Most children infected with HIV-1 develop CD4 cell depletion during the first few years of life, unless antiretroviral therapy is initiated within the first few months of life, in which case normal CD4 cell counts may be preserved . HIV-infected children with moderate or severe immunosuppression, as defined by Centers for Disease Control criteria for CD4 cell count and percentage , are susceptible to opportunistic infections. HIV infection also can affect CD4 cell function in HIV-infected children. Of particular concern, HIV-infected children generally do not have demonstrable CD4 helper cell responses to HIV antigens, an impairment that may contribute to their inability to control viral replication .
Some of the immunologic deficits caused by HIV infection may be reversed by administration of highly active antiretroviral therapy (HAART). Several studies have shown impressive rises in both naive and memory CD4 cell counts in HIV-infected children receiving HAART [4–8]. On the other hand, the effect of HAART on immunologic function, as measured by assays for lymphocyte proliferation to specific antigens, remains controversial [3,8]. Of particular importance, CD4 helper cell responses to HIV antigens did not improve in most HIV-infected children receiving HAART .
Immune-based therapy in conjunction with HAART might cause more rapid immune reconstitution in HIV-infected children. Of various potential immune modulators available for clinical use, recombinant interleukin-2 (rIL-2) appeared to be the most attractive for use in HIV-infected children. Several previous studies had shown that HIV-infected children have impaired production of type 1 cytokines, including interleukin-2 (IL-2) [9–12], and that addition of rIL-2 to lymphocyte cultures in vitro enhanced lymphocyte proliferation responses to recall antigens such as tetanus . Furthermore, controlled studies have shown significantly higher CD4 cell count rises in HIV-infected adults receiving intravenous or subcutaneous rIL-2 plus antiretroviral therapy (ART) compared with those receiving ART alone [14–24]. In these studies, rIL-2 was tolerated, although side effects were common, and in some cases treatment had to be discontinued because of severe adverse effects.
The present study was designed to determine the tolerated dose of rIL-2 when given to HIV-infected children as a continuous intravenous infusion for 5-day cycles every 8 weeks and to determine the safety and immunologic effects of the tolerated dose in a cohort of HIV-infected children.
This open-label study was conducted at nine Pediatric AIDS Clinical Trials Group (PACTG) sites. Children were enrolled between March 1997 and January 2000.
The objective of part A of this study was to define the tolerated dose of rIL-2 in HIV-infected children. For this purpose, groups of four HIV-infected children, 3–12 years of age, were to receive rIL-2 by continuous intravenous infusion at doses of 1 × 106 IU/m2 per day, 4 × 106 IU/m2per day, or 8 × 106 IU/m2 per day for 5 days every 8 weeks for three cycles. Dose escalation occurred if all four subjects receiving a dose level tolerated therapy without evidence of grade 3 or higher toxicity attributable to study treatment. If one of four subjects at any dose level experienced grade 3 or higher toxicity attributable to study treatment, two additional subjects were to be enrolled at that dose level. If one of these two additional subjects experienced grade 3 or higher toxicity attributable to study treatment, the previous dose level was considered the tolerated dose. Similarly, if two or more of the four subjects receiving a dose level experienced grade 3 or higher toxicity attributable to study treatment, the previous dose level was considered the tolerated dose.
The objective of part B of this study was to determine the safety and immunologic effects of the tolerated dose of rIL-2 when given to a cohort of HIV-infected children who had not participated in part A. Children enrolled in part B received 1 × 106 IU/m2 per day of rIL-2 by continuous intravenous infusion for 5 days every 8 weeks for six cycles.
Children enrolled in parts A and B had to be between 3 and 12 years of age and to have previously documented HIV infection defined as a positive culture or PCR result on at least two occasions or a positive ELISA and a confirmatory Western blot. Subjects had to have symptomatic HIV infection defined as Centers for Disease Control categories A, B, or C. Children in part A had to be receiving the best available ART, generally two nucleoside reverse transcriptase inhibitors (NRTI), while children enrolled in part B had to be receiving combination ART with at least three drugs, one of which had to be a protease inhibitor (PI). According to study protocol, subjects enrolled in part B also had to have a viral load < 400 copies/ml within 30 days of entry, however three subjects with viral load > 400 copies/ml were allowed to enroll after discussion with the study chair. Institutional review boards at each site approved the study. Written informed consent was obtained from the parents and guardians of all children.
Recombinant IL-2 was provided by Chiron Corporation (Emeryville, California, USA), as a sterile lyophilized powder in glass vials containing 22 × 106 IU per vial and was reconstituted with 1.2 ml of sterile water.
Safety monitoring and treatment discontinuation
The following screening tests were completed within 14 days prior to entry: complete blood count and differential count, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen BUN, creatinine, electrolytes, urinalysis and, for females of childbearing potential, a urine or serum pregnancy test. In addition, each subject had a chest radiograph, electrocardiogram and ECHO cardiogram within 60 days prior to entry. Hematologic and biochemical studies were obtained on days 1, 3, and 5 of each cycle, and urinalyses were obtained on days 1 and 5 of each cycle. These tests also were obtained 4 and 20 weeks after the last cycle in parts A and B, respectively. Neurologic examinations and neurodevelopmental assessments were performed at baseline and weeks 20 and 44 in part A, and at baseline and weeks 44 and 60 in part B. In both parts of the study, mental status evaluations were performed on days 1, 3 and 5 of each cycle.
We used the Division of AIDS Toxicity Table to grade the severity of adverse effects. A grade of 0 indicates the absence of an adverse effect, while a grade of 1 indicates a mild adverse effect, a grade of 2 a moderate adverse effect, a grade of 3 a severe adverse effect, and a grade of 4 a life-threatening adverse effect. Criteria for treatment discontinuation included grade 3 or 4 toxicity (except fever and shaking) or a decline in CD4 cell count of ≥ 25% from baseline.
Quantitation of lymphocyte subpopulations
CD4 cell percentages and absolute counts were determined in PACTG-certified immunology laboratories according to the PACTG consensus protocol for flow cytometry. In part A, lymphocyte subpopulations were quantified at weeks 0 (days 1 and 5 of cycle), 4, 8 (days 1 and 5 of cycle), 12, 16 (days 1 and 5 of cycle), 20, 28, 36, and 44, while in part B, these measurements were obtained at weeks 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 60, on day 1 of corresponding cycles.
Plasma HIV-1 RNA
In part A, HIV-1 plasma RNA levels were determined at weeks 0 (days 1, 2 and 5 of cycle), 4, 8 (days 1, 2 and 5 of cycle), 12, 16 (days 1, 2 and 5 of cycle), 20 and 44. In part B, HIV-1 plasma RNA levels were determined at weeks 0, 12, 20, 28, 36, 40, 44 and 60. Plasma was stored at −70°C, and HIV-1 RNA was measured using a quantitative assay (Amplicor, Roche Diagnostics, Nutley, New Jersey, USA) with a lower limit of quantification of 400 copies/ml. All RNA assays were performed in a single PACTG-certified virology core laboratory.
The baseline characteristics were summarized using descriptive statistics. The statistical significance of changes in CD4 cell count and percent from week 0 to week 20 and week 44 in parts A and B, respectively, were assessed using the Wilcoxon Sum Ranks test. The median changes in CD4 cell count and percent were displayed graphically. For subjects who did not complete treatment due to intolerance or toxicity, CD4 data for the time points subsequent to the treatment discontinuation were not used in the analyses.
The study was approved by the responsible ethical committee at each participating site and informed consent was obtained for all participants.
The baseline characteristics of children enrolled in parts A and part B are shown in Table 1. Ten children were enrolled in part A, and 10 different children were enrolled in part B. All of these children had acquired HIV infection as a result of mother-to-infant transmission. The baseline characteristics of these children were similar, except for the higher median plasma HIV-1 RNA titer in those who received 4 × 106 IU/m2 per day in part A. Subjects enrolled in part A were receiving a variety of antiretroviral regimens, ranging from two NRTI to two NRTI plus a PI with or without a non-nucleoside reverse transcriptase inhibitor (NNRTI). The median duration of the current antiretroviral regimen was 5 months (range, 7 weeks to > 12 months). All subjects enrolled in part B, with one exception, were receiving at least two NRTI plus a PI. One subject was receiving two NRTI only and two subjects were receiving an NNRTI in addition to two NRTI and a PI. The median duration of the current antiretroviral regimen for part B subjects was > 12 months (range, 3 months to > 12 months).
Toxicity, tolerated dose, and treatment discontinuations
The most common side effect, fever, occurred in 50% of subjects receiving 1 × 106 IU/m2 per day and in 100% of subjects receiving 4 × 106 IU/m2 per day. The second most common side effect, vomiting, occurred in 31% of subjects receiving 1 × 106 IU/m2 per day and 75% of subjects receiving 4 × 106 IU/m2 per day. Of the first four subjects enrolled in part A to receive 1 × 106 IU/m2 per day, one subject developed grade 3 memory loss by history, although this finding could not be confirmed when a neuropsychologist examined the patient several days later. Two additional subjects were enrolled at this dose level, neither of whom developed grade 3 or higher toxicity related to study drug. Dose-escalation was allowed to proceed, and four subjects were enrolled to receive 4 × 106 IU/m2 per day. Of these four subjects, one developed grade 3 fever (> 40°C) during the first cycle, but did not have recurrence of grade 3 fever during the subsequent two cycles with pre-treatment with antipyretics. Another subject developed grade 3 vomiting. Both grade 3 toxicities were thought to be related to administration of rIL-2. Since two of the four subjects enrolled at this dose level developed grade 3 toxicity, the tolerated dose of rIL-2 was considered to be 1 × 106 IU/m2 per day. Of the 10 subjects enrolled in part B who received 1 × 106 IU/m2 per day, one developed grade 3 fever. None of the subjects developed grade 4 signs or symptoms related to administration of rIL-2. One subject enrolled in part B developed grade 4 neutropenia (absolute neutrophil count, 203 × 106/l) at week 16, however, this was thought to be due to concomitant medications.
Treatment was discontinued early in two subjects in part A and five subjects in part B. The two early treatment discontinuations in part A were for memory loss (week 4) and vomiting (week 16). The five early treatment discontinuations in part B were for new onset lupus erythematosus (week 8); request of parent because of difficulty in maintaining intravenous access (week 20); request of parent owing to development of eye pain on day 4 and 5 of cycle 4 (week 28); CD4 cell count decrease > 25% from baseline (week 40); and wheezing, diaphoresis and a possible hive on the cheek (week 40, day 4 of cycle 6).
Plasma HIV-1 RNA levels
Plasma HIV-1 RNA levels were obtained for 19 of the 20 subjects enrolled in the study, the exception being one subject in part A who received 1 × 106 IU rIL-2/m2 per day. The median change from baseline in HIV RNA log10 copies/ml was 0.0, 0.0, and 0.0 for part A 1 × 106 IU rIL-2/m2 per day (n = 5), −0.2, −0.35, and −0.7 for Part A 4 × 106 IU rIL-2/m2 per day (n = 4), and 0.0, 0.0, and 0.0 for part B (n = 10 week 12 and 20, n = 9 week 44) at weeks 12, 20, and 44, respectively. At the last time point on study all subjects had plasma HIV RNA levels no more than 0.2 log10 copies/ml above their baseline value.
Subjects enrolled in part A had HIV RNA determinations during the rIL-2 infusions on days 1, 2, and 5 as well as at 4-week intervals after the infusions (Fig. 1a and b). Transient increases in viral load between infusion day 1 and day 2 or 5 occurred during at least one of the infusions for two of five and four of four subjects receiving rIL-2 at 1 × 106 IU/m2 per day and 4 × 106 IU/m2 per day, respectively (range 0.1 to 0.7 log10 copies/ml). Viral loads declined by 4–8 weeks after the infusion. Two of five subjects receiving rIL-2 at 1 × 106 IU/m2 per day had decreases in plasma HIV RNA during the infusions.
Of the 10 subjects enrolled in part B, six had < 400 copies/ml of plasma HIV-1 RNA at baseline and throughout the study (data not shown). Viral loads for the other four subjects in part B are shown in Fig. 1c. Of the three subjects with viral loads of > 400 copies/ml at baseline, two had transient increases detected 4 weeks after one or two cycles of rIL-2, respectively. These viral load changes did not exceed +1 log10 copies/ml, and in both cases viral loads returned to baseline by week 60. One subject with < 400 copies/ml at baseline had viral loads up to a maximum of 3.5 log10 copies/ml during rIL-2 therapy but the viral load decreased to < 400 copies/ml at week 60.
CD4 cell percentages and absolute counts
Median changes in CD4 cell absolute counts and percentages from baseline to week 44 in part A and from baseline to week 60 in part B are shown in Figs 2 and 3. In part A of the study, in which CD4 cell parameters were studied before and after 5-day infusions of rIL-2, transient rises were observed in both parameters, particularly in subjects receiving 4 × 106 IU/m2 per day. For the five subjects who completed therapy at a dose of 1 × 106 IU/m2 per day in part A, CD4 cell counts increased from baseline to week 20 in four (range, +79 × 106 to +801 × 106/l) and decreased in one (−132 × 106/l). The median change in CD4 cell count to week 20 (4 weeks after the last infusion) in all subjects was +344 × 106/l (Table 2; P = 0.19, Wilcoxon test). Over this same time period, the median increase in CD4 cell percentage was +4 (P = 0.13, Wilcoxon test). After the three cycles were completed, the median CD4 cell count fell considerably by week 28, but remained above baseline for the remainder of the observation period. In contrast, the CD4 percentage rose to +7 at week 28 and dropped only slightly thereafter.
For the four subjects who received 4 × 106 IU/m2 per day of rIL-2, CD4 cell counts rose in three (range, +560 × 106 to +1618 × 106/l) and fell in one (−16 × 106/l). The median change in CD4 cell count to week 20 (4 weeks after the last infusion) in all subjects was +563 × 106/l (Table 2; P = 0.25, Wilcoxon test), while the median change in CD4 cell percentage was +6.5 (P = 0.13, Wilcoxon test). The median CD4 cell count and percentage fell after the completion of three cycles, but both remained above baseline levels for the remainder of the study.
Owing to treatment discontinuations, CD4 cell parameters were available at week 44 (4 weeks after the last cycle) for only five of the subjects who enrolled in part B of the study. For these five, CD4 cell counts rose in four (range, +93 × 106 to +498 × 106/l) and fell in one (−317 × 106/l). This latter subject was discontinued from study treatment 8 weeks after the fifth cycle of rIL-2 (at week 40) due to a > 25% decrease in CD4 cell count from baseline. The median change in CD4 cell count to week 44 for all subjects was +109 × 106/l (Table 2; P = 0.31, Wilcoxon test). The median change in CD4 percentage over this time period was +6 (P = 0.06, Wilcoxon test). The median CD4 cell count and percentage both fell by week 60 (20 weeks after the last cycle), but remained above baseline levels.
HIV-infected children receiving rIL-2 in this study developed side effects similar to those observed previously in HIV-infected adults. The majority of children developed fever, but in only two cases did the height of the fever exceed 40°C. Vomiting was another prominent side effect, particularly in subjects receiving the higher dose of 4 × 106 IU/m2 per day. In the dose escalation part of the study, two of four subjects receiving 4 × 106 IU/m2 per day developed grade 3 toxicity, fever in one subject and vomiting in the other; however, both of these side effects might have been prevented by administration of anti-pyretics or anti-emetics before rIL-2 therapy. The dose of 1 × 106 IU/m2 per day was considered the tolerated dose of rIL-2 in this study although higher doses have been used in studies of rIL-2 in children with malignancies .
The number of children who were discontinued from rIL-2 therapy was greater than anticipated. Most of the discontinuations were due to possible or probable side effects of rIL-2 or to difficulties in maintaining intravenous access. Development of lupus erythematosus in one child may have been related to rIL-2 therapy, which has been associated with exacerbation of pre-existing or initial presentation of autoimmune disease. While the sample size was small, and the rate of discontinuations might be lower in a larger study, our results underscore the difficulties of administering an intravenous drug with significant side effects to children over a long period of time. Of note in this regard, CD4 cell parameters remained above baseline levels for several months after completion of rIL-2 therapy, and six cycles did not appear to boost CD4 cell parameters more than three cycles. These observations suggest that after three cycles, HIV-infected children may need rIL-2 therapy only on an intermittent basis to maintain CD4 cell parameters, as has been observed in HIV-infected adults receiving subcutaneous rIL-2 . Administration of rIL-2 via the subcutaneous route also would be an attractive option for future studies, particularly since studies in HIV-infected adults have shown comparable increases in CD4 cell count after intravenous or subcutaneous administration of rIL-2 [17,27].
Transient small rises in plasma HIV-1 RNA levels were observed in some subjects during rIL-2 therapy, as has been observed previously in HIV-infected adults. Since this study did not have a control group, and transient small rises in plasma HIV-1 RNA levels can be observed in HIV-infected children receiving ART or HAART, some of the transient rises observed may not have been associated with rIL-2 therapy. Most of the small rises were observed in subjects with > 400 copies/ml plasma HIV-1 RNA at baseline, including those receiving combination therapy with a PI, suggesting that incomplete viral control increases the risk of increased viral replication during rIL-2 therapy. These transient rises in plasma HIV-1 RNA levels occurred sporadically, and in all cases levels returned quickly to baseline or near baseline. In part B of the study, in which subjects were receiving combination therapy including a PI, only one of seven subjects who had < 400 copies/ml plasma HIV-1 RNA at baseline developed > 400 copies/ml during therapy, suggesting that viral replication is less likely to be stimulated during rIL-2 therapy, if subjects are receiving potent combination therapy and have < 400 copies/ml at baseline. We did not examine effects of rIL-2 therapy on viral replication and latency in other body sites.
CD4 cell count and percentage increased substantially in HIV-infected children who received rIL-2. In part B of the study, the observed increase in CD4 percentage was similar to that observed in part A. Subjects enrolled in part B appeared to reach a maximal increase in CD4 percentage after the fourth infusion. The median rises in CD4 cell count and percentage in part A and B did not achieve statistical significance, possibly owing to the small sample size, but the results nevertheless suggest that rIL-2 therapy, even at a relatively low dose, may result in substantial rises in peripheral blood CD4 cells in some HIV-infected children. Data to be reported elsewhere on naive and memory CD4 cell subsets, and on T helper cell responses to recall antigens in the present study, also support a potential role for rIL-2 in HIV-infected children.
Based on the results of this open-label phase I/II study, controlled trials of rIL-2 in HIV-infected children are warranted. For convenience and perhaps increased safety, subcutaneous administration rIL-2 should be considered in future trials. Recombinant IL-2 therapy might be of particular value in HIV-infected children whose CD4 cell parameters do not improve after HAART.
This paper is dedicated to the memory of Dr. Jane Pitt, deceased April 16, 2003. The authors thank the study patients and their parents and guardians for their participation.
Sponsorship: Supported by the Pediatric AIDS Clinical Trials Group of the National Institute of Allergy and Infectious Diseases, the Pediatric/Perinatal HIV Clinical Trials Network of the National Institute of Child Health and Human Development, by General Clinical Research Centers funded by the National Center for Research Resources, and by Chiron Corporation.
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The PACTG 299 Study Team
E. Smith, National Institute of Allergy and Infectious Diseases, Bethesda, MD; J. Moye, National Institute of Child Health and Human Development, Bethesda, MD; B. Nowak, Frontier Science and Technology Research Foundation, Amherst, NY; R. McEvoy, Denver Children's Hospital, Denver, CO; C. Vincent, The Children's Hospital of Philadelphia, Philadelphia, PA; S. Estep, National Institute of Allergy and Infectious Diseases, Bethesda, MD, A.M. Duliege, Chiron Corporation, Emeryville, CA.
Additional study participants
Boston Children's Hospital; Children's Hospital of Philadelphia (R. Rutstein, G. Koutsoubis); Long Beach Memorial Medical Center (L. Melton); Texas Children's Hospital; Tulane University Health Sciences Center (R.B. Van Dyke, M Silio, C. Borne-Lepree, O.R. Turner); University of California at San Francisco; University of Chicago (J. Englund, C. Elsen, R. Oram, P. Lofton); University of Colorado Health Sciences Center (A. Weinberg, M. Abzug, C. Salbenblatt); University of Florida Health Sciences Center/Jacksonville (A. Khayat, M. Matti, E. Harkey, L.M. Eagle).