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Evidence of Ongoing Immune Reconstitution in Subjects with Sustained Viral Suppression following 6 Years of Lopinavir-Ritonavir Treatment
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Clinical Infectious Disease March 1, 2007
Alan Landay,1 Barbara A. da Silva,3 Martin S. King,3 Mary Albrecht,4 Constance Benson,5 Joseph Eron,7 Marshall Glesby,9 Roy Gulick,5 Charles Hicks,8 Harold Kessler,1 Robert Murphy,2 Melanie Thompson,10 A. Clinton White, Jr.,11 Peter Wolfe,6 Florence I. McMillan,3 and George J. Hanna3
1Rush University Medical Center and 2Northwestern University, Chicago, and 3Abbott Laboratories, Abbott Park, Illinois; 4Harvard University, Boston, Massachusetts; 5University of California, San Diego, and 6Pacific Oaks Research, San Francisco, California; 7University of North Carolina, Chapel Hill, and 8Duke University Medical Center, Durham, North Carolina; 9Weill Medical College of Cornell University, New York, New York; 10AIDS Research Consortium of Atlanta, Atlanta, Georgia; and 11Baylor College of Medicine, Houston, Texas
".....In summary, this study is the first to demonstrate continuous immune reconstitution [mean Cd4 increase was 528 after 6 years] for up to 6 years in subjects with virological suppression who are receiving a lopinavir-ritonavir-based HAART regimen. In addition, the level of CD4+ T cell immune reconstitution is similar for all subjects, regardless of baseline CD4+ T cell count. Furthermore, levels of CD4+ and CD8+ T cell activation appear to normalize by year 6 of therapy. Our findings support a strong association of potent and durable viral suppression with improved and continued immune reconstitution...... This study represents the longest follow up (6 years) of patients on a lopinavir-ritonavir-containing regimen and demonstrates that, in subjects with sustained virologic suppression, continued immune reconstitution is possible. Even in subjects in the lowest CD4+ T cell count strata of <50 cells and 50-199 cells, there were increases in CD4+ T cell counts similar to those for subjects in the higher CD4+ T cell count strata. The significant increases in CD4+ T cell count observed in this study were accompanied by stable CD8+ T cell counts, except for subjects in the lower CD4+ T cell count strata, for whom CD8+ T cell counts increased. This led to an overall significant increase in the CD4+ : CD8+ T cell count ratio, from a baseline value of 0.38 to 0.96 at week 312. In addition to the T cell subset changes, we observed normalization of B cell and NK cell populations. Furthermore, activation levels (HLA-DR and CD38 positive) of both CD4+ T cells (mean value, 4.1%) and CD8+ T cells (mean value, 7.7%) normalized by week 312...."
Introduction
HAART is associated with significant immune reconstitution and reduction in mortality in HIV-1-infected patients [1]. Few data exist on how the time course of immunologic reconstitution over several years of therapy differs between patients who start HAART with low versus high CD4+ T cell counts. Studies that have followed cohorts of patients receiving diverse HAART regimens for longer than 3 years [2-4] have suggested that a plateau in the CD4+ T cell increase occurs after 3 years of therapy, even when therapy was initiated at CD4+ T cell counts <200 cells/uL. However, the lack of uniformity in viral suppression reported in these studies limits their interpretation. Few studies have assessed the effect of HAART on long-term immune reconstitution of >5 years [5-12].
To assess the impact of long-term antiretroviral therapy, we analyzed immune reconstitution in a group of antiretroviral-naive subjects enrolled in a phase II multicenter study of therapy with lopinavir-ritonavir, stavudine, and lamivudine. This study represents, to our knowledge, the longest and largest study to evaluate immune reconstitution in subjects with sustained suppression of plasma HIV-1 RNA to <50 copies/mL during treatment with a specific antiretroviral regimen.
ABSTRACT
Background. We evaluated the immunologic impact of highly active antiretroviral therapy in subjects who maintained human immunodeficiency virus type 1 (HIV-1) suppression through 6 years of receiving a lopinavir-ritonavir-based regimen.
Methods. A total of 100 antiretroviral-naive subjects with any CD4+ T cell count initiated therapy with lopinavir-ritonavir, stavudine, and lamivudine. Sixty-three subjects who remained in the study for 6 years were assessed. Laboratory measurements included plasma HIV-1 RNA levels, multiparameter flow cytometry of immune cells, and markers of maturation and activation.
Results.
After 6 years, 62 of 63 subjects had plasma HIV-1 RNA levels <50 copies/mL.
