Immune correlates of unusual control of viral replication after cessation of HAART Ellen Van Gulck 1, Leo Heyndrickx 1, Céline Merlin 1, Sandra Coppens 1, Derek Atkinson 1, Eric Florence 2, Anne Buvé 3, Guido Vanham 1,4. 1 Virology unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp (ITMA), Antwerp, Belgium. 2 HIV/STD unit, Department of Clinical Sciences, ITMA, Antwerp, Belgium. 3 STI/HIV Epidemiology and Control Unit, Department of Microbiology, ITMA, Antwerp, Belgium 4 Department of Medicinal Chemistry, University of Antwerp (UA); Faculty of Medicine and Pharmacy, Free University of Brussels (VUB) and Department of Gynecology and Obstetrics, Catholic University of Leuven (KUL), Belgium. Institute of Tropical Medicine, Antwerp Nationalestraat 155, B-2000 Antwerp, Belgium Tel Fax Abstract Background: So far highly active antiretroviral treatment (HAART) has not resulted in “cure” of HIV infection mainly because of latent virus reservoirs. Cessation of HAART typically results in viral rebound within days or weeks after treatment interruption. We have defined 4 patients who were treated for progressive HIV infection, but spontaneously controlled their VL for at least 6 months after cessation of HAART. We labeled these patients “secondary controllers” (SC). We hypothesized that the combined selection pressure of HAART and immunity had resulted in the induction of T-cell responses that prevent the archived provirus from growing out after treatment interruption. Here we present immune characteristics of these patients. Methods: We compared the SC with 4 control groups (of 4 patients each) including elite controllers (EC); patients under HAART; treatment naïve patients; and non-controllers. All patients were matched on % CD4 T-cells. Proviral load, intracellular unspliced and multiple spliced HIV mRNA in CD4 T-cells was measured by real-time PCR. Magnitude of T cell response against HIV was measured using IFN-  ELISPOT. Neutralizing activity of IgG was measured using TZMbl assay. The capacity to prevent infection by CD8+ T-cells was measured using a viral inhibition assay. Purified CD4+ T-cells were infected at MOI 10-3 with labstrains or autologous virus. Four hours after infection autologous CD8+ T-cells were added. P24 was monitored every 3 days, for 14 days. Results: Proviral load in SC and EC was much lower compared to the other groups and correlated with the amount of unspliced and multiple spliced mRNA. There was no difference between the groups in the magnitude of induced HIV-specific responses. However, there was a significant difference in the capacity of CD8+ T-cells to inhibit infection. CD8+ T-cells from all EC and SC could inhibit infection of CD4+ T-cells while those from others could not. Broad cross neutralizing antibodies were lacking in all patients. However, in contrast to the control patients, SC showed neutralization against autologous virus. Conclusion: SC had a normal disease progression before starting HAART but behave like EC after cessation of HAART. Low intracellular HIV DNA and RNA, together with strong virus inhibitory capacity are the most discriminating parameter of controllers versus non-controllers. These findings should be taken into account when designing and evaluating new immuno-therapeutic strategies. Patient Characteristics at enrolment of study GroupPatientHIV-1 subtypesexoriginageYears since HIV diagnosis Days on HAART Viral load (copies/ml) CD4 T cell count (cells/µl) SC P1BMBelgium <50250 P2DFBelgium < P3BMVenezuela364Unknown<50244 P4A1MBelgium < EC P5BMBelgium3670<50633 P6BMBelgium62130<50614 P7BMBelgium6190<50437 P8BMBelgium5990<50675 SNC P9DFCongo P10BMBelgium P11BMBelgium P12BFBelgium TN P13BMBelgium P14BMBelgium P15BMBrazil P16BMBelgium HAART P17CRF02_AGFBelgium <50707 P18BMBelgium <50973 P19BMBelgium <50806 P20BMBelgium <50438 Results: 1) Magnitude and breadth2) Virus inhibition assay3) Autologous neutralization4) Real time PCR Materials and methods Blood HIV-1 Donor plasma PBMC Isolation IgG Isolation total RNA Stimulation peptides Stimulation bispecific monoclonal AB Neutralization assay against autologous gp160 (result 3) Real time PCR for ribosomal, unspliced and multiple spliced RNA (Result 4) Real time PCR for proviral DNA and beta actin (Result 4) Virus inhibition assay (Result 2) ELISPOT (Result 1) PBMC from different patient groups were stimulated with peptide pools encoding the whole HIV genome. A)Represented the mean number of peptide pools inducing pos ELISPOT responses B)The mean number of IFN-  spot forming cells per million PBMC Purified CD4+ T-cells from SC (A) and from EC (B) were infected with different virus isolates and cultured in the absence or presence of CD8+ T-cells (1:1 ratio). On day 13 the ratio of p24 production in the supernatant of cultures with CD8+ T-cells and without CD8+ T-cells was calculated and the % inhibition of viral production is represented. Pseudoviral constructs were generated from the plasma samples. The same plasma was also used to purify IgG that was used in the PV-TZMbl neutralization assay to look for autologous neutralization. This assay was performed for SC (A), SNC (B), TN (C) and HAART patients (D). Percent neutralization is shown over a concentration range of individual patient IgG. Each patient is represented by one symbol. Boston, – CROI – 18th Conf on Retrovir & Opport Infect