Because the primary objective of this study was to examine the effect of IL-2 on preserving CD4+ T-cell count in this relatively immunologically healthy group of patients, rather than to examine the effect of IL-2 on increasing CD4+ T-cell counts, the protocol-specified primary analysis was based on differences by treatment arm in the number of patients who ever had a CD4+ T-cell count <75% of baseline (therefore equal to a ≥25% drop) over the first 24 weeks. Three patients (2 on ART alone and 1 on ART + IL-2) did not have an entry or preentry CD4+ T-cell count and therefore are omitted from this analysis. Of the 55 patients on ART alone with baseline and follow-up values, 15 (27%) had a drop of ≥25% in their CD4+ T-cell count at some time over the 24 weeks. Of the 56 patients on ART + IL-2, 23 (41%) had a CD4+ T-cell count drop of ≥25% at some time over the first 24 weeks of treatment; this difference was surprising but not statistically significant (P = 0.16). Additionally, the difference in time to first drop in CD4+ T-cell count by ≥25% is not statistically significant (P = 0.11, Fig. 3).
However, a parallel analysis of the number of patients who ever had a CD4+ T-cell count increase over the first 24 weeks (using the definition of at least a 25% increase over baseline since this is analogous to the protocol-defined decrease specified in the primary analysis) revealed differences that were statistically significant. On ART alone, 18 of 55 patients (33%) had a ≥25% increase in CD4+ T-cell count at some time during the first 24 weeks, whereas 39 of 56 of patients (70%) on ART + IL-2 had a CD4+ T-cell count increase of ≥25% over the same period (P = 0.0001). The time to first increase of ≥25% in CD4+ T-cell count was also significantly different between treatment arms (P = 0.001, Fig. 4).
To compare the changes in both directions in CD4+ T-cell numbers in the 2 treatment groups, we analyzed overall change from baseline to the last CD4+ T-cell count observed, assigning patients into 1 of 3 categories: ≥25% decrease, <25% change in CD4+ T-cell count, and ≥25% increase. For ART + IL-2, the percents of patients in these 3 categories were 18%, 48%, and 34%, respectively. For ART alone, the percents of patients in these 3 categories were 7%, 80%, and 13%, respectively; the difference was significant (P = 0.03).
Stratifying patients by single vs. combination nucleoside-based treatment at entry and then evaluating for CD4+ T-cell count change revealed a similar pattern of greater splay in the CD4+ T-cell count in the ART + IL-2–treated groups (both more increases and more decreases in CD4+ T-cell count) regardless of the type of nucleoside treatment, except in the stratum of patients who had been treated with nucleoside-based combination regimens for only 2–6 months at study entry. An equal distribution of CD4+ T-cell count decreases and increases in patients on both arms of the study was seen in that stratum alone.
Based on the first 24 weeks of the study, the estimated annual slope of CD4+ T-cell count (based on a random effects analysis) for ART-only patients was an increase of 1 cell/mm3 vs. an estimated annual slope of 57 cells/mm3 for ART + IL-2-treated patients. Again, however, this difference was not significant, even when corrected for initial CD4+ T-cell count (P = 0.18). This analysis assumes the between-patient variances of slope in CD4+ T-cell counts are equal in the 2 treatment groups, but as previously discussed, the variance of change in CD4+ T-cell counts in patients treated with ART+IL-2 was about twice that for the patients treated with ART alone, so the slope would also have different variances.
Changes in Plasma HIV-1 RNA
The interim analysis of plasma HIV RNA in the first 20 patients was based on a bDNA assay with lower level of detection of 10,000 copies/mm3 and showed no significant treatment difference. The final analysis of plasma HIV RNA was based on a bDNA assay with lower level of detection of 50 copies/mm3. The differences between treatment arms for changes in plasma HIV RNA from baseline over the study period were not significant at any time point. Table 2 shows mean and median change in log10 plasma HIV RNA by bDNA: for each arm at each follow-up time; the mean change was never >0.07 (corresponding to a 17% increase or 15% decrease in viral load). Although there were no significant differences between the treatments in viral load change, ART + IL-2 was always associated with a more beneficial direction of viral load change (bigger decrease or smaller increase) than ART alone.
Serologic Responses to Vaccine
Comparing serologic titers after immunization in the 2 groups assessed the effect of low-dose IL-2 on enhancing responses to immunization in these immunocompromised patients. Patients eligible for vaccination (see “Methods”) had baseline prevaccination serologies compared with 4 weeks postvaccination and end-of-study serologies. Depending on vaccine type, serologic results were available for analysis in 47–98% of those vaccinated. The protocol did not define serologic response, but for purposes of the analysis, response was defined as a 4-fold rise in titer. Only 8 patients on ART only and 10 on ART + IL-2 had pneumococcal serologies, and none of them had a response; the results of the other serologies are given in Table 3. There were no statistically significant differences between the treatment arms in the proportions of responders to any vaccine antigen except for 1 influenza strain (for which ART alone was associated with a larger response percent). However, the power to detect reasonable treatment differences in these proportions (e.g., 40% vs. 60%) was quite low (6% for pneumococcal, 18% for tetanus and diphtheria, 12% for influenza, and 32% for meningococcal serologies).
Skin Test Responses
Improvements in DTH responses to recall antigens after treatment with IL-2 would be expected with improvement in CD4+ T-cell numbers, function, or both. DTH response was therefore measured in response to PPD, tetanus, mumps, and 2 doses of Candida at study entry and week 16. Only 5 patients ever had a reaction to PPD, so results were not analyzed for this antigen. Depending on which skin test antigen was analyzed, between 4 and 32 patients per arm had negative responses at baseline and also had a skin test result recorded at 16 weeks. Between 2 and 13% of patients with initial negative responses had positive responses at 16 weeks. For tetanus and mumps, only 10 and 8% of patients, respectively (overall), had an increase in induration ≥5 mm between baseline and week 16. For the lower dose of Candida, 30% of ART-only patients and 21% of ART + IL-2 patients had an increase of induration ≥5 mm; for the higher dose of Candida, 26% of ART-only patients and 23% of ART + IL-2 patients had an increase of induration ≥5 mm. There were no significant differences discerned in the frequency of or changes in skin test responses between treatment arms. However, the power of these comparisons was again very low (e.g., in the comparison of conversion rates from a baseline negative skin test response to positive skin test response at 16 weeks, the power to detect a difference between arms that have true conversion rates of 10% and 30% was 41% for tetanus, 30% for mumps, and 6% for Candida).
Serum Activation Markers
Serum activation markers at baseline, prior to initiating IL-2, were similar in the 2 randomized groups, with means of 2.06 and 1.98 mg/L for β2-microglobulin, 9.62 and 8.96 nml/L for neopterin and 3.01 and 3.02 ng/mL for sTNF-RII (for the ART and ART + IL-2 arms, respectively). After starting IL-2, for all 3 activation markers measured, the median and mean changes from baseline were downward at most time points in the ART-only group, and upward in the ART + IL-2 group at all time points (weeks 2, 4, 8, 16, and 24). Using the 2-sided Wilcoxon rank sum test, there were significant differences between the groups at each time point. For all time points, values were P ≤ 0.01 for β2-microglobulin, P &le 0.0009 for neopterin, and P ≤ 0.0001 for sTNF-RII.
Of the 56 patients entered on the intensive immunologic substudy, 27 patients had baseline lymphoproliferative assays to tetanus and Candida performed. Eighteen patients had baseline lymphoproliferative assays performed to 2 HIV envelope proteins and 9 patients to an HIV p24 antigen labeled Chi p25; fewer had follow-up assays performed. Results were analyzed both by the difference in counts per minute (CPM) in the presence of antigen or medium alone (delta CPM) and by stimulation index (SI). Over the duration of the study there was little change in response to any antigen for either treatment arm. However, the power to detect reasonable differences (e.g., a 3-fold median increase in SI on one arm and a total lack of change in SI on the other arm) was small (53% for tetanus or Candida and 34% for the HIV envelope proteins if standard deviations were about the same size as means).
Natural Killer Cell Assays
Measurements of NK and LAK cell activities were performed on 16 patients per arm at 12 weeks and 13 patients per arm at 24 weeks. There were no significant differences by treatment arm in the difference between follow-up and baseline results of these assays using effector to target ratios from 6.25 to 50 for assays done with peripheral blood mononuclear cells (PBMCs) alone or with PBMCs + IL-2 (although the power to detect an increase of 1000 cells in one arm and 500 cells in the other arm if standard deviations were equal to means was only 38% at 12 weeks and 32% at 24 weeks).
An interim safety analysis performed using data received on 54 patients followed for 6 months revealed no significant differences between treatment arms in the occurrence of grade 3 or worse toxicity, and there was no protocol interruption or modification. By the end of the study at 24 weeks, only 2 grade 4 toxicities had occurred: one grade 4 hypertriglyceridemia in the ART + IL-2 group and one case of agitation in the same group. Table 4 shows percent of patients with grades 2 and 3 toxicities by treatment arm; some of these are associated with ART therapies rather than with IL-2. In comparing grade 2 or worse toxicity, the ART + IL-2 group had significantly more toxicity only for the category “general body,” and that was primarily due to more grade 2 discomfort and fatigue.
Clinical Events and Deaths
New clinical diagnoses seen in ≥5 patients while on study included lymphadenopathy, sinusitis or rhinitis, upper respiratory infection or “strep” throat, bronchitis, oral hairy leukoplakia, and herpes simplex. There were no significant differences between the treatment arms in the occurrence of these events. One patient died at week 38 due to sepsis, with invasive aspergillosis and HIV disease progression as contributing causes. He had been assigned to the ART-only treatment arm.
