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Translational Research

Reversal of Viral Latency and Induction of Gag-Specific T-Cell Responses in HIV-1-Infected Adults Through Cyclic Treatment Interruption of Rosuvastatin: A Proof-of-Concept Study

Hsieh, Szu-Min MD; Pan, Sung-Ching MD, PhD; Huang, Yu-Shan MD; Chang, Shan-Chwen MD, PhD

Author Information
JAIDS Journal of Acquired Immune Deficiency Syndromes: April 1, 2021 - Volume 86 - Issue 4 - p 500-508
doi: 10.1097/QAI.0000000000002577

Abstract

INTRODUCTION

Latent infection of CD4+ T cells provides lifelong persistence of HIV-1, even in patients on effective combination antiretroviral therapy (cART).1 Interventions to activate the latent reservoir to induce immune responses that help destroy the viral reservoir, and then to achieve a functional cure are being investigated, the so-called “shock and kill” strategy.2–6 Clinical trials examining the use of latency-reversing agents, such as histone deacetylase inhibitors (including romidepsin and vorinostat), have been conducted.2,3,5 However, the results of the first randomized clinical trial, the RIVER study, showed no obvious impact on measures of HIV reservoir compared with cART alone.5 Thus, the strategy has been considered to be in need of revision,4 and other interventions aimed at achieving a functional cure for HIV-1 infection remain needed, such as strategies using other latency-reversing agents,7 broadly neutralizing antibodies,8 toll-like receptor agonists,9 gene therapy,10 or mimetics of second mitochondrial-derived activator of caspases.11

Statins, lipid-lowering agents, are known to have immunomodulatory effects,12 by reducing the production of proinflammatory cytokines, including interferon (IFN)-γ and tumor necrosis factor-α, through an HMG-CoA-dependent pathway that involves inhibition of the nuclear factor (NF)-kappa(κ) B pathway,13–16 or induction of T-cell Krüppel-like factor 2 gene expression.17 Statins may also increase the frequency of circulating CD4+FOXP3+ regulatory T cells with the potential to inhibit T-cell responses.18 Modification or rebound of inflammatory reactions and immune responses have also been noted shortly after interruption of statins.19,20

Increased cardiovascular events in patients with high cardiovascular risk or pre-existing myocardial infarction have been observed after statin withdrawal.21 However, 20% of patients with hyperlipidemia may develop intolerance to statins, leading to inadequate adherence or even withdrawal, and physicians may discontinue the medication because of muscle-related adverse events.22

This prospective, single arm, proof-of-concept study was conducted to test the hypothesis that the immune rebound during statin withdrawal in a cyclic treatment-interruption (CTI) of rosuvastatin may trigger the reversal of latent reservoir in CD4+ T cells. We longitudinally assessed and analyzed the levels of cell-associated RNA (as a marker of activity of viral reservoirs3,23) and proviral DNA (as a marker of the size of latent reservoirs3,23) in CD4+ T cells and HIV-1 Gag-specific CD4+ and CD8+ T-cell responses in the HIV-1-infected adults on rosuvastatin with CTI.

METHODS

Study Design and Participants

A long-term observational cohort has been established at National Taiwan University Hospital (NTUH), one of the major medical centers for HIV/AIDS care and research in Taiwan. In this proof-of-concept study, HIV-1-infected Asian men with hyperlipidemia were considered to be eligible for enrollment if they would like to adhere to CTI of rosuvastatin with a fixed 12-week interval (ie, temporary discontinuation for 12 weeks followed by restarting treatment for 12 weeks) for 72 weeks (ie, 3 cycles), since Feb 2017. The eligible subjects could be enrolled if they fulfilled all of the following criteria, including an age less than 40 years, receiving cART for at least 3 years with undetectable plasma HIV RNA, receiving rosuvastatin for hyperlipidemia, no history of any cardiovascular event, no smoking, no family history of early-onset cardiovascular disease, and body mass index <25 kg/m2. Lifestyle modification to help lipid control was strongly suggested.

