While the introduction of highly active antiretroviral regimens has been associated with a significant decrease in the morbidity and mortality of HIV infection, it has not resulted in viral eradication [1–4]. Furthermore, many patients experience virological failure in the face of such therapy and are often left without effective therapeutic options [5–8]. This has led to the search for alternative or complementary approaches to treatment, including immune-based therapies . Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an immodulatory agent that has demonstrated potential therapeutic benefit on viral load and CD4 cell count in recent clinical studies [10,11].
GM-CSF has pleiotrophic effects on the human immune system, enhancing the number and function of both myeloid-derived and lymphoid-derived cells [12–17]. GM-CSF production is significantly reduced in persons with AIDS [18,19]. Thus, replacement therapy may improve neutrophil, monocyte/macrophage, and dendritic cell function, and potentially prevent or clear opportunistic infections (OI). GM-CSF treatment of cell lines chronically infected with HIV in vitro has been reported to upregulate viral replication . However, this activity is blocked by the addition of low doses of zidovudine because GM-CSF significantly increases the intracellular concentration of active metabolites of this agent. [21,22] GM-CSF has recently been shown to downregulate HIV co-receptor expression by monocyte-derived macrophages in vitro and to render these cells resistant to infection with HIV-1 [23–25]. Thus, GM-CSF may prove to have a role in HIV treatment as an adjunct to antiretroviral therapy. Two randomized, placebo-controlled trials, one a pilot trial of 20 subjects, and the other a Phase II study of 105 subjects, have demonstrated that GM-CSF administered two or three times weekly may augment the effects of antiretroviral therapy on CD4 cell counts and viral load, and may delay the evolution of zidovudine resistant genotypes. [10,11]. The present study was initiated before our current understanding of these effects of GM-CSF on HIV replication, and was designed to investigate the potential role of this agent in the prevention of OI in and death of patients with advanced HIV infection.
HIV seropositive individuals with either a prior OI and CD4 cell count ≤ 100 × 106/l, or no prior OI and CD4 cell count ≤ 50 × 106/l were enrolled. Subjects were required to have received at least 28 days of a stable antiretroviral regimen by study entry; however, during the study regimen change was permitted by protocol using defined criteria from the 1996 NIH-Consensus guidelines . Requisite laboratory values at entry included hemoglobin > 6 g/dl, platelet count ≥ 50 000/μl, absolute neutrophil count (ANC) > 750× 106/l serum creatinine ≤ 2.0 mg/dl, total bilirubin ≤ 2.5 mg/dl and aspartate transaminase and alkaline phosphatase less than five times the upper limit of normal. Concurrent use of other colony stimulating factors, cytokines, vaccines or other investigational immunomodulators was not allowed in the 4 weeks prior to and during study. Institutional Review Board approval was obtained at each site, and written informed consent was required of all eligible subjects before enrollment.
This Phase III, multicenter, double-blind, randomized, placebo-controlled trial was designed to evaluate the efficacy and safety of GM-CSF treatment in subjects with advanced HIV disease. Subjects were stratified based upon viral load at baseline (≤ 30 000 copies/ml versus > 30 000 copies/μl) and randomized to receive either GM-CSF (sargramostim, Immunex, Seattle, Washington, USA) 250 μg or placebo subcutaneously three times per week for 24 weeks. A voluntary extension phase permitted delivery of blinded drug for up to 20 months. All subjects underwent evaluation at baseline (within 2 weeks prior to entry), days 15, 29, 85, and 169 of therapy, as well as months 9, 12, 15, and 18 for subjects enrolled in the extension phase. Each evaluation included a medical history, physical examination, serum chemistry panel, hematology panel, CD4 lymphocyte counts by flow cytometry, and cryopreservation of plasma at −70 °C for batch analysis of HIV-RNA. All viral load measurements were performed using Amplicor (lower limit of detection 400 copies/ml) in a central laboratory (Lab Corp, Burlington, North Carolina, USA) by trained personnel blinded to study assignment. HIV-RNA levels ≤ 400 copies/ml were entered as 400 for purposes of data analysis. Toxicity was graded on a 5-point scale (0–4) based on the Common Toxicity Criteria of the National Cancer Institute. All subject records were reviewed by the Data Safety and Monitoring Board. All data regarding clinical events (bacterial pneumonia, OI and deaths) were collected at each site and then formally reviewed to affirm the assigned diagnosis by a Clinical Events Committee (CEC). The CEC assignment occurred prior to unblinding of the database. The diagnosis and severity of infections other than OI were assigned by investigators and entered into the database separately.
The randomization schedule was computer generated in a block design, and subjects were sequentially assigned to treatment within the appropriate stratum according to the subject's baseline viral load.
GM-CSF was supplied as a lyophilized powder in vials containing 500 μg of recombinant human (rhu) GM-CSF protein; 40 mg mannitol; 10 mg sucrose; and 1.2 mg tromethamine. The placebo supplied was identical except for the absence of rhu GM-CSF protein. Both were reconstituted with 1.0 ml of bacteriostatic water for injection, containing 0.9% benzyl alcohol.
The primary endpoint of this trial was the incidence of clinical events, specifically Centers for Disease Control and Prevention-defined opportunistic infections, bacterial pneumonia or death. Overall infections were also evaluated and included all clinical events and any other infections reported by investigators regardless of type or severity. The trial was sized assuming a 25% incidence of clinical events among placebo subjects over 6 months , with 150 subjects per treatment group providing 82% power to detect a 40% reduction in this rate with GM-CSF treatment with a 12-month accrual period. All subjects receiving at least one dose of blinded study medication were included in the intent-to-treat analyses of efficacy and safety. Time-to-event data were compared between treatments using the log-rank test stratified by baseline HIV-RNA. The proportion of subjects experiencing infections, whether clinical events or overall infections, during study were compared between treatments using the Mantel–Haenszel test stratified by baseline HIV-RNA. All other comparisons between treatment groups were made using the likelihood ratio chi-square test. Fisher's exact test was applied when the likelihood ratio chi-square test may be inappropriate due to low expected cell frequencies. Treatment comparisons of changes from baseline in CD4 cell counts, HIV-RNA (log10 scale), ANC and maximum intensity of infection were made using stratified rank tests. A last observation carried forward (LOCF) approach was used in the summary and analysis of HIV-RNA and CD4 cell count data through 24 weeks. To examine the impact of changes in antiretroviral therapy during study, HIV-RNA data was analyzed, both with all data and with data censored for change in antiretroviral regimen. In the censored analyses, all evaluations subsequent to the first change in antiretroviral therapy were treated as missing, and the last evaluation prior to the change was carried forward. All P values are two-sided.
Between November 1996 and August 1998, 309 subjects were enrolled at 48 USA and Canadian centers. Baseline demographics were balanced between groups as shown in Table 1. The type and duration of antiretroviral therapy were similar between groups. Prophylaxis against opportunistic infections was administered to nearly all subjects at baseline and was similar between the two groups.
Overall, 70% of subjects completed 24 weeks of therapy. Within each treatment group, similar proportions of subjects remained on study at 1 month (94% placebo versus 95% GM-CSF), 3 months (86% placebo versus 86% GM-CSF), and 6 months (71% placebo versus 69% GM-CSF). Approximately 45% of subjects in each group continued on into the blinded extension phase for an additional median duration of 6.2 months.
GM-CSF three times a week produced a significant increase in total lymphocyte and CD4 lymphocyte counts by 1 month that almost doubled by 6 months (Fig. 1). More than 45% of individuals receiving GM-CSF had CD4 cell counts > 100 × 106/l at 6 months. GM-CSF subjects continuing on the extension phase demonstrated a progressive increase in CD4 cell count from baseline; yielding mean counts by 12 months of 102 + 15 × 106/l for placebo versus 152 + 18 × 106/l for GM-CSF. Mean changes from baseline in total lymphocyte counts also increased with therapy paralleling the rise in CD4 cell counts over the 6 months of study (150 ± 45 × 106/l placebo versus 295 ± 9 × 106/l GM-CSF). In addition the GM-CSF group also demonstrated an increase in mean change in CD4 percentage relative to placebo by 6 months, but this did not achieve statistical significance (+ 0.9% versus + 2.0%;P = 0.39).
ANC also increased within 2 weeks of GM-CSF therapy initiation by a median of approximately 800 × 106 cells/l, and then remained stable from 2 weeks to 6 months compared with no increase in the placebo group; however, no subject was withdrawn because of leukocytosis or eosinophilia. In fact, the range of median ANC values by visit for both groups up to 6 months remained within the normal range (1877–2084 × 106 cells/l placebo versus 1769–2909 × 106 cells/l GM-CSF), thus limiting any potential unblinding by patient/provider knowledge of ANC values.
HIV-RNA values decreased with time on study in both treatment and placebo groups; however, no significant difference in viral load change from baseline was observed between the two groups overall, or when analyzing only the high viral load stratum (> 30 000 copies/ml). In subjects with < 30 000 copies/ml at baseline, lower median values were observed at all time points in the GM-CSF treatment group and these differences became more pronounced when subjects were censored after a change in antiretroviral therapy (Table 2).
Among subjects who entered the treatment phase with viral load levels below the limit of detection (< 400 copies/ml), significantly more GM-CSF-treated subjects maintained viral loads below the limit of detection at 6 months [15/28 (54%) placebo versus 24/29 (83%) GM-CSF;P = 0.02, LOCF analysis) regardless of regimen changes, and at both 3 months [19/28 (68%) placebo versus 27/29 (93%) GM-CSF;P = 0.01, LOCF analysis] and 6 months [14/28 (50%) placebo versus 25/29 (86%) GM-CSF, P = 0.003, LOCF analysis] when subjects were censored for regimen change.
Changes in antiretroviral therapy may have obscured a preferential decrease in viral load with GM-CSF therapy. Therefore, regimen changes in response to increases in viral load as defined in the protocol were analyzed. In the low viral load stratum, significantly fewer changes in antiretroviral therapy were observed in the GM-CSF group compared with controls (38% placebo versus 19% GM-CSF;P = 0.03), and time to regimen change was significantly longer for the GM-CSF group (P = 0.01, Fig. 2). First quartile and median time were estimated to be 137 days and 337 days for the placebo group, but could not be estimated for the GM-CSF group due to the low incidence of subjects changing treatment for virological failure. No difference was observed for the incidence of treatment change in the high viral load stratum (62% placebo versus 62% GM-CSF;P = 0.68).
The incidence of OI, bacterial pneumonia and deaths, and the incidence of any specific OI during study did not differ statistically between the placebo and treatment groups (Tables 3 and 4). The event rate in the placebo arm was 18%, which was 28% lower than the anticipated rate of 25%. No difference in OI incidence was observed between groups based on viral load stratum or CD4 cell count. In the GM-CSF subjects without a prior history of OI there was a non-statistically significant difference in these events [7/27 (26%) placebo versus 2/28 (7%) GM-CSF;P = 0.08]. Prophylaxis for cytomegalovirus, Pneumocystis carinii pneumonia, or Mycobacterium avium complex was discontinued at the investigators’ discretion during the study in more GM-CSF subjects (6% placebo versus 12% GM-CSF;P = 0.07), perhaps biasing the results toward more OI in the GM-CSF group. OI developed subsequently in one placebo- and four GM-CSF-treated subjects in whom prophylaxis had been discontinued.
The incidence of overall infections (OI and non-OI) or death during study was significantly lower in the GM-CSF group relative to placebo [120/154 (78%) placebo versus 104/155 (67%) GM-CSF;P = 0.03] (Table 4). The median time to first infection or death was significantly prolonged in the GM-CSF group (56 days placebo versus 97 days GM-CSF;P = 0.04). The mean maximum intensity score of the infections assigned by investigators on a 0 (none) to 4 (death) scale, also showed a trend to be lower in the GM-CSF group (1.55 placebo versus 1.33 GM-CSF;P = 0.09).
GM-CSF was safe and well-tolerated when administered to subjects with advanced HIV disease receiving antiretroviral therapy. No difference was observed between groups for hospitalizations (42 placebo versus 45 GM-CSF) or deaths (seven placebo versus six GM-CSF), none of which were related to study drug. Adverse events in both groups were mainly mild in intensity (grade 1/2). Only two events, injection site reactions [6/154 (4%) placebo versus 39/155 (25%) GM-CSF;P = 0.001], and weight loss ≥ 5% of body weight [15/154 (10%) placebo versus 28/155 (18%) GM-CSF;P = 0.03] occurred more frequently in the GM-CSF group. Injection site reactions were mild and occurred only infrequently.
In vitro studies have demonstrated that GM-CSF increases the resistance of macrophages to HIV infection both by down-regulation of CCR5 and CXCR4 chemokine receptor expression on monocyte-derived macrophages and by inducing monocytes/macrophages to secrete beta-chemokines that competitively inhibit HIV entry in bystander CD4 T cells [23–25], and augments the activity of antigen-presenting cells, neutrophils, macrophages, and lymphocytes [13–17]. GM-CSF also increases the intracellular concentration of azido-nucleoside active metabolites [21,22]. Recent randomized trials of GM-CSF in individuals who are HIV positive have demonstrated effects in vivo including increased CD4 cell counts, decreased HIV-RNA and decreases in the number of persons with detectable zidovudine-resistant viral genomes [10,11].
To our knowledge the present study in more than 300 patients represents the first demonstration of a favorable immunological, virological, and clinical response to an immune-based therapy in a randomized, placebo- controlled trial in HIV-infected individuals. Thrice weekly low dose GM-CSF was associated with increased CD4 cell counts, increased time of viral load suppression, and delayed change in antiretroviral therapy for virological failure. Also, a decrease in the overall infection rate was observed in individuals with late stage disease treated with GM-CSF. These results were observed in subjects receiving community standard of care antiretroviral therapy which included a wide variety of regimens that were balanced for specific agents between arms by randomization. This study indicates that GM-CSF has a beneficial effect in advanced HIV disease that is not specific to the use of a particular antiretroviral regimen.
The maintenance of viral suppression is a primary objective of antiretroviral therapy. Although no significant difference in viral load was observed overall between the two groups in this study, GM-CSF use delayed virological failure among persons with HIV-RNA < 400 copies/ml at baseline, and decreased the need to change regimens in subjects with HIV-RNA levels between 400 and 30 000 copies/ml at baseline. Thus, GM-CSF may provide additional inhibition of HIV replication sufficient to prevent breakthrough viral replication or the development of resistant genotypes. A prior study utilizing twice weekly GM-CSF demonstrated decreases in viral load over a 3–6 month period in persons with HIV-RNA ranging from < 400 copies/ml to > 500 000 copies/ml and receiving mono or dual nucleoside therapy . This is clinically relevant if GM-CSF treatment results in the modification of susceptible cells to resist infection during brief periods of non-adherence to complex antiretroviral regimens or use of less optimal therapy.
The use of highly active antiretroviral therapy in persons with CD4 cell counts < 100 cells/μl often fails to produce significant increases in CD4 cell counts over 6–12 months of therapy [28,29]. GM-CSF-treated subjects clearly demonstrated significant increases in CD4 lymphocyte count by 1 month of therapy, which progressively increased over 12 months. The favorable effect on suppression of plasma viremia may partially explain the CD4 T-cell increase observed, however CD4 T-cell increases were also observed in subjects with HIV-RNA > 30 000 copies/ml. Alternatively, GM-CSF may increase proliferation and differentiation of early bone marrow progenitors. Subjects in this study demonstrated increases in several lineages, including ANC and total lymphocyte count, and pro-lymphocytes which are known to express the GM-CSF receptor.
Increased lymphocyte and neutrophil cell counts were associated with a significant decrease in overall infection rate and time to first infection, even though no difference in predefined clinical events (OI, pneumonia, and death) was observed. This may be explained by the fact that general infections were observed in a majority of subjects while the incidence of predefined clinical events was substantially lower than anticipated, probably due to the introduction of multiple protease inhibitors. Despite the overall low incidence of predefined clinical events one subgroup of subjects without a history of OI demonstrated a trend toward fewer OI. This was also observed in a previous trial .
The ability of GM-CSF to maintain the suppression of plasma viremia in subjects on antiretroviral therapy presents another option in the management of HIV-positive individuals. Although viral suppression can usually be achieved in 70–80% of individuals initiating their first potent antiretroviral regimen, sustained responses are only achieved in 40–50% of treatment-experienced individuals on their second protease inhibitor-based regimen [31–36]. Virological failure may be delayed considerably in these individuals by the addition of GM-CSF to an effective antiretroviral regimen. Whether the ability of GM-CSF to maintain viral suppression as observed in the advanced HIV population will translate to individuals with earlier stages of HIV disease remains to be evaluated. Additional studies to evaluate further the efficacy of GM-CSF in individuals with advanced HIV who are receiving antiretroviral regimens are warranted.
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