S Kinloch-de Loes, F C Lampe, L Perrin, D Cooper, B Hoen, L Goh, J Andersson, A Sonnerberg, D Smith C Tsoukas, B Autra

1. QUEST study: Follow-up of primary HIV infection subjects on long-term ART randomized to therapeutic immunization versus placebo at one year post-stopping treatment S Kinloch-de Loes, F C Lampe, L Perrin, D Cooper, B Hoen, L Goh, J Andersson, A Sonnerberg, D Smith C Tsoukas, B Autran, R El Habib, G Theofan, A N Phillips for the QUEST study group

2. Background It is unclear whether therapeutic immunization can increase virological control in the long-term after discontinuation of ART initiated in acute HIV infection. The rationale for such an intervention includes the absence of chronic immunosuppression, limited reservoirs of integrated proviral DNA and the potential for preservation of HIV-specific T cell responses. Therapeutic immunization may further boost these immune responses which tend to decrease with long-term ART.

3. Background The QUEST trial assessed the impact of therapeutic immunization of two vaccines in addition to ART versus ART alone on HIV plasma viremia (VL) 24 weeks after treatment interruption, in subjects who had initiated ART (Combivir®-abacavir-amprenavir bd) at the time of primary HIV infection (PHI).

4. Quest Study Design The QUEST study design is shown in Figure 1. PHI subjects (n=79) on chronic ART for >72 weeks were randomized to one of three arms: Arm A: ART + vaccine placebo Arm B: ART + ALVAC-HIV vCP1452 Arm C: ART + ALVAC-HIV vCP1452 + Remune™ After 24 weeks subjects discontinued ART. Subjects were followed with regular laboratory and clinical monitoring for an additional 24 weeks when the primary end-point was measured (percentage of subjects with plasma VL < 1000 HIV copies/mL).

5. Randomisation HAART + Placebo vaccines >72 weeks HAART: durable viremia <50c/mL HAART + Alvac HIV vCP 1452 + Placebo Remune™ PHI Quadruple HAART (n = 79) HAART + Alvac vCP 1452 + Remune™ W0 W4 W8 W12 W16 W20 Rm Rm Rm Rm VCP VCP VCP VCP Rm = Remune™ VCP = ALVAC-HIV vCP1452 Figure 1. Quest Study Design Week24 STOP ART

6. Quest findings at 24 weeks post-stopping • Results at week 24 post-stopping ART showed: • No difference in the percentage of subjects with viral load <1000 HIV copies(c)/mL between the pooled ART+ vaccine arms (B/C) and the ART+ placebo vaccine arm (A) [primary efficacy endpoint]. • Immunogenicity in vaccine arms by IFN-gamma CD4 and CD8 ELISPOT analysis at the end of the immunization phase. • Absence of correlation between immunogenicity at the end of the immunisation phase and viremia levels at week 24 post-stopping ART.

7. Objective The Quest cohort has been followed up since assessment of the primary endpoint at 24 weeks post-stopping ART (DSMB recommendation). The follow-up will assess the impact of our therapeutic strategy on VL, CD4, ART use and clinical events in the longer term. The current analysis extends the follow-up of the 79 randomised patients to 48 weeks post-stopping ART.

8. Methods Individualized forms sent to enrolling centers in Europe, Canada and Australia recorded patient status, laboratory parameters (viral load and CD4 count), antiretroviral treatment details and clinical AIDS-defining events. Data analysis was performed centrally (Royal Free and University College Medical School, London).

9. Methods Proportions of subjects with VL  1000 HIV-1 copies/mL at week 48 post-stopping ART were assessed andcompared between arms A and B/C using Fishers exact test and an intention to treat approach (subjects with missing VL, those who did not stop ART and those who restarted ART were classified as failure). Absolute values of VL and CD4 count at week 48 post-stopping ART were compared between arms A and B/C using the Mann-Whitney test. Subjects with missing values and those who restarted treatment were included using ‘last observation carried forward’, providing this was at least 28 days after stopping ART.

10. Results Follow-up information was received for 60 of 79 patients. No deaths or AIDS events were reported. Table 1 shows numbers of patients who did not stop ART, restarted ART and had missing information at weeks 24 and 48 post-stopping ART. Overall, 15 subjects restarted ART prior to week 48. Last VL (c/mL) prior to ART-restart was: 50 (n=1); 10,000-100,000 (n=2); >100,000 (n=12). No significant differences were found between arms A and B/C for numbers restarting ART, primary VL end-point (Table 2), or median VL and CD4 count at week 48 post-stopping ART (Table 3).

11. Table 1. Patient status

12. Table 2. VL distribution and primary endpoint

13. Table 3. Median VL and CD4 count Using ‘last observation carried forward’ if at least 28 days after stopping ART

14. Conclusion The safety of our intervention was demonstrated by the preservation of CD4 counts and the absence of deaths or AIDS events in subjects who initiated ART at PHI with or without the addition of therapeutic immunization and later interrupted ART. The re-initiation of ART during this additional follow-up occurred mainly because of high VL. The proportions of subjects fulfilling the primary efficacy end-point (<1000 c/mL) decreased at week 48 compared to week 24 post-stopping ART, with no major impact of immunization on VL or CD4 count over the present follow-up period.

