In order to identify correlates of SC status, we compared clinical characteristics, viral factors, genetic traits and immune activation markers in the two SC and patients who rebound after treatment cessation. We used the following criteria to define SC and rebounders: (1) being HIV-1 infected for at least 3 years; (2) HAART started in the chronic progressive phase; (3) HAART discontinued for the first time; and (4) VL below the detection limit of 1.6log at treatment interruption. Pregnant women taking HAART to prevent mother-to-child transmission and patients who started treatment in the acute phase of infection were excluded. SC were defined as patients who met the above criteria and who kept their VL below 3log for at least 6 months after treatment cessation. In order to make sure that the SC were not taking antiretroviral drugs, plasma samples were tested for the presence of protease inhibitors (PI) and non nucleoside reverse transcriptase inhibitors at the Bioanalytical Laboratory of the Catholic University of Louvain.
The following parameters were assessed: HIV subtype, CD4 count, VL and number of days on HAART and were compared in SC and rebounders. In addition, we evaluated plasma markers of immune activation, including: neopterin; beta-2 microglobulin (B2M) (Demiditek, Germany); soluble CD14 (sCD14) (R&D, United Kingdom); and lipopolysaccharide (LPS) (Lonza, Belgium). These measurements were done on stored plasma samples taken before starting HAART (T1), during HAART (T2) and 6 months after stopping HAART (T3). Soluble markers in plasma were measured instead of activation markers on cells (Roberts AIDS 2010), because at our clinic only plasma samples of patients are stored.
In order to assess viral factors, the SC provided 100 ml of blood for purification of CD4+ T-cells. One part was used to extract DNA, as described elsewhere , for sequence analysis of Gag, Pol and Env. The other part was used to cultivate the virus .
In addition, we evaluated the relative replication capacity of P1. To assess viral fitness phytohemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) from 3 healthy donors were infected at multiplicity of infection (MOI) 10-3 with the viruses, IIIB and BAL as reference strains (NIH, Germantown, MD), and with virus isolated from SC patient 1 and from 4 different rebounders. P24 concentrations were measured by ELISA over time.
This study was approved by the Institutional Review Board of ITM and the Ethics Committee of the University Teaching Hospital in Antwerp. Informed consent was obtained from all patients.
When searching our database on 1700 patients in regular follow-up, we found that a total of 160 patients had stopped HAART for the first time, after a mean of 25 months of successful viral suppression. Treatment was stopped under medical guidance, mostly because of toxicity and/or because the patient wanted a drug holiday. At least two VL measurements after treatment cessation were available for 124 patients, which enabled us to classify them as "rebounders" or "SC". Median time to rebound (VL > 3log) was 7 weeks and median time to re-starting treatment was 4.5 months. Sixty-eight patients remained off treatment for at least 6 months. By that time all but 2 patients (the SC) had a VL that exceeded 3log. Both SC were negative on the drug assays.