Elite controllers are individuals who naturally control HIV replication in the absence of therapy [1,2]. They count for approximately 1%  of all HIV-positive subjects. In most individuals, the control of HIV replication requires the use of a continuous combined antiretroviral therapy (cART) as discontinuation of cART results in a rapid rebound of infection  due to the replication-competent HIV that persists in latent reservoir. Here, we describe a new controversial phenomenon: individuals who started and remained on cART for several years, and who stopped therapy failing to exhibit the expected viral rebound. These post-treatment controllers (PTCs) are frequently described among individuals treated during the early phases of infection [5–11]; however, there have been scattered reports of individuals who started cART during chronic infection and who subsequently interrupted it exhibiting some degree of control of plasma viremia [12,13]. We report three of these cases.
Patients are included in a large cohort (2700 subjects) of HIV-positive individuals treated in a reference Center of Northern Italy. All patients were treated and monitored according to current Italian guidelines for the management of HIV infection.
HIV-RNA was quantified by routine tests and was reported according to the limit of detection of each system. In the last years, we have been using a test with a lower limit of detectability of 3 copies/ml .
Since 2011, at each lab evaluation, immune-activation was measured by computing the percentage of CD8+CD38−HLA*DR+ cells and of CD4+CD38−HLA*DR+ cells. For the description of cases, we performed a few analyses not routinely used in clinical practice. Low-resolution (two-digit) major histocompatibility complex class I and class II genotyping (HLA) was performed by using sequence-specific PCR primers.
We quantified the total HIV-DNA level, both integrated and nonintegrated, by real-time PCR with amplification of the LTR gene in peripheral blood mononuclear cells [5,15]. Values have been adjusted for the number of CD4+ T cells. We tested patients at least once for plasma levels of ARV drugs, so to rule out the possibility of unprescribed drug use.
Patient 26636, female, IVDU, was diagnosed HIV positive [Center for Diseases Control (CDC) class A1] in February 1991. She presented a hepatitis C virus genotype 3 coinfection. At that time, HIV-RNA test was not available. Her first CD4+ cell count was 916 cells/μl, so we delayed therapy until October 1993 when her CD4+ cell count dropped to 555 cells/μl (Fig. 1c). At the age of 24 years, she started zidovudine (AZT) monotherapy that was maintained until July 1994 when she performed a voluntary stop and then again from December 1994 to June 1996. In August 1997, she started AZT + didanosine (DDI). She performed her first HIV-RNA test in March 1998 which was below the 500 copies/ml limit of detection (Fig. 1). The viral load varied between 50 and 500 copies/ml thereafter until December 2000 and was steadily less than 50 copies/ml until August 2002 when she enrolled in a structured treatment interruption trial . After therapy withdrawal, HIV-RNA rebounded up to 710 copies/ml and then spontaneously decreased below 50 copies/ml (and often <3 copies/ml) until January 2015 (13 years) when cART was restarted (in the absence of measurable HIV-RNA) because of a drop in CD4+ (337 cells/μl) (Fig. 1). Over the time off therapy, the percentage of CD8+CD38−HLA*DR+ cells varied from 23.7% to 52.3% and that of CD4+CD38+HLA*DR+ cells from 1.9% to 8.2%. CD8+CD38−HLA*DR+ cells lowered between 6% and 20.2% after therapy was resumed. When tested for HLA class I and II alleles, she was HLA−A*02 *29, HLA−B*18 *52, HLA−C*07 *12 and HLA−DRB1*11. T-cell-associated HIV-DNA, tested soon before restarting cART, was 3.78 log copies/106 CD4+.
Patient 93016, male, IVDU, was diagnosed HIV positive (CDC class A2) in August 2005. He had a previous negative HIV serology 15 months before. At time of diagnosis, HIV-RNA was 131458 copies/ml, and his first CD4+ cell count was 245 cells/μl. In October 2005, at the age of 27 years, he started cART [DDI + lamivudine (3TC)+ efavirenz], his HIV-RNA dropped less than 50 copies/ml by December 2005 and was always below this threshold until August 2008 when he decided to stop therapy. Despite therapy withdrawal, HIV-RNA remained steadily less than 50 copies/ml until August 2009 and less than 3 copies/ml thereafter (11 years) (Fig. 1b) with CD4+ values steadily well above 500 cells/μl (Fig. 1). During the time off therapy, the percentage of CD8+CD38−HLA*DR+ cells varied from 1.0% to 4.1% and that of CD4+CD38+HLA*DR+ cells from 0.7% to 1.9%. When tested for HLA class I and II alleles, he was HLA−A*01 *03, HLA−B*18 *35, HLA−C*04 *12 and HLA−DRB1*08 *11. T-cell-associated HIV-DNA was 3.48 log copies/106 CD4+ after 11 years off cART.
