Immunity, inflammation and reservoir in patients at an early stage of HIV infection on intermittent ART (ANRS 141 TIPI Trial)

Abstract

Introduction

To alleviate the constraints and reduce the long-term toxicity and cost of continuous ART, intermittent ART (I-ART) has been proposed as an alternative strategy provided it can maintain immunological status, but most trials have shown more morbidity/mortality with this approach than with continuous ART.

These conclusions could be tempered when reconsidering the population and the strategy studied. Several studies focusing on patients starting treatment with high CD4 cell counts showed no significant difference in clinical outcomes between continuous ART and I-ART.^1,2 In the SMART trial, patients with a high nadir or current CD4 cell count on I-ART had a 2.5-fold higher risk of poor clinical outcomes than patients on continuous ART, but they took ART for <20% of their follow-up period.^3–5 In addition, no clinical events occurred within 6 months after ART interruption. Furthermore, the relationship between clinical outcome during ART interruptions and immune activation, inflammation and coagulation activity has not been specifically assessed in patients with high CD4 cell count.

We postulated that I-ART ensuring sufficient exposure to antiretroviral drugs with a high genetic barrier may maintain CD4 cell counts in patients with early-stage HIV infection.

Methods

Study design and population

The ANRS 141 TIPI Trial was a 2 year, Phase II, non-comparative multicentre trial. HIV-1-infected patients were eligible if they met the following criteria: ART-naive, current CD4 cell count ≥500/mm³ and ≥15%, HIV susceptibility to the study drugs at screening, no active opportunistic infection, negative hepatitis B surface antigenaemia and absence of chronic hepatitis C requiring therapy. They were ineligible if they were HIV-2-infected, had a history of cardiovascular events or splenectomy, had any previous CD4 cell count <400/mm³, were pregnant or breast-feeding or desired to become pregnant within 2 years, or had recent HIV primary infection. Other non-inclusion criteria were haemoglobin concentration <8 g/dL, neutrophil count <750/mm³, platelet count <100 000/mm³, creatinine clearance <50 mL/min and AST or ALT or total bilirubin >3× the upper limit of the normal range.

All patients gave their written informed consent, and the protocol was approved by an ethics committee (Comité de Protection des Personnes Est-I, reference number 2008/32) and the competent health authority (Agence Française de Sécurité Sanitaire des Produits de Santé). The trial was conducted in accordance with the Declaration of Helsinki, and is registered with ClinicalTrials.gov, number NCT 00820118.

Study treatment and procedures

The treatment scheme consisted of 6 month periods on a once-daily boosted-PI-based regimen, alternating with 6 month periods without ART. The recommended regimens were atazanavir boosted with ritonavir (300/100 mg), plus a fixed combination of either abacavir and lamivudine (600 + 300 mg) or emtricitabine and tenofovir (200 + 245 mg). Lopinavir/ritonavir, saquinavir/ritonavir, fosamprenavir/ritonavir and zidovudine + lamivudine were also allowed with a lower level of preference.

Clinical examinations and laboratory analyses were performed at the screening visit, the inclusion visit at month 0 and the visits at week 2 and months 1, 3, 6, 9, 12, 13, 15, 18, 21 and 24. CD4 cell counts, HIV-1 RNA assay and genotypic resistance tests were performed in local laboratories. HIV-1 DNA load was determined in a centralized laboratory with a test previously described (Biocentric, Bandol, France).⁶ Markers of inflammation were assayed every 6 months in a centralized laboratory with enzyme immunoassay kits from R&D Systems (Minneapolis, MN, USA) for high sensitivity C-reactive protein (hsCRP), IL-6 and vascular cell adhesion molecule 1 (VCAM-1) and from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA) for serum amyloid A (SAA).

Clinical and laboratory abnormalities were graded according to the French National Institute for Health and Medical Research (INSERM-ANRS) scale for grading severity of adverse events in adults. Grade 3 or 4 clinical adverse events that were drug related and non-AIDS serious clinical events were reviewed by an events validation committee.

Outcome measures

The primary outcome was the rate of immunological success, defined as an average CD4 cell count at month 21 and month 24 no lower than the average count at screening and inclusion, and no values below 400/mm³.

Secondary outcomes were the proportion of patients experiencing AIDS and non-AIDS serious adverse events, the proportion of clinical and biological adverse events, and the proportion of patients who complied with the trial strategy (no ART regimens other than those allowed, no prolongation of ‘on’ periods by more than 30 days, and no ART interruption for more than 30 days during ‘on’ periods).