The mean increase in CD4+ T cell count was 528 cells/L (P < .001), and 81% of subjects had CD4+ T cell counts >500 cells/L, compared with 21% of subjects at baseline.
The mean ratio of CD4+ T cell count to CD8+ T cell count increased from 0.38 at baseline to 0.96 at year 6 (P < .001).
The percentage of subjects with cell counts below the lower limit of normal at year 6, compared with at baseline, was significantly decreased for total T cells, B cells, and natural killer cells.
At year 6, the median CD4+ T cell activation level was 3.4%, and the median CD8+ T cell activation level was 5.8%.
Conclusions. The receipt of a lopinavir-ritonavir-based regimen resulted in ongoing immune reconstitution through 6 years of therapy in a cohort of HIV-1-infected, antiretroviral-naive subjects with suppressed HIV-1 RNA levels. Normalization of activation marker expression on CD4+ and CD8+ T cell subsets was demonstrated.
MATERIALS AND METHODS
Study design. Details of the study design and results through 4 years have been reported elsewhere [13, 14]. The study enrolled 100 antiretroviral-naive HIV-1-infected adults with a baseline plasma HIV-1 RNA >5000 copies/mL and any CD4+ T cell count to receive combination therapy with lopinavir-ritonavir, stavudine, and lamivudine. All subjects who were enrolled in the study provided written, informed consent, and all sites received institutional review board approval. Sixty-three subjects remained enrolled in the study for 312 weeks (6 years). Plasma HIV-1 RNA levels were quantitated using the Roche Amplicor HIV-1 Monitor assay with a lower limit of detection of 400 copies/mL (at least every 12 weeks) or 50 copies/mL (at least yearly).
Flow cytometric studies for lymphocyte subsets. The lymphocyte subset analysis was performed on fresh samples using 4-color flow cytometric analysis with a Becton Dickinson FACSCalibur Instrument using multiset software. CD4+ and CD8+ T cell counts, B cell (CD19+ cell) count, natural killer (NK) cell (CD16+ and/or CD56+ cell) count, and total T cell (CD3+ cell) count were obtained at each study visit. At the year 6 visit, naive (CD45RA+, CD62L+) and memory (CD45RO+, CD45RA-) CD4+ T cell counts and activation markers (HLA-DR and CD38 positive) were also determined.
Statistical analysis. All analyses were conducted using data from the subjects who remained in the study for at least 6 years (n = 63), with the exception of summaries of baseline characteristics and certain sensitivity analyses. Mean changes from baseline to each visit and/or mean values over time were assessed for CD4+ T cell counts, CD8+ T cell counts, and ratio of CD4+ T cell count to CD8+ T cell count (CD4+ : CD8+ T cell count ratio) by strata of baseline CD4+ T cell count. Because baseline measures of activation markers for CD4+ and CD8+ and memory and naive CD4+ T cells were not available for this study, values from week 312 were compared with historical control values from a set of 38 HIV-1-infected, antiretroviral-naive subjects in another study [15] who had comparable plasma HIV-1 RNA levels and CD4+ T cell counts (data on file). The association of demographic or baseline characteristics with the change from baseline through week 312 in CD4+ T cell count was assessed by mixed-effects repeated-measures models.
RESULTS
Baseline characteristics and plasma HIV-1 RNA response. Baseline characteristics for all subjects and for those remaining in the study are summarized in table 1. Following 312 weeks of treatment, 62 (98%) of 63 subjects (using on-treatment analysis) had plasma HIV-1 RNA levels <50 copies/mL. Subjects who discontinued treatment prematurely generally had an undetectable HIV-1 RNA level at discontinuation of therapy; no subject cited virologic failure as a reason for discontinuation.
BASELINE CHARACTERISTICS
N=100; 61 subjects completed 6 yrs of treatment (97% men); at baseline 29/100 were Black, 6/100 Hispanic, 65/100 White. Median HIV RNA: 4.9 log c/ml; 45% >100,000 c/ml. Median CD4: 338. Of interest, the median baseline CD4 at baseline for patients completing 6 yrs treatment was 245, while the median baseline CD4 for subjects discontinuing treatment before year 6 was 463 (p= .002).
CD4+ and CD8+ T cell recovery.
The mean CD4+ T cell count increase from baseline to week 312 was 528 cells/L (figure 1A). CD4+ T cell count increases for subjects who discontinued treatment prematurely were comparable (during comparable amounts of time in the study) with those for subjects who completed the study (data not shown). Corresponding median (25th-75th percentile) increases through week 312 were 488 cells/uL (328-614 cells/L).