Patient 50293, male, heterosexual infection, was diagnosed HIV positive (CDC class B2, oral thrush) in April 2006 at the age of 70 years. At time of diagnosis, HIV-RNA was 45499 copies/ml and his first CD4+ cell count was 388 cells/μl. In August 2006, he started an atazanavir-boosted-based cART that continued without interruptions until December 2014 with different backbones (emtricitabine/tenofovir; DDI + 3TC; AZT/3TC and abacavir/3TC) because of a concomitant chronic kidney insufficiency and dyslipidemia. In December 2014, he simplified cART to 3TC + boosted-darunavir, and in April 2015 further changed to 3TC + dolutegravir. His HIV-RNA dropped less than 50 copies/ml by October 2006 and was always below this threshold until July 2009 and less than 3 copies/ml thereafter. In January 2016, he decided to stop therapy. Despite therapy withdrawal, HIV-RNA remained steadily less than 3 copies/ml (18 months) (Fig. 1a) with CD4+ values steadily well above 500 cells/μl (Fig. 1). During the time off therapy, the percentage of CD8+CD38−HLA*DR+ cells varied from 1.2% to 5.0% and that of CD4+CD38+HLA*DR+ cells from 0.3% to 0.9%. When tested for HLA class I and II alleles, he was HLA−A*11 *68, HLA−B*51, HLA−C*14 *15 and HLA−DRB1*04 *13. T-cell-associated HIV-DNA was 3.13 log copies/106 CD4+, 18 months after cART withdrawal.
All the best characterized post-treatment controllers started therapy during early HIV infection.
The most comprehensive description of PTCs is based on individuals (14 subjects) included in the VISCONTI cohort . Since then, to date, there have been additional observational data and case reports of individuals able of achieving prolonged periods of virologic control after cART interruption [6–13]. Estimating prevalence of PTCs is difficult; nonetheless, the VISCONTI cohort, as well as other early reports, suggests that prevalence could be much higher than that of elite controllers and could be as high as 5–15% .
Early treatment of HIV infection seems to be an important factor on becoming a PTC, although it does not ensure post-treatment control .
A good example may be a recent pediatric case from the French team who described the VISCONTI cohort . A female infant, infected at birth, received AZT prophylaxis after birth for 6 weeks and started cART, at the age of 3 months, when plasma HIV-RNA became positive. The child was lost to follow-up between 5.8 and 6.8 years and stopped treatment during that time. Upon re-engagement into care and off ART, the plasma HIV-RNA was undetectable. At 18 years of age, she remains off therapy and has always had undetectable plasma HIV-RNA except for two small blips.
The degree to which post-treatment control is possible in a setting of chronic disease is controversial, and the prevailing opinion is that such outcomes are not possible. However, there have been sporadic reports of individuals who started cART in the chronic state and that subsequently successfully interrupted therapy. Belgian Authors  reported four patients who maintained plasma viral loads less than 50 copies/ml at 6 months after cessation of therapy, even if two of them had an initial, detectable rebound. During an extended 3 years follow-up, one patient had a viral blip of about 1000 copies/ml; another multiple blips and a third controller restarted cART, while virologically suppressed, due to infectious complications.
In a treatment interruption study performed in France, 95 patients, who started treatment during chronic infection and were successfully treated, stopped cART . Seven of them had a HIV-RNA less than 400 copies/ml 12 months after treatment interruption, and four maintained viral suppression up to 36 months.
It is difficult, especially in the observational setting, to describe the characteristics of PTCs, and the risk exists that individuals considered as PTCs more likely reflect poorly characterized controllers who received and stopped cART and that cART really did not alter the virus/host interactions.