There were 2 substudies. For longer duration of observation after 72 weeks, subjects could choose freely to continue the CTI of rosuvastatin and participated in an extended observation. For case-matched comparison, two 1:1 matched groups (one group with continuously regular use of rosuvastatin and the other one group without using any statin) were enrolled by matching age (within a difference of 2 years), sex (same sex), duration of cART with stable viral control (with a difference of 0.5 years), and nadir CD4+ T-cell counts (within a difference of 25 cells/µL).

Ethics

This study has been approved by the Institutional Review Board of NTUH and registered with ClinicalTrials.gov (ClinicalTrials.gov ID: NCT04068441). All patients provided written informed consent.

The levels of proviral DNA were monitored every 12 weeks, and the plasma HIV-1 RNA, CD4+ T-cell counts, HIV-1 Gag-specific T-cell responses, and levels of cell-associated RNA in CD4+ T cells were checked every 12 weeks, and 2 weeks after stopping rosuvastatin in every cycle because of the potential for rebound of immune responses within 2 weeks.19,20

Preparation of Antigen-Presenting Monocyte-Derived DCs

The method to prepare antigen-presenting dendritic cells (DCs) to assess DC-driven antigen-specific T-cell responses has been described previously.24–26 Briefly, monocytes were negatively isolated from PBMCs (Dynabead Untouch Human Monocytes Kit; Invitrogen, Carlsbad, CA) and suspended in complete RPMI (Gibco, Grand Island, NY) supplemented with recombinant human IL-4 (1000 U/mL, PharMingen, San Diego, CA) and GM-CSF (50 ng/mL, PharMingen) in 24-well plates (1 mL/well) in triplicate. These monocytes showed a phenotype of immature DCs after 6 days of culture. The immature DCs were pulsed with HIV-1 Gag p24 (5 μg/mL; Research Diagnostics, Flanders, NJ) or cytomegalovirus (CMV) antigen (as a recall antigen, 1:8 diluted solution from a commercially prepared lysate of CMV-infected fibroblasts; BioWhittaker, Walkersville, MD, at a concentration of 10 μg/mL) for another 24 hours to achieve a state of complete maturation as antigen-presenting DCs (confirmed by staining with a mAb against CD83 (FITC-conjugated HB15a, mouse IgG2b, Immunotech, Marseille, France).

Cell Isolation

CD4+ or CD8+ T cells were positively isolated from PBMCs and detached from beads using an immunomagnetic method (CD4 or CD8 Positive Isolation Kit; Dynabeads plus DETACHaBEAD; Invitrogen) according to the instructions supplied by the manufacturer. The resulting purity was 99%, and the viability was 95%.

Antigen-Specific DC-Driven CD4+ T-Cell Response

CD4+ T-cell responses were determined by incubating isolated CD4+ T cells with autologous DCs in a final volume of 200 μL RPMI (2 × 105 CD4+ T cells with 2 × 104 DCs per well in a 96-well plate in triplicate) for 3 days. The levels of IFN-γ were determined using a commercial ELISA kit (Quantikine; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Antigen-specific DC-driven IFN-γ production from CD4+ T cells was defined as [the levels of IFN-γ in supernatants from CD4+ T cells incubated with antigen-presenting DCs] − [the levels of IFN-γ in supernatants from CD4+ T cells incubated with immature DCs without being pulsed with antigen].