In this prospective, randomized, controlled, multicenter trial, the addition of low-dose subcutaneous daily IL-2 to ART treatment comprising largely single- or dual-nucleoside therapy for 6 months showed no significant benefit in preserving CD4+ T-cell counts in a population of relatively immunologically healthy patients. Although the study was primarily designed to evaluate only preservation of baseline CD4+ T-cell counts, we observed that a significantly larger proportion of patients treated with this schedule of IL-2 had a >25% increase from baseline in CD4+ T-cell counts than did patients receiving ART therapy alone. However, the frequency of individuals experiencing a decrease in CD4 + T-cell counts by ≥25% was nonsignificantly greater in recipients of IL-2. Importantly, the variation in change in CD4+ T cell counts over time was significantly greater in the patients randomly assigned to the IL-2 arm.
ART therapy in these patients was primarily single or dual nucleoside based, which suppressed viral replication to <50 copies/mL in only 11% of patients as assessed by bDNA assay at baseline. Median plasma HIV RNA on ART therapy obtained at baseline by bDNA was 3.63 log10 in the ART-alone arm and 3.41 log10 in the ART + IL-2 arm. Plasma HIV RNA was unchanged by the addition of IL-2, and no baseline characteristic, including plasma HIV RNA at baseline, predicted a >25% drop in CD4+ T-cell counts over the study period.
Although our study addresses some aspects of IL-2 therapy in HIV disease, several questions remain, including the types of immunologic responses generated at different doses and its utility in different patient populations. In patients with lower CD4+ T-cell counts, it is unclear whether IL-2 boosts CD4+ T-cell counts primarily by expansion of the pool of preexisting CD4+ T cells, 6 by increasing thymic generation of naive cells, 7 or by selective expansion and prolonged survival of a unique subset of naive CD4+CD25+ T cells. 8 In addition, increases in CD4+ T-cell numbers in these patients may not restore a diverse repertoire of T-cell clones once their distribution has been perturbed.
Efforts to maximize immunologic activity and to minimize toxicity led to trials of lower doses given daily 9,10 and to trials of subcutaneous or intradermal administration. 11–13 Indeed, some investigators have proposed that lower-dose IL-2 administered daily is more physiologic since lower concentrations of IL-2 will bind only to T cells with high-affinity receptors and not to NK cells with lower-affinity receptors. 14 Lower doses may also limit toxicities associated with activation of cytokine cascades and limit the extent of rebound cytopenias. It has been argued therefore that “less is more” in IL-2 dosing. 15,16 A recently published randomized trial of highly active antiretroviral therapy (HAART) + daily subcutaneous IL-2 at a starting dose of 1.2 mIU/m2 /d in patients with CD4+ T-cell counts <300 cells/mm3 reported significantly greater increases in the number of CD4+ T cells, in the proportion of naive cells, and in the number of NK cells in the IL-2–treated group at weeks 4 and 8 compared with these indices in HAART-alone–treated controls, but this difference was not sustained at weeks 16 and 26 of the study. 9 There were also substantial changes in antiviral therapies during the trial period. Higher IL-2 doses and route and frequency of dosing were addressed in a randomized, controlled clinical trial in France of IL-2 added to background dual-nucleoside therapy, which showed comparable significant improvements in CD4+ T-cell counts and proliferative responses in the intermittent IV and in the high-dose daily subcutaneous groups. 17
Our study does not address the question of whether this schedule of daily low-dose IL-2 administration would benefit patients on potent ART therapy who have substantial virologic suppression with viral loads below the limit of detection. Higher doses of IL-2 administered intermittently in conjunction with daily ART therapy have shown benefit in increasing CD4+ T-cell counts in subjects whose plasma HIV RNA is below the limit of detection. 18,19 For example, Katlama et al. 20 analyzed CD4+ T-cell area under the curve minus baseline in patients with very low CD4+ T-cell counts who were treated with HAART alone vs. HAART + IL-2 administered as 4 cycles of 9 million IU of IL-2 daily for 5 days every 6 weeks and found significant and sustained increases in CD4+ T-cell area under the curve out to 80 weeks.
The population of patients participating in this study was quite healthy from an immunologic standpoint and by current guidelines might not begin ART therapy. “Adjunctive” therapy to further increase CD4+ T-cell counts might not be necessary, but administration of IL-2 might delay the decline in CD4+ T-cell counts as disease progresses and possibly prolong the time before HAART is initiated.
