A randomized pilot trial to evaluate the benefit of the concomitant use of atorvastatin and Raltegravir on immunological markers in protease-inhibitor-treated subjects living with HIV.

OBJECTIVE: Optimization of antiretroviral therapy and anti-inflammatory treatments, such as statins, are among the strategies aimed at reducing metabolic disorders, inflammation and immune activation in people living with HIV (PLWH). We evaluated the potential benefit of combining both strategies.
DESIGN: Forty-two PLWH aged ≥40 years receiving a protease inhibitor (PI)-based regimen were randomized (1:1) to switch from PI to Raltegravir (n = 20), or to remain on PI (n = 22). After 24 weeks, all patients received atorvastatin 20mg/day for 48 weeks.
METHODS: We analyzed plasma inflammatory as well as T-cell maturation, activation, exhaustion and senescence markers at baseline, 24 and 72 weeks.
RESULTS: Plasma inflammatory markers remained unchanged. Furthermore, no major changes on T-cell maturation subsets, immunoactivation, exhaustion or immunosenescence markers in both CD4 and CD8 T cell compartments were observed. Only a modest decrease in the frequency of CD38+ CD8 T cells and an increase in the frequency of CD28-CD57+ in both CD4 and CD8 T-cell compartments were noticed in the Raltegravir-switched group.
CONCLUSIONS: The study combined antiretroviral switch to Raltegravir and Statin-based anti-inflammatory strategies to reduce inflammation and chronic immune activation in PLWH. Although this combination was safe and well tolerated, it had minimal impact on inflammatory and immunological markers. CLINICAL TRIALS REGISTRATION: NCT02577042.

RCT Entities:   Population Interventions Outcomes

Introduction

The incidence of non-AIDS-defining and age -related comorbidities is increasing among people living with HIV (PLWH) and seems to be higher than in the uninfected control population [1-4]. Although the potential pathogenic mechanisms behind this observation are diverse, three main factors might explain the accentuated aging in this population [5]. Firstly, antiretroviral therapy has enlarged the life expectancy of PLWH, increasing in turn the number of elderly individuals in this population. Nowadays, approximately one-half of the PLWH in the United States are aged 50 or older [6]. Aging itself is a condition associated with a chronic inflammation and immune senescence (inflammaging), which are major contributors to increased prevalence of morbidities [7]. Secondly, natural inflammaging is exacerbated in treated PLWH by the persistent inflammatory status and activation of the immune system induced by HIV infection per se [8, 9], increasing the risk of age-related morbidities [10-13]. Finally, the continuous exposure to antiretrovirals and their toxicities, mainly but not exclusively associated to former antiretroviral drugs [14], may contribute to accelerate the emergence of some of age-related diseases such as dyslipidemia, cardiovascular events, renal damage or low bone mineral density [15].

Consequently, one of the current aims of the clinical management of PLWH is the control of chronic non-AIDS-related comorbidities in an increasingly older and complex population [5]. Among clinical challenges, the maintenance of viral suppression is a premise, as HIV undetectability is a key factor to reduce the systemic inflammation [16], and can be now safely achieved by using newest and less toxic antiretroviral drugs, particularly in the elderly population. However, virological suppression is not sufficient to eliminate chronic inflammation, as appropriately treated PLWH still show higher levels of inflammation and immune activation markers in comparison with general population [8]. Among these markers, IL-6 seems to be a major player of inflammaging [17] and a determinant factor of survival in PLWH [12].

Additional strategies have been evaluated to curtail the chronic inflammatory status. These strategies encompass therapy intensification to reduce residual viral replication [18], switch antiretrovirals to safer profiles to reduce related toxicities [19], and addition of immunomodulatory therapies to specifically reduce inflammation or immune activation, including anti-CMV drugs [20], immunosuppressors [21] or statins [22]. Despite these wide clinical efforts, no conclusive data have been generated, and only some switch strategies but not immune-focused approaches have impacted current clinical guidelines.

A beneficial effect has been demonstrated with raltegravir, an integrase inhibitor. It exhibits a rapid, potent and durable antiretroviral activity with a better impact on bone mineral density and lipid and renal profiles than from a protease inhibitors (PIs) [23-26]. In addition, integrase inhibitors might have a benefit on immune and inflammatory parameters in comparison with other antiretroviral classes [27-29]. Similarly, statins are lipid-lowering drugs that also exert anti-inflammatory effects, and have immune-modulatory properties. Recent studies in HIV-infected population have suggested that statins impact inflammatory markers [22, 30–35], and that its use is associated with a lower risk of non-AIDS defining morbidities and malignancies and mortality [36-39]. Nonetheless, data are limited and mainly emerged from retrospective or cohort studies. Currently, a prospective, randomized, placebo-controlled trial (REPRIEVE study) is ongoing to assess a statin strategy for the primary prevention of major cardiovascular events [40]. Although around 7,500 patients have already been included from over 120 clinical sites across 12 countries, final results will be available in several years.

