Caroline P Passaes
email: caroline.passaes@cea.fr

Asier Saez-Cirion
email: asier.saez-cirion@pasteur.fr

HIV cure research: advances and prospects

Keywords: HIV cure, eradication, remission of HIV infection, HIV reservoirs, reactivation of latent virus, repression of provirus, control of infection. Highlights

Preamble

The introduction of improved combined antiretroviral therapy (cART) has dramatically improved the clinical outcome and life expectancy of HIV infected patients. The development of drugs that inhibit different steps of viral replication allows clinicians to successfully manage the disease, improving immunologic parameters, and avoiding drug resistance and side effects. Due to advances in antiretroviral development, HIV infection can nowadays be handled as a complex chronic disease [START_REF] Deeks | The end of AIDS: HIV infection as a chronic disease[END_REF]. Nonetheless, despite all of the clinical benefits provided by drug therapy, cART alone is not able to eradicate the virus, which persists in reservoirs that are thought to be the source for viral reemergence after treatment interruption (Chun et al., 2010;[START_REF] Chun | Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy[END_REF]. In addition, cART does not fully restore healthit increases the risk of non-AIDS disorders such as cardiovascular, kidney, liver and neurological diseases. Moreover, persistent immune dysfunction and inflammation increase the risk of non-AIDS morbidity and mortality [START_REF] Clifford | HIV-associated neurocognitive disorder[END_REF][START_REF] Lake | Metabolic disease in HIV infection[END_REF]. Undoubtedly, a cure for HIV infection is needed to bypass the limitations of the current therapy and restore health. Although a vaccine against HIV remains an unachieved aspiration, several recent findings suggest that either a cure or a durable remission of infection might be possible.

Is a cure for HIV possible?

Since the isolation and characterization of the HIV as the etiologic agent of AIDS [START_REF] Barre-Sinoussi | Isolation of a Tlymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS)[END_REF], the search for a cure has been considered a major research priority. The description of a unique case of HIV cure after hematopoietic stem cell (HSC) transplantation has boosted remarkable optimism in the field and rekindled the hope that a cure for HIV infection is possible. In 2007, the so-called "Berlin patient" -HIV positive and diagnosed with acute myeloid leukemia -received double allogeneic HSC transplant from an HLA-matched and unrelated donor screened for homozygosity for the CCR5 ∆32 allele [START_REF] Hutter | Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation[END_REF]. This patient discontinued ART the day before the first transplantation and, after 6 years of follow up in the absence of therapy, he shows no trace of HIV in blood and tissue samples (Figure 1A), revealing no evidence for persistent HIV. Levels of HIV-specific antibodies have also declined, suggesting that HIV antigen stimulation was very low or absent after transplantation [START_REF] Allers | Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation[END_REF][START_REF] Hutter | Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation[END_REF][START_REF] Yukl | Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient[END_REF].

Several studies have aimed to use autologous or allogeneic HSC transplantation in association with antiretroviral therapy as a strategy to eradicate HIV in seropositive patients diagnosed with leukemia and/or lymphomas (Table 1). Nevertheless, in most of these studies HIV was detected after transplantation, either following therapy withdrawal or because the therapeutic regimen was not able to completely eliminate the viral reservoirs. In addition, in several cases, the patients died after transplantation. The most recent study investigating the impact of cART on viral reservoirs in two patients who received HSC transplantation -known as "the Boston patients"described an important reduction of HIV DNA in long term, reaching undetectable levels [START_REF] Henrich | Longterm reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation[END_REF]. These observations are in agreement with another study which described that HIV DNA levels at month 24 post transplantation in HSC transplanted patients are lower than those observed at baseline [START_REF] Simonelli | Immune recovery after autologous stem cell transplantation is not different for HIV-infected versus HIV-uninfected patients with relapsed or refractory lymphoma[END_REF]. Altogether, these data suggest that HSC transplantation in association with cART may reduce viral reservoirs in HIV infected patients. However, a recent report during the 6th International Workshop on HIV Persistence, Reservoirs and Eradication Strategies revealed that the Boston patients experienced a strong rebound in plasma viremia several months after analytic treatment interruption (ATI) (Figures 1B and1C)( http://es.scribd.com/doc/189931630/12-06-13-Statement-From-Dr-Henrich-Regarding-HIV-Patients). These results suggest that HIV hides in yet unidentified sanctuaries or at levels that are not detectable by current available techniques.

So far, HSC transplantation to eradicate the virus was successful only in the Berlin patient. The mechanisms involved in HIV eradication in this patient are not yet fully understood. This patient underwent severe particular transplant conditions [START_REF] Hutter | Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation[END_REF] (Figure 1), such as double HSC transplantation from the same CCR5 ∆32 donor, conditioning regimen (total body irradiation) and the development of graftversus-host disease (GVHD), which may have contributed to cure. Comparison with the protocols applied in the case of the Boston patients [START_REF] Henrich | Longterm reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation[END_REF] suggests that total body irradiation or the engraftment with cells from a CCR5 ∆32 donor may have been critical differential aspects in the case of the Berlin patient. As the homozygous CCR5 ∆32 deletion offers a natural resistance to HIV infection [START_REF] Dean | Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study[END_REF], this unique successful experience has encouraged the search for new CCR5-based therapeutic approaches for HIV cure interventions (see below). It is interesting to notice that the Berlin patient and both Boston patients were originally heterozygous for the CCR5 ∆32 allele, but this did not confer any advantage to the Boston patients once treatment was interrupted [START_REF] Henrich | Longterm reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation[END_REF][START_REF] Hutter | Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation[END_REF]. A recent effort to cure HIV infection in a 12-year old boy with acute lymphoblastic leukemia consisted of an allogenic transplantation of HSC obtained from cord blood. Similar to the Berlin patient, the donor was screened for homozygosity for the CCR5 ∆32 allele. This procedure was performed at the University of Minnesota in April 2013, but the pediatric patient died two months after transplantation by a severe GVHD (http://www.healthtalk.umn.edu/2013/07/13/pediatric-patient-dies-after-undergoinghistoric-transplant-at-u-of-m/).

Despite the impact of HSC transplantation on reducing HIV reservoirs, this kind of treatment is not a viable option for the majority of HIV infected patients, since it is a risky and expensive procedure that is recommended only for those who develop cancer. Furthermore, CCR5 ∆32 homozygous donors are rare (1% of caucasians) [START_REF] Lucotte | Frequencies of 32 base pair deletion of the (Delta 32) allele of the CCR5 HIV-1 coreceptor gene in Caucasians: a comparative analysis[END_REF], what makes the search for a compatible donor with the protective genotype an additional challenge.

How can HIV infected patients achieve durable remission of infection?

"HIV controllers" or "elite controllers" correspond to a small percentage of HIV infected patients that can naturally control viral replication below the levels of detection with standard clinical assays. Consequently, this group of patients is considered an important model to understand the mechanisms underlying control of infection in the absence of treatment [START_REF] Hamimi | Will it be possible to live without antiretroviral therapy?[END_REF]Saez-Cirion and Pancino, 2013). The aim of a so-called "functional cure" would be similarly to HIV controllers, that is, to allow HIV infected patients to achieve viral remission in which HIV remains in the body at low levels and it is controlled by the host in the absence of cART. This status may be achieved by 5-15% of patients treated very early during primary HIV infection (PHI) for long periods of time who experience treatment interruption afterwards (Goujard et al., 2012b;[START_REF] Hocqueloux | Long-term immunovirologic control following antiretroviral therapy interruption in patients treated at the time of primary HIV-1 infection[END_REF][START_REF] Lisziewicz | Control of HIV despite the discontinuation of antiretroviral therapy[END_REF][START_REF] Lodi | Immunovirologic control 24 months after interruption of antiretroviral therapy initiated close to HIV seroconversion[END_REF]Saez-Cirion et al., 2013;[START_REF] Salgado | Prolonged control of replication-competent dual-tropic human immunodeficiency virus-1 following cessation of highly active antiretroviral therapy[END_REF][START_REF] Steingrover | HIV-1 viral rebound dynamics after a single treatment interruption depends on time of initiation of highly active antiretroviral therapy[END_REF]. These patients are known as "post-treatment controllers".

The VISCONTI study (Viro-Immunological Sustained CONtrol after Treatment Interruption) investigated whether the characteristics of 14 post-treatment controllers were similar to those observed in HIV controllers that spontaneously control HIV replication (Saez-Cirion et al., 2013). In this study, cART duration at after primary infection was 3 years, and after treatment interruption, post-treatment controllers presented a sustained control for a median of 7 years. During acute phase, posttreatment controllers had higher viremia levels and lower CD4+ T cell counts than patients who naturally control infection afterwards. In addition, these groups of patients have a different genetic backgroundwhile HIV controllers cohorts are enriched by the protective HLA-B*27 and B*57 [START_REF] Migueles | HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors[END_REF][START_REF] Pereyra | Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy[END_REF][START_REF] Saez-Cirion | HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype[END_REF], the risk alleles B*07 and B*35 [START_REF] Carrington | HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage[END_REF][START_REF] Itescu | HLA-B35 is associated with accelerated progression to AIDS[END_REF]Pereyra et al., 2010;Peterson et al., 2013b) were prevalent among the 14 post-treatment controllers. HIV-1 specific CD8+ T cell responses and immune activation differ between post-treatment controllers and HIV controllers (post-treatment controllers have weak CD8+ T cell responses and a low level of immune activation during the control phase).

Another recent incident that suggests that a very early treatment may lead to HIV remission is the case identified as 'the Mississipi baby" [START_REF] Persaud | Absence of detectable HIV-1 viremia after treatment cessation in an infant[END_REF]. This infant born to a seropositive mother started receiving cART 30 hours after birth. HIV- [START_REF] Ananworanich | Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection[END_REF][START_REF] Gianella | Effect of early antiretroviral therapy during primary HIV-1 infection on cell-associated HIV-1 DNA and plasma HIV-1 RNA[END_REF][START_REF] Hecht | A multicenter observational study of the potential benefits of initiating combination antiretroviral therapy during acute HIV infection[END_REF] and the duration of cART [START_REF] Fidler | Short-course antiretroviral therapy in primary HIV infection[END_REF][START_REF] Stohr | Duration of HIV-1 viral suppression on cessation of antiretroviral therapy in primary infection correlates with time on therapy[END_REF] might play a key role in the achievement of a durable HIV control. In addition, baseline viral load and immune activation may predict success [START_REF] Volberding | Antiretroviral therapy in acute and recent HIV infection: a prospective multicenter stratified trial of intentionally interrupted treatment[END_REF]. Whether the therapeutic regimen used during PHI may impact the outcome is still to be investigated. In any case, in the absence of markers predicting success after treatment interruption, this procedure is not recommended outside structured protocols for patients undergoing suppressive cART.

Treatment during PHI seems to result in broad and strong HIV-1 specific immune responses [START_REF] Hecht | A multicenter observational study of the potential benefits of initiating combination antiretroviral therapy during acute HIV infection[END_REF][START_REF] Kaufmann | Comprehensive analysis of human immunodeficiency virus type 1-specific CD4 responses reveals marked immunodominance of gag and nef and the presence of broadly recognized peptides[END_REF][START_REF] Lisziewicz | Control of HIV despite the discontinuation of antiretroviral therapy[END_REF]Moir et al., 2010;[START_REF] Ortiz | HIV-1-specific immune responses in subjects who temporarily contain virus replication after discontinuation of highly active antiretroviral therapy[END_REF][START_REF] Oxenius | Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T lymphocytes[END_REF][START_REF] Rosenberg | Immune control of HIV-1 after early treatment of acute infection[END_REF], reduced immune activation [START_REF] Volberding | Antiretroviral therapy in acute and recent HIV infection: a prospective multicenter stratified trial of intentionally interrupted treatment[END_REF], immune restoration in the gastrointestinal mucosa [START_REF] Guadalupe | Viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection[END_REF] and limited viral evolution [START_REF] Chamberland | Effect of antiretroviral therapy on HIV-1 genetic evolution during acute infection[END_REF]. Additionally, initiation of cART during PHI may limit the establishment of viral reservoirs (Archin et al., 2012b;[START_REF] Chun | Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication[END_REF]Gianella et al., 2011;[START_REF] Pires | Initiation of antiretroviral therapy during recent HIV-1 infection results in lower residual viral reservoirs[END_REF][START_REF] Schmid | Profound depletion of HIV-1 transcription in patients initiating antiretroviral therapy during acute infection[END_REF][START_REF] Wyl | Early antiretroviral therapy during primary HIV-1 infection results in a transient reduction of the viral setpoint upon treatment interruption[END_REF]. Central memory CD4+ T cells (T CM ) are a key component of the long-lasting HIV reservoir [START_REF] Chomont | HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation[END_REF], and recent studies have demonstrated that very early cART limits the seeding of the HIV reservoir in long-lived T CM (Figure 2) (Ananworanich et al,20th Conference on Retroviruses and Opportunistic Infections, 2013, San Francisco, USA). Reservoirs levels in post-treatment controllers during the control phase were very low and it was mostly associated to the transitional memory CD4+ T cell subset, due to a skewed distribution of quiescent CD4+ T cell subsets in these patients (Saez-Cirion et al., 2013)(Figure 2).

Treatment interruption in patients who started cART during chronic phase leads to viral rebound within weeks, with viral loads frequently reaching pretreatment set-point levels [START_REF] Fernandez Guerrero | Long-term followup of asymptomatic HIV-infected patients who discontinued antiretroviral therapy[END_REF][START_REF] Steingrover | HIV-1 viral rebound dynamics after a single treatment interruption depends on time of initiation of highly active antiretroviral therapy[END_REF][START_REF] Wit | Safety of long-term interruption of successful antiretroviral therapy: the ATHENA cohort study[END_REF].

Notwithstanding, some patients that started cART during chronic phase were also described to maintain controlled levels of viremia after therapy discontinuation [START_REF] Van Gulck | Control of viral replication after cessation of HAART[END_REF]. For all these studies describing cases of a potential "functional cure", long period follow-up is needed to evaluate whether long HIV control can be maintained and the mechanisms involved in the long-term suppression after treatment interruption. Collectively, these studies suggest that HIV remission is possible to be achieved with the help of therapeutic interventions in patients without a favorable genetic background to naturally control HIV infection. Certainly, additional studies will be needed to understand the mechanisms that may lead to a long-lasting viral remission in post-treatment controllers.

Is it better if we intensify ART?

Therapeutic intervention during PHI appears to be valuable in inducing viral remission in a certain number of HIV infected patients. However, just a small percentage of patients are diagnosed during PHI. Most of them will know their seropositive status only during the chronic phase of HIV infection. For the great majority of HIV infected patients, other strategies to achieve viral remission must be developed. Combined antiretroviral therapy dramatically reduces viremia to levels below the detection limit of current assays (<50 copies/mL), but low-level residual viremia persists and is usually detectable by ultra-sensitive tests in patients undergoing successful ART [START_REF] Palmer | Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy[END_REF]. Residual viremia may partially be the cause of persistent immune activation [START_REF] Corbeau | Immune reconstitution under antiretroviral therapy: the new challenge in HIV-1 infection[END_REF], which increases the risk of non-AIDS morbidity and mortality in treated patients [START_REF] Clifford | HIV-associated neurocognitive disorder[END_REF][START_REF] Lake | Metabolic disease in HIV infection[END_REF]. There are two probable sources of residual viremia [START_REF] Doyle | Low-level viraemia on HAART: significance and management[END_REF]: (i) long-lived, latently infected cells whose provirus became transcriptionally active, with intermittent or continuous viral release; (ii) ongoing low-levels of HIV replication with de novo viral infection, due to partial suppression and/or to inadequate drug penetration. In the latter case, therapy intensification should reduce the residual viremia and associated persistent inflammation. Moreover, in the long term, it would be expected to decrease HIV reservoir levels.

Previous studies of therapy intensification with protease and reverse transcriptase inhibitors were contradictory in determining whether or not ongoing replication is the source of persistent viremia [START_REF] Dinoso | Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy[END_REF][START_REF] Havlir | Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years[END_REF][START_REF] Ramratnam | Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication[END_REF]. The development of new classes of antiretroviral drugs allowed the exploration of the impact of treatment intensification with drugs targeting other steps of HIV replication cycle. Treatment intensification with Raltegravir (RAL), a potent integrase inhibitor, offered a unique opportunity to study the dynamic of ongoing lowlevel replication. As RAL blocks HIV integration, residual replication could be assessed by the increase of 2-LTR circles [START_REF] Schacker | New tools to track HIV[END_REF]. Most studies of RAL treatment intensification showed no impact on residual viremia, immune activation or in promoting CD4+ T cell reconstitution. Collectively, these data suggested that residual viremia does not originate from ongoing cycles of HIV replication (Gandhi et al., 2012;[START_REF] Gandhi | The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial[END_REF][START_REF] Hatano | A randomized, controlled trial of raltegravir intensification in antiretroviral-treated, HIV-infected patients with a suboptimal CD4+ T cell response[END_REF][START_REF] Mcmahon | Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy[END_REF][START_REF] Negredo | Early but limited effects of raltegravir intensification on CD4 T cell reconstitution in HIVinfected patients with an immunodiscordant response to antiretroviral therapy[END_REF]. In contrast, the study of Buzon and colleagues [START_REF] Buzon | HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAARTsuppressed subjects[END_REF] identified a transient increase in the levels of 2-LTR HIV DNA within two to four weeks after intensification, but no significant decrease in low-level viremia was observed. In addition, only those patients with high levels of immune activation at baseline presented a decrease in these markers. These findings strongly suggested that RAL intensification was blocking new rounds of infection in these patients and that consequently ongoing replication was taking place. Nonetheless, other studies failed to detect 2-LTR increase in RAL intensified-treated patients [START_REF] Besson | Short-course raltegravir intensification does not increase 2 long terminal repeat episomal HIV-1 DNA in patients on effective antiretroviral therapy[END_REF][START_REF] Gandhi | No effect of raltegravir intensification on viral replication markers in the blood of HIV-1-infected patients receiving antiretroviral therapy[END_REF]. It is possible that differences in patients' characteristics, cART regimens at baseline, and/or the time of therapy intensification with RAL had an impact in the discrepancies observed among these studies. An important observation in the Buzon et al, study was the fact that 61% of the patients with detectable 2-LTR were taking a protease inhibitor-based regimen (PI). More recently,

Hatano and colleagues also detected the transient increase of 2-LTR, which was also more accentuated in patients taking PI [START_REF] Hatano | Increase in 2-long terminal repeat circles and decrease in D-dimer after raltegravir intensification in patients with treated HIV infection: a randomized, placebo-controlled trial[END_REF]. In addition, a significant reduction in D-Dimer levels, a coagulation biomarker, was observed, indicating that RAL intensification may lead to reduction in inflammation/activation markers.

Because HIV-1 might replicate in tissue reservoirs where drug concentrations are suboptimal [START_REF] Fletcher | Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues[END_REF], such as lymph nodes, gut associated lymphoid tissues, bone marrow and the central nervous system, some RAL intensification studies assessed the immunologic and virologic responses in tissues. Yukl et al showed reduction in unspliced HIV RNA, T cell activation and a trend towards CD4+

T cell increases in ileum, suggesting that this tissue may be an important site for ongoing replication in some patients on cART [START_REF] Yukl | Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy[END_REF]. In contrast, therapy intensification with RAL did not impact cerebrospinal viral loads [START_REF] Dahl | Raltegravir treatment intensification does not alter cerebrospinal fluid HIV-1 infection or immunoactivation in subjects on suppressive therapy[END_REF] or isolated HIV semen shedding [START_REF] Osborne | Impact of antiretroviral therapy duration and intensification on isolated shedding of HIV-1 RNA in semen[END_REF].

Based on previous observations that Maraviroc (MVC), a CCR5 antagonist, promotes a gain of CD4+ T cells in viremic HIV-infected patients [START_REF] Cooper | Maraviroc versus efavirenz, both in combination with zidovudine-lamivudine, for the treatment of antiretroviral-naive subjects with CCR5-tropic HIV-1 infection[END_REF][START_REF] Wilkin | The relationship of CCR5 antagonists to CD4+ T-cell gain: a meta-regression of recent clinical trials in treatment-experienced HIV-infected patients[END_REF], it was hypothesized that the immunomodulatory effects of this drug could decrease immune activation and improve CD4+ T cell counts in patients under suppressive cART with insufficient immunological restoration. As observed in RAL intensification trials, no impact in residual viremia or HIV reservoir was achieved.

Data regarding CD4+ T cell gain was also controversial in these trials [START_REF] Cuzin | Maraviroc intensification of stable antiviral therapy in HIV-1-infected patients with poor immune restoration: MARIMUNO-ANRS 145 study[END_REF][START_REF] Hunt | The immunologic effects of maraviroc intensification in treated HIV-infected individuals with incomplete CD4+ T-cell recovery: a randomized trial[END_REF][START_REF] Rusconi | Maraviroc as intensification strategy in HIV-1 positive patients with deficient immunological response: an Italian randomized clinical trial[END_REF][START_REF] Wilkin | A pilot trial of adding maraviroc to suppressive antiretroviral therapy for suboptimal CD4(+) T-cell recovery despite sustained virologic suppression: ACTG A5256[END_REF]. Some unexpected effects, however, were observed in MVC intensification trials, such as increased activation levels in blood and rectal mucosa [START_REF] Hunt | The immunologic effects of maraviroc intensification in treated HIV-infected individuals with incomplete CD4+ T-cell recovery: a randomized trial[END_REF][START_REF] Rusconi | Maraviroc as intensification strategy in HIV-1 positive patients with deficient immunological response: an Italian randomized clinical trial[END_REF], increase in plasma LPS, sCD14 and sCD163 [START_REF] Gutierrez | Intensification of antiretroviral therapy with a CCR5 antagonist in patients with chronic HIV-1 infection: effect on T cells latently infected[END_REF][START_REF] Hunt | The immunologic effects of maraviroc intensification in treated HIV-infected individuals with incomplete CD4+ T-cell recovery: a randomized trial[END_REF] and increased 2-LTR levels [START_REF] Gutierrez | Intensification of antiretroviral therapy with a CCR5 antagonist in patients with chronic HIV-1 infection: effect on T cells latently infected[END_REF], suggesting that MVC intensification might induce viral replication. The mechanisms underlying these effects are still unclear, but some evidences suggest that MVC promotes an increase in the circulating levels of CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES), natural ligands of CCR5, which may activate T cells and monocytes/neutrophils through CCR3/CCR4 and CCR1 signaling, respectively [START_REF] Guan | Natural truncation of the chemokine MIP-1 beta /CCL4 affects receptor specificity but not anti-HIV-1 activity[END_REF][START_REF] Sarau | Characterization of functional chemokine receptors (CCR1 and CCR2) on EoL-3 cells: a model system to examine the role of chemokines in cell function[END_REF]. Further studies are necessary to carefully evaluate the immunomodulatory effects of MVC in vivo and the benefits of therapy intensification with CCR5

antagonists in HIV-infected patients.

Despite some evidence that therapy intensification may have positive effects on residual replication, its potential to eradicate the virus appears limited. These results highlight the importance of designing new strategies to eliminate persistent HIV reservoirs.

HIV persistence as a challenge for a cure

HIV replication and persistence depend on the integration of the viral DNA in the host genome. Although most proviruses integrate in transcriptionally active genomic regions, in most cases these proviruses remained repressed [START_REF] Ho | Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure[END_REF]. Our knowledge of the complex mechanisms underlying establishment and maintenance of HIV latency has been enormously improved in the last years (see [START_REF] Mbonye | Transcriptional control of HIV latency: Cellular signaling pathways, epigenetics, happenstance and the hope for a cure[END_REF] in this issue of Virology for a thorough review). Repressed HIV proviruses integrated in long-lived cells constitute the persistent HIV reservoir, which is not recognized by immune responses, not eliminated under cART, and is the main obstacle to achieving an HIV cure. Since the description of HIV reservoirs despite long-term cART, attempts to purge this reservoir have been tried. The first one consisted in the infusion in three patients under cART of the anti-CD3 OKT3 antibody in the presence of IL2, in an effort to mimic in vivo a well-established in vitro reactivation protocol [START_REF] Prins | Immunoactivation with anti-CD3 and recombinant human IL-2 in HIV-1-infected patients on potent antiretroviral therapy[END_REF]. OKT3+IL2 treatment was accompanied by very strong activation of T cells, and transient increases of HIV RNA levels were observed in plasma or lymph nodes of these patients. However, no decrease in the frequency of infected cells could be reported and the treatment had many undesirable effects, associated with exacerbated immune activation and cytokine release, induction of anti-OKT3 antibodies and severe long-lasting depletion of CD4+

T cells [START_REF] Prins | Immunoactivation with anti-CD3 and recombinant human IL-2 in HIV-1-infected patients on potent antiretroviral therapy[END_REF][START_REF] Van Praag | OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective longlasting CD4+ T cell depletion[END_REF].

HDAC inhibitors were next proposed as optimal candidate to flush HIV reservoirs due A first study reported a decrease in the frequency of circulating resting CD4+ T cells carrying replication competent HIV-1 in four patients receiving cART after 16 weeks of additional treatment with VPA [START_REF] Lehrman | Depletion of latent HIV-1 infection in vivo: a proof-of-concept study[END_REF]. However, subsequent randomized clinical studies could not confirm these results and no significant effects of VPA were evidenced [START_REF] Archin | Antiretroviral intensification and valproic acid lack sustained effect on residual HIV-1 viremia or resting CD4+ cell infection[END_REF][START_REF] Routy | Valproic acid in association with highly active antiretroviral therapy for reducing systemic HIV-1 reservoirs: results from a multicentre randomized clinical study[END_REF][START_REF] Sagot-Lerolle | Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir[END_REF][START_REF] Steel | No change to HIV-1 latency with valproate therapy[END_REF]. Failure of VPA was eventually associated to a poor capacity of this molecule to inhibit HDAC3, the HDAC isoform that is thought to play a preponderant role in HIV latency [START_REF] Huber | Inhibitors of histone deacetylases: correlation between isoform specificity and reactivation of HIV type 1 (HIV-1) from latently infected cells[END_REF]. In a proof of concept study, the more potent HDAC inhibitor vorinostat has shown capacity to increase levels of HIV mRNA transcripts after a single dose, without affecting overall HIV DNA cellular levels (Archin et al., 2012a). Preliminary results of the NCT01365065 trial presented in the 20th Conference on Retrovirus and Opportunistic Infections (Lewin S, et al 2013, San Francisco, USA) also reported an increase of cell associated unspliced HIV mRNAs in 15/17 patients on cART who received daily doses of vorinostat for 14 days. Another clinical trial (NCT01680094) is evaluating the impact on HIV reservoirs of panobinostat, which has been described as an even more potent HDAC inhibitor [START_REF] Rasmussen | Comparison of HDAC inhibitors in clinical development: effect on HIV production in latently infected cells and T-cell activation[END_REF].

The use of HDAC inhibitors in the context of HIV cure face several hurdles. Among them, the lack of specificity of HDAC inhibitors may impact the transcription of a large fraction of all cellular genes [START_REF] Glaser | Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines[END_REF]. Also, it has been recently shown [START_REF] Shan | Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation[END_REF]) that viral reactivation with HDAC inhibitors will not necessarily be followed by viral-induced cell death, and that immune responses may need to be boosted in parallel to recognize and eliminate these new viral-producing cells (see below). It is also unlikely that HDAC inhibition by itself will be able to induce efficient viral reactivation in the absence of the factors necessary to ensure full viral transcription. The current belief is that it will be necessary to combine treatment of HDAC inhibitors with other molecules able to provide the additional stimuli required for HIV transcription. PKC agonists, such as prostratin [START_REF] Kulkosky | Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART[END_REF][START_REF] Reuse | Synergistic activation of HIV-1 expression by deacetylase inhibitors and prostratin: implications for treatment of latent infection[END_REF] and bryostatin [START_REF] Perez | Bryostatin-1 synergizes with histone deacetylase inhibitors to reactivate HIV-1 from latency[END_REF], or TLR agonists [START_REF] Novis | Reactivation of latent HIV-1 in central memory CD4(+) T cells through TLR-1/2 stimulation[END_REF], that activate NF-kB have been proposed as good potential combinatorial candidates.

Purging viral reservoirs is undoubtedly the only solution to achieve HIV eradication.

However, letting aside the mechanistic issues, activating every single latently infected cell in the organism still appears a gargantuan endeavor. Taking the example of natural HIV controllers or the post-treatment controllers from the VISCONTI study, durable HIV remission may be a more reasonable goal in the medium term. In this setting, limiting and controlling the viral reservoirs would be the main objectives. With this in mind, our current knowledge, albeit incomplete, of HIV latency and transcription might be turned to devise strategies aiming to repress, rather than activate, viral transcription. As alternatives to HDAC inhibitors, HAT inhibitors such as garcinol derivatives [START_REF] Mantelingu | Specific inhibition of p300-HAT alters global gene expression and represses HIV replication[END_REF] and curcumin [START_REF] Zhang | HDAC1/NFkappaB pathway is involved in curcumin inhibiting of Tat-mediated long terminal repeat transactivation[END_REF] [START_REF] Mousseau | An analog of the natural steroidal alkaloid cortistatin A potently suppresses Tat-dependent HIV transcription[END_REF]. This promising molecule showed potent inhibitory capacity in vitro at very low concentrations (nM-pM). Zincfinger domains, small protein domains that can be designed to bind specific DNA sequences, can be fused to appropriate effector domains of transcription factors to specifically regulate the expression of targeted genes. Zinc-finger transcription factors targeting different regions of the LTR promoter and carrying the repression domain of the KOX1 protein were able to repress transcription from the LTR in infected cells in vitro [START_REF] Reynolds | Repression of the HIV-1 5' LTR promoter and inhibition of HIV-1 replication by using engineered zincfinger transcription factors[END_REF]. Overall these results suggest that different opportunities exist and may be exploited to specifically repress HIV transcription.

Gene therapy

The apparent cure of the Berlin patient after receiving HSCT from a CCR532 homozygous donor and the impossibility of applying this protocol in a large scale have inspired attempts to generate HIV-resistant cells through gene therapy (Peterson et al., 2013a). One of the most promising gene therapy strategies against HIV infection aims to disrupt the CCR5 gene by expressing an engineered zinc-finger nuclease (ZFN). Evidence that ZFN could inactivate CCR5 in primary human CD4+

T cells and in CD34+ hematopoietic stem cells limiting HIV replication was first obtained in mouse models [START_REF] Holt | Human hematopoietic stem/progenitor cells modified by zincfinger nucleases targeted to CCR5 control HIV-1 in vivo[END_REF][START_REF] Perez | Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases[END_REF]. The results of a development of these studies, ZFNs have been designed to be combined and simultaneously target CCR5 and CXCR4. This approach resulted in human primary CD4+ T cells that were resistant to R5 and X4 viruses in vitro and also in vivo when introduced in humanized mice [START_REF] Didigu | Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection[END_REF]. This strategy would tackle the risk of tropism switching.

Other strategies consist of transducing CD34+ or CD4+ T cells with vectors expressing (i) shRNA interfering with CCR5 or other key factors associated with the HIV life cycle [START_REF] Kiem | Hematopoietic-stem-cell-based gene therapy for HIV disease[END_REF], including its promoter [START_REF] Suzuki | Promoter Targeting shRNA Suppresses HIV-1 Infection In vivo Through Transcriptional Gene Silencing[END_REF]; (ii) small peptides with capacity to inhibit HIV entry into the cells [START_REF] Younan | Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model[END_REF]. Another encouraging gene therapy approach directly targets the viral reservoir and is based on the ability of a tailored site-specific recombinase (Tre) to excise HIV provirus from host genome [START_REF] Sarkar | HIV-1 proviral DNA excision using an evolved recombinase[END_REF]. Recently, successful expression of Trerecombinase in human cells was demonstrated. In addition, an important antiviral effect was observed in humanized mice, suggesting that this approach may be valuable for future HIV eradication strategies [START_REF] Hauber | Highly significant antiviral activity of HIV-1 LTRspecific tre-recombinase in humanized mice[END_REF].

Innovative approaches to eliminate HIV reservoirs

Several experimental and proof-of-concept studies aim to interfere with cell cycle and survival of HIV infected cells to facilitate their elimination or avoid their persistence by homeostatic proliferation. Rapamycin is an immunosuppresor that blocks the mammalian target of rapamycin (mTOR), a protein kinase that control cell cycle, proliferation and survival, and plays a critical role in the differentiation of T cells.

Rapamycin has been shown to be able to block HIV infection in vitro [START_REF] Donia | Potential use of rapamycin in HIV infection[END_REF] by decreasing the expression of CCR5, and also interferes with the synthesis of HIV transcripts [START_REF] Roy | The immunosuppressant rapamycin represses human immunodeficiency virus type 1 replication[END_REF]. Through its capacity to suppress activation and proliferation of T cells, rapamycin has also the potential to block HIV infection by reducing the number of target cells and to dodge the maintenance of viral reservoirs through homeostatic proliferation. Rapamycin is commonly used to avoid organ rejection in kidney transplantation when there are complications associated with calcineurin inhibitors. Interestingly, a clinical report on a group of 14 HIV infected patients who received kidney transplantation showed that the patients who switched to rapamycin monotherapy after transplantation were able to better control HIV replication than those who kept calcineurin inhibitors [START_REF] Di Benedetto | First report on a series of HIV patients undergoing rapamycin monotherapy after liver transplantation[END_REF].

Another recent study, which analyzed a larger group of HIV-infected kidney recipients, has shown that those who received rapamycin experienced, two years after transplantation, some reduction in the levels of cell associated DNA when compared to patients receiving other immunosuppresors (Stock P,et al,19th International AIDS Society conference, 2013, Kuala Lumpur, Malaysia).

Another compound with cytostatic activities that has been tried in the context of HIV infection is hydroxyurea. Hydroxyurea is an inhibitor of ribonucleotide reductase (RNR) [START_REF] Koc | Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools[END_REF], a key enzyme responsible for dNTP synthesis that has been shown to play an important role in regulating HIV infection [START_REF] Allouch | p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway[END_REF].

Hydroxyurea, other than maintaining cells in a quiescent state, reduces the levels of intracellular dNTPs thus inhibiting HIV DNA synthesis during reverse transcription.

Although the impact of hydroxyurea in HIV reservoirs has not been evaluated, hydroxyurea was shown to act synergically with the nucleoside analogue didanosine to reduce HIV viral loads in patients [START_REF] Lori | Optimal suppression of HIV replication by low-dose hydroxyurea through the combination of antiviral and cytostatic ('virostatic') mechanisms[END_REF][START_REF] Lori | Rationale for the use of hydroxyurea as an anti-human immunodeficiency virus drug[END_REF].

Despite its promises, hydroxyurea treatment was shown to provoke complications, such as blunted CD4+ T cell recovery and severe toxicity, in HIV infected patients [START_REF] Bloch | The role of hydroxyurea in enhancing the virologic control achieved through structured treatment interruption in primary HIV infection: final results from a randomized clinical trial (Pulse)[END_REF][START_REF] Zala | Higher rate of toxicity with no increased efficacy when hydroxyurea is added to a regimen of stavudine plus didanosine and nevirapine in primary HIV infection[END_REF]. Recent studies suggest that these undesirable effects were due to a too high dose of the drug, and that both the antiviral and the cytostatic activities of hydroxyurea may contribute to control HIV when used at lower concentrations [START_REF] Lori | VS411 reduced immune activation and HIV-1 RNA levels in 28 days: randomized proof-of-concept study for antiviral-hyperactivation limiting therapeutics[END_REF].

JQ1 is an agonist of the bromodomain protein BRD4, which promotes cell cycle progression [START_REF] Yang | Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression[END_REF]. BRD4 is also a positive regulator of P-TEFb that competes with Tat for binding to this complex at the HIV promoter [START_REF] Bisgrove | Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription[END_REF]. JQ1 dissociates BRD4 from the HIV promoter and allows Tat recruitment and subsequent HIV elongation [START_REF] Li | The BET bromodomain inhibitor JQ1 activates HIV latency through antagonizing Brd4 inhibition of Tat-transactivation[END_REF]. In addition, JQ1 appears also to alter chromatin organization and downregulates T cell activation genes [START_REF] Banerjee | BET bromodomain inhibition as a novel strategy for reactivation of HIV-1[END_REF].

A different strategy has been explored with auranofin, a gold-based compound that has been evaluated in vivo in SIV infected macaques. This compound was shown in vitro to promote differentiation of CD27+ memory CD4+ T cells to more terminal short-lived stages [START_REF] Lewis | Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension[END_REF] preceding increased cell death. Administration of auranofin to a group of SIVmac251 infected rhesus macaques with viral loads suppressed by cART, induced indeed a decreased in the frequency of T CM and T TM CD4+ T cells, the cells thought to contribute the most to persistent reservoirs [START_REF] Chomont | HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation[END_REF]. Interestingly, auranofin treatment was accompanied by a transient decrease of viral reservoirs and a delayed rebound of viremia after interruption of auranofin and antiretroviral treatment [START_REF] Lewis | Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension[END_REF]. In a followup study, auranofin treatment in two macaques combined with cART and buthionine sulfoximine, a glutathione-depleting agent that has been proposed to favor selective death of infected cells in vitro [START_REF] Savarino | Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence[END_REF], favored control of infection at relatively low levels after treatment interruption [START_REF] Shytaj | Investigational treatment suspension and enhanced cell-mediated immunity at rebound followed by drug-free remission of simian AIDS[END_REF].

How these interventions may impact (positively or negatively) T cell immune defenses against HIV has not been thoroughly addressed, and the translation of current formulations of these drugs to clinical intervention is unlikely due to toxicity and possible harmful unspecific effects. However, the proof-of-principle studies described above suggest that targeting T cell proliferation and survival deserves further attention in the search for an HIV cure.

Immunotherapies to treat HIV infection

There is a consensus in the scientific community on the need to potentiate immune responses in the context of combinatory approaches to reach an HIV cure. Such responses would contribute to either control or eliminate HIV infected cells. A study performed by the Siliciano group showed that reactivation of latent viruses would not automatically entail the elimination of the infected cells by viral-replication-driven apoptosis [START_REF] Shan | Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation[END_REF]. Efficient CD8+ T cell responses, such as those found in HIV controllers, react to very small amounts of antigens and eliminate HIV producing cells [START_REF] Almeida | Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover[END_REF][START_REF] Saez-Cirion | HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype[END_REF][START_REF] Shan | Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation[END_REF]. In contrast, responses from non-controller patients would not have such capacity. Antiexhaustion strategies try to reverse negative signaling provided by immunoregulatory molecules such as PD-1 to restore T cell function, in particular, the capacity to kill infected cells [START_REF] Porichis | Role of PD-1 in HIV pathogenesis and as target for therapy[END_REF]. Blockage of PD-1 interactions with PD-1 ligands on SIV-infected macaques resulted in the expansion of CD8+ T cells with improved functionality, longer survival, lower viral loads and delayed viral rebound after antiretroviral treatment interruption [START_REF] Velu | Enhancing SIV-specific immunity in vivo by PD-1 blockade[END_REF]. However, another report suggests that mere expansion of polyfunctional CD8+ T cell responses through PD-1 blockade may not suffice to sustainably decrease viremia [START_REF] Amancha | In vivo blockade of the programmed cell death-1 pathway using soluble recombinant PD-1-Fc enhances CD4+ and CD8+ T cell responses but has limited clinical benefit[END_REF]. A key aspect may be the association of the efficient responses found in HIV controllers with the selection of particular TCR clonotypes in these cells. In this sense, PD-1 blockade may be a useful adjuvant in vaccination protocols [START_REF] Finnefrock | PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination[END_REF] helping to induce efficient responses. This review will not summarize therapeutic vaccine strategies currently in development. However, recent results showing that (i) non-conventional CD8+ T cell responses were associated with control of infection and viral clearance in a group of macaques vaccinated with a cytomegalovirus vector (Hansen et al., 2013a;Hansen et al., 2013b) despite profound viral dissemination during primary infection; and that (ii) passive transfer of broadly neutralising antibodies allows control of infection in chronically SIV-infected macaques [START_REF] Barouch | Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys[END_REF], suggest that therapeutic vaccines should be an important element in the global plan to achieve HIV cure or remission.

So far, interventions based on the administration of gamma-chain cytokines such as IL-2, IL-7 or IL-15, which tried to improve T cell function and restore T cell homeostasis [START_REF] Leone | IL-2, IL-7 and IL-15 as immuno-modulators during SIV/HIV vaccination and treatment[END_REF][START_REF] Levy | Enhanced T cell recovery in HIV-1-infected adults through IL-7 treatment[END_REF][START_REF] Levy | Effects of recombinant human interleukin 7 on T-cell recovery and thymic output in HIV-infected patients receiving antiretroviral therapy: results of a phase I/IIa randomized, placebo-controlled, multicenter study[END_REF][START_REF] Vanham | Can immunotherapy be useful as a "functional cure" for infection with Human Immunodeficiency Virus-1?[END_REF], have not shown, at least by themselves, significant benefits in HIV treatment. Indeed, improved immune reconstitution with IL-7 is also accompanied by a significant increase in the total number of HIV-harbouring CD4+ T cells [START_REF] Levy | Enhanced T cell recovery in HIV-1-infected adults through IL-7 treatment[END_REF][START_REF] Vandergeeten | Interleukin-7 promotes HIV persistence during antiretroviral therapy[END_REF]. It is not excluded, however, that these cytokines may have an important adjuvant effect in vaccine strategies. Therapies based on IFN administration have been shown to decrease viremia during chronic infection and reduce viral reservoirs after treatment interruption [START_REF] Asmuth | Safety, tolerability, and mechanisms of antiretroviral activity of pegylated interferon Alfa-2a in HIV-1-monoinfected participants: a phase II clinical trial[END_REF][START_REF] Azzoni | Pegylated Interferon alfa-2a monotherapy results in suppression of HIV type 1 replication and decreased cell-associated HIV DNA integration[END_REF][START_REF] Pillai | Role of retroviral restriction factors in the interferon-alphamediated suppression of HIV-1 in vivo[END_REF]. However, these effects were not observed in all situations [START_REF] Boue | Alpha interferon administration during structured interruptions of combination antiretroviral therapy in patients with chronic HIV-1 infection: INTERVAC ANRS 105 trial[END_REF]Goujard et al., 2012a), which may be due to the dual role that IFN (and immune activation) may play at different stages of infection.

Immune activation is a major determinant of HIV pathogenesis, and increased levels of inflammation markers are associated with faster progression to disease and CD4+

T cell loss [START_REF] Liovat | Acute plasma biomarkers of T cell activation set-point levels and of disease progression in HIV-1 infection[END_REF] including in HIV controllers with undetectable viremia [START_REF] Hunt | Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy[END_REF][START_REF] Noel | Elevated IP10 levels are associated with immune activation and low CD4+ T-cell counts in HIV controller patients[END_REF]. Therefore, targeting the harmful immune activation appears to be a necessary element in ensuring a long-life remission of HIV infection. Some of the molecules targeting HIV reservoirs described in this review have also immunosuppressive effects, and their impact is being evaluated. Other molecules such as chloroquine analogues or statins specifically target inflammation.

So far, studies employing these and other anti-inflammatory molecules have rendered contrasting results [START_REF] Hatano | Immune activation and HIV persistence: considerations for novel therapeutic interventions[END_REF]. Thus, chloroquine administration has been shown to decrease levels of T cell activation [START_REF] Murray | Reduction of immune activation with chloroquine therapy during chronic HIV infection[END_REF] but produce faster CD4+ T cell loss when used for longer periods of time [START_REF] Paton | Effects of hydroxychloroquine on immune activation and disease progression among HIV-infected patients not receiving antiretroviral therapy: a randomized controlled trial[END_REF]. A better knowledge of the best timing to implement interventions aimed at decreasing persistent immune activation is warranted.

Concluding remarks

Achieving HIV cure at a global scale through the eradication of HIV reservoirs seems still far off. In contrast, durable HIV remission with low levels of infected cells being controlled by host mechanisms seems more plausible at medium-term horizon. The first step in such strategy would be to limit the size of the viral reservoirs. Early treatment initiation has been shown to provide immunological and virological benefits to HIV infected patients, and early treated patients may be more prone to positively respond to cure therapies. Moreover, treatment is prevention [START_REF] Cohen | Prevention of HIV-1 infection with early antiretroviral therapy[END_REF] and, thus, early diagnosis and treatment initiation appears as a priority in the global fight against HIV. Approaches trying to purge the viral reservoirs, including activation with HDAC inhibitors or PKC agonists face the difficulty of trying to circumvent complex mechanisms of repression with non-specific drugs that may cause undesirable effects. An alternative strategy may be to further reinforce latency and repression mechanisms with specific molecules targeting Tat-dependent transcription of HIV products. Gene therapy offers also the possibility to target specific genes to render cells resistant to HIV infection and even to excise integrated provirus.

Enhancing susceptibility to apoptosis of HIV infected cells or impairing homoeostatic proliferation and persistence of these cells are also proposed as potential strategies to tackle HIV reservoirs. However, such approaches need to carefully evaluate the impact they may have in immune responses of treated individuals. HIV remission will require efficient host responses to control infected cells and to prevent harmful HIVrelated inflammation. These areas of research need to be explore in parallel to strategies targeting the reservoirs.

  to their potential to activate HIV transcription without inducing generalized of the histones that constitute the core of the nucleosomes.Histone acetyl transferases (HAT) mediate the addition of acetyl groups to histones, reducing chromatin condensation and promoting transcription. Histone deacetylases (HDAC) remove the acetyl groups and repress transcription. The balance of HAT/HDAC activities is thus thought to be a key component of HIV latency. The first molecule with HDAC inhibitory activity to be assayed in vivo was valproic acid (VPA).

  have been shown to inhibit HIV transcription in vitro. Transcriptional elongation of the viral promoter depends on the recruitment of the HIV transactivator factor Tat to the transactivation-responsive element (TAR) located at the end of initial HIV transcripts (D'Orso and Frankel, 2010). Compounds such as Celastrol (Narayan et al., 2011) directly block Tat function by altering its structure. An analogue of Cortistatin A, a natural steroidal alkaloid, has shown to block HIV transcription initiation and elongation by binding specifically to the TAR-binding domain of Tat and impairing the interaction of Tat with viral RNAs

  Phase I clinical trial showed that HIV-infected patients treated with ZFN CCR5 modified CD4+ T cells (SB-728-T) have sustained increases in the CD4+ T cells, mostly T CM and T TM with low PD-1 expression. Viremia was controlled after treatment interruption for some patients, but cART was resumed by most of them due to dualtropic infection or viral load rebound (Ando D. et al 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, 2013, Denver, CO, USA). Phase II clinical trials are currently ongoing to evaluate the efficacy of SB-728-T. In a recent

Figure 1 :

 1 Figure 1: Schematic representation of the clinical course before and after

Figure 2 :

 2 Figure 2: Schematic representation of the HIV acute infection and the

Table 1 :

 1 Hematopoietic stem cell transplantation in HIV positive patients : acute lymphoblastic leukemia; AML: Acute myeloid leukemia; BL: Burkitt lymphoma; BM: bone marrow; F: Female; HL: Hodgkin lymphoma; M: male; NHL: non-Hodgkin lymphoma; NA: not available; PBMC: Peripheral blood mononuclear cells; PCR: polymerase chain reaction; TI: treatment interruption; VL: viral load.

	Study Reference		Patients		Diagnosis	Graft type	Strategy against HIV infection	Effect on HIV persistence	Clinical Outcome
		N	Gender	Age
	(Holland et al., 1989)	1	M	41	NHL	Allogeneic (bone marrow)	High-dose zidovudine for 2 weeks before transplantation. Lower maintenance dose transplantation. zidovudine after	No detectable HIV RNA and DNA at day 32 after autopsy. transplantation and at	Death 47 days after transplantation.
	(Contu et al., 1993)	1	F	25	NA	Allogeneic (bone marrow)	Zidovudine, IFN-alpha 2 and anti-HIV-1-specific T cell clones.	No detectable HIV RNA at day 30 after transplantation and at autopsy.	Death 10 months after transplantation.
	(Sora et al., 2002)	1	F	33	AML	Allogeneic (bone marrow)	cART before and after transplantation, with interruptions due to side-effects.	No detectable HIV RNA on cART from day 210 after transplantation.	Alive after 42 months of follow up.
	(Gabarre et al., 2004)	14	M/F	27-53	BL, HL, NHL	Autologous	cART before and after transplantation.	No detectable HIV RNA on cART in three patients who survived.	Five patients were alive.
								On cART
								No detectable HIV RNA after
								transplantation (two
	(Resino et al., 2007)	4	NA	31-58	BL, HL, NHL	Autologous	cART before and after transplantation.	patients), viral load rebound (two patients). Detectable HIV DNA at	The four patients were alive after 12 months of follow up.
								month 12 after
								transplantation for all
								patients.
	(Avettand-Fenoel et al., 2007)	1	M	17	BL,AML	Allogeneic (bone marrow)	cART before and after transplantation, with interruptions due to side-	Undetectable RNA and DNA on cART. Detectable HIV RNA and DNA at day 16	Death 191 days after transplantation.

ALL

Acknowledgments

Our work receives financial support from the ANRS, Sidaction, amfAR and Total Foundation. C.P.B.P. has postdoctoral funding from the ANRS.


Nicolas Burrus

Thierry M Bernard

Adaptive Vision Leveraging Digital Retinas: Extracting Meaningful Segments

In general, the less probable an event, the more attention we pay to it. Likewise, considering visual perception, it is interesting to regard important image features as those that most depart from randomness. This statistical approach has recently led to the development of adaptive and parameterless algorithms for image analysis. However, they require computer-intensive statistical measurements. Digital retinas, with their massively parallel and collective computing capababilities, seem adapted to such computational tasks. These principles and opportunities are investigated here through a case study: extracting meaningful segments from an image.

Introduction

Designing robust vision algorithms is a serious challenge. The infinite variability of natural images and the difficulty to find precise rules specifying how to solve a -generally trivial for a child-vision problem are greatly contributing to this complexity.

Dynamically adapting algorithms to images they encounter is certainly a way to overcome part of this complexity. Being robust also requires a strict management of algorithm parameters, by relating them to a physical quantity, deducing them from image properties, learning them, selecting them to optimize further treatments through a closed loop process, etc.

Recently, an almost parameterless statistical framework has been proposed [START_REF] Desolneux | Maximal meaningful events and applications to image analysis[END_REF], relying on the so-called Helmholtz principle, which states that meaningful events are events whose probability to appear in a purely random environment is very low. It seems that human perception follows this rule to some extent, and this framework has been applied notably to gestalts detection with some success [START_REF] Desolneux | Meaningful Alignments[END_REF][START_REF] Desolneux | A grouping principle and four applications[END_REF]. The absence of parameters mainly comes from the fact that no generative probability model of events has to be defined, but only a rarity measure in a well-defined random environment. In addition, some properties of the image can be taken into account to define the random model in which the rarity of other properties will be evaluated, enabling adaptation to the analyzed image.

Such kind of approaches generally requires a considerable number of potentially meaningful events to be evaluated, making real-time processing difficult or impossible. Adaptivity also requires the computation of global quantities, such as probability distributions over the image, which are time-and power-consuming to obtain using a standard computer. The reason is that pixel data have to be transfered many times, for each pixel, from memory to processor.

To ease such global computations, less standard architectures are needed, that better combine processor and memory, in a more distributed fashion. The latter issue is addressed for more general reasons by the computer architecture community (e.g. [START_REF] Taylor | Evaluation of the raw microprocessor: An exposed-wire-delay architecture for ILP and streams[END_REF]). But, in this paper, we focus on artificial retinas (also known as vision chips [START_REF] Moini | Vision Chips[END_REF]), which mix processor and memory in an extreme way. Indeed, these are smart imaging sensors, with processing ressources in each pixel, thus making massively parallel image array processors without input bottleneck.

On the output side, many retinas are fitted with a global adder (analog as [START_REF] Bernard | Vision through the power supply of the NCP retina[END_REF] or digital as [START_REF] Komuro | A dynamically reconfigurable SIMD processor for a vision chip[END_REF]), able to quickly provide the sum of pixel data over the whole image. The global adder has been used to measure image moments, e.g. for extracting the position of a target. More powerfully, it has been used in a feedback scheme to allow image capture with automatic histogram equalization [START_REF] Ni | Histogram-equalization-based adaptive image sensor for real-time vision[END_REF]. We believe that this feedback scheme is worth being systematically extended to image processing : sums provided by a global adder can be surely taken advantage of to better control the way in which images are processed. In particular, it can provide at low cost statistical measures about images in order to make algorithms more adaptive, therefore more robust, as we are looking for. Of course, this only makes sense with programmable retinas, that is retinas with a programmable processor in each pixel -thus allowing versatile image processing -such as the digital retinas we design in our lab [START_REF] Paillet | Second generation programmable artificial retina[END_REF][START_REF] Gies | Asynchronous regional computation capabilities for digital retinas[END_REF].

In the present paper, our goal is two-fold: show the potential of these general principles through a case study: meaningful segment detection; use this experience to improve algorithm/architecture adequation, by motivating future evolutions of both vision system architectures and statistical methods.

In the following, we deal with segment detection in natural images, a standard primitive which can be interesting in artificial environments and which drastically reduces the information contained in an image, while keeping important features. We are looking for an adaptive, parameterless algorithm taking advantage of retina capabilities.

After a global overview of the proposed algorithm in Section 2, Section 3 focuses on segment candidates extraction on digital retinas, then Section 4 gives statistical criteria to decide whether the candidates are meaningful or not. Finally, quantitative results are given in Section 5 and questions raised by this study are discussed in Section 6.

2.1 What is a segment? Definition 1. A segment in a cone C is a one-pixel thick connected set of pixels, such that:

each pixel has a local gradient direction in C; for each non-extremity pixel, the direction of the vector formed by its two neighbors is also in C.

Gradient vectors are computed using a Sobel operator. To keep cone belonging easy to check, only eight cones are considered, corresponding to the possible angles in a 5x5 discrete neighborhood, as shown in Figure 1. The main steps of the algorithm are as follows:

1. groups of pixels conforming to the definition of segments are extracted as briefly described in Section 3 and their properties (length, mean of pixels gradient magnitude, etc.) are attached to one of their extremities; 2. the extremities are selected by an a contrario statistical criterion, as will be detailed in Section 4. The criterion takes into account global image measures and for each segment answers the question: "could a segment with the same properties possibly be observed in a purely random environment?"; 3. segments for which the answer is "no" are reconstructed from their representative extremity, resulting in a binary image of meaningful segments. Segment extraction, the first step of the overall algorithm (see Section 2) is itself performed in three steps, as illustrated in Figure 2: 1. Eight binary images are computed, each representing a direction cone in which segments will be looked for. Any pixel with a determined gradient direction is marked as white in the direction image(s) of which it matches the direction. 2. In each direction image, connected sets of white pixels are made one-pixel thick, such that the remaining pixels lie where the image gradient magnitude is maximal in the orthogonal direction. 3. In each direction image, independently, connected groups of white pixels which match our segment definition are reduced to one of their extremities, to which segments properties are attached. This step is performed by iterative segment erosion: for e.g. horizontal segments, left extremities are removed at each iteration. Before removal, extremities transfer all the information gathered so far to their right neighbor. At the end of this step, extremities support the needed properties of their associated segment.

The extremities are now ready to go through the selection step.

4 Candidates selection

About the Number of False Alarms (NFA)

The question addressed in this section is: observing a segment with some properties, how to decide whether this segment is meaningful or whether it is just an artefact or coincidence? Two segment properties will be considered, the mean of the gradient magnitude of the segment pixels in Section 4.2 and the length of the segment in Section 4.3. In the spirit of [START_REF] Desolneux | Maximal meaningful events and applications to image analysis[END_REF], we chose to reason a contrario, i.e. instead of computing the probability to observe such a segment in a natural image, we try to answer the question: could the observed segment possibly appear in a noise image? If not, it must be due to a real world phenomenon: object, shadow, etc. To quantify this a contrario likelihood, we recall the definition of the number of false alarms of an event.

Definition 2. The number of false alarms of an event E is the expected number of occurrences of E in a random environment.

Using the NFA, the notion of -meaningfulness may be defined, with a strictly positive (possibly 1) real number.

Definition 3. An -meaningful event E is an event such as N F A(E) < . A 1-meaningful event is simply called a meaningful event.

In practice, choosing = 1 means that the event is expected to appear less than once in a random context. It is a sound choice as the NFA generally has a exponential behavior w.r.t. event properties, so the dependence on is rather a log-dependence.

An example is provided in Figure 3 to illustrate how deviations from a random model make events perceptually meaningful.

Finally, to decide whether an event is meaningful in this framework, we need three elements, chosen a priori :

1. What kind of events are we looking for? 2. Which event's property should be analyzed? 3. What is the relevant a contrario random model? For segments detection, we have already answered question 1 with Definition 1. Question 2 and 3 will be shortly addressed in Section 4. Fig. 3. Illustration of the number of false alarms. Unlike the more complex segments matching Definition 1, the events considered here are simply "perfectly horizontal segments". The property associated to the event is the length and the a contrario random model is an image whose black pixel density is the same as in the original image but where points are spatially independent and uniformly distributed. A segment of 6-pixel length has been artificially added to each image, which otherwise follows the a contrario model. As the density of black pixels gets smaller, the number of false alarms of the 6-pixel segment decreases and it becomes an increasingly important deviation from the a contrario model. Our perception seems to follow similar rules: the segment becomes detectable with a NFA of 0.5 and obvious for a NFA of 10 -3 .

Selection based on a gradient magnitude criterion

A natural criterion to start with is based on the contrast between the segment and its neighborhood, an approach comparable to [START_REF] Desolneux | Edge detection by helmholtz principle[END_REF]. Whereas [START_REF] Desolneux | Edge detection by helmholtz principle[END_REF] was interested in the minimal gradient intensity value along level lines, here we consider the mean gradient value along the segment to be less sensitive to outliers. The gradient magnitude is computed using 2x2 finite differences to avoid creating artificial correlation between pixels (see [START_REF] Cao | Extracting Meaningful Curves from Images[END_REF] for detailed explanations).

The a contrario model we choose is a white uniform noise with the same gradient magnitude distribution as in the original image, but where pixels are spatially independent and uniformly distributed. This makes the model adapted to the global gradient properties of the image, while making pixel spatial organization in the original image the source of large deviations. The rarity of a segment will not come from the fact of observing pixels with high gradients in itself, but from the fact that a group of adjacent pixels contains many high gradient values.

Under these assumptions, we can compute a number of false alarms for segments.

Definition 4. Let µ g and σ g be respectively the gradient magnitude mean and standard deviation on the whole image. Let N segments be the number of candidate segments detected in the image. Let µ(S) be the mean gradient value of a segment S and L(S) its number of pixels. Then

N F A(S) = N segments × (1 -normcdf (µ(S), µ g , σ g L(S) ))
where normcdf (x, µ, σ) is the normal cumulative distribution function with mean µ and deviation σ applied to x.

This definition comes from the central limit theorem. Under the a contrario assumption, a segment can be seen as a collection of L(S) independent and identically distributed samples of the image, thus the mean of their gradient magnitude should follow a normal law if L(S) is big enough, according to the central limit theorem. Since we have N segments candidates, the expected number of segments having a deviation from the random model at least as large as the one observed for S is the N F A(S) of Definition 4.

We have implemented this selection criterion on a standard computer, but not on digital retinas because of some limitations of the current generation. This is discussed in more details in Section 6. This has led us to consider a different criterion as defined in Section 4.3, enabling a fast implementation on our retina.

Selection based on segment length

Instead of considering the gradient values along the segment, one might wonder what minimal length is required for a segment to be meaningful, whatever its contrast. The question becomes: in a direction image I d , how many chains of pixels of length l matching Definition 1 would be expected under an a contrario random model?

The choice of the a contrario model is somewhat similar to the one of Section 4.2. Taking I d , the a contrario image is an image whose white pixels density is the same as I d , but where pixels are spatially independent and uniformly distributed. This way, the selection adapts to the global density of pixels sharing the same local directions, and large deviations correspond to large groups of adjacent white pixels.

Unfortunately, even under these fairly simple assumptions, a NFA is analytically difficult to compute. This complexity comes from the rather particular connectivity induced by our definition of segments, which makes the number of candidates difficult to count. Still, we have to find the minimal length above which the NFA will be less than one, depending on the direction image white pixel density. This can be evaluated by stochastic Monte Carlo simulation, that is, by analyzing the actual statistical distribution of the lengths of segments occurring in randomly generated images.

Let I d be a direction image of size N × N , and p white its white pixel density #whitepixels N ×N

. The following procedure is repeated M times: 1. Generate a random black and white image of size N × N by drawing independently for each pixel the value white or black according to p white ; 2. Apply on it the segment extraction algorithm of Section 3 ; 3. Store the histogram of the segment lengths.

This results, for one p white value, into a collection of M samples of segment lengths histograms, as depicted in Figure 4. We are looking for the length threshold L min which ensures N F A(L(S)) < 1 whenever L(S) ≥ L min . N F A(L(S)) is the expectation of the number of occurrences of segments with length greater than L(S) in random images. It can be estimated from the simulations. Having a N F A < 1 means the expected maximal segment length in a random image must be less than L min .

From the M simulations above, one can compute a confidence bound on the expected maximal length. Let X i the maximal length observed in random image i. The empirical mean µ and empirical deviation σ of the maximal length are then:

µ = 1 M M i=1 X i σ 2 = 1 M -1 M i=1 (X i -µ) 2
Let µ true be the real expectation of the maximal segment length. When M is big enough, the random variable Y = (µ-µtrue) √ M σ follows a Student law with M -1 degrees of freedom. We construct a bound on µ true such that:

P (Y < t) = α
with α the confidence we want. We can get α arbitrarily close to 1 by increasing M and t. Note that α gets exponentially closer to 1 with respect to t, so for M = 1000 and t = 3.1 the Student law gives α = 0.999, leading to:

P (µ true < µ + 3.1 σ √ 1000 ) = 0.999
Thus, choosing L min = µ + 3.1 σ √ 1000 ensures P (N F A(L(S)) < 1) = 0.999 whenever L(S) ≥ L min . Figure 4 shows the typical exponentially decreasing distribution of maximal lengths values.

Finally, running simulations for different p white gives a table of minimal lengths thresholds. Then, the selection algorithm becomes, for each direction image:

1. Estimate p white from the direction image using the global adder; 2. Lookup in a table the corresponding minimal length threshold; 3. Remove extremities associated to segments having a too small length.

Quantitative results

Meaningful segment extraction based on the segment length criterion (see Section 4.3) has been successfully implemented on Pvlsar34, a home-made digital retina of 200x200 pixels, each containing a pixellic Boolean processor with 42 bits of local memory, under SIMD control. To evaluate our algorithm, we bypassed Pvlsar34 capture abilities by transferring standard images into retina memory, scaled to 200x200 pixels and reduced to 64 gray levels to save retina memory. Figure 5 shows an example of segment extraction on an interior scene. Note that there is no free parameter to set in the method, since the segment length thresholds are automatically derived from the direction images densities. Figure 6 clearly illustrates the benefits of context adaptation. If meaningful segments had been selected on the "house" image with the same threshold as the one derived for the "desk" image, a lot of false alarms would have been obtained, as shown on Figure 6(c).

The meaningful segment extraction algorithm runs in real-time on our digital retina Pvlsar34, at video rate. It runs 10 times slower on an up-to-date personal computer, with 2 images processed per second. This factor of ten seems ridiculously small considering the massive parallelism (40k processors operating together) available in Pvlsar34. One of the reasons is that Pvlsar34 is operated at a low frequency of 5MHz, which ensures a very low power consumption of a few tens milliwatts, 3 to 4 orders of magnitude as small as that of a PC! Another reason is examined in the next section. 6 Discussion

About statistical criteria

We try to avoid ad-hoc parameters. But there are still a number of choices which are subject to discussion: the definition of the considered properties, the chosen a contrario models, etc. Of course, ideally, those assumptions should be replaced by objective measurements or justifications. What should be noted however is that the nature of the remaining a priori is never quantitative but only qualitative. This means the a priori decisions always rely on reasoning, never on numerical, empirical values. Another limitation of current a contrario approaches is the global nature of the statistics from which large deviations are measured. There is an underlying assumption of image spatial stationarity, which is obviously not true in the general case. A direct consequence is the so-called blue sky effect, where a very flat zone in the image influences detection in other, shaky, parts. In [START_REF] Cao | Extracting Meaningful Curves from Images[END_REF], relying on level lines nesting properties, local meaningful level lines are detected using the statistics of the region associated to their closest surrounding meaningful level line. However, this is not easily applicable in our case.

Finally, we notice that our meaningful segment extraction algorithm does not use so much the retina abilities to compute global statistics though global summations. Using it much more intensively could provide interesting algorithmic and statistical innovations in the future.

About candidates and properties extraction

Whereas using a digital retina fitted with a fast global adder is a source of algorithmic inspiration, implementing algorithms on it suggests architectural improvements. Here, what are lessons to draw? Whereas gradient and directionrelated computations are fast, information propagation toward extremities takes most of the computation time. Indeed, these propagations are done synchronously in Pvlsar34, and only a few pixels (the extremities) are actually performing useful computations at each iteration. This is clearly under-exploiting massive parallelism. To drastically reduce propagation delays and energy consumption, asynchronous retinas (e.g [START_REF] Gies | Convergent micro-pipelines: a versatile operator for mixed asynchronous-synchronous computations[END_REF][START_REF] Ducourthial | Parallel asynchronous computation for image analysis[END_REF]) have been proposed, allowing efficient computing of regional quantities, which are typical of middle level vision. For example, the computation of a gradient magnitude mean over a segment would become tractable, thereby enabling more complex properties to be statistically analyzed. More generally, to cope with statistical detection of big groups of pixels, we believe asynchronism will play an important role and we are currently working on the realization of the model described in [START_REF] Gies | Asynchronous regional computation capabilities for digital retinas[END_REF].

Conclusion

This paper shows a first step towards more adaptive, parameterless and statistically founded algorithms taking advantage of digital retinas philosophy and capabilities. We have developed an original algorithm for the detection of meaningful segments. On the presented images, detected segments indeed seem to be the important ones. These encouraging results are obtained in spite of the relative simplicity of the statistical segment model we have chosen. Implementation on our home-made digital retina has allowed real-time operation but has recalled the limitations of its synchronous SIMD character for middle level vision.

Fig. 1 .

 1 Fig. 1. Top row: the eight overlapping direction cones. Bottom row: illustration of Definition 1 for the first cone (horizontal). Dark pixels must have a local gradient direction in the cone. Dashed lines show the direction induced by the neighbors of each point. Among the three sets of dark pixels, only the left and central ones are segments according to Definition 1, since all dashed lines are within the cone. The right one is not a valid segment since the white dashed line lies outside the cone (too large angle).

Fig. 2 .

 2 Fig. 2. Overview of the segment extraction algorithm. Step 1 produces eight binary direction images by analyzing local gradient directions in the original image. Each image corresponds to a direction cone and white pixels are pixels whose local direction fits in the cone. Step 2 and 3 are illustrated on portions of the horizontal direction image. Step 2 makes connected sets of white pixels one-pixel thick by only keeping pixels lying where the image gradient magnitude is maximal. Step 3 iteratively propagates segment information (length in the example) from the left extremity to the right one, following the rules of Definition 1. Finally, only right extremities will be fed to the selection step.

  2 and Section 4.3. P (black) = 0.5 P (black) = 0.23 P (black) = 0.1 N F A(E) 63 N F A(E) 0.5 N F A(E) 10 -3

Fig. 4 .

 4 Fig. 4. Horizontal axes are segments length. Vertical axes are the number of occurrences. (a) Histogram of maximal segments lengths for 1000 different uniform noise images with p white = 0.1 (b) Histogram of segments lengths for 100 different uniform noise images with p white = 0.1, only segments of length greater than four are considered.

Fig. 5 .

 5 Fig. 5. (a) "Desk" image (b) Horizontal direction image, localized on gradient maxima, p white = 0.09 (c) In white: segments which have a meaningful length.

Fig. 6 .

 6 Fig. 6. (a) "House" image (b) Horizontal direction image, localized on gradient maxima, p white = 0.2 (c) In white: horizontal segments which would have a meaningful length if the same threshold as for the desk image was used (d) In white: meaningful horizontal segments according to the new threshold.