Changes in HIV-1 RNA, HIV-1 DNA and CD4 cell values were noted during the ‘on’ and ‘off’ periods. HIV-1 genotypic resistance testing was performed systematically at screening, month 9 and month 24. HIV-1 genotypic resistance testing was also performed at mid-period and at the end of each 6 month ‘on’ period if HIV-1 RNA load was ≥50 copies/mL.

Changes in inflammation and endothelial activation were monitored by measuring six biomarkers: hsCRP, IL-6, SAA, VCAM-1, D-dimers and fibrinogen. Some of these markers were also analysed as categorical variables, using reported threshold values associated with the lowest and highest clinical risks: 1 and 5 mg/L for hsCRP (risk of opportunistic disease, death);^7–9 1.6 and 3.8 pg/mL for IL-6 (AIDS events, death);^7,9–12 3.19 and 4.06 mg/L for fibrinogen (death);⁸ and 0.18 and 0.34 mg/L for D-dimers (cardiovascular events, serious non-AIDS events, death).^12–14

Adherence was assessed with a patient-administered questionnaire at month 1, at month 13 and at the end of each ‘on’ period. Self-reported symptoms were assessed every 6 months by using a validated self-reported symptoms questionnaire,¹⁵ and changes during the phases ‘on’ and ‘off’ ART were described.

An immunological substudy was conducted in a subset of 13 patients, focusing on T cell and B cell phenotypic distribution, and their activation statuses based on the expression of CCR5 for CD4 cells, and CCR5, HLA-DR and CD38 for CD8 cells. T cell function was assessed in terms of proliferation tests to recall antigens (HIV p24, cytomegalovirus, candidine and purified protein derivative) and IFN-γ secretion in response to HIV or Epstein-Barr virus (EBV) stimulation.

Statistical analysis

We estimated that virological suppression would be achieved after 6 months on treatment in 75% of the patients, and that 10% of the patients would stop the strategy. A success rate of 70% at month 24 was considered acceptable and a rate of 50% unacceptable. To be able to conclude that the response rate at month 24 was above 50%, with a 5% α risk, 80% statistical power and a maximal 5% rate of loss to follow-up, the trial strategy could fail in no more than 14 of 39 patients (Fleming method for an exact test). The primary efficacy analysis was based on a modified intent-to-treat approach, which included all enrolled patients who received at least one dose of ART. A ‘missing equals failure’ strategy was applied to missing CD4 cell counts at month 21 and month 24. Additionally the primary outcome was assessed in patients who fully respected the trial strategy.

Secondary analyses included changes in the CD4 cell count, HIV-1 RNA and HIV-1 DNA loads, genotypic resistance mutations, clinical and biological adverse events, proportions of patients having experienced AIDS- and non-AIDS-related serious adverse events at month 12 and month 24, changes in markers of inflammation and endothelial activation and immunological markers, and treatment adherence.

Differences were assessed using Fisher's exact test for categorical variables and Student's t-test or the Wilcoxon test, as appropriate, for continuous variables. Correlations between qualitative or quantitative variables were tested using the Pearson or Spearman correlation coefficients. All statistical analyses were carried out with SAS version 9.1.3.

Results

Main characteristics

Sixty-seven patients were screened from May 2009 through June 2010, and 45 were included in the study (Figure 1). One patient did not start ART as planned and was excluded from the statistical analysis. Baseline characteristics of the 44 included patients are shown in Table 1. Initial ART consisted of atazanavir/ritonavir plus emtricitabine and tenofovir in 34 patients (77%), atazanavir/ritonavir plus abacavir and lamivudine in 9 patients (21%) and lopinavir/ritonavir plus emtricitabine and tenofovir in 1 patient (2%).

Figure 1.

Flow chart.

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Thirty-one patients (71%) fully adhered to the trial strategy. Of the 13 who did not, two patients received a prohibited antiretroviral drug (darunavir) and five patients stopped ART, three for personal reasons and two for adverse effects (grade 3 toxidermia; and grade 3 asthenia-insomnia and digestive disorders). Six patients did not stop ART after 6 months as planned, because of personal reasons in two cases, clinical events in three cases (acute hepatitis C; Burkitt's lymphoma; and grade 3 asthenia and arthralgia) and a high risk of HIV transmission in one case. Seven of these 13 patients prematurely withdrew from the study. One patient died, at month 11 from septic shock after chemotherapy for Burkitt's lymphoma diagnosed at month 10 (CD4 493/mm³).