Meanwhile, based on these data, we explored the potential benefits of combining both approaches in the Ralator study, which includes a double strategy to reduce systemic inflammation and improve lipid profile in chronic HIV population by i) switching the PI to Raltegravir; and ii) adding atorvastatin for one year, owed their potential anti-inflammatory effect.

Methods

Study design and population

Ralator is a 72-week, active-controlled, single site, randomized, open-label, pilot study in PLWH aged 40 years or over (registered under the code NCT02577042 at the Clinical trials website). Candidates to be included in the study were PLWH, aged ≥ 40 years, receiving a PI-based antiretroviral regimen combined with tenofovir/emtricitabine or abacavir/lamivudine for at least 6 months and maintaining undetectable plasma HIV-1 RNA (VL < 50 copies/mL) for at least 12 months. Exclusion criteria were history of virological failure to integrase inhibitors and suspected or documented resistance mutations to the integrase or NRTI, systemic concurrent processes (e.g. active coinfection with hepatitis C or B, acute systemic infection within the last 4 months, neoplasia or chronic inflammatory process), treatment with anti-inflammatory, anticoagulant or antiplatelet drugs (e.g. corticosteroids, aspirin or other) and therapy with statins and raltegravir within the last 6 months. The ethics committee of the Hospital Germans Trias i Pujol and the Spanish Medicines Agency (AEMPS) approved the protocol, and the study was performed in accordance with the principles of the Declaration of Helsinki. All participants provided written informed consent before inclusion.

At randomization, patients were stratified according to: 1) the baseline levels of LDL-cholesterol, cutoff value 160 mg/dL since a LDL-cholesterol 130–159 mg/dL is considered borderline high for people without cardiovascular risk factors and 2) the nucleoside drugs used, tenofovir/emtricitabine or abacavir/lamivudine. Patients were randomly assigned in a 1:1 ratio to: 1) continue with the same PI-based regimen, including tenofovir/emtricitabine or abacavir/lamivudine, for 24 weeks (Control group); or 2) switch the PI to Raltegravir (400mg/12 hours), plus tenofovir/emtricitabine or abacavir/lamivudine, for 24 weeks (Raltegravir group). The assignation was centralized and performed by telephone. After 24 weeks, atorvastatin (20mg/day), was added to both Control and Raltegravir groups for 48 weeks (Fig 1). All data was collected at the Hospital Germans Trias i Pujol.

Fig 1

Trial design.

A. Consolidated Standards of Reporting Trials (CONSORT) flow diagram for the trial showing Participant flow in Control and Raltegravir groups. B. Schematic representation of intervention, patients were randomized to the Control and Raltegravir groups and, after 24 weeks, atorvastatin was added to all participants. Follow up visits are indicated for each group.

Study objective and endpoints

The primary objective of the study was to compare changes in plasma levels of IL-6 between Control and Raltegravir groups. To assess the effect of switching to raltegravir, differences between groups at week 24 and intragroup longitudinal changes at week 24 from baseline were assessed. In addition, we compared both groups at week 72 to assess the effect of adding atorvastatin. Furthermore, we analyzed intragroup longitudinal changes at week 72 from baseline and from week 24.

Secondary objectives were to compare changes in the above-mentioned time periods in: i) other plasma inflammatory/coagulation markers: soluble CD14 (sCD14), C-reactive protein (CRP) and D-Dimer; ii) CD4 and CD8 T-cell maturation/activation/exhaustion and immunosenescence markers; and iii) plasma lipid and lipoprotein parameters (total, HDL-, LDL-cholesterol and triglyceride levels). In addition, participants were followed-up for virological and immunological response by assessing plasma viral load and circulating CD4 and CD8 T cell counts at the indicated timepoints (Fig 1).

Demographic and HIV infection-related data were collected in order to characterize the study population (gender, age, time since HIV diagnosis, time since ART treatment, route of transmission of HIV).

Plasma inflammation markers

Plasma levels of several markers were measured using ELISA kits following the manufacturer’s instructions: C-reactive protein (CRP) and D-dimer from Ray Biotech; soluble CD14 (sCD14) from Diaclone and IL-6 from ThermoFisher.