We checked our patients during the off-therapy period for the presence of unprescribed ARV drugs and all tested negative.
We can reasonably affirm that our patients started therapy during chronic infection. Patient 26636 started her first ARV drug 2 years after diagnosis, and patients 93016 and 50293 started cART 2 and 5 months after the first positive HIV test, with high viral loads and low CD4+ cell counts (namely 245 and 388 cells/μl) testifying a much longer persistence of infection. The high viral loads of the last two patient is also in contrast with the hypothesis that these two individuals could be spontaneous controllers [5,18,19]. We did not have a baseline HIV-RNA of patient 26636 and afterward during treatment interruptions her viral loads never exceeded 1000 copies/ml; in her case, the possibility of a misinterpretation of her status cannot therefore completely ruled out, also because her higher levels of immune-activations stand in favor of being an elite controller [2,20,21]. However, none of our patients present any of the protective class I HLA alleles, particularly HLA−B*57 and to a lesser degree HLA−B*27, that are overexpressed in elite controllers [20,21] and much less common in PTCs . Patient 26636 shows one of the five most frequent HLA haplotype described in a large Italian casuistry , and patient 93016 has the HLA−B*35 allele that appears to be enriched in the PTCs and is associated with more rapid HIV progression [5,12,23]. Furthermore, HLA−B*18 and HLA−C*12 appear to be enriched in these patients, as they are expressed by 18.6% and 12.7% of the Italian population , respectively.
Compared with other reports [5,12,13] on PTCs, our patients during the off-therapy period presented a slightly higher HIV-DNA levels. However, it must be underlined that in the French experience previously cited  of the seven individuals with HIV-DNA levels below the limit of detection, only four maintained plasma viral loads less than 400 copies/ml to month 12, suggesting that other factors contributed to viral control other than just low HIV-DNA levels. HIV-DNA levels in our patients were consistent with levels previously reported by our group in subjects on chronic therapy with persistent absence of any measurable viremia (HIV-RNA constantly <3 copies/ml) . Significantly, patients 93016 and 50293 who off-therapy steadily maintained their virus suppressed below 3 copies/ml showed lower HIV-DNA levels than patient 26636 that presented viral blips less than 50 but more than 3 copies/ml. Patient 26636 restarted cART after 13 years of therapy because of a slight decline of her CD4+ cell count. The dynamic of CD4+ loss, in this patient, was comparable with that previously reported  in similar settings and can be explained, at least partially, by the presence of a residual low-level viremia. On the other hand, patient 50293, who currently has a much shorter follow-up off therapy, in the first 18 months without cART was absolutely comparable with patients 93016 in terms of HIV-RNA spontaneous control.
We did not measure HIV-specific immune response, but evaluated immune activation. Post-treatment controllers have lower levels of CD8+ T-cell activation compared with elite controllers and those who are on effective cART [5,25,26]. It has been reported that an elevated level of CD38 antigen expression on CD8+ cells is a predictor that HIV disease will progress to AIDS and that the prognostic power of elevated CD38 expression on CD8+ cells is greater than that of any other activation marker and greater than that of CD4+ cells number or percentage [27,28]. Patients 93016 and 50293 had very low levels of CD8+CD38−HLA*DR+ cells and their values were much lower than those of patient 26636.
Finally, we checked our patients during the off-therapy period for the presence of unprescribed ARV drugs, and all tested negative.
One must consider carefully the ethical implications of treatment interruptions after the SMART study  demonstrated the increased risks associated with Structured Treatment Interruptions. However, any evaluation of interventions aimed at functional cure will require a treatment interruption . Cases like ours, although sporadic, may advance our understanding of the characteristics for which individuals may be more likely to achieve ART-free remissions. Furthermore, our patients are among the few, so far described in patients who started cART during chronic infection extending the hope that a functional cure is possible even in this setting.
Author contributions: F.M. followed patients, performed unscheduled exams, analyzed the data and wrote the paper. E.D. followed patients, searched for literature and revised the paper. L.C. followed patients, tabulated results and revised the paper. A.C. performed virological exams, searched for literature and revised the paper. All discussed results and article layout.
Conflicts of interest
There are no conflicts of interest.
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