Primary outcome

At 2 years, 24 patients (55%; 95% CI 40%–69%) had a mean month 21-month 24 (‘final’) CD4 cell count (676/mm³, IQR 590–821/mm³) higher than the mean screening-month 0 (‘baseline’) count (596/mm³, IQR 556–635/mm³), and no counts were <400/mm³. Eleven of the other 20 patients had a final mean CD4 cell count (622/mm³, IQR 519–803/mm³) below the mean baseline CD4 cell count (706/mm³, IQR 547–837/mm³), with a median difference of −35 cells (IQR −107 to −20 cells). Two other patients had one CD4 cell count below 400/mm³. Seven patients had missing month 21-month 24 values.

Immuno-virological outcomes

The median difference between the baseline and final CD4 cell counts was +48/mm³ overall and +62/mm³ in the 37 patients who completed the whole study (Figure 2). The median CD4/CD8 ratio remained stable, from 0.6 (IQR 0.4–0.9) at month 0 to 0.7 (0.5–0.9) at month 24.

Figure 2.

CD4 cell count and HIV-1 RNA and HIV-1 DNA changes in the ANRS 141 TIPI Trial. Circles, CD4 cells (/mm³); stars, HIV-1 RNA (log₁₀ copies/mL); squares, HIV-1 DNA (log₁₀ copies/10⁶ leucocytes).

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All but three patients who adhered to the treatment strategy had HIV-1 RNA values below 50 copies/mL after each 6 month ‘on’ period. HIV-1 RNA values returned to baseline levels during the ‘off’ periods.

HIV-1 genotypic resistance mutations occurred in three patients, but they had no consequences for the trial strategy. In two cases, the new mutations were transient and unrelated to the ART regimen (protease mutations I15V and M36I in one case, and reverse transcriptase mutation 215A in the other case); HIV-1 RNA concentration was <50 copies/mL at the end of each ‘on’ period in these two patients. In the third case, the reverse transcriptase mutation 184I conferring resistance to lamivudine/emtricitabine was detected at month 15 and month 18 in a patient on atazanavir/ritonavir plus abacavir and lamivudine; HIV-1 RNA concentrations were 75 and 73 copies/mL at the end of each ‘on’ period.

Median HIV-1 DNA loads were, respectively, 2.8 (IQR 2.4–3.3), 2.4 (2.0–2.8), 2.6 (2.4–3.1), 2.3 (1.8–2.6) and 2.6 (2.3–2.9) log₁₀ copies/10⁶ leucocytes at months 0, 6, 12, 18 and 24. There was no significant virological difference between month 0 and month 24 (P = 0.29). There was no significant difference in HIV-1 DNA loads between patients with and without immunological success: respectively, 2.9 (2.7–3.3) versus 2.7 (2.3–3.0) log₁₀ copies/10⁶ leucocytes at month 0 (P = 0.29) and 2.7 (IQR 2.4–3.0) versus 2.5 (1.8–2.7) log₁₀ copies/10⁶ leucocytes at month 24 (P = 0.15).

Adverse events and safety

Forty-one patients (93%) reported at least one adverse event, and 11 patients (25%) experienced 18 grade 3-4 events, mostly nephrolithiasis and psychiatric disorders (insomnia, suicidal ideation or attempts). Six grade 3-4 clinical adverse events were considered drug related, namely toxidermia, insomnia, asthenia, jaundice and renal colic (n = 2). Only the first three events resulted in treatment discontinuation.

One patient experienced a non-AIDS serious clinical event (viral purpura at month 21 during the second phase ‘off’ ART—HIV load 4.57 log₁₀ copies/mL at the time of diagnosis) and one a new AIDS-defining event (Burkitt's lymphoma at month 9 during the first phase ‘off’ ART—HIV load 4.49 log₁₀ copies/mL at the time of diagnosis). This patient was the one of the study who died, despite chemotherapy. No HIV primary-like infection was observed. Twelve patients (27%) reported 15 episodes of sexually transmitted infections (7 syphilis, 3 gonorrhoea, 3 condylomatosis and 2 chlamydia infections), of which 11 were diagnosed on treatment and 4 off treatment. One patient developed acute hepatitis C during the first ‘on’ period.

Ten patients (23%) experienced grade 3-4 laboratory events, and seven patients reported hyperbilirubinemia related to atazanavir, none of which required atazanavir withdrawal.