We were not able to detect an effect of this dose of IL-2 on other immunologic endpoints such as skin test responsiveness, serologic responses to the administration of vaccines, lymphoproliferative responses, and assays of LAK and NK cell activity. In the setting of incomplete control of HIV replication, administration of daily low-dose IL-2 in this study provides very little evidence of immune enhancement. Since responsiveness to immunization is often impaired in HIV infection, immune-based strategies to enhance vaccine responses would have important clinical implications. The utility of low-dose daily or twice weekly subcutaneous or intradermal IL-2 as adjunctive therapy to restore or preserve immunologic function in HIV infection was studied previously in a few small, uncontrolled trials. In these studies, enhanced skin test reactivity suggested an immunologic benefit. However, a recent paper suggests that high-dose intermittent IL-2 administered to patients on HAART does not show evidence of enhancing vaccination responses when compared with responses seen in persons receiving HAART alone, despite inducing significant increases in CD4+ T-cell number. 21
In conclusion, this regimen of daily low-dose subcutaneous IL-2 was well tolerated. The observation that IL-2 was associated with both a more frequent increase and a more frequent decrease in CD4+ T-cell numbers than seen in subjects receiving nucleosides alone is difficult to explain. Nonetheless, a substantial proportion of the patients treated with adjunctive low-dose IL-2 compared with those not treated with IL-2 experienced an increase of ≥25% in CD4+ T-cell count from baseline. Our conclusions are supported to some degree by other studies, in which study protocols monitored CD4+ T-cell counts as IL-2 doses were escalated and showed evidence for a dose-response association between IL-2 and CD4+ T-cell counts. 17–19,22 Phase 3 clinical endpoint trials of higher-dose intermittent IL-2 therapy are in progress and the results of these studies should provide a definitive answer about the clinical benefit of the admittedly more toxic but possibly more active regimen of IL-2 administration for the treatment of HIV disease.
Without the patients who so willingly provided blood samples, learned injection techniques, and returned for frequent follow-up visits, this study could not have been done and the authors thank them for their participation.
Critical concept review, manuscript review, and study support were provided by Anne-Marie Duliege, Chiron Corp. Influenza serologies were performed by Alexander Klimov, PhD, Influenza Branch, Centers for Disease Control, Atlanta, GA. Meningococcal serologies were performed by Mike Bybel, Aventis Pharmaceuticals. Pneumococcal serologies were performed by Daniel Musher, Baylor College of Medicine, Veterans Affairs Medical Center, Houston, TX. β2-Microglobulin, neopterin, and sTNF-RII assays were performed by John Fahey (University of California, Los Angeles, School of Medicine). HIV-1 bDNA assays were performed and funded by Chiron Corp. IL-2 (aldesleukin, rhIL-2, Proleukin) was provided by Chiron Corp. Tetanus and diphtheria toxoids, adsorbed (for adult use) (Td), Meningococcal Polysaccharide Vaccine (Menomune-A/C/Y/W-135), and Influenza Virus Vaccine (Fluzone, 1995–1996 season) were provided by Connaught Laboratories. Pneumococcal vaccine, polyvalent (Pneumovax 23) was provided by Merck Research Laboratories.
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ACTG 248 Contributors
Dawn Bell, BSc (NIAID, NIH)
Margarita Vasquez, RN, Victoria Rosenwald, RN, MPH (NYU/Bellevue)
Holly Ingelfinger-Lopez, RN, and Paul Stockdill, RPh (SUNY-Buffalo, Rochester)
David Mushatt, MD (Tulane University)
Michael F. Giordano, MD (Cornell University)
Michael Chance, RN (Case Western Reserve University)
Mitchell Goldman, MD, Kristin Todd, RN, MSN, CCRC (Indiana University)
Charles van der Horst, MD, and Barbara Longmire, RN (UNC) (University of North Carolina)
Virginia A. Waite, BSN (University of Colorado Health Sciences Center, Denver)
Janice Jacovini, RN, and Chris Helker, RN (University of Pennsylvania, Philadelphia)
Janet Pientka and Elizabeth McCann (Thomas Jefferson University Hospital, University of Pennsylvania, Philadelphia)
Cyndi Frank (Yale University)
Study concept and design: Dr. Teppler, Dr. Pomerantz, Dr. Gelman
Acquisition of data: all participating ACTG sites
Drafting of manuscript: Dr. Vogler, Dr. Gelman
Critical revision of manuscript for important intellectual content: Dr. Lederman, Dr. Valentine, Dr. Teppler, and Dr. Gonzalez
Statistical expertise: Dr. Gelman, Ms. Cherng
Obtained funding: ACTG
Administrative, technical, or material support: Ms. Mahon, Ms. Schock, Ms. Bell
Study supervision: Dr. Teppler, Dr. Vogler, Dr. Pomerantz, Dr. Pollard
Keywords:© 2004 Lippincott Williams & Wilkins, Inc.
HIV infection; interleukin-2; IL-2; immunotherapy; immune function; CD4