Immunophenotyping and activation markers in peripheral CD4+ and CD8+ T cells

Cryopreserved PBMC (10x106 cells) were thawed, sequentially washed in RPMI medium (Invitrogen) containing 60%, 20% and 10% of Fetal Bovine Serum (FBS, Invitrogen) and incubated for 15 minutes at 37°C in RPMI medium supplemented with 10% FBS. PBMC were incubated with Fixable Viability Stain 780 (BD Bioscience, final dilution 1:4000) as described [19]. Then, cells were stained with the following antibodies: CD3 BV605 (clone SK7), CD4 FITC (clone RPA-T4), CD8 BV510 (clone SK1), CD45RA Alexa Fluor 700 (clone HI100), CD197 PE-CF594 (clone 150503), CD57 APC (clone NK-1), HLA-DR BV650 (clone G46-6), CD279(PD-1) BV421 (clone EH12.1), CD38 PE (clone HIT2), CD28 PerCP Cy-5.5 (clone L293) and CD27 BV786 (clone L128) all from BD Bioscience (San Jose, CA). After 15 minutes incubation in the dark at room temperature, cells were washed twice in PBS containing 1% FCS and fixed in formaldehyde 1% in PBS. All samples were acquired in a BD LSRFortessa flow cytometer (BD Bioscience) and further analyzed with FlowJo software (Tree Star Software). Briefly, a time gate was defined to ensure homogenous cell acquisition, then singlets and lymphocyte gates were defined by morphological parameters. Viable CD3+ cells were gated for CD4 and CD8 compartment analyses gated as CD4+CD8- or CD8+CD4-, while double-positive cells and double-negative cells were excluded from the analysis. Further automated detection of marker cutoffs based in fluorescence controls was performed using OurFlow, a custom pipeline running concatenated R packages [19]. Gating strategy is described in S1 Fig. T-cell maturation was analyzed using CD45RA and CD197 (CCR7) expression to define naïve (TN, CD45RA+CCR7–), central memory (TCM, CD45RA–CCR7+), Effector (TEF, CD45RA–CCR7–) and effector memory RA+ cells (TEMRA, CD45RA+CCR7–); CD27 was used to differentiate TEF cells in transitional memory (TTM, CD27+CD4+ or CD27–CD8+) and effector memory (TEM, CD27–CD4+ or CD27+CD8+). CD4 and CD8 T cells were also analyzed for the expression of HLA-DR and CD38 to define activated cells (% of HLA-DR+CD38+cells), the expression of PD-1 to measure exhaustion (% of PD-1+ cells) and the expression of CD57 and CD28 to measure immunosenescence (% of CD28-CD57+ cells).

Statistical analyses

Sample size determination was guided by the number of candidates who could be enrolled in the study according to our patient’s data base attended in our HIV Unit, whereby 60 patients were planned to be included into the study. Since this is a pilot study, power analysis was not conducted in order to calculate sample size.

A general descriptive analysis of all the study variables was done by using mean and standard deviation for normally distributed variables or median and interquartile range for the non-parametric variables; relative frequencies was used for categorical variables.

Differences between groups and longitudinal changes where represented and evaluated using parametric (t-test and paired t-test) and non-parametric tests (Mann-Whitney and Wilcoxon signed-rank tests), according to the variable’s distribution, and linear mixed-effects models that were fitted for the most relevant markers. For categorical variables, independence between groups was studied using the Chi-Square or Fisher exact test and time effect was assessed by means of a McNemar tests. The Spearman correlation test was used to evaluate the association between quantitative variables. Longitudinal comparisons were performed by linear mixed effects models.

Results

Patient characteristics and clinical follow-up

The recruitment period of the study was 32 weeks. The start date of recruitment was February 2015. The follow-up period started on November 2015 and the end of study date was June 2018. A total of 42 participants were included; of these, 20 were randomized to switch to Raltegravir (Raltegravir group) and 22 to continue the same PI/r-based cART regimen (Control group) (Fig 1). The main characteristics at baseline of both groups are summarized in Table 1. No significant differences among groups were found. The overall mean (standard deviation, SD) age of participants was 50.6 (6.2) years and the mean time with HIV infection was 18.7 (7.2) years. The overall mean (SD) CD4 T cell count was 610 (223) cells/μL and the mean nadir CD4 count was 202 (128) cells/μL (Table 1).

Table 1

Baseline characteristics of participants.