Biomarkers of inflammation and endothelial activation

The numbers (percentage) of patients with all marker values below the lowest thresholds associated with a higher clinical risk were 0/40 (0%), 4/38 (11%), 2/39 (5%), 4/38 (11%) and 3/36 (8%) at months 0, 6, 12, 18 and 24, respectively (Figure 3). The numbers (percentage) of patients with at least one biomarker above the threshold associated with a higher clinical risk were 21/40 (53%), 15/38 (39%), 18/39 (46%), 14/38 (37%) and 18/36 (50%) at months 0, 6, 12, 18 and 24, respectively. Two patients (6%) had at least one biomarker value above the highest threshold at all timepoints. There was no significant correlation between the primary endpoint and the concentration of hsCRP (r = −0.35, P = 0.06), IL-6 (r = 0.10, P = 0.60), fibrinogen (r = −0.30, P = 0.17) or D-dimers (r = 0.01, P = 0.96).

Figure 3.

Inflammation markers in the ANRS 141 TIPI Trial.

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T cell differentiation and activation status

As we present in Figure 4, median naive CD4 cell numbers tended to fall between month 0 and month 24, from 263/mm³ (39%) to 189/mm³ (24%) (P = 0.08), whereas effector memory CD4 RA+ cell values increased significantly from 47/mm³ (9%) to 87/mm³ (13%) (P < 0.03). Naive, central and effector memory CD8 cells and B cell subpopulations remained roughly stable throughout the study. CD4 CCR5+, CD8 CCR5+, CD8 HLA-DR+ and CD8 CD38+ cell numbers fluctuated throughout the study. CD8+CCR5+ and CD8+CD38+ cell counts fell between month 0 and month 24 (from 36% to 18%, P = 0.08; and from 60% to 29%, P = 0.04), whereas CD8+ HLA-DR+ cell numbers did not vary significantly (47% at month 0, 35% at month 24, P = 0.29). No significant correlation was observed in the HIV-1 DNA concentrations. CD4 cell proliferative responses to HIV, candidine and cytomegalovirus antigens did not change significantly between month 0 and month 24. By contrast, their response to purified protein derivative fell significantly, from 50% to 0% (P = 0.04). The strength of anti-HIV response was not significantly modified between month 0 and month 24 (from 1628 to 1429 cpm/10⁶ PBMCs, P = 0.28), as was the anti-EBV response (from 1017 to 1377 cpm/10⁶ PBMCs, P = 0.62).

Figure 4.

Immunological changes in the ANRS 141 TIPI Trial. CM, central memory; EM, effector memory.

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Adherence and self-reported symptoms

At least 61% of the patients reported a good or moderate treatment adherence at each timepoint, with no statistical difference between those with immunological success and failure, except at month 18 (P = 0.04).

There was no change in terms of quality of life between month 0 and month 24: the median number of reported symptoms was 3.5 (IQR 2.0–7.5) at month 0 and at month 24 (IQR 1.5–11.0) (P = 0.83). The median number of self-reported symptoms did not vary significantly between the ‘off’ and ‘on’ phases: 2 (IQR 1.0–3.5) during ‘on’ phases and 2 (IQR 1.0–3.0) during ‘off’ phases (P = 0.84).

Discussion

I-ART with alternating 6 month periods on and off therapy was immunologically and virologically safe in this 2 year trial. Even though the demanding study objective of higher CD4 cell counts at 2 years than at baseline in 70% of patients was not achieved, the median CD4 cell count increased overall, and all the patients had a last count above 500/mm³. HIV-1 RNA load was undetectable at the end of each 6 month period on treatment in all but three of the adherent patients. In contrast to the findings of studies involving shorter ‘on’ ART periods and/or antiretroviral drugs with a lower genetic barrier,^16–22 an HIV resistance mutation (M184I) occurred in only one patient. There were few significant and unexpected clinical events, except for one case of Burkitt's lymphoma that led to death despite chemotherapy. That this death would not have occurred on continuous therapy cannot be ruled out. In addition, 15 sexually transmitted infections occurred despite strong advice on measures to prevent HIV transmission, confirming that intermittent therapy is not an option for treatment-as-prevention. Intermittent therapy might also not be an attractive strategy for all patients in real-life settings, as five (11%) of our patients stopped therapy prematurely because of a lack of motivation.