Antigen-Specific DC-Driven Cytotoxic Capacity of CD8+ T Cells

Isolated CD8+ T cells (106 cells/well) were prepared as effector cells by incubation with autologous antigen-pulsed DCs (as APCs, at a ratio of 10:1) in a final volume of 1 mL of complete RPMI 1640 in 24-well plates for 48 hours. Autologous monocytes isolated from PBMCs were prepared as target cells (106 cells/mL) by pulsing with Gag or CMV antigen in 24-well plates for 24 hours. A nonradioactive lactate dehydrogenase (LDH)-releasing cytotoxicity assay kit (CytoTox 96 Non-Radioactive Cytotoxicity Assay; Promega, Madison, WI) was used with an effector cell:target cell (E:T) ratio of 10:1. Released LDH in supernatant was measured with a 30-min coupled enzymatic assay (provided by CytoTox 96 kit) that resulted in the conversion of a tetrazolium salt (INT) into a red formazan product. The percentage of cytolysis was calculated as {[(experimental release − background) − (spontaneous target release − background) − (spontaneous effector release − background)]/(maximal target release − spontaneous target release)} × 100%. The percentage of antigen-specific cytolytic activity was calculated as [percentage of cytolytic activity of CD8+ T cells induced by antigen-pulsed DCs] − [percentage of cytolytic activity of CD8+ T cells induced by immature DCs without being pulsed with antigen].

Quantitation of Proviral DNA

The procedures for total HIV-1 proviral DNA measurement were performed as previously described with modifications.3,23 Briefly, DNA was extracted from isolated CD4+ T cells using a QIAamp DNA Blood Mini Kit (QIAGEN, Germantown, MD), and all forms of intracellular HIV-1 DNA were detected and quantified by amplification of a specific conserved sequence using the QuantStudio 5 RealTime PCR System by AmpliTaq Gold (Applied BioSystem) (The PCR protocol and list of primers in Methods, Supplemental Digital Content, https://links.lww.com/QAI/B575). We estimated our limit of detection was 50 copies/106 CD4+ T cells. To know the individual dynamic changes in the 3 cycles of treatment interruption, we determined the values of the 7 samples for HIV DNA from the 72-week follow-up side-by-side simultaneously for each subject in duplicate and showed the average value.

Quantitation of Cell-Associated RNA

The procedures for quantifying cell-associated (CA) HIV RNA, were performed as previously described with modifications.2,3,23 Briefly, isolated CD4+ cells were lysed, and lysates were stored at −80°C until RNA was extracted (Allprep isolation kit; Qiagen, Valencia, CA). The cDNA template was prepared using the miScript II RT kit (Qiagen). The amplification reactions were performed in a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) (The PCR protocol and list of primers in Methods, Supplemental Digital Content, https://links.lww.com/QAI/B575). The means of 2 replicated wells were used in all analyses. The limit of detection was estimated to be 20 copies/106 CD4+ T cells. To know the individual dynamic changes in the 3 cycles of treatment interruption, we determined the values of the 10 samples for CA HIV RNA from the 72-week follow-up side-by-side simultaneously for each subject in duplicate and showed the average value.

Cardiovascular Events

During the 72-week follow-up period, the occurrence of a cardiovascular event was determined when the patient history was recorded during the regular clinical follow-up visit, and the medical and emergency ward records were also routinely reviewed.

Statistical Analysis

The statistical significance of paired data was determined by Wilcoxon signed-rank test. The linear correlations were determined using Pearson correlation coefficient calculations. All tests were two-tailed. P < 0.05 was considered statistically significant. The statistical analyses were performed with SAS 9.3 (North Carolina).

RESULTS

Demographic Characteristics

From Feb 2017 to Dec 2019, 10 subjects were successfully enrolled and completed 72-weeks follow-up (flow diagram, shown in Fig. 1). All of the subjects were Asian men with a median age of 36 years when enrolled. The median nadir CD4+ T-cell count is 493 cells/μL, and 2 of them had nadir CD4+ T-cell counts below 350 cells/µL (295 in subject C and 302 in subject E, respectively), but all of them had CD4+ T-cell counts > 350 cells/µL when enrolled. Baseline demographic characteristics are shown in Table 1. During the 72-week follow-up period, the lipid levels of all subjects were well-controlled with lifestyle modification and CTI of rosuvastatin. Not any cardiovascular event was noted or recorded in all subjects.

F1
FIGURE 1.:
The enrollment process.
TABLE 1. - Baseline Characteristics of the Ten Subjects When Enrolled
Baseline Characteristics n = 10
Asian, n (%) 10 (100%)
Male gender, n (%) 10 (100%)
Age (yr), median (range) 36 (31–39)
Years since HIV diagnosis, median (range) 4.3 (3.2–6.4)
Duration of cART (yr), median (range) 4.2 (3.1–6.3)
cART regimen
 DTG/ABC/3TC as fixed dose combination, n (%) 5 (50%)
 EVG/c/TAF/FTC as fixed dose combination, n (%) 3 (30%)
 RPV/TDF/FTC as fixed dose combination, n (%) 2 (20%)
Years with HIV RNA < 50 copies/mL, median (range) 4.0 (2.9–6.0)
Nadir CD4+ cell count per μL, median (range) 493 (295–623)
CD4+ cell count per μL when enrolled, median (range) 591 (503–813)
Lipid levels when enrolled
 Total cholesterol (mg/dL), median (range) 243 (223–278)
 HDL (mg/dL), median (range) 41 (31–48)
 LDL (mg/dL), median (range) 162 (150–176)
Lipid levels at the end of 72-week follow-up
 Total cholesterol (mg/dL), median (range) 217 (210–228)
 HDL (mg/dL), median (range) 50 (46–59)
 LDL (mg/dL), median (range) 136 (130–148)

Pilot Phase

In the pilot phase (the first 14 weeks, including 12 weeks of regular rosuvastatin use and 2 weeks after stopping rosuvastatin, Fig. 1, Supplemental Digital Content, http:// links.lww.com/QAI/B575) to test the feasibility, the Gag-specific lymphoproliferative responses, Gag-specific NF-kB signaling, and frequencies of CD69 expression were found to be significantly upregulated in CD4+ T cells isolated from PBMCs from the 10 subjects shortly after stopping rosuvastatin (see Figs. 1A–D, Supplemental Digital Content, https://links.lww.com/QAI/B575). In addition, the percentage of regulatory T cells (Treg, CD4+CD25+FoxP3+ expression) in CD4+ T cells was decreased. Therefore, the initial assessment supported the possibility that the activity of CD4+ T cells may have a phenomenon of rebound shortly following the treatment interruption of rosuvastatin.

Changes of CD4+ T-Cell Count, Plasma HIV RNA, and CA HIV RNA

During the whole 72-week follow-up, their CD4+ T-cell counts did not have significant changes (median, 591 cells/μL [range, 503–813] at week 0 vs. 520 cells/μL [520–794] at week 72, P = 0.332). There was no significance difference in CD4+ T-cell counts 2 weeks after stopping rosuvastatin during the 3 cycles of CTI of rosuvastatin (Fig. 2A). All of the 10 determinations of plasma HIV-1 RNA in all subjects showed the levels were persistently below 50 copies/mL in the 72-week follow-up (Fig. 2B).

F2
FIGURE 2.:
A, The changes of CD4+ T-cell counts (B) the levels of CA HIV RNA in CD4+ T cells and plasma HIV RNA in 10 subjects in the 72-week follow-up. P values were calculated to compare the values at week 12 vs. week 14, week 36 vs. week 38, and week 60 vs. week 62, by Wilcoxon signed rank test. Ten triangles indicate the timing of determination for plasma HIV RNA. All of the determinations of plasma viral RNA in 10 subjects showed the levels were below 50 copies/mL. The gray zones indicate the periods of rosuvastatin use, and the white zones indicate the periods of rosuvastatin withdrawal. Black horizontal dash line indicates the detectable level of CA HIV RNA (20 copies/106 CD4+ T cells).

However, the levels of CA HIV RNA had significant dynamic changes during the CTI of rosuvastatin. The data showed the significant increases in the levels of CA HIV RNA were detected 2 weeks after stopping rosuvastatin during every cycle of CTI of rosuvastatin (Fig. 2B). The levels of CA HIV RNA at week 14, 38, and 62 in subject C and E seemed to be relatively low when compared with those in other subjects.

Dynamic Changes in DC-Driven HIV-1 Gag-Specific T-Cell Responses

The reversal of viral latency may result in subsequent viral replication and antigen re-expression.3,27 To know whether the increase of levels of CA HIV RNA during rosuvastatin withdrawal is associated with the induction of HIV-1-specific T-cell responses, we assessed DC-driven Gag-specific T-cell responses at the same time points of CA HIV RNA determination. Consistent to the dynamic changes of CA HIV RNA during the 3 cycles of CTI of rosuvastatin, the dynamic changes in Gag-specific IFN-γ production from CD4+ T cells showed remarkable but transient increases shortly after stopping rosuvastatin (Fig. 3A). The dynamic changes of Gag-specific CD8+ cytotoxic T-lymphocyte (CTL) activity were also consistent with those of the Gag-specific CD4+ T-cell responses (Fig. 3B). Similarly, the levels of Gag-specific IFN-γ production from CD4+ T cells and CD8+ CTL activity at week 14, 38, and 62 in subject C and E seemed to be relatively low when compared with those in other subjects.

F3
FIGURE 3.:
DC-driven Gag-specific T-cell responses during the 72-week follow-up period. A, HIV-1 Gag-specific IFN-γ production from CD4+ T cells; (B) HIV-1 Gag-specific CD8+ cytotoxic activity. P values were calculated to compare the values at week 12 vs. week 14, week 36 vs. week 38, and week 60 vs. week 62, by Wilcoxon signed rank test. The gray zones indicate the periods of rosuvastatin use, and the white zones indicate the periods of rosuvastatin withdrawal.

Despite the obvious dynamic changes during the 72-week follow-up, Gag-specific CD4+ or CD8+ T-cell activity had no significant change from the beginning of enrollment to the end of the observation period (IFN-γ production, 121 vs. 114 pg/mL, P = 0.652; cytolytic activity, 12.7% vs. 13.3%, P = 0.646, by Wilcoxon signed rank test).

Dynamic Changes in DC-Driven CMV-Specific T-Cell Responses

To determine whether the transient increases in CD4+ and CD8+ T-cell reactivity shortly after stopping rosuvastatin also occurred in response to stimulation by other antigens, we used the CMV antigen as a recall antigen and prepared CMV-presenting DCs to stimulate T cells. As a result, the CMV-specific IFN-γ production from CD4+ T cells and the CMV-specific CD8+ cytotoxic capacity showed no significant dynamic changes related to the CTI of rosuvastatin (see Fig. 2, Supplemental Digital Content, https://links.lww.com/QAI/B575).

Dynamic Changes in Levels of Proviral DNA

The levels of proviral DNA in CD4+ T cells were determined every 12 weeks in the 72-week follow-up (Fig. 4A). Significant declines were observed during the period of rosuvastatin withdrawal in every cycle of CTI of rosuvastatin. After 3 cycles of CTI of rosuvastatin, the levels of proviral DNA decreased significantly from week 0 [median, 512 copies/106 CD4+ T cells (range, 323–965)] to week 72 [median, 262 copies/106 CD4+ T cells (range, 85–413), P = 0.005 by Wilcoxon signed rank test] with a median fold decrease of 2.63 [range, 1.41–4.82].

F4
FIGURE 4.:
Changes of proviral DNA levels in CD4+ T cells. A, The levels of proviral DNA declined significantly during the period of rosuvastatin withdrawal in every cycles of CTI of rosuvastatin. There was a median 2.63-fold decrease in proviral DNA levels in 10 subjects during the 72-week follow-up. P values were calculated to compare the values at week 12 vs. week 24, week 36 vs. week 48, and week 60 vs. week 72, by Wilcoxon signed rank test. The gray zones indicate the periods of rosuvastatin use, and the white zones indicate the periods of rosuvastatin withdrawal. B, Significant linear correlation between the nadir CD4+ T cell counts and fold decrease in proviral DNA levels in 10 subjects during the 72-week follow-up. The Pearson correlation coefficient (R) was 0.808 and P = 0.004. C, No significant linear correlation between the CD4+ T-cell counts when enrolled and fold decrease in proviral DNA levels in 10 subjects during the 72-week follow-up (R = 0.168 and P = 0.643). D, Kinetics of decay of the latent reservoir size with one-phase exponential correlation in 3 subjects that completed 120-week follow-up, determined by Pearson correlation of logarithm of proviral DNA levels with time (duration of rosuvastatin CTI). In subject A, the predicted time of proviral DNA levels in CD4+ T cells decreasing below the limit of detection (50 copies/106 CD4+ T cells, dashed line) would be 183.1 weeks; in subject B, it would be 225.7 weeks, and in subject D, 177.7 weeks.

Only 2 subjects had a fold decrease of proviral DNA levels below 2.0 during the 72-week follow-up (1.41 in subject C and 1.58 in subject E, respectively, Fig. 4A). We also noted subject C and subject E had relatively lower levels of CA HIV RNA, Gag-specific IFN-γ production from CD4+T cells, and Gag-specific CD8+ CTL activity at week 14, 38, and 62 (Figs. 2 and 3). Furthermore, among the 10 subjects, only subject C and subject E had nadir CD4+ T-cell counts below 350 cells/μL. Therefore, we hypothesized the impact of CTI of rosuvastatin on the reversal of viral latency may be related to the nadir CD4+ T-cell counts. Linear correlation in all of the 10 subjects was determined and the data showed a highly significant correlation between the nadir CD4+ T-cell counts and the fold decrease in proviral DNA levels during the 72-week follow-up (Pearson correlation coefficient R = 0.808, P = 0.004, Fig. 4B). However, no significant linear correlation was found between the CD4+ T-cell counts when enrolled and the fold decrease in proviral DNA levels during the 72-week follow-up (R = 0.168, P = 0.643, Fig. 4C).

Extended Observation Study

After completion of the 72-week follow-up (3 cycles of CTI of rosuvastatin), 3 subjects (subject A, B, D) agreed to participate the extended observation study to 120 weeks (5 cycles of CTI of rosuvastatin). We found the kinetics of decay of the proviral DNA levels had significant one-phase exponential correlations with time [Pearson correlation of logarithm of proviral DNA levels with time (the duration of rosuvastatin CTI), Fig. 4D]. The predicted time of proviral DNA in CD4+ T cells decay below the limit of detection (50 copies/106 CD4+ T cells) was 183.1 weeks, 225.7 weeks, and 177.7 weeks, respectively.

Case-Matched Comparison

In the case-matched comparison study, 10 matched subjects with continuous rosuvastatin use and 10 matched subjects without using any statin were enrolled. Baseline demographic characteristics of the 2 groups were listed in Appendix (see Tables 1 and 2, Supplemental Digital Content, https://links.lww.com/QAI/B575). The proviral DNA levels in the subjects with continuously rosuvastatin use did not have a significant change in the 72-week follow-up [median (range), 520 (342–972) vs. 517 (367–985) copies/106 CD4+ T cells, at week 0 and week 72, respectively, P = 0.682]. The proviral DNA levels in the subjects without using any statin did not have a significant change in the 72-week follow-up either [median (range), 525 (278–996) vs. 517 (280–872) copies/106 CD4+ T cells, at week 0 and week 72, respectively, P = 0.542].

DISCUSSION

Here, we show the results of a proof-of-concept study applying a novel approach that may help achieve the aim of functional cure. In the HIV-1-infected adults with stable cART, the activity of CD4+ T cells and the latent viral reservoir may be activated through the CTI of rosuvastatin; HIV-1 Gag-specific CD4+ and CD8+ T cell responses may undergo a transient but remarkable upregulation shortly after stopping rosuvastatin in every cycle of CTI of rosuvastatin. These dynamic changes were associated with the decline in levels of proviral DNA in CD4+ T cells during the 72-week follow-up. The fold decrease in proviral DNA levels had a significant linear correlation with nadir CD4+ T-cell counts and may have a one-phase exponential correlation with time (duration of rosuvastatin CTI). Therefore, the data suggest that it is possible to awaken the latent HIV-1 reservoir in CD4+ T cells, to reboot HIV-1-specific T-cell reactivity, to destroy the viral reservoir, and to achieve a decline in the size of the viral reservoir through CTI of rosuvastatin.

Our pilot study data showed that the activity of NF-κB signaling may have been suppressed during rosuvastatin treatment but significantly upregulated shortly after rosuvastatin withdrawal. The critical role of NF-κB signaling in the reversal of HIV-1 latency has also recently been demonstrated through the application of AZD5582, an activator of the NF-κB signaling pathway, in animal models.11 Previous trials have also shown that the transient reversal of viral latency may restart viral replication that can awake HIV-1-specific T-cell responses perhaps through re-expression of viral antigens on cell surface.3,27 That may explain our observation why the dynamic changes of HIV-1-specific CD4+ or CD8+ T-cell responses were consistent with those of the levels of transcription of CA HIV RNA in CD4+ T cells. Furthermore, the awakened HIV-specific immune response may destroy the viral reservoir and decrease the levels of proviral DNA, as shown in our data.

HIV-1-infected adults who start cART at lower baseline CD4 levels (ie, with lower nadir CD4+ T-cell counts) have been demonstrated to have relatively poor recovery of immune function.28–31 A large-scale randomized clinical trial has emphasized the importance of starting cART as early as possible.32 In our study, although the subject number was small, we found the fold changes in proviral DNA levels after 3 cycles of CTI of rosuvastatin were linearly correlated with the nadir CD4+ T-cell counts with a high statistical significance. Therefore, the immune potential to reboot HIV-1-specific T-cell responses to decrease the size of the viral reservoir through CTI of rosuvastatin may also be related to the nadir CD4+ T-cell counts.

The proviral DNA levels declined significantly but remained detectable in all subjects during the 72-week follow-up period. Finzi et al1 found a long half-life of the latent reservoir with stable cART. Through one-phase exponential correlation in extended observation study, we found that the predicted time for the reservoir size below the limit of detection would be approximately 4 years. However, the levels of proviral DNA decay below the limit of detection may not indicate the clearance of viral persistence, and the clinical significance remains to be determined. Longer duration of observation is needed.

Rosuvastatin interruption may result in an increased risk of cardiovascular events in patients with high cardiovascular risks.21,22 Thus, only patients with low cardiovascular risk could be enrolled in this proof-of-concept study. Cyclic statin interruption should not be allowed in patients with high cardiovascular risks or pre-existing myocardial infarction even intolerance to statin or any other reason.

This study has several limitations. First, this study is not a randomized trial and only very small number of subjects were enrolled in this single-arm proof-of-concept study. The subject number was too small to evaluate the impact of different cART regimens or duration of HIV-1 infection on the study results. Second, the CA HIV RNA and the proviral DNA levels in peripheral CD4+ T cells may not necessarily be the ideal surrogate markers to estimate the activity of latency reversal and the size of the viral reservoir respectively,27 and we had no data focusing on the tissue reservoir. Third, all of the enrolled subjects were Asian men with ages below 40 years old thus the results of this study should not be generalized to other population so far. Furthermore, this intervention should not be applied in HIV-infected persons who are not diagnosed with hyperlipidemia.

In conclusion, the data of this proof-of-concept study showed that it may be possible to reverse viral latency, awake HIV-1-specific CTL, reduce the size of the viral reservoir, and help achieve the aims of functional cure through CTI of rosuvastatin in HIV-1-infected Asian men with stable cART, especially in those with nadir CD4+ T-cell counts >350 cells/μL. As a next step, we will conduct a randomized trial to evaluate the size of the viral reservoir with more surrogate markers (such as outgrowth assay) in addition to proviral DNA and to determine the potential of the latency-reversing effect of CTI of rosuvastatin over a longer duration of follow-up with larger subject numbers.

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Keywords:

HIV-1; rosuvastatin; viral latency; proviral DNA; viral reservoir

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