	Control group *(n = 22)	Raltegravir group *(n = 20)
Age (Years)	51.8 (8.2)	49.5 (3.5)
Gender (% Male)	77%	85%
Route of HIV infection: (%)
MSM	50%	35%
HSM	18%	40%
Injecting drug use	23%	15%
Other	9%	10%
Time from HIV diagnosis (Years)	19.2 (7.9)	18.2 (6.6)
Current protease inhibitor: (%)
Atazanavir or atazanavir/ritonavir	27%	20%
Lopinavir/ritonavir	18%	15%
Darunavir/ritonavir oe cobicistat	55%	60%
Other	0%	5%
Hepatitis C (%)	18%	10%
CD4 count
Current (cells/μL)	640 (253)	579 (184)
Current (% of lymphocytes)	29 (8)	29 (8)
Nadir CD4 count (cells/μL)	191 (143)	211 (116)
CD8 count
Current (cells/μL)	1003 (409)	898 (349)
Current (% of lymphocytes)	46 (10)	43 (9)
LDL-cholesterol > 160 mg/dL (%)	9%	5%
Lipid profile (mg/dL)
Total cholesterol	181 (31)	189 (35)
LDL cholesterol	104 (27)	108 (31)
Triglycerides	177 (133)	159 (106)

*All parameters are expressed as mean and standard deviation (SD) except for discrete variables that are expressed as percentage (%). No significant differences were found among groups.

LDL, Low-density lipoprotein.

Eight participants discontinued the study protocol early, 5 were from the Raltegravir group: study interruption for personal reasons (week 12), Hepatitis C treatment (week 12), virologic failure secondary to a poor adherence (week 24), interruption of treatment due to patient’s decision (week 36) and interruption of atorvastatin due to a grade 4 elevation of liver enzymes and gastrointestinal intolerance (week 48); and 3 from the Control group: interruption of treatment due to PI-related gastrointestinal discomfort (week 12), study interruption for personal reasons (week 24) and interruption of treatment due to patient’s decision (week 60). The remaining randomized individuals completed the study protocol (Fig 1).

Three participants ─ all in Raltegravir arm ─ showed a grade 3–4 adverse events possibly related to the study medication: 2 patients showed a grade 3 and grade 4 increases of creatine kinase, which improved without interrupting therapy, and, as mentioned above, one participant showed a grade 4 increase of liver enzymes that resolved after interrupting the atorvastatin.

Changes in soluble plasma markers of inflammation

Four different inflammation plasma markers (IL-6, sCD14, CRP and D-dimer) were measured at baseline, week 24 and week 72 using commercial ELISA kits. Results, summarized in Table 2, clearly showed that either PI switch to Raltegravir or atorvastatin treatment failed to modify IL-6 plasma levels (primary study objective). Indeed, no differences between groups were observed at week 24 (to evaluate the impact of therapy switch) or at week 72 (to evaluate the impact of statin treatment). Furthermore, no longitudinal intragroup differences in IL-6 plasma concentrations were noticed in the Control or Raltegravir groups (Table 2). Similarly, the analysis of D-dimer, sCD14 and CRP plasma levels, showed minimal and no differences between groups at 24 and 72 weeks and a lack of longitudinal effect intragroup (Table 2). Thus, therapy switch, atorvastatin treatment or the combination of both strategies failed to modify plasma levels of IL-6 and other soluble markers of inflammation and coagulation in our study.

Table 2

Changes in plasma soluble markers.

		Control group		Raltegravir group
	Baseline	W24	W72	Baseline	W24	W72
	n = 22	n = 21	n = 19	n = 20	n = 18	n = 15
IL-6 (pg/mL)	40.0	41.1	41.7	42.5	39.3	43.2
Median (IQR)	(36.9–53.7)	(36.1–53.2)	(34.7–51.5)	(34.5–50.1)	(34.6–49.8)	(36.8–52.9)
D-Dimer (ng/mL)	1990	1917	1868	1743	1844	2051
Median (IQR)	(1574–2523)	(1552–2244)	(1438–2175)	(1459–1894)	(1547–2340)	(1656–2414)
sCD14 (ng/mL)	7588	7736	8342	7579	7368	7658
Median (IQR)	(7121–8419)	(7307–8465)	(7333–8 856)	(7039–8726)	(7131–9056)	(7039–8355)
CRP (ng/mL)	8587	7531	10268	6744	7059	8334
Median (IQR)	(6700–16735)	(5056–15069)	(5695–21228)	(4461–15211)	(4729–10868)	(5134–17392)

Changes in T-cell maturation, activation, exhaustion and immunosenescence in PBMCs