This study also provides some insights into HIV pathogenesis, despite the limited sample size. First, the patients' high current and nadir CD4 cell counts and low HIV-1 DNA load may have contributed to their immunological stability throughout the study.²³ Intermittent therapy did not increase the viral reservoir. HIV-1 DNA values at the end of the ‘on’ ART periods were close to those reported after 1.5 to 4 years of continuous ART started during primary HIV infection.^24,25 They returned to baseline at the end of the ‘off’ ART periods, but were lower than reported in patients who began treatment later.²⁵ HIV-1 DNA values were of the same order as those associated with the lowest risk of clinical progression.^26,27

Second, markers of inflammation and endothelial activation did not vary substantially. This lack of significant fluctuation of inflammatory biomarkers was also observed in another I-ART trial, with 8 week periods ‘on’ and ‘off’ ART.²⁸ In our trial with longer periods, half of the patients had one or more biomarkers above the thresholds associated with the highest clinical risk and only two (6%) had all biomarkers above them at month 24. The clinical risk associated with the I-ART strategy tested here thus seems to be low and stable. Overall, inflammation appears more likely a consequence of an irreversible pro-inflammatory environment (potentially constituted gut damage from HIV replication, or host genetics), or of non-directly HIV-related consequences (e.g. coinfections), considering the contrast between constant inflammation and fluctuating immune activation.

Indeed, T cell activation status fluctuated throughout the study, as did the CD4 cell count, HIV-1 RNA load and HIV-1 DNA load. The observed correlation with HIV-1 RNA loads suggests that CD4 cell activation is a direct consequence of viral replication. However, the CD4 CCR5+ cell count/percentage was lower than usually reported,²⁹ suggesting that the CD4 cell count may also influence CD4 cell activation.^30,31 A stable high CD4 cell count might ensure that activation remains at a low level, even during short periods of uncontrolled HIV replication. Increased proliferative capacity of activated CD4 cells could be a first major response to HIV-induced CD4 cell depletion,³⁰ naive CD4 cells playing a particular role in this compensatory mechanism. The decline in naive CD4 cells, their differentiation towards a memory phenotype, and the decline in proliferative responses we observed after 2 years of I-ART suggest that this mechanism might be overwhelmed in cases of repeated treatment interruptions.

By contrast, though CD8 T cell activation increased during the ‘on’ ART periods and decreased during the ‘off’ ART periods, its level was roughly halved after 2 years, reaching values similar to those observed in patients on continuous ART started early in HIV infection and lower than those observed when treatment starts later.³² None of our patients reached an immunological set point. Despite the irreversible pro-inflammatory environment linked to cumulative exposure to HIV replication,^30,32 our data suggest that the level of CD4 is also important in determining the level of CD8 cell activation and its reversibility, possibly via an action of regulatory CD4 T cells.³³ The reported role of the HIV reservoir in fuelling immune activation³⁴ was not observed here. As each 10% increase in activated CD8 T cells after 6 months of suppressive ART has been found in a previous study to be associated with a 1.6-fold increased risk of subsequent death,³⁵ the decrease we observed may be clinically relevant.

In conclusion, given the better tolerability and acceptance of current ART regimens, it is unlikely that I-ART offers enough advantages to envisage further evaluation or wider use. Nevertheless, our results could help to anticipate what might occur during ‘treatment holidays’ in patients treated early with boosted-PI-based ART. In particular, discontinuing virologically effective treatment for no longer than 6 months after controlling HIV replication would appear to have limited consequences. In addition, while immune activation seems strongly related to HIV replication in patients with high nadir and current CD4 cell counts, inflammation seems to evolve independently and may require specific attention and care.

Other members of the ANRS 141 TIPI Trial Study Group

Members of the Scientific Committee (Xavier Anglaret, François Boué, Marie-Josèphe Commoy, Sandrine Couffin-Cadiergues, Alpha Diallo, Christophe Piketty, Marie Préau, Anne-Marie Taburet), the Data Safety Monitoring Board (Florence Buseyne, Dominique Costagliola, Constance Delaugerre, Bruno Hoen, Olivier Lortholary, Fabrice Pilorgé), the Events Validation Committee (Cédric Arvieux, Christine Jacomet, Catherine Sgro), INSERM U866 (Laurence Loïodice) and INSERM U897 (Sofiane Ait-Ouferoukh, Alex Assuied, Céline Colin, Françoise Couturier, Elodie Rouch, Anne-Aygline Soutthiphong, Audrey Taïeb).

Funding

The French National Institute for Health and Medical Research–France Recherche Nord & Sud Sida-HIV Hepatites (INSERM-ANRS) is the sponsor and funder of the trial.

Transparency declarations

None to declare.

Acknowledgements

This work was presented at the Twentieth Conference on Retroviruses and Opportunistic Infections, Atlanta, GA, USA, 2013 (Abstract PB562).

We thank the ANRS 141 TIPI study participants and their partners, families and caregivers and the staff from all the centres taking part in the trial.

We also thank all those who contributed, in particular those listed below.

References

Author notes