When subjects were stratified according to baseline CD4+ T cell count (<50 cells/uL, 50-199 cells/uL, 200-349 cells/uL, 350-499 cells/uL, or 500 cells/uL), the mean CD4+ T cell increases from baseline to week 312 were similar (553 cellsu/L, 491 cells/uL, 581 cells/L, 521 cells/uL, and 490 cells/uL, respectively)(figure 1).
CD4+ T cell counts increased at the highest rate in the first 12 weeks of the study (mean increase in CD4+ T cell count, 40.5 cells/uL per month; 95% CI, 32.9-47.9 cells/uL per month; P < .001). Over each successive period of 2 years, significant increases were also observed, although the rate of increase decreased over time: week 12 to year 2 (mean CD4+ T cell count increase, 10.0 cells/L per month; 95% CI, 7.6-12.4 cells/uL per month; P < .001); year 2 to year 4 (mean CD4+ T cell count increase, 4.9 cellsu/L per month; 95% CI, 2.9-6.8 cells/uL per month; P < .001); year 4 to year 6 (mean CD4+ T cell count increase, 4.1 cells/uL per month; 95% CI, 2.2-6.0 cells/L per month; P < .001). After 6 years of treatment, 60 (95%) of 63 patients had a CD4+ T cell count >350 cells/uL, compared with 43% at baseline (figure 2).
The mean CD8+ T cell count increase from baseline to week 312 was 179 cells/uL (P = .003). Increases were apparent primarily in subjects with low baseline CD4+ T cell counts. For subjects with baseline CD4+ T cell counts <50 cells/uL or 50-199 cells/L, mean increases from baseline to week 312 in CD8+ T cell count were 420 cells/uL (P < .001) and 307 cells/L (P = .11), respectively, whereas no significant changes from baseline were observed for other strata of baseline CD4+ T cell count (200 cells/L).
Evaluation of CD4+ : CD8+ T cell count ratio also demonstrated a significant increase from a baseline level of 0.38 to 0.96 at 312 weeks. The change in CD4+ : CD8+ T cell count ratio was statistically significant and of comparable magnitude within each stratum of baseline CD4+ T cell counts.
Total T cell, B cell, and NK cell recovery. Statistically significant mean increases from baseline to week 312 were observed for total T cell count (1074 cells/L to 1778 cells/uL), B cell count (141 cells/uL to 334 cells/uL), and NK cell count (144 cells/uL to 232 cells/uL).
Evaluation of CD4+ and CD8+ T cell markers of activation and maturation.
Control values measured in 38 antiretroviral-naive, HIV-1-infected subjects from an earlier study [15] were used to assess whether the week 312 values observed in the present study were likely to represent changes from baseline values. Historical control subjects had mean plasma HIV-1 RNA levels (4.7 log10 copies/mL) and CD4+ T cell counts (259 cells/uL) that were comparable with baseline values from subjects in the current study.
Mean (median) activation marker levels (HLA-DR and CD38 positive) at week 312 were 4.1% (3.4%) for CD4+ T cells and 7.7% (5.8%) for CD8+ T cells. These values were considerably lower than historical control values (mean values of 23.1% and 39.3% for CD4+ and CD8+ T cells, respectively), suggesting that the week 312 values likely represent significant decreases from baseline (figure 3). Mean week 312 CD4+ T cell activation levels were significantly higher among subjects with baseline CD4+ T cell counts <200 cells/uL than among subjects with baseline CD4+ T cell counts 200 cells/uL (5.1% vs. 3.3%; P = .001). No differences were observed for CD8+ T cell activation markers. Four subjects were identified whose activation marker levels were >2 SDs above the mean CD4+ T cell (>8.4%) or CD8+ T cell (>19.7%) activation levels; these subjects had higher baseline plasma HIV-1 RNA levels (mean value, 5.31 log10 copies/mL) and lower baseline CD4+ T cell counts (mean value, 83 cells/uL; all 4 subjects had values <200 cells/L). Mean increases in CD4+ T cell count through week 312 were robust in this group (mean value, 418 cells/uL), although this group included the only subject whose CD4+ T cell count remained <200 cells/uL at week 312.
Mean (median) week 312 values for naive (CD45RA+, CD62L+) and memory (CD45RO+, CD45RA-) CD4+ T cells were 275 cells/uL (223 cells/uL) and 507 cells/uL (458 cells/uL), respectively. These values were higher than historical control values for untreated HIV-1-infected subjects (mean values of 68 cells/L and 178 cells/L for naive and memory cells, respectively), suggesting that the week 312 values likely represent significant increases from baseline.
Predictors of CD4+ T cell count increase through week 312. On the basis of a mixed-effects repeated-measures model, the change in CD4+ T cell count over 6 years was significantly associated with baseline HIV-1 RNA level (mean value, 67 additional cells/uL per additional 1.0 log10 copies/mL at baseline; 95% CI, 15-119 additional cells/uL per additional 1.0 log10 copies/mL at baseline). Other variables, including race, sex, age, time to HIV-1 RNA level <400 copies/mL, and baseline CD4+ T cell count, did not demonstrate a significant association with change from baseline in CD4+ T cell count.
DISCUSSION
This study represents the longest follow up (6 years) of patients on a lopinavir-ritonavir-containing regimen and demonstrates that, in subjects with sustained virologic suppression, continued immune reconstitution is possible. Even in subjects in the lowest CD4+ T cell count strata of <50 cells/uL and 50-199 cells/uL, there were increases in CD4+ T cell counts similar to those for subjects in the higher CD4+ T cell count strata. The significant increases in CD4+ T cell count observed in this study were accompanied by stable CD8+ T cell counts, except for subjects in the lower CD4+ T cell count strata, for whom CD8+ T cell counts increased. This led to an overall significant increase in the CD4+ : CD8+ T cell count ratio, from a baseline value of 0.38 to 0.96 at week 312. In addition to the T cell subset changes, we observed normalization of B cell and NK cell populations. Furthermore, activation levels (HLA-DR and CD38 positive) of both CD4+ T cells (mean value, 4.1%) and CD8+ T cells (mean value, 7.7%) normalized by week 312.
CD4+ T cell changes through 6 years were modestly but statistically significantly higher for subjects with higher baseline HIV-1 RNA levels. The association with baseline HIV-1 RNA level has been observed in other studies [2, 16-18]. In contrast to earlier studies [2, 18-20], we observed no diminution of CD4+ T cell count increase with increasing age, although the relatively small number of older subjects (only 6% of our subjects were >50 years old at baseline) may have limited the power of our analysis.
In this study, we evaluated immune reconstitution of virologically suppressed subjects across the entire spectrum of CD4+ T cell counts and noted consistently robust increases across all CD4+ T cell count strata. Previous studies (generally of shorter duration) that have examined other HAART regimens have shown significant reductions in viral replication both in blood and in tissue sites, but CD4+ T cell count increases have not appeared to be as robust [21-25]. One study that followed subjects receiving an indinavir-based regimen for 6 years noted an increase in mean CD4+ T cell count of 268 cells/mm3 from baseline to year 6, whereas our study showed a mean increase of 528 cells/L [12]. There are several possible reasons for differences in findings between our study and earlier studies: study populations differed, as did the specific treatment regimens, and nearly all subjects in our study had continued plasma HIV-1 RNA levels <50 copies/mL, in contrast to other studies. This supports the hypothesis that a more robust CD4+ T cell increase may be produced with a regimen better able to induce significant and durable HIV-1 suppression.
Immune cell activation may also be important for this continued CD4+ T cell response. Numerous investigations have evaluated subjects for immune reconstitution for at least 4 years of study [2-4, 6-9, 26, 27]. A recent study of subjects followed up for 6 years who were receiving suppressive antiretroviral therapy demonstrated significant recovery of naive and memory CD4+ T cells, similar to our study, but found an increase in CD4+ and CD8+ T cell activation levels between years 3 and 6 [2]. An important predictor of CD4+ T cell reconstitution is the level of immune activation that contributes to CD4+ T cell death via apoptosis [28], as has been demonstrated in animal models in which the lack of immune activation induced by simian immunodeficiency virus is associated with maintenance of CD4+ T cell counts [29, 30]. Therefore, the normalization in both CD4+ and CD8+ T cell activation levels noted in our study may relate to more potent inhibition of HIV-1, allowing improved CD4+ T cell reconstitution in this study cohort.
In summary, this study is the first to demonstrate continuous immune reconstitution for up to 6 years in subjects with virological suppression who are receiving a lopinavir-ritonavir-based HAART regimen. In addition, the level of CD4+ T cell immune reconstitution is similar for all subjects, regardless of baseline CD4+ T cell count. Furthermore, levels of CD4+ and CD8+ T cell activation appear to normalize by year 6 of therapy. Our findings support a strong association of potent and durable viral suppression with improved and continued immune reconstitution.
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