Control of viral replication after cessation of HAART

Highly active antiretroviral treatment (HAART) has improved life expectancy and quality of life of HIV infected patients [1]. However, so far it is not possible to "cure" HIV infection mainly because latent reservoirs persist even in patients who are on effective combination treatment [2]. Cessation of HAART results in viral rebound within days or weeks and pre-treatment levels of viral load (VL) are typically reached within one year after treatment interruption [3, 4].

In 2008 we identified a patient who, after stopping HAART, had a VL below the detection limit of 1,6log for more than 2 years. After searching the data base of 1,700 HIV infected patients under follow-up at the clinic of the Institute of Tropical Medicine, another patient was identified, who spontaneously controlled VL after cessation of HAART for at least 6 months. We labeled these two patients "secondary controllers" (SC) as opposed to "elite controllers" (EC) who spontaneously control viral replication but are treatment naive.

Patient 1 was diagnosed with HIV infection in 1985. In 1993 he started treatment with AZT, when his CD4 count was 228/μL. VL testing was unavailable at that time and no plasma samples from 1993 were available at the clinic in Antwerp. The first VL measurement dated from 1999 when the patient was already taking AZT monotherapy. It was 3.4 log and a PI based HAART regimen was started and continued till 2001, when the patient was switched to Nevirapine and 2 nucleoside reverse transcriptase inhibitors (NRTI's). He took this combination till treatment interruption in April 2005, when his CD4 count was 458/μl. In July 2005 the VL was 3.9log but from October 2005 until now it remained below the detection limit. The CD4 count, however, has been decreasing to 205/μL in February 2010 but the patient refuses to be restarted on treatment.

Patient 2 was found to be HIV positive in 1997, but was probably infected since 1991. In 1998 her CD4 count was 377/μL and her VL 4.6log. She was started on HAART with a PI based regimen. In 1999 she switched to Nevirapine and 2 NRTIs. Treatment was stopped in March 2006. The VL remained undetectable after treatment interruption, though in April 2009 there was a blip in the plasma VL of 2,9log, which was detected with the Cobas ampliprep/cobas Taqman (Roche, New Jersey, USA). The patient is still off treatment with a stable CD4 count above 1000/μL.

Virus measured as p24 in culture supernatant of purified CD4+ T-cells from patients under HAART, typically emerges after 3 to 6 days. In contrast, in patient 1 it emerged on day 21, while in patient 2 virus culture failed despite three attempts. In 2009, however, the latter patient had a blip in plasma virus suggesting that the virus is not completely defective. Moreover the sequences of Gag, Pol and Env in the two SC did not show any obvious defects, and no CD8 T-cell associated escape mutations could be detected. Remarkably, phylogenetic analysis of env from patient 1 revealed the presence of two related but distinct viruses. Both viruses were lacking the lentivirus lytic peptides (LLP) 1 region, but could still be cultured.

Autologous viruses from patient 1 could be cultured making it possible to investigate whether the virus of this patient was less fit in comparison to reference strains or virus of 4 different rebounders. Figure 1 clearly shows that the virus of the SC replicates less as compared to the reference strains and viruses of rebounders since the amount of p24 produced in PHA stimulated PBMC remains much lower over time in comparison to viruses of rebounders and the reference strain.

We identified two HIV-1 infected patients with an exceptional clinical course after stopping HAART. One patient kept stable and high CD4 T-cell counts for more than 4 years after treatment cessation, while the other patient had progressive disease with decreasing CD4 T-cell counts. Such discrepancy of evolution has also been described in elite controllers [7–9].

To our knowledge there is only one previous study that had identified patients who maintained an undetectable viral load for several months after treatment cessation. Skiest et al mentioned 2 patients who had an undetectable viral load for 6 months after treatment was stopped as part of a study on the effects of treatment interruption. However, they just mention them without comparing them with patients whose viral load rebounded [10]. We are aware of only one study that has explored factors predictive of "non-rebounder" status [11]. Bedimo et al defined non-rebounders as patients who had a VL below 3,7log at least 6 months after treatment interruption while we defined SC as patients who had a VL below 3log. In Bedimo's cohort 27% of patients were non-rebounders and peak VL prior to HAART was predictive of non-rebounder status. In contrast, the SC we identified had a VL prior to starting HAART that was in the range of VL of rebounders which we defined as having a VL > 3 log 6 months after HAART cessation. In our study VL before HAART initiation seemed thus not to be associated with control of viral replication after treatment cessation.

One possible explanation for the SC status is that deleterious mutations have been induced in the virus by HAART. However, sequencing of the major structural genes of the 2 available viruses did not reveal any obvious defects. Virus could be cultivated in patient 1 only, but patient 2 had a spontaneous "in vivo" blip. We hypothesize that these replication competent viruses are less fit and/or are kept under control by the patient's immune system. Indeed, as shown in Figure 1, virus from patient 1 was less fit than the viruses from rebounders and the two reference strains BAL and IIIB. The fact that the virus from patient 2 could not be cultivated in vitro under conditions that easily allow virus growth in all other patients is also an implicit proof of lower fitness.

Markers of immune activation are more disturbed in patients with rapid progression and closer to normal in EC [12, 13]. Neopterin and β2 microglobulin have also been shown to predict disease progression, especially in late stage HIV-1 infection [14–16]. However, we did not find any differences between SC and rebounders, in levels of immune activation before initiating therapy, during therapy and after treatment cessation, suggesting that immune activation does not play a role in SC status.

Research on EC is very active because these patients may give clues for novel approaches to the development of a preventive HIV vaccine. Research on immune function in the secondary controllers in this study is ongoing; as we believe that they might provide new insights about correlates of protection and be useful for the development of therapeutic vaccines.

Written informed consent was obtained from the patients for publication of this case report. A copy of the written consent is available for review by the Editor-in -chief of this journal.

EVG is a postdoc at the Institute of Tropical medicine. During her PhD she was trying to develop a therapeutic vaccination against HIV based on dendritic cells electroporated with mRNA encoding HIV Gag. In this studies patients on HAART were vaccinated with these DC. After vaccination it is the goal that they stop treatment and control the virus. Therefore these patients are so special because they can do it without vaccination. If we can find out why than we know to which parameter we have to look for protection in a therapeutic vaccination. AB, EF, GV are clinicians. EF is really working in the clinic and seeing patients. AB and GV are more interested in research. For EF is it also important if we can unravel why these patients control the virus after treatment interruption, maybe more patients can go on drug holiday.