Subjects were eligible to enroll into AIDS Clinical Trials Network Protocol 5192 if they had a CD4+ T-cell count ≥ 300 cells/mm3, plasma HIV-1 RNA ≥ 5000 copies/mL, and were ART-naïve or were ART-experienced but currently off therapy for at least 12 weeks. The patients must have been negative for HBV surface antigen and HCV antibody and have less than grade 1 transaminase levels at entry. Exclusion criteria included a history of severe psychiatric illness or any history of a chronic illness, such as cardiopulmonary disorder, that could be worsened by interferon therapy. All participants expressed a willingness to defer initiation (or re-initiation) of ART until after the completion of the study, though a safety clause for study withdrawal for CD4+ T-cell count ≤ 200 cells/mm3 was stipulated in the toxicity management section of the protocol. Filgrastim (Neupogen; provided by Amgen), a granulocyte colony-stimulating factor analog, was available for treatment of neutropenia through the study for providers to use per local standard of care practices. Written informed consent was obtained from all subjects.
The primary endpoints were change in plasma HIV-1 RNA from baseline to week 12, and safety and tolerability of PegIFN (provided by Roche Pharmaceuticals) at 180 μg given subcutaneously weekly by study personnel for 12 weeks. Post hoc, a decision was made to include analyses at weeks 1 and 2, when the largest decreases in HIV-1 RNA were seen. Secondary objectives included: assessment of HIV-1-specific CD4+ T-cell immunity while on treatment compared with baseline levels by lymphocyte proliferation response (LPA) to p24Ag and whole inactivated HIV-1 antigen; measurement of weekly and end of study serum trough levels of PegIFN and 2’,5’, oligoadenylate synthetase (OAS); durability of the virologic and immunologic responses to peginterferon alfa-2a therapy 6 weeks after discontinuation of study drug (week 18); and correlations among baseline and concurrent week-specific changes in plasma HIV-1 RNA, CD4+ T-cell count and OAS, and concurrent absolute concentrations of peginterferon alfa-2a and OAS. Post hoc examinations of the effect of missed doses on viral load change were performed. After the correlations between OAS and HIV-1 viral load changes were observed, a substudy was designed to explore the induction of IFIG.
Viral load measurements were performed by the Amplicor HIV-1 Monitor PCR assay (Roche Diagnostics, Indianapolis, IN) at pre-entry and entry and then during weeks 1, 2, 3, 4, 6, 8, 10 and 12 just prior to each weekly injection of study drug as well as at weeks 13 and 18 (2 and 7 weeks after therapy had been discontinued). With the exception of week 1, CD4+ T-cell counts were measured at the same times.
Conventional LPA were performed on freshly obtained PBMC within 24 hours of collection using the following stimulants: tetanus, Candida, phytohemaglutinin (PHA), HIV-1 p24 antigen, and whole inactivated HIV-1 antigen. LPA is described in terms of the stimulation index (SI), which is defined as the median counts per minute (CPM) in the stimulated replicates divided by the median CPM in the appropriate control replicates.
PegIFN concentrations were measured by Quest Pharmaceutical Services (QPS) [Newark, DE] using an enzyme-linked immunosorbent assay that has been used previously, with lower limits of quantification of 0.250 ng/mL for alfa interferon (18
). The assay is specific for pegylated interferon and does not recognize non-pegylated (endogenous) interferon up to a concentration of 25 ng/mL. Post-treatment interferon levels reported as below the lower limit of quantification (LLQ, 0.250 ng/mL) were assigned the value 0.125 ng/mL (one-half the LLQ). Average steady-state trough interferon concentration was calculated as the mean of concentrations at weeks 6, 8, 10 and 12 (a priori
definition). For subjects for whom week 0 and at least one other PegIFN concentration were available, area under the pegylated interferon trough concentration-time curve (AUC) was calculated from week 0 to 12 using the linear trapezoidal rule as an estimate of total drug exposure after multiple weekly doses. PegIFN clearance was calculated assuming complete absorption from subcutaneous injections as the dosing rate (180 μg/week, 135 μg/week for subjects with reduced doses) divided by average steady-state trough concentration, divided by 168 to obtain L/h. Weight-adjusted clearance was calculated as clearance divided by the subject’s pre-treatment weight (kg).
The activity of 2’–5’OAS in serum was measured in duplicate by QPS using a radioimmunoassay assay (RIA) kit obtained from EIKEN Chemical Co LTD, Japan and distributed by ALPCO Diagnostic, that measures the amount of ATP converted into oligoadenylate. OAS levels reported as ‘undetectable’ (10 pmol/dL) were assigned the value 5 pmol/dL. Three distinct isoforms of OAS exist in human cells, small, medium and large. Based on the principle of the RIA assay used, it is assumed that this assay measures total activity of OAS.
PBMC stored at weeks 0, 3, 6, 12 and 18 were used to measure IFIG. Quantitation was performed using a novel customized bDNA multiplex assay capable of detecting the expression of 35 genes (11
). The expression of individual genes was measured in relation to housekeeping genes, i.e., in a one-to-one relationship. The IFIG levels reported herein represent the average mean fluorescence intensity (MFI) across the individual measured genes.
The study was designed to provide 80% power, with twelve evaluable subjects, to detect a 0.56 log10 change in viral load at week 12, assuming use of a one-sided t-test with alpha set to 5% and a standard deviation of 0.88 log10. After study design, the decision was made to use nonparametric methods and two-sided confidence intervals (CIs) with significance level 0.10 without adjustments for multiple testing. Continuous measures are summarized by medians and associated 90% CI. Rank-based Spearman correlations, adjusted for bias using Fisher’s z-transformation, assessed associations between continuous variables. Baseline viral load was defined as the average of pre-entry and entry levels.