15. References 1. Rosenberg ES et al. Science.1997;278:1447-50. 2. Oxenius A et al. Proc Natl Acad Sci USA. 2000;97:3382-7. 3. Altfeld M et al. J Exp Med. 2001;193:169-80. 4. Kinloch-de Loes S et al. J Infect Dis 2005; 192: 607-17.

16. Acknowledgements Quest Study Group:Recruiting centres: Australia: M. Bloch (Holdsworth House General Practice, Darlinghurst, New South Wales), D Baker (407 Doctors Clinic, Surry Hills, Sydney NSW 2010), R Finlayson (Taylor Square Private Clinic, Surry Hills, Sydney NSW 2010), P Grey, D Smith, D Cooper (National Centre in HIV Epidemiology and Clinical Research, Sydney). Belgium: Ph Hermanns, K Kabeya, N Clumeck (Department of Infectious Diseases, St Pierre Hospital, Brussels). Canada: M Harris, J Montaner (John Ruedy Immune Deficiency Clinic, St Paul’s Hospital, University of British Columbia, Vancouver), C Tsoukas (Immune Deficiency Treatment Centre, Montreal General Hospital, Montreal). France: B Hoen (Department of Infectious Diseases, Besancon University Hospital, Besancon), P M Girard (Department of Infectious and Tropical Diseases, Rothschild Hospital, Paris), J Modai (Department of Infectious Disease St Louis Hospital, Paris), Ph Canton, T May (Department of Infectious Diseases, CHU Nancy, Vandoeuvres-les-Nancy), D Sereni (Department of Internal Medicine, St Louis Hospital, Paris), C Katlama (Department of Infectious Diseases, Pitié-Salpetrière Hospital, Paris). Germany: S Staszewski (Klinikum der JW Goethe Universitat. Zentrum der Inneren Medizin, Frankfurt), H J Stellbrink (Medizinische Poliklinik, Universitatsklinikum Eppendorf, Hamburg). Italy: G Tambussi, A Lazzarin (Clinic of Infectious Diseases, San Raffaele Scientific Institute, Milan). Sweden: H Gaines, S Lindback, A Blaxhult (Department of Infectious Diseases, Karolinska University Hospital, Stockholm). Switzerland: M C Bernard, B Hirschel, L Perrin (Division of Infectious Diseases, University Hospital, Geneva), K Wolf, M Battegay (Division of Infectious Diseases, Basle University Hospital, Basle), P Vernazza (DIM, Kantonsspital, St Gallen), R Weber (Department fur Innere Medizin, Zurich). UK: S Kinloch, T Drinkwater, Z Cuthbertson, P Byrne, M Youle, M Tyrer, S Bhagani, M A Johnson (Ian Charleson Centre, Royal Free Hospital, London), C Higgs, D Hawkins, B Gazzard (Kobler Centre, Chelsea & Westminster Hospital, London), A. Friedman (University Hospital of Wales, Cardiff), M Fisher (Claude Nicol Centre Research Department, Royal Sussex Country Hospital, Brighton). GlaxoSmithKline QUEST Team (Greenford, UK): V Mallet, S Turkish, S Fortes, H Maseruka, H Steel, D Thorborn, H McDade, L E Goh. GlaxoSmithKline monitors: Australia (M Haberl, J Young); Belgium (D Luyts, I van Steenberg); Canada (S Pratt, T Russell); France (L Beauvais, J M Vauthier); Germany (M Sikora); Italy (C Gussetti, C M Anghileri, V Piva, D Fendt); Sweden (G Larsson); Switzerland (C Python, I Schauwecker, E Gremlich); UK (K Studdard, P Humphreys, U Loughrey). The Immune ResponseCorporation: (Carlsbad, California, USA) - R Moss, G Theofan. Aventis Pasteur (Lyon, France) - D Blanc, C di Vita, V Mazarin, B Mouterde, R El-Habib. DSMB - M Schechter (Canada), I Weller (United Kingdom), R Luethy (Switzerland), J M Molina (France). Quest Core Group:S Kinloch, D Cooper, L Perrin, B Hoen, A Sonnerborg, C Tsoukas, J Andersson, F C Lampe, A N Phillips, B Autran. Statistical analysis: F C Lampe, N Carter, A N Phillips. Data management: J Puradiredja Roche Molecular Systems (Alameida, CA, USA): B Dale, A Capt. Laboratory support:L Wegmann, S Yerly, L Perrin (Laboratory of Virology, Geneva University Hospital, Geneva, Switzerland). A Samri, B Autran (Cellular and Tissue Immunology Laboratory, Inserm U543, Pitié-Salpetrière Hospital, Paris, France). J Zaunders, P Cunningham, A Kelleher (Centre for Immunology, St Vincent’s Hospital, NCHECR, University of New South Wales, Sydney, Australia). S Martins, G Janossy (HIV Immunology Laboratory, Department of Immunology and Molecular Pathology, Royal Free and University College Medical School, London, UK). A L Spetz, J Andersson (Karolinska University Hospital, Sweden). G Tambussi, A Lazzarin, A Galli (Clinic of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy).