccine protection needs to be critically assessed by appropriately designed epidemiological studies and clinical trials.

15. WHO-UNAIDS HIV Vaccine Initiative 15 HIV Variability and HIV Vaccine Development -Identification of correlates of immune protection -Multiple vaccine strategies and vaccine candidates based on epidemiologically relevant HIV strains -Development of reliable animal models, e.g. SHIV challenges -Clinical trial design (matched vs mismatched trials) -Development and validation of assays for use in clinical trials, e.g. binding and NAb, T-cell assays, VL measurement

16. WHO-UNAIDS HIV Vaccine Initiative 16 Examples of the first matched vaccine phase III efficacy trials rgp120 BB (AIDSVAX B/B) (VaxGen) –USA, Canada, the Netherlands –5.418 volunteers, mostly MSM with only a small control group involving women with high risk for HIV – Start: June 1998 – Completed: February 2003 rgp120 BE (AIDSVAX B/E ) (VaxGen) –Thailand –2.545 volunteers (IDUs) – Start: March 1999 – Completed: January 2004 Don Francis Phil Berman

17. WHO-UNAIDS HIV Vaccine Initiative 17 Opportunities provided by large scale trials: on-going Thai phase III trial, STEP/Phambili trials rgp120 BE (VaxGen) + ALVAC (Pasteur Merieux) –16,000 volunteers –Expected efficacy: 50% protection against HIV infection or reduced viral load set point –Start: September 2003 –End: mid-2009

18. Samarskaya oblast (n=25) Yamal-Nenetsky region (n=101) Irkutsk (n=80) Molecular epidemiology of HIV-1 in Russia (1997-2004) Kaliningrad (n=91)

19. IDU-A B C F1 CRF01_AE A1 1997-2004 2006 IDUHeterosexHomo/Bisex Distribution of HIV-1 genetic forms in St. Petersburg according to risk category (2006)

20. Phylogenetic tree of HIV-1 pol sequences from Russia SIVcpz.US 0.02 95 IDU-A variant 95% 100% 74% 85% H F B D G C 03_AB PR-V77I cluster St Petersburg cluster Yamal-Nenetsky cluster A1 (E. Afr.) 99% Irkutsk 92% Samarskaya 23% Yamal 12% St. Petersburg 46% St. Petersburg 34% Yamal-Nenetsky

21. WHO-UNAIDS HIV Vaccine Initiative 21 Impact of HIV variability on definition of efficacy and validation of assays for use in clinical trials Prevention of infection/disease: Neut/binding Ab, T-cell assays and VL measurement Decrease infectiousness/transmission: Highly sensitive subtype specific assays Better response to ARV treatment: Drug resistance markers Efficacy against different virus subtypes: Large throughput assays for determination of HIV subtypes and CRFs Efficacy in different populations (transmission routes & host genetics: Mucosal and innate immunity; MHC/HLA/epitope mapping

22. WHO-UNAIDS HIV Vaccine Initiative 22 HIV Neutralization Assay Variables TARGET CELLS –Type of cell Transformed cells (CEM) PHA-stimulated PBMCs Resting PBMCs GHOST cells TZM cells –Receptor density –Adhesion molecules –”Drift” of cell lines –Biology of donors of PBMCs VIRUS – T cell line-adapted – Primary isolates Freshly isolated High or low passaged Recent infection Tier 1 or 2 – Cloned Biological clone Moleluclar clone Which clone? How constructed? ANTIBODY - specificity - isotype - avidity/affinity - valency - poly/mono-clonal

23. WHO-UNAIDS HIV Vaccine Initiative 23 EC/WHO NeutNet Project Good agreement between pseudovirus (PSV) assays Good agreement between PBMC assays PSV assays more sensitive than PBMC assays (?) Variation was dependent on both the reagents (AB & sCD4) and the virus PSV assay can measure IC50; PBMC assay requires >IC50 for precise measurements (?) However, no single assay could depict the full spectrum of HIV neutralization and the results were complementary

24. WHO-UNAIDS HIV Vaccine Initiative 24 HIV Neutralization Assay Future Goals a better understanding of the mechanisms of neutralization a more standardized approach to the PBMC assays guidelines for tests to assess HIV-1 neutralizing ABs statistical evaluation of neutralization assays. a panel of HIV-1 isolates representative of the major genetic subtypes in epidemiologically relevant areas of the world. a panel of monoclonal/polyclonal Abs with defined activity

25. WHO-UNAIDS HIV Vaccine Initiative 25 An Effective HIV Vaccine is the Ultimate Goal of Molecular Epidemiology Studies of HIV Development of an effective Global HIV Monitoring and Surveillance Network to guide HIV vaccine development, produce relevant reagents and panels of well characterized HIV isolates for vaccine production Establish correlation between genetic subtypes/CRFs and potential immunotypes of HIV Development of broadly-reactive HIV vaccines or their combinations which are effective against all major HIV subtypes and CRFs

26. WHO-UNAIDS HIV Vaccine Initiative 26 Acknowledments Participating laboratories of the WHO Network for HIV Isolation and Characterization Rafael Najera, Miguel Thomson, Aza Rakhmanova, E. Vinogradova et al. Members of the EC/WHO NeutNet Project (PI: Gabriella Scarlatti) Peter Ghys, Eleanor Gouws, Joris Hemelaar