Controlled viral replication under highly active antiretroviral therapy (HAART) allows restoring circulating CD4+ T-cell counts, leading to major enhancement of life expectancy. However, in 15–30% of HAART-treated individuals, CD4+ T-cell counts fail to reconstitute despite long-term control of viral replication upon effective antiviral treatment .
Several mechanisms have been associated to poor immunological responder (PIR) profile, such as residual viral replication , poor thymic production , naïve T-cell depletion because of X4 virus infection , Treg expansion [5,6], or NKP44L overexpression by CD4+ T-cells .
Suboptimal CD4+ T-cell restoration has been correlated to increased risk to develop opportunistic infection and to higher mortality, which highlight the need for additional treatments for these individuals [8,9]. Recombinant human IL-7 (rhIL-7) was used in various clinical trials, for its activity on both central and peripheral T-cell homeostasis, in order to help PIR participants reconstituting their CD4+ T-cell compartment [10,11]. These trials allowed to conclude that IL7-based therapy stimulates CD4+ T-cell reconstitution as measured in circulating blood, as a consequence of increased thymic output  and enhanced survival and proliferation of both naïve and memory T-cells [10,11,13]. More importantly, rhIL-7 therapy allows reconstituting T-cell populations in gut-associated lymphoid tissue , as a consequence of its capacity to stimulate local chemokine expression in tissues , as well as homing receptor and integrin expressions by circulating T-cells [15,16]. Neither in PIR-associated trials nor in untreated simian immunodeficiency virus (SIV)-infected macaques was IL-7 therapy followed by any significant enhancement of circulating viral loads [10,11,13,17]. Transient viral replication (blips) was only observed during rhIL-7 therapy in a small proportion of participants and was associated to increased frequency of IFN-γ and/or IL-2 producing CD4+ T-cells . Nevertheless, the applicability of rhIL-7 therapy in HIV-infected individuals remains debated because of its possible effect on viral reservoir size. Indeed, in-vitro studies have associated IL-7 stimulation to viral production by sorted CD4+ T-cells  and to sustained permissiveness to cellular infection [19–21]. Furthermore, IL-7 stimulates NF-κB activation and SAMHD1 phosphorylation, possibly leading to reactivation of latent viral reservoirs [22–24]. In the present study, we explored the evolution of HIV reservoir in rhIL-7-treated participants from INSPIRE 3 clinical trial.
Materials and methods
Forty-one rhIL-7 treated participants and 16 controls from the INSPIRE 3 (CLI-107–14) trial were selected for HIV reservoir analysis according to the availability of blood samples at baseline (D0), D28 and month 3 (M3) following the initiation of rhIL-7 therapy (Table 1). In this trial, treated participants received 3 weekly subcutaneous administrations of rhIL-7 (20 μg/kg of body weight) and were followed up quarterly .
Total HIV-DNA was quantified on total white blood cell (WBC) lysates as previously described . Total HIV-DNA, was chosen as the most representative measurement of viral reservoir in chronically infected patients under HAART treatment for more than 1 year . Oligonucleotides specific for conserved regions of both gag and env genes were defined on consensus sequences of either HIV-1 clades A/C for South African participants (A/C-envOUT5′-CATGCCTGTGTACCCACAGA; A/C-envOUT3′- CTCTTCTTCTGCTAGACTG/ACCAT; A/C-gagOUT5′- CAGACAGGAACAGAG/AGAACT; A/C-gagOUT3′-ATTCTGCAGCTTCCTCATTGAT; A/C-envIN5′-AGACCCCAACCCACAAGAA; A/C-envIN3′-ACCAGCTGGGGCACAATAAT; A/C-gagIN5′-ATAGAGGTAAAAGACACCAAGGA; A/C-gagIN3′-GGGTGGCTCCTTCTGATAAT) or HIV-1 clade B for European participants (B-envOUT5′-GCCACACATGCCTGTGTACCCACA; B-envOUT3′-TTTAGAATCGCAAAACCAGCCG; B-gagOUT5′-CCCTTCAGACAGGATCAGAA; B-gagOUT3′-ATTCTGCAGCTTCCTCATTGAT; B-envIN5′-AGACCCCAACCCACAAGAA; B-envIN3′-TGGACAGGCCTGTGTAATGA; B-gagIN5′-TAGAGGTAAAAGACACCAAGGA; B-gagIN3′-ATGTCCCCCCACTGTGTTTA) [28,29]. CD3γ chain was used as housekeeping gene . HIV-DNA concentration was calculated as per milliliters and per CD4+ T-cell using blood cell counts.
CD4+ cell purification
PBMCs sampled at D0 and M3 were stained with an anti-CD4-PerCP-Cy5.5 (clone L200, BD-Biosciences, Le Pont de Claix, France) and CD4+ cell population was sorted on a FACS ARIA III (BD-Biosciences). HIV-DNA load was then quantified on purified CD4+ cells, as described above.
Statistical analyses were performed using nonparametric Wilcoxon signed-rank test and Spearman rank-order correlations with Real Statistics using Excel software (http://www.real-statistics.com).
In order to assess the consequences of rhIL-7 based therapy on HIV reservoirs, total HIV-DNA loads were quantified on peripheral blood cells sampled at D28 and M3 following the initiation of rhIL-7 therapy. Quantified on total WBC, HIV-DNA concentration very significantly increased during the first 28 days of rhIL-7 therapy [median = 357 HIV copies/106 WBC (interquartile range, IQR: 105–697) at D28 as compared with 104 (IQR: 50–189) at D0; p < 10−6; Fig. 1a). Despite a substantial decrease between D28 and M3, HIV-DNA copies/106 WBC remained significantly higher, 3 months after the initiation of rhIL-7 therapy (M3) than at baseline, with a median 46% increase as compared with D0 [median = 140 HIV copies/106 WBC (40–317); p < 10−3; Fig. 1a]. Similarly, the concentration of HIV-DNA copies adjusted per milliliters of blood significantly rose at D28 [2876 HIV copies/mL (676–4710); p < 10−7; Fig. 1b] and despite significant reduction during subsequent follow-up, remained higher at M3 than before therapy [819 (245–2042) and 500 (144–1044) HIV copies/mL at M3 and D0, respectively; median increase = 86%; p < 10−4; Fig. 1b]. In contrast, no significant change in HIV-DNA loads was observed in the control group, whether the results were expressed in HIV copies/106 WBC [median = 63 (35–195), 77 (2–277) and 83 (32–135) HIV copies/106 WBC at D0, D28 and M3 respectively; n = 16] or in HIV copies/mL [median = 250 (142–881), 493 (7–1809) and 401 (108–632) HIV copies/mL at D0, D28 and M3, respectively; n = 16].
To further assess the impact of rhIL-7 treatment on neoinfection in vivo, we extrapolated HIV-DNA loads to circulating CD4+ T-cell counts in all tested samples. Adjusted to CD4+ T-cell counts, HIV reservoir increased in size during the first month of rhIL-7 therapy [median = 3105 (884–5661) HIV-DNA copies/106 CD4+ T-cell at D28 versus 1910 (601–5130) at D0; p = 0.05; Fig. 1c]. Interestingly, an inverse correlation was observed between changes in CD4+ T-cell counts and HIV-DNA/106 CD4+ T-cells in rhIL-7-treated individuals (r = −0.45; p = 0.003; Fig. 1d), suggesting either that rhIL-7-induced viral replication impairs the reconstitution of CD4+ T-cell compartment or that participants who responded to rhIL-7 treatment by high increases in CD4+ T-cells demonstrated a stronger control of HIV replication than participants poorly responding to rhIL-7 therapy. In contrast to HIV-DNA load measured per WBC or per mL, HIV-DNA load per circulating CD4+ T-cell eventually decreased at M3 in a majority of treated individuals [1645 (310–3146) HIV-DNA copies/106 CD4+ T-cells; median 26% decrease as compared with baseline values; p = 0.01; Fig. 1c]. In the control group, no significant evolution was observed [median = 1238 (600–2105), 2321 (37–7737) and 1537 (387–1710) HIV-DNA/106 CD4+ T-cells at D0, D28 and M3, respectively; n = 16].
In order to confirm this observation, we quantified HIV-DNA in purified CD4+ cells from 19 rhIL-7-treated and 9 control participants sampled at D0 and M3 (Fig. 1e). In this subgroup, measured HIV copies/106 CD4+ cells strongly correlated with calculated values (r = 0.72, p < 10−9), confirming the relevance of our initial calculation to estimate HIV-DNA load in CD4+ cells. Very similar results were observed with a decline in HIV-DNA load in 14 rhIL-7 treated individuals [median = 1615 (753–2327) and 1117 (623–1934) HIV copies/106 CD4+ cells at D0 and M3, respectively; 22% mean decline; p = 0.018; Fig. 1e]. No significant modification of HIV-DNA load was observed in the control group [median = 2447 (632–2760) copies/106 CD4+ cells at D0 as compared with 2284 (738–2467) at M3; 3% mean decline; Fig. 1e].
In the INSPIRE 3 clinical trial, enlarged HIV-DNA reservoirs accompanied the massive expansion of T-cell compartments triggered by IL-7 during the first 4 weeks of therapy. This expansion of the HIV reservoir is most likely because of CD4+ T-cell proliferation elicited by the high IL-7 concentrations reached both in plasma and, more importantly, in tissues. Indeed, tissue IL-7 concentrations reached after injection of pharmacological doses of the cytokine are sufficient to locally trigger massive T-cell proliferation . Yet, to increase the frequency of infected CD4+ T-cells, as observed at D28 (Fig. 1c), infected CD4+ T-cells should proliferate more than uninfected ones. However, during the first 4 weeks of therapy, rhIL-7-dependant expansion was greater in naïve CD4+ T-cells than in central and effector memory CD4+ T-cells  the latter accounting by far, for most of the infected T-cells [26,31,32]. Moreover, it seems highly improbable that HIV-infected memory cells proliferate or survive more than uninfected cells upon IL-7 stimulation.
Accordingly, the increase in HIV-DNA frequencies within the CD4 compartment observed during the first 4 weeks of rhIL-7 therapy argues against IL-7-dependent cell proliferation being the only factor impacting circulating infected CD4+ T-cell frequencies and suggests increased neoinfection despite antiretroviral treatment. Indeed, IL-7 is known to stimulate HIV transcription as a consequence of NF-κB activation and SAMHD1 phosphorylation [22–24,33], leading to partial reactivation of latent viruses, but fails to reduce the size of the reservoir in vitro[22,33]. This is in line with the increased activation of circulating CD4+ T-cells previously observed during the first 4 days of rhIL-7 therapy  and with residual viral replication in tissue of PIR patients . Of note, viral blips were occasionally observed in both INSPIRE 3 and previous trials [10,25,34]. However, in virally suppressed patients, IL-7 alone barely stimulates viral replication in latently infected cells, but enhances residual levels of viral replication in productively infected cells [18,20,35]. Moreover, IL-7-mediated enhancement of both HIV transcription and permissiveness of uninfected cells could lead to increased cell-to-cell virus transmission in lymphoid structures, despite antiretroviral treatment . Indeed, as a direct consequence of local IL-7-dependent chemokines expression, and their activation [15–17], IL-7 therapy may increase local CD4+ T-cell concentration and favor cell-to-cell transmission in lymphoid tissues. However, during the first 28 days of therapy, the observed negative correlation between gain in CD4+ T-cell and gain in HIV-DNA load (per/106 CD4+ T-cell; Fig. 1d) could also be a consequence of greater expansion of the naïve T-cell compartment in participants presenting the strongest response to IL-7, whereas participants with lower response preferentially expand memory compartments. Such a possible dichotomy deserves further investigation.
Three months after rhIL-7 therapy, despite significant decrease in total HIV-DNA loads (per mL and per WBC) as compared with D28, these parameters remained higher than at baseline. In contrast, the frequency of infected cells within the CD4+ compartment not only returned to baseline levels, but also decreased in a majority of participants. During this period, the evolution of HIV-DNA load neither correlated with that of CD4+ or CD8+ T-cell counts nor depended upon initial T-cell counts or HIV-DNA load. Such a decline could be a consequence of either preferential expansion and/or survival of the barely infected naïve CD4+ T-cell population or specific elimination of infected cells. In the INSPIRE 2 clinical trial, naïve T-cell counts strongly declined during this period, whereas memory T-cells plateaued , the latter containing most of HIV-infected cells. Similar results were observed on a subgroup of participants from the INSPIRE 3 trial (n = 14; data not shown). These data suggest that the decrease in HIV-DNA load between D28 and M3 was not a consequence of the dilution of infected memory cells in larger amounts of uninfected naïve T-cells. Accordingly, one can reason that rhIL-7 therapy could allow better elimination of HIV-expressing cells through improvement of HIV-specific immunity. Indeed, IL-7 stimulation triggers expression of viral proteins in vitro, which should allow their recognition by specific CTLs. However, the impact of such a shock and kill strategy was not detected in immunological responder patients under treatment intensification  and remained limited in PIR patients as infected cell frequency only declined by 25% during the 3 months follow-up period in our study. Accordingly, even though IL-7 therapy alone will probably not be efficient in a shock and kill strategy, the effect of its combination with other compounds susceptible to stimulate HIV expression deserves further investigation.
Considering that viral reservoirs constitute the major barrier to HIV eradication and has been associated to virological control after HAART treatment discontinuation , understanding the molecular pathways and immunovirological parameters triggered by rhIL-7 treatment is required to apprehend HIV-DNA fluctuations observed in IL-7-treated PIR patients. Furthermore, the impact of rhIL-7 therapy on tissue HIV reservoirs remains to be addressed. Finally, considering the large proportion of defective proviruses in patient's quasispecies, the evolution of replication-competent reservoirs upon IL-7 therapy should be further investigated.
This work was carried out in partial fulfillment of S.L.'s PhD thesis at Université Paris Descartes, Paris, France. This work was supported by Inserm-ANRS (Agence Nationale de Recherches sur le SIDA et les Hépatites Virales), ANRT (Association Nationale de la Recherche et de la Technologie), in agreement with Cognate Bioservices and RevImmune Inc, owners of the samples from INSPIRE 3 clinical trial. Inserm, CNRS and Université Paris Descartes also supported this work. Cytheris S.A. (now Revimmune Inc.), Inserm-ANRS and Université Paris Descartes supported S.L.'s PhD thesis. The authors would like to thank Caroline Janot Sardet, Caroline Codron and Anne Gregoire for their help in the management of the project.
Author's Contributions: S.L., M.R., S.R., S.F-M and B.C-d.M. performed the experiments. S.B. and R.C. designed the experiments. S.L., M.R., ACC and R.C. analyzed the data. R.C. wrote the manuscript. G.T. directed the steering committee of the INSPIRE 3 clinical trial. All the authors discussed the results and commented the manuscript.
Conflicts of interest
S.B. and S.R. were employees of Cytheris S.A. (now Revimmune Inc.). S.L.'s PhD thesis was supported, in part, by a CIFRE (Convention Industrielle de Formation par la Recherche) fellowship co-funded by the French government and Cytheris S.A., now Revimmune Inc. Cytheris S.A., now Revimmune Inc., which was developing recombinant human IL-7 as an immune enhancer, partly supported this work. The remaining authors declared no competing financial interests.
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