No changes in absolute counts or percentages of CD4 and CD8 T cells, either intragroup or intergroup were observed at any time point. Immunophenotyping of T cells was performed at baseline, week 24 and week 72. First, we explored changes in T cell maturation, by defining five T-cell maturation subsets: naïve (TN), central memory (TCM), transitional memory (TTM), effector memory (TEM) and effector memory RA+ (TEMRA) based on the combination of CD197, CD45RA and CD27 markers (see Methods). In CD4+ T cells, we observed that the Control group tended to show higher levels of TN cells and lower levels of TTM cells at all timepoints, including baseline, in comparison with Raltegravir group; (Fig 2A). Longitudinally, minimal changes were observed, although a decrease in TCM at week 24 and 72 from baseline in Control group was noticed (Fig 2A).

Fig 2

of maturation subsets in CD4 and CD8 T cells.

Analysis Figure shows median values for the frequency of the maturation subsets (Naïve, Central Memory, Effector Memory, Transitional Memory and Effector Memory RA+) in CD4 (panel A) or CD8 T cells (Panel B) in Control (greyscale) and Raltegravir (blue) groups at three different time points: week 0 (start of the study), week 24 (treatment follow-up), and week 72 (48 weeks after atorvastatin treatment). Maturation stages were defined based on the combination of CD45RA, CD197 (CCR7) and CD27 as described in Methods. Asterisks denote p<0.05 either intergroups or longitudinally intragroup as indicated.

The analysis of maturation subsets in CD8 T cells also showed minimal differences between groups at baseline, although a lower frequency of TTM cells was noticed in the control group. Longitudinal analysis showed a decrease in TCM frequency between baseline and week 24 in the Control group. In addition, the Raltegravir group showed a transient decrease in the frequency of TEMRA CD8 T cells at week 24, that vanished at week 72 in (Fig 2B).

Next, we analyzed changes in immune activation (defined by CD38 expression or CD38 and HLA-DR co-expression), immune exhaustion (defined by PD-1 expression) and immunosenescence (defined by the frequency of CD28-CD57+ cells). In CD4 T cells, consistent with the lower frequency of TN cells, CD38 expression (which is constitutive in TN cells) tended to be lower in the Raltegravir group. Longitudinally, no changes were observed in activation (CD38+HLA-DR+) and exhaustion (PD-1+) markers in any group, although an unexpected increase in immunosenescence (CD28-CD57+) was specifically observed in the Raltegravir group at week 72 (Fig 3).

Fig 3

Evaluation of the changes in activation, CD38+ expression, immunosenescence and exhaustion levels in CD4 and CD8 T cells.

Measure of changes in mean activation levels (defined by HLA-DR+CD38+ coexpression, panels A and E) CD38+ cells (panels B and F) senescence (CD57+CD28–, panels C and G) and exhaustion (PD-1+, panels D and H) levels in CD4+ T cells (panels A-D) and CD8 T cells (panels E-H) comparing both study groups. These differences were evaluated between w0–w24, w0–w72 and w24–w72. Control group is represented in grey and the Raltegravir group in blue. Asterisks denote p<0.05.

Similarly, minimal changes were noticed in activated (CD38+HLA-DR+) and exhausted (PD-1+) CD8 cells, which did not show any difference inter or intragroup throughout the study. However, CD38 expression decreased between baseline and week 72 in Raltegravir group (p = 0.06, Fig 3), In contrast, we found an increase in the frequency of immunosenescent (CD28-CD57+) CD8 T cells between week 24 and week 72 in the Raltegravir group that was not observed in the Control group (Fig 3).

To ascertain the impact of Raltegravir switch (change at week 24 from baseline) and atorvastatin treatment (change at week 72 from week 24) on the different parameters we have properly transformed data to normalize and analyze them using linear mixed-effects models. These models were fitted in different ways, parameterizing the time variable as continuous and as categorical time points and including or excluding interaction effects. The initial analysis failed to confirm a significant change overtime in the frequency CD28-CD57+ CD4 or CD8 T cells, but confirmed the decay of CD38 expression in CD8 T cells.

Changes in lipid profile

A decrease in triglyceride levels was seen at week 24 from baseline in the Raltegravir group. At week 72 from baseline (effect of switch antiretroviral and adding atorvastatin), a decrease was seen in both groups in all lipid parameters, as well as at week 72 from week 24 (effect of atorvastatin) (S1 Table). No differences were seen between groups at week 72 in any lipid parameter.

