Intensification of Antiretroviral Therapy With ...

MAJOR ARTICLE

Intensification of Antiretroviral Therapy With Raltegravir or Addition of Hyperimmune Bovine Colostrum in HIV-Infected Patients With Suboptimal CD41 T-Cell Response: A Randomized Controlled Trial

1National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, Australia; 2Queensland Health, AIDS Medical Unit, Brisbane, Australia; 3University of Melbourne, Australia; 4University of Western Australia, Perth, Australia; 5Infectious Diseases Unit, Alfred Hospital, Melbourne, Australia; 6Department of Medicine, Monash University, Melbourne, Australia; 7Centre for Virology, Burnet Institute, Melbourne, Australia; 8St Vincent's Centre for Applied Medical Research, Sydney, Australia; and 9Royal Prince Alfred Hospital, Sydney, Australia

Background. Despite virally suppressive combination antiretroviral therapy (cART), some HIV-infected patients exhibit suboptimal CD41 T-cell recovery. This study aimed to determine the effect of intensification of cART with raltegravir or addition of hyperimmune bovine colostrum (HIBC) on CD41 T-cell count in such patients. Methods. We randomized 75 patients to 4 treatment groups to receive raltegravir, HIBC, placebo, or both raltegravir and HIBC in a factorial, double-blind study. The primary endpoint was time2weighted mean change in CD41 T-cell count from baseline to week 24. T-cell activation (CD381 and HLA-DR1), plasma markers of microbial translocation (lipopolysaccharide, 16S rDNA), monocyte activation (soluble (s) CD14), and HIV-RNA (lowest level of detection 4 copies/mL) were monitored. Analysis was performed using linear regression methods. Results. Compared with placebo, the addition of neither raltegravir nor HIBC to cART for 24 weeks resulted in a significant change in CD41 T-cell count (mean difference, 95% confidence interval [CI]: 3.09 cells/lL, 214.27; 20.45, P = .724 and 9.43 cells/lL, 27.81; 26.68, P = .279, respectively, intention to treat). There was no significant interaction between HIBC and raltegravir (P = .275). No correlation was found between CD41 T-cell count and plasma lipopolysaccharide, 16S rDNA, sCD14, or HIV-RNA. Conclusion. The determinants of poor CD41 T-cell recovery following cART require further investigation. Clinical Trials Registration. ClinicalTrials.gov identifier: NCT00772590, Australia New Zealand Clinical Trials Registry: ACTRN12609000575235.

Received 8 March 2011; accepted 24 June 2011; electronically published 19 September 2011. Presented in part: 22nd Annual Conference for the Australasian Society for HIV Medicine, Sydney, New South Wales, Australia, 20–22 October; 18th Conference on Retroviruses and Opportunistic Infections, Boston, Massachusetts, 27 February– 2 March 2011. Correspondence: Helen Byakwaga, MD, Kirby Institute, University of New South Wales, 45 Beach St, Coogee NSW 2034, Australia ([email protected]. edu.au). The Journal of Infectious Diseases 2011;204:1532–40 Ó The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected] 0022-1899 (print)/1537-6613 (online)/2011/20410-0010$14.00 DOI: 10.1093/infdis/jir559

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Combination antiretroviral therapy (cART) in human immunodeficiency virus (HIV)–infected individuals is associated with control of viral replication and immune recovery. In the majority of patients, this response is associated with increases in CD41 T cells in peripheral blood [1]. However, the extent of CD41 T-cell recovery following cART is highly variable [2, 3]. Despite achieving reliable and sustained suppression of viral replication, up to 30% of patients exhibit suboptimal increase in CD41 T-cell count [2, 4, 5]. Disconcertingly, a greater risk of non-AIDS-related complications is consistently observed in patients with lower CD41 T-cell counts on suppressive therapy [6, 7].

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Helen Byakwaga,1 Mark Kelly,2 Damian F. J. Purcell,3 Martyn A. French,4 Janaki Amin,1 Sharon R. Lewin,5,6,7 Hila Haskelberg,1 Anthony D. Kelleher,1,8 Roger Garsia,9 Mark A. Boyd,1 David A. Cooper,1,8 and Sean Emery1 for the CORAL Study Group

METHODS Study Participants

Patients were recruited from 20 Australian sites between March and September 2009. Eligibility criteria at randomization were

as follows: HIV-1 infection, age $18 years, cART exposure $12 months (without change for a minimum of 6 months), 2 documented consecutive plasma HIV-RNA measurements ,50 copies/mL in the preceding 9 months, CD41 T-cell count ,350 cells/lL for the preceding 6 months, and a CD41 T-cell count rise of ,50 cells/lL in the preceding 12 months. Exclusion criteria at randomization included use of immune-modulating medications within preceding 60 days, current cART containing an integrase inhibitor, allergy to cow’s milk, pregnancy or breastfeeding, or a known cause of impaired CD41 T-cell gain, for example, splenomegaly or a current cART regimen containing both tenofovir and didanosine. The study received ethics approval from the Human Research Ethics Committee at the University of New South Wales and at each participating site. All participants provided written informed consent. Study Design and Interventions

This was a randomized factorial design, double-blind, placebocontrolled, multicenter study. Eligible patients were randomized to 1 of 4 treatment groups: group I, HIBC 1 raltegravir; group II, HIBC 1 raltegravir placebo; group III, HIBC placebo 1 raltegravir; and group IV, HIBC placebo 1 raltegravir placebo. Randomization was stratified by site and screening CD41 T-cell count (#200, .200 cells/lL). The active substance in HIBC is freeze-dried bovine colostrum powder. It contains approximately 40% bovine immunoglobulins (predominantly immunoglobulin [Ig] G and IgA with small amounts of IgM and IgE) that have high binding activity against the LPS and cell wall antigens of Gram-negative bacteria [28, 32]. HIBC contains bovine transforming growth factor b and insulin-like growth factor. These cytokines are bioactive in humans and are expected to assist in quelling immune activation [27] and reducing intestinal permeability to microbial products [29]. HIBC tablets contain an excipient that includes milk-derived casein and calcium carbonate to buffer stomach acid. A small amount of bovine IgG makes its way into the bloodstream with an estimated half-life of 15 hours observed in mice [33]. Immunoglobulins are excreted intact or in the form of immunoglobulin fragments in stool [34]. A dose of three 600-mg tablets was taken twice daily. The dosage of raltegravir was 400 mg (1 tablet) twice daily. Clinical Assessments and Laboratory Testing

Assessments performed during the screening visit and weeks 0 (randomization visit), 4, 8, 12, 24, 36, and 48 included measurement of CD41 T-cell count, T-cell immune activation markers (CD41 and CD81 T-cell expression of CD381 and HLA-DR1), plasma microbial translocation markers (LPS and 16S rDNA), soluble CD14 (sCD14; marker of monocyte activation), plasma HIV-RNA, routine safety biochemistry and hematology, assessment for adherence (estimated patient selfreport), and adverse events. All adverse events were graded for

Interventions for Poor CD41 T-Cell Response

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The factors that have been consistently associated with limited CD41 T-cell recovery in patients on cART include advanced age [2, 8, 9], viral coinfection [10, 11], and a lower nadir CD41 Tcell count [9, 12]. Persistent immune activation is thought to play an important role in poor immunological response to cART [13–15]. Two potential mechanisms have been proposed as a continuous trigger of immune activation in untreated chronic HIV infection. First, HIV-associated damage to intestinal mucosal integrity may result in increased microbial translocation into the circulation from the gastrointestinal lumen, though this observation is not consistently demonstrated [16–19]. Second, persistent low-level HIV replication, even in the setting of suppressive cART, may result in persistent immune activation [20, 21]. The observation of higher levels of proviral DNA in memory and naive CD41 T cells of patients with poor CD41 Tcell recovery on suppressive cART supports the hypothesis that HIV antigen–driven CD41 T-cell activation may play a key role in continuous loss of CD41 T-cell loss in these individuals [22]. Currently, no consensus has been reached on the most efficacious management for suboptimal immunological response to cART. Raltegravir is a potent HIV-1 integrase inhibitor, and it blocks viral replication through a mechanism different from more commonly used antiretroviral agents like protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and reverse transcriptase inhibitors [23, 24]. Therefore, it provides an opportunity to investigate the hypothesis that intensification of cART might decrease ongoing virus replication [25], and, as a result, improve CD41 T-cell response. Hyperimmune bovine colostrum (HIBC) containing antilipopolysaccharide immunoglobulin offers protection against infection with a wide number of enteric pathogens, mainly through passive immunization in the mucosa of the gut and suppression of gut-associated inflammation with promotion of mucosal repair and regeneration [26, 27]. The activity of HIBC is thought to occur through luminal binding and neutralizing of toxin [28]. Bovine colostrum was shown to reduce acute nonsteroidal anti-inflammatory drug-induced increase in intestinal permeability [29] and to lower lipopolysaccharide (LPS) levels in surgical patients [30, 31], suggesting it may have an effect on microbial translocation. Therefore, HIBC may reduce translocation of bacterial products, diminishing immune activation and resulting in improved CD41 T-cell recovery. The CORAL study aimed to determine the effect of cART intensification with raltegravir or addition of HIBC on CD41 T-cell count in HIV-infected patients with suboptimal CD41 T-cell response despite prolonged viral-suppressive cART.

Statistical Considerations

A comparison of HIBC or raltegravir versus placebo with an expected mean CD41 T-cell change of 75 cells/lL (SD: 100 cells/lL) for participants receiving HIBC or raltegravir and 0 (zero) cells/lL (SD: 80 cells/lL) in the placebo arm would require 32 participants on HIBC and 32 participants on raltegravir to give a 90% power. An additional 10% (6) of patients would provide for modest losses to follow-up over 24 weeks. This resulted in a recruitment objective of 72 participants (18 participants in each of 4 study arms). These samples sizes assumed no interaction between HIBC and raltegravir. The primary analysis was conducted when all participants had completed at least 24 weeks or had permanently discontinued from the study. The primary efficacy endpoint was the timeweighted area under the curve for mean change in CD41 T-cell count from baseline to week 24 for main effects: HIBC (treatment groups I 1 II) versus placebo (treatment groups III 1 IV), and raltegravir (treatment groups I 1 III) versus placebo (treatment groups II 1 IV)dintention-to-treat (ITT) population. The ITT population included all randomized participants 1534

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who received at least 1 dose of study medication and had 1 follow-up visit. For the mean change analyses, ITT analysis was conducted by carrying forward the last observed value if the week 24 result was missing. For binary outcomes with missing data, participants were imputed to be failures in the ITT analysis. For secondary outcomes, data were analyzed on both the ITT and per protocol (PP) populations. The PP population was all participants included in the ITT population censored when or if randomized therapy was stopped. Comparison of mean change from baseline to study week of interest of outcomes for main effects was assessed by ANOVA; differences in proportions were calculated using exact methods. Linear regression was used to test for interaction between raltegravir and HIBC on continuous outcomes and exact linear regression was used for binary outcomes. Comparison of proportions was assessed by v2 tests. We examined the relationship between CD41 T cell count, microbial translocation, immune activation, and HIV plasma viremia at baseline and at week 24 using using Spearman’s rho. In all analyses, 2-sided a was considered statistically significant at .05. SAS software version 9.2 was used for analysis of all continuous outcomes; Stata software version 10.1 was used for analysis of all binary outcomes.

RESULTS Study Participants and Baseline Characteristics

Of 100 patients screened, 75 were randomized (Figure 1). Two participants withdrew consent, (1 before commencing study drugs and the other shortly after commencing drugs) and their data were not included in the primary or secondary analyses. The ITT population comprised 73 participants (3 of these completed 24 weeks on study but were off study medications). Primary analysis results showed no benefit of study drugs to the patients; therefore, all participants were advised to discontinue study drugs immediately. At this point, 70 participants (64 on randomized therapy) had completed week 36, and 43 participants (38 on randomized therapy) had completed week 48. Baseline demographic and clinical characteristics were similar across randomized arms (Table 1). The mean (SD) estimated duration of infection was 11.6 (7.5) years, and mean age was 53.1 (10.2) years. At baseline, 41 (57%) of participants had a detectable HIV-RNA level by the ultrasensitive assay used in this study (lowest level of detection: 4 copies/mL). Effect of Addition of HIBC or Raltegravir to cART on CD41 T-Cell Count

The addition of HIBC or raltegravir resulted in no significