Clinical and Diagnostic Laboratory Immunology, May 1999, p. 311-315, Vol. 6, No. 3
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Recombinant Human Gamma Interferon in Human
Immunodeficiency Virus-Infected Children: Safety,
CD4+-Lymphocyte Count, Viral Load, and Neutrophil
Function (AIDS Clinical Trials Group Protocol 211)
William T.
Shearer,1,*
Mark W.
Kline,1
Stuart L.
Abramson,1
Terence
Fenton,2
Stuart E.
Starr,3 and
Steven D.
Douglas3
Departments of Pediatrics and Microbiology
and Immunology, Baylor College of Medicine,1 and
Department of Allergy and Immunology, Texas Children's
Hospital,1 Houston, Texas; Frontier
Science and Technology Research Foundation, Brookline,
Massachusetts2; and Children's Hospital
of Philadelphia, Department of Pediatrics, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania3
Received 16 October 1998/Returned for modification 30 November
1998/Accepted 22 January 1999
 |
ABSTRACT |
Nineteen children with human immunodeficiency virus (HIV) infection
were treated with recombinant human gamma interferon (rIFN-
) (50 µg/m2 subcutaneously three times each week during weeks 1 through 12 and 100 µg/m2 subcutaneously three times each
week during weeks 13 through 24) in a phase I/II clinical trial. All
children continued to receive previously prescribed therapy with oral
zidovudine or didanosine. Children were assessed clinically and with
laboratory studies during 24 weeks of study treatment and for 12 weeks
after completion of rIFN- therapy. In general, rIFN- therapy was
well tolerated. There were two clinical or laboratory adverse events thought to be possibly or probably study drug associated. One child
developed acute pancreatitis; another child developed granulocytopenia. Median CD4+-lymphocyte counts and plasma HIV RNA
concentrations did not change significantly during therapy. In vitro
neutrophil bactericidal activity against Staphylococcus
aureus and superoxide production were not significantly affected
by rIFN- therapy. We conclude that rIFN- therapy in HIV-infected
children receiving single-agent antiretroviral therapy is safe and does
not produce consistent changes in CD4+-lymphocyte count,
plasma HIV RNA concentration, or in vitro neutrophil function.
| |
INTRODUCTION |
Human immunodeficiency virus (HIV)
infection is associated with important perturbations of normal cytokine
production. Decreased production of type 1 cytokines (gamma interferon
[IFN-] and interleukin 2 [IL-2]) and increased production of
type 2 cytokines (IL-4 and IL-10) among children with vertical HIV
infection have been described (20). An important role for
type 1 cytokines in HIV disease pathogenesis has been suggested by the
finding that in vitro T-cell-receptor-induced programmed
CD4+-cell death (apoptosis) can be blocked by addition of
exogenous IFN- and IL-2 (4). In addition, IFN- can
reduce in vitro HIV infectivity in human monocytes (7, 8,
10). Decreased neutrophil function in HIV-infected children has
been described (9, 16).
In vitro addition of IFN- to HIV-positive-patient-derived monocytes
or mixed lymphocyte populations can correct deficient activity against
Toxoplasma gondii (5) and Cryptococcus
neoformans (11), two important pathogens of patients
with AIDS. In vivo treatment with IFN- can restore the
toxoplasmastatic activity of monocytes from AIDS patients
(6), and case reports suggest a beneficial role for
adjunctive therapy with IFN- in patients with AIDS and disseminated
Mycobacterium avium-M. intracellulare complex infection
(18). Taken together, these studies suggest a possible role
for IFN- in the prophylaxis or treatment of complicating infections
in individuals with HIV infection.
| |
MATERIALS AND METHODS |
Patients and study design.
The study was approved by the
institutional review boards of each participating institution, and
institutional human experimentation guidelines were followed. Informed
consent was obtained from each child's parent or legal guardian. In
the case of children 7 years of age or older, assent of the minor
subject also was obtained. Eligible subjects included children and
adolescents, aged 1 to 17 years, with symptomatic HIV infection as
defined by the U.S. Centers for Disease Control and Prevention in 1987 (3). All children had been receiving therapy with zidovudine
or didanosine for more than 6 weeks at the time of study enrollment.
Thirteen patients were recruited at Texas Children's Hospital,
Houston, and six were recruited at Children's Hospital of Philadelphia.
All children continued to receive previously prescribed therapy with
oral zidovudine or didanosine at standard doses for the duration of the
study. Therapy with recombinant IFN- (rIFN-) (Genentech, Inc.,
South San Francisco, Calif.) (50 µg/m2 subcutaneously
three times each week during study weeks 1 through 12; 100 µg/m2 subcutaneously three times each week during study
weeks 13 through 24) was initiated on study day 1. Dose interruption
and reduction were prescribed in the protocol for a variety of
potential drug-associated toxicities. All patients received prophylaxis
for Pneumocystis carinii pneumonia, and nutritional support
and antibiotic therapy were prescribed as needed. The use of
immunomodulators (other than intravenous immunoglobulin) or
antiretroviral agents (other than zidovudine or didanosine) was prohibited.
Clinical and routine laboratory monitoring.
Children were
evaluated clinically at baseline and every 4 weeks through study week
36. A complete blood count, absolute neutrophil count (ANC), and
routine blood chemistries (aspartate aminotransferase [AST], alanine
aminotransferase [ALT], blood urea nitrogen, creatinine, and total
bilirubin) were monitored at baseline and every 4 weeks through study
week 36. Immunologic monitoring included lymphocyte phenotyping (ACTG
[AIDS Clinical Trials Group] certified laboratories at Baylor College
of Medicine and Children's Hospital of Philadelphia) at baseline and
at weeks 12, 24, and 36. Virologic evaluations included the measurement
of plasma HIV RNA concentrations at baseline and at weeks 12, 24, and
36. Complete HIV RNA measurements were performed at Baylor by using
spin-column technology and chemiluminescence assays of PCR products
(13). At Children's Hospital of Philadelphia, nucleic-acid-sequence-based amplification was employed (Organon Teknika, Durham, N.C.).
Assessment of phagocyte function. (i) Cell preparation.
Neutrophils were obtained from venous peripheral blood (weeks 0, 8, 20, and 32 for bactericidal activity measurements and every 4 weeks for
superoxide production assays) and purified by dextran sedimentation and
hypotonic lysis of erythrocytes (14). Bactericidal activity
and superoxide production studies were performed within 2 h of
cell separation.
(ii) Bactericidal assay.
Neutrophil killing of
Staphylococcus aureus was assessed only for the 13 Baylor
patients by a colorimetric technique modified from that used by Stevens
et al. (19). Bacteria were grown overnight in a shaking
incubator at 37°C, washed once with Dulbecco's phosphate-buffered saline plus 0.2% glucose, and adjusted to a concentration of 5 × 107/ml. Opsonization of bacteria was performed via 1 h
of incubation at 37°C with bovine antistaphylococcus serum and pooled
normal human adult serum; 5 × 105 neutrophils were
placed in each microtiter plate well (control and patient separately);
to establish a dose-titration response, control neutrophils from
healthy adults were incubated with various amounts of S. aureus (0 × 106 to 5 × 106/well). After bacteria and cells were incubated for
1 h, 0.3% saponin (S-1252; Sigma) was added to each well. Fifty
microliters of a 2-mg/ml concentration of MTT dye (M-2128; Sigma) was
added to each well and incubated for 15 min at 37°C. Plates were then centrifuged for 10 min at 1,800 × g. The supernatant
was aspirated, and 150 µl of isopropanol was added. The wells were
then mixed with a multichannel pipette, sealed with an adhesive cover,
and stored overnight at 4°C. The next day, the wells were remixed and
read in a 96-well plate reader at 560 nm. Linear regression analysis
was performed on the kill curve, and the percentages of bacteria killed
by patient samples and control samples were calculated. The assay was
performed at baseline and at 8 weeks into each of the three 12-week
therapeutic divisions of the protocol (receiving dose level 1 rIFN-,
receiving dose level 2 rIFN-, and off rIFN-). A control sample
was run at each assay point along with 1 to 3 patient samples.
(iii) Superoxide production.
The rate of superoxide
production was determined by the superoxide dismutase-inhibitable
reduction of ferricytochrome c, as previously described
(1). Cells were incubated with the reaction solution
containing 150 µM ferricytochrome c type VI (Sigma) in Hanks balanced salt solution, pH 7.3 to 7.4, at 37°C with 100 µg of
catalase and 100 ng of phorbol myristate acetate per ml with or without
100 mg of superoxide dismutase per ml. Microtiter plates were incubated
at 37°C, and change in absorbance was measured at 550 nm, with
absorbance at 505 nm as the reference standard. Measurements were done
in triplicate at 10-min intervals up to 1 h. Results were
expressed as nanomoles of superoxide produced by 106 cells
per 10 min.
Statistical analysis.
The statistical significance of
changes over time was tested by the Wilcoxon matched-pairs signed-ranks
test. Binomial probabilities were used to test the statistical
significance of the tendency for the patient values on the bactericidal
assay to fall below those of the daily controls at each time point.
| |
RESULTS |
Baseline patient characteristics.
Nineteen children were
enrolled in the study between August 1993 and July 1995. Selected
characteristics of the study population are shown in Table
1. All of the children had vertically
acquired HIV infection.
Safety.
Therapy with rIFN- was well tolerated by most
children (Table 2). Four children
developed a fever between 38.5 and 40.0°C which lasted for less than
48 h after one or more rIFN- injections. Moderately severe
vomiting and anorexia were observed in a 6-year-old girl who developed
pancreatitis of unknown etiology. One patient developed mild vomiting
and one developed anorexia during the course of the study. Two children
developed liquid diarrhea; one case was associated with mild
dehydration.
Adverse events observed in laboratory evaluations during the course of
the study included moderate or more severe (toxicity grade 2 or higher)
abnormalities of ANC, blood hemoglobin concentration, platelet
concentration, serum bilirubin concentration, serum ALT concentration,
and serum AST concentration (Table 2).
There was no apparent relationship between rIFN- dosage or duration
of drug exposure and occurrence of clinical or laboratory adverse
events. Only two clinical or laboratory adverse events were assessed by
the investigators as possibly or probably study drug associated. A
6-year-old girl developed pancreatitis of unknown etiology at study
week 4. Therapy with both didanosine and rIFN- was permanently
discontinued, and the pancreatitis resolved after a severe illness and
a protracted course of hospitalization. An 18-month-old boy with a
baseline ANC of 1,300/µl developed ANC values as low as 380/µl
while receiving zidovudine and rIFN-. Treatment with rIFN- (but
not zidovudine) was interrupted temporarily at study weeks 6 and 20 and
discontinued permanently at study week 24 without an obvious effect on
subsequent ANC values. Two other children discontinued study treatment
prematurely due to HIV disease progression and parental request.
Clinical observations.
Compared with age-appropriate normal
values (17), the median 6-month weight-growth velocity for
the 15 children evaluable after receiving rIFN- for 24 weeks reached
the 15th percentile. Seven children had 24-week weight-growth
velocities below the 10th percentile for their age.
No AIDS-defining symptoms were observed during the course
of study treatment. A 2-year-old girl developed
Streptococcus pneumoniae bacteremia during study week 16 and
pulmonary tuberculosis during study week 24. A 20-month-old girl
developed what was thought to be HIV-associated pancytopenia during
study week 12 and died shortly thereafter of hemorrhagic brain infarction.
Immunologic and virologic observations.
Exploratory
longitudinal analyses of CD4+-lymphocyte counts and plasma
HIV RNA concentrations did not reveal consistent changes (Tables
3 and 4).
The median baseline absolute CD4+-lymphocyte count for the
13 children with complete data for the 36 weeks of study was 331/µl
(range, 0 to 1,047/µl). The same children had median absolute
CD4+-lymphocyte counts of 298, 191, and 232/µl at study
weeks 12, 24, and 36, respectively. Analysis of change in
CD4+-lymphocyte count was restricted to patients with
complete data from study weeks 0, 12, 24, and 36. Table 3 indicates
that there was no essential change between weeks 0 and 12, there was a
nonsignificant decline between weeks 12 and 24, and there was an
apparent gain during the follow-up period between weeks 24 and 36. A
Wilcoxon sum ranks test indicated that the gain after discontinuation
of rIFN- therapy was statistically significant, but given the
overall pattern of changes, this increase may represent a chance
occurrence. CD8+-T-cell counts and T-cell subset
percentages did not differ.
The median baseline plasma HIV RNA concentration among the 10 children
at Baylor with complete data for the 36 weeks of study was 14,470 copies/ml (range, 1,512 to 496,800). The same children had median
plasma HIV RNA concentrations of 12,850, 14,580, and 10,040 copies/ml
at study weeks 12, 24, and 36, respectively. Analysis of change in
plasma HIV RNA concentration was restricted to a subgroup of 10 patients from Baylor whose results came from the same assay and who had
complete data from study weeks 0, 12, and 24. Table 4 shows that these
patients had modest decreases in plasma HIV RNA concentration, with a
median decline of 0.22 log10 copies/ml from baseline to the
end of treatment at week 24. A Wilcoxon matched-pairs test indicated
that the observed changes were not significant. Plasma HIV RNA
concentrations also were measured in the eight subjects at Children's
Hospital of Philadelphia, but because the values were obtained with a
different RNA assay, only the Baylor data are shown. The RNA values
obtained on these eight subjects did not show significant change with
rIFN- therapy.
Neutrophil function.
No significant change was observed during
the course of the study in neutrophil bactericidal activity against
S. aureus relative to week 0 (Table 5; Fig.
1). Because
of the decrease in the median value at week 8, there appears to be an
increase in bactericidal activity at weeks 20 and 32. It is possible
that a study with additional subjects would clarify whether this
apparent increase is real. Variability between controls was
considerably lower, with almost all controls recording 70 to 95%
killing (data not shown). At weeks 20 and 32 only, one patient had a
value of killing that was less than 50% of the control value, as
contrasted to several patient values less than 50% of the control
value at weeks 0 and 8. Patient values were significantly less than
control values at all time points (Table 5).
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|
FIG. 1.
Neutrophil bactericidal activity in 13 patients treated
with rIFN-. Values are the percentages of the neutrophil killing of
S. aureus by controls. Subjects were given subcutaneous
rIFN- (50 µg/m2 during weeks 1 to 12 and 100 µg/m2 during weeks 13 to 24). The median values are
indicated by horizontal lines in the columns.
|
|
Patients were assessed every 4 weeks for non-receptor-mediated (i.e.,
phorbol myristate acetate-induced) superoxide production. Values ranged
from 0.6 to 58.9 nmol/106 neutrophils/10 min. IFN-
failed to produce significant changes in values (Table
6) over the course of the study.
| |
DISCUSSION |
There is theoretical support for use of immune modulators,
including rIFN-, for restoration or correction of HIV-induced immune
dysfunction. A previous phase I/II trial of rIFN- (10 µg/m2 intramuscularly three times each week for 16 weeks)
in adults with AIDS-related complex did not lead to either beneficial
or detrimental changes in immunologic or virologic markers of HIV disease status (2). The present study was conducted to
derive preliminary information on the tolerance, safety, and
immunologic and virologic effects of rIFN- in HIV-infected children
receiving long-term zidovudine or didanosine therapy.
Therapy with rIFN- generally was well tolerated in this population
of children with symptomatic HIV infection. However, we found that the
necessity of injections was a disincentive to enrollment and led to
premature discontinuation of study treatment in three cases. Only two
clinical or laboratory adverse events were judged to be possibly or
probably associated with rIFN- treatment, but it is also possible
that these events were attributable to underlying HIV disease or
treatment with other medications. For example, there is a
well-recognized association between didanosine therapy and the
occurrence of acute pancreatitis (15), which might have accounted for one of these events.
This study was not designed to assess the clinical efficacy of rIFN-
therapy, and all clinical findings must be interpreted in the context
of advanced HIV disease. One child developed two serious bacterial
infections during the course of study treatment, and a surprisingly
large number of children experienced growth failure. Among HIV-infected
children, 6-month weight-growth velocities below that of the 10th
percentile for age and gender are associated with higher rates of
mortality (12). Since rIFN- therapy was added to an
antiretroviral therapeutic regime (monotherapy with zidovudine or
stavudine) which we now know to be suboptimal in controlling HIV-1
viral burden, it is difficult to ascribe this growth failure to
rIFN-, since inadequate primary treatment of HIV-1 is known to be
associated with growth failure.
Examination of median absolute CD4+-lymphocyte counts
revealed a decline over the 24 weeks of study treatment that was not statistically significant, with a modest rebound in count after discontinuation of rIFN- therapy. Significant changes in median plasma HIV RNA concentrations were not observed, and the mechanism for
any possible effect of rIFN- on CD4+-lymphocyte counts
is unclear. Phagocyte function was evaluated by assessment of the
bactericidal activity of HIV-infected-patient neutrophils and by their
ability to produce superoxide. Neither neutrophil function was altered
by giving study subjects three-times-weekly subcutaneous injections of
rIFN-. Whether the shift in some patients' bactericidal activity to
more than 50% of control killing at 20 weeks of rIFN- therapy and
at 8 weeks after stopping therapy has any in vivo significance is unknown.
In summary, this phase I/II trial of rIFN- in symptomatic
HIV-infected children has demonstrated that the cytokine is safe and
well-tolerated. Whether the modest changes in
CD4+-lymphocyte counts which were observed during the trial
are related to the study drug is unknown. Neutrophil bactericidal
activity and superoxide production were not changed by therapy.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge R. Nelson Bennett, Pamela Bouquin,
Donald Campbell, Peter Chvany, John G. Curd, Yih J. Danels, Courtney V. Fletcher, Richard Gelber, Elizabeth Hawkins, Linda M. Page, Deborah H. Schaible, Cara Simon, and Dennis Weller for technical and scientific
assistance. Genentech, Inc., supplied recombinant human
interferon-gamma.
This work was supported by Pediatric AIDS Clinical Trials Group grants
UO1-AI27551 and UO1-AI32921 from the National Institutes of Health
(NIH) National Institute of Allergy and Infectious Diseases, NIH grants
MO1-RR00188 and MO1-RR00240 from the General Clinical Research Centers
program, NIH grant P30-AI 36211 from the Center for AIDS Research
program, and grant 50425-15-PG from the Pediatric AIDS Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Texas
Children's Hospital, Department of Allergy and Immunology, 6621 Fannin
St., MC 1-3291, Houston, TX 77030. Phone: (713) 770-1274. Fax: (713)
770-7131. E-mail: wshearer{at}bcm.tmc.edu.
| |
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Clinical and Diagnostic Laboratory Immunology, May 1999, p. 311-315, Vol. 6, No. 3
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