Discussion

We have evaluated the anti-inflammatory effect of atorvastatin in a prospective clinical trial measuring changes in inflammatory, immune activation and immunosenescence markers after adding atorvastatin, at intermediate dose (20 mg per day), to a Raltegravir- or a PI-based regimen. The selection of atorvastatin was guided by the low risk of drug-drug interactions between this statin and boosted-PI, as well as its low cost. The intermediate dose (20 mg per day) was selected to avoid toxicity.

Four soluble plasma markers that have been shown to predict mortality in treated HIV infection in different clinical settings [41-43] were selected to determine changes on overall inflammation: IL-6, CRP, D-dimer and sCD14. These markers cover pyrogenic inflammation, acute-phase proteins, coagulation and monocyte activation, respectively. Moreover, those markers have been shown to be impacted by at least one of these strategies (statin addition or Raltegravir intensification or switch): Raltegravir has been reported to reduce D-dimer plasma levels [44], specifically in patients on a PI-based regimen [8], while statins (pravastatin or pitavastatin) reduced sCD14 levels [24] or CRP levels [31] in antiretroviral-treated individuals. Despite those reported effects, all selected biomarkers remained unchanged through our study in both arms.

To fully analyze the immunological effects of antiretroviral switching and atorvastatin treatment, we have also evaluated the impact on immunological markers of immune activation, exhaustion and immunosenescence. Consistent with the minimal impact on inflammation, most cellular immunological markers remained unchanged throughout the study. Actually, only minimal changes in the frequency of maturation CD4 and CD8 T cell subsets were noticed, that were consistent with previous reported effects of integrase inhibitors switch [19] but unrelated to the reported effects of Raltegravir intensification [27, 28]. In contrast, among our patients, the PI switch to Raltegravir and the addition of atorvastatin had minimal effect on reducing CD38 expression on CD8 T cells and on increasing the frequency of immunosenescent CD4 and CD8 T cells. Regarding CD38 expression on CD8 T cells, this effect seems to be consistent with previously reported beneficial effects of Raltegravir intensification [27, 45]. However, this effect has not been observed in PI to integrase inhibitor switch strategies [19], suggesting a direct effect of atorvastatin, that was not fully supported by statistical modelling of changes in CD38 expression. Statins have shown the ability to reduce CD8 activation in PLWH either untreated [22] or under antiretroviral drugs [33]. However, this effect is still controversial, as different studies failed to observe relevant effects of statins in immune activation [46]. Our data are on agreement with these latter reports, although the dose and the specific statin used in the different studies are confounding factors that may impact on the final effect, thus contributing to the conflicting data reported.

Regarding immunosenescence measured by the frequency of CD28-CD57+ cells, we observed an unexpected increase at week 72 compared to week 24 in the CD8 T cell compartment and a similar increase at week 72 compared to baseline in CD4 T cells, both in the Raltegravir group, suggesting an effect of the concomitant use of raltegravir and atorvastatin. Although the changes were low in magnitude, they were consistent in both T cell subsets. This marker is increased in HIV individuals and it is associated with several comorbidities [47]. However, its modulation by antiretroviral therapy has not been fully characterized, and no specific effects of Raltegravir have been identified. Indeed, impact of HIV infection on this marker is qualitatively different from other chronic infection such as cytomegalovirus [48].

Our study has some limitations, including the low sample size and the loss of follow-up of a considerable number of patients. Some study criteria (aged ≥ 40 years, receiving a PI) could explain the high difficulty of achieving and maintaining patients in the study since the high genetic barrier makes this antiretroviral class the best option at that time for subjects with not optimal treatment adherence. As well, the low statin dose used to avoid toxicity may also contribute to the lack of impact of the intervention. In addition, the lack of a study arm without statin treatment impedes the direct identification of separate effects of Raltegravir switch and statin treatment. We have overcome this limitation by using longitudinal linear models to evaluate the ultimate responsible for the changes observed, suggesting a direct role of atorvastatin. However, those changes are small and their clinical impact on the long-term uncertain.

Therefore, considering our data, switching from a PI to Raltegravir together with low dose atorvastatin did not provide significant immunological benefits in terms of a reduction in inflammatory or immune activation markers. However, the design of the REPRIEVE study including a large number of patients with a long follow-up will help us to elucidate this issue.

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13 Mar 2020

PONE-D-20-01200

Limited Benefit of the Concomitant Use of Atorvastatin and Raltegravir on Immunological Markers in Treated Subjects living with HIV.

PLOS ONE

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: Negredo et al present a pilot trial to assess the concomitant use of atorvastatin and raltegravir on immunological markers in treated subjects living with HIV switching from PI.

The idea to combine two potentially effective strategies options is original and merit consideration for publication, regardless negative results in reducing inflammation and immune activation.

Some issues should be considered to improve understanding of methods and overall discussion.

1. Title should mention that PLWH were PI-experienced.

2. Inclusion criteria should specify if all the patients were Raltegravir and statine naïve.

3. Table 1 should mention the cardiovascular risk profile of the patients and the proportion of the individuals offered statine regardless of dyslipidemia or low CVR.

4. It should be mention why a LDL cut off of 160 mg was chosen for stratification and be consistent through the paper when using mg of mmol units .

5. Line 284-294 is in fact part of the background and can be summarized in this section.

6. Background should mention the relevance of using IL6 as the primary end point of the study

7. It is not clear why mentioning data regarding BMD

8. Limitation should mention not using hsCRP and the possible low dose of statine which has been used (it was mentioned the fear of statines’ toxicities)

9. Line 256 which discuss the choice of using a mix linear iodel should be anticipated in statistical methods

10. Figured have low resolution and are difficult to be read.

All together the paper is well written and contributes to the research in the field of inflammaging in HIV

Reviewer #2: The authors evaluated the impact of switching from a Protease Inhibitor based regimen to raltegravir and addition of atorvastatin on metabolic disorders, inflammation and immune activation in people living with HIV.

There was no power calculation done as this was a pilot study. That in itself is not a problem as long as the authors are aware that they can only describe their results without attempting formal comparisons between groups. However the authors have tried to demonstrate differences between the two arms despite the study not being powered to do so.

Table 1: Change ‘drug users’ to either drug use or injecting drug use

Line 199: sentence relating to CD4 count should be revised for clarity

Line 201 Authors stated: Eight participants early discontinued the study protocol

This should be revised to ‘eight participants discontinued the study protocol early

Line 203: bad adherence should be changed to poor adherence

Line 224 & Table 2, line 527 authors should avoid comments like ‘no SIGNIFICANT differences were observed between groups or time points’. The study was not powered to detect differences between groups. The presentation of the results should be purely descriptive without need of any formal statistical tests to assess differences between groups

Lines 231/232: No significant changes in absolute counts or percentages of CD4 and CD8 T cells, either intragroup or intergroup were observed at any time point. The authors should remove the word significant.

I would not read too much meaning into the significant difference reported in lines 239, 254 and 259.

Line 266: The sample size is too small to examine for any meaningful interaction. OK, the authors pointed this out, so no need to even report this in the paper.

Lines 275 to 277: This should be reported as differences in lipid parameters rather than whether significant or not.

Sample is too small to detect any significant change in BMD.

The discussion of the findings should be presented bearing in mind that lack of a difference does not mean a difference does not truly exist but the study was not powered to detect any difference. Any significant differences observed with some measured parameters could have just been due to chance. I suggest the discussion is moderated accordingly.

**********

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Reviewer #1: Yes: Guaraldi Giovanni

Reviewer #2: No

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27 Jul 2020

Reviewer #1:

Negredo et al present a pilot trial to assess the concomitant use of atorvastatin and raltegravir on immunological markers in treated subjects living with HIV switching from PI.

The idea to combine two potentially effective strategies options is original and merit consideration for publication, regardless negative results in reducing inflammation and immune activation.

We thank the reviewer for his/her comments.

Some issues should be considered to improve understanding of methods and overall discussion.

1. Title should mention that PLWH were PI-experienced.

Authors have included this information in the title.

2. Inclusion criteria should specify if all the patients were Raltegravir and statine naïve.

The following exclusion criterium was already included in Methods: “.... and therapy with statins within the last 6 months”. Following the reviewer’s comment, authors have added raltegravir as a exclusion criteria: “.... and therapy with statins and raltegravir within the last 6 months” (Methods section).

3. Table 1 should mention the cardiovascular risk profile of the patients and the proportion of the individuals offered statine regardless of dyslipidemia or low CVR.

Table 1 already includes information about lipid profile. In addition, the proportion of individuals with baseline LDL-cholesterol >160mg/dL, according to the study group, has been included in Table 1, following the reviewer suggestion. Only 2 (9%) patients from the Control group and 1 (5%) patient from the Raltegravir group presented LDL>160 at baseline, that was always <170 during the follow up. Unfortunately, no data about proportion of patients with hypertension are available.

4. It should be mention why a LDL cut off of 160 mg was chosen for stratification and be consistent through the paper when using mg of mmol units.

LDL cut off of 160 mg was chosen for stratification since LDL cholesterol 130-159 mg/dL is considered borderline high and >160 mg/dL is considered high for people without cardiovascular risk factors. This information has been added to the Methods section.

All units have been changed to mg/dL in Table 1.

5. Line 284-294 is in fact part of the background and can be summarized in this section.

This entire paragraph has been moved at the end of the Introduction to justify the purpose of the current study.

6. Background should mention the relevance of using IL6 as the primary end point of the study

A brief sentence highlighting the key role of IL-6 has been included in the Introduction section with a reference to justify it.

7. It is not clear why mentioning data regarding BMD.

Following the suggestion of both reviewers, sentences describing changes in BMD have been removed from methods and result sections since no additional data were provided.

8. Limitation should mention not using hsCRP and the possible low dose of statine which has been used (it was mentioned the fear of statines’ toxicities).

We have reinforced this limitation by including the reason why we used low doses of atorvastatin (to avoid toxicities) in the paragraph describing the limitations of the study.

9. Line 256 which discuss the choice of using a mix linear iodel should be anticipated in statistical methods

A brief description of methods has been included in the Methods section, as follow: “Longitudinal comparisons were performed by linear mixed effects models.”

10. Figured have low resolution and are difficult to be read.

The resolution of figures has been improved.

All together the paper is well written and contributes to the research in the field of inflammaging in HIV

Reviewer #2:

The authors evaluated the impact of switching from a Protease Inhibitor based regimen to raltegravir and addition of atorvastatin on metabolic disorders, inflammation and immune activation in people living with HIV.

There was no power calculation done as this was a pilot study. That in itself is not a problem as long as the authors are aware that they can only describe their results without attempting formal comparisons between groups. However the authors have tried to demonstrate differences between the two arms despite the study not being powered to do so.

Table 1: Change ‘drug users’ to either drug use or injecting drug use.

This term has been changed. Thanks.

Line 199: sentence relating to CD4 count should be revised for clarity.

In text, the overall mean CD4 T cells count and nadir values of all participants has been described while in Table 1, we described the mean values in each study group. Sentence has been modified for clarity.

Line 201 Authors stated: Eight participants early discontinued the study protocol. This should be revised to ‘eight participants discontinued the study protocol early.

Authors have changed the sentences following the reviewer’s comment.

Line 203: bad adherence should be changed to poor adherence.

Authors have changed the sentences following the reviewer’s comment.

Line 224 & Table 2, line 527 authors should avoid comments like ‘no SIGNIFICANT differences were observed between groups or time points’. The study was not powered to detect differences between groups. The presentation of the results should be purely descriptive without need of any formal statistical tests to assess differences between groups Lines 231/232: No significant changes in absolute counts or percentages of CD4 and CD8 T cells, either intragroup or intergroup were observed at any time point. The authors should remove the word significant. I would not read too much meaning into the significant difference reported in lines 239, 254 and 259.

Following the reviewer comment, the word significant has been removed when describing difference between groups throughout the manuscript.

Line 266: The sample size is too small to examine for any meaningful interaction. OK, the authors pointed this out, so no need to even report this in the paper.

The sentence describing the analysis of interaction has been removed.

Lines 275 to 277: This should be reported as differences in lipid parameters rather than whether significant or not.

Following the reviewer comment, the word significant has been removed when describing difference between groups throughout the manuscript.

Sample is too small to detect any significant change in BMD.

Following the suggestion of both reviewers, the sentences describing changes in BMD have been removed from methods (Objectives) and result sections.

The discussion of the findings should be presented bearing in mind that lack of a difference does not mean a difference does not truly exist but the study was not powered to detect any difference. Any significant differences observed with some measured parameters could have just been due to chance. I suggest the discussion is moderated accordingly.

Discussion has been modified by removing any reference to significant changes between groups, and highlighting the minimal longitudinal changes observed (as mention in the conclusions).

Submitted filename: Reviewers RALATOR.docx

Click here for additional data file.

20 Aug 2020

A Randomized Pilot Trial to Evaluate the Benefit of the Concomitant Use of Atorvastatin and Raltegravir on Immunological Markers in Protease-Inhibitor-Treated Subjects living with HIV

PONE-D-20-01200R1

Dear Dr. Negredo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Alan Winston

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

4 Sep 2020

PONE-D-20-01200R1

A Randomized Pilot Trial to Evaluate the Benefit of the Concomitant Use of Atorvastatin and Raltegravir on Immunological Markers in Protease-Inhibitor-Treated Subjects living with HIV.

Dear Dr. Negredo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Alan Winston

Academic Editor

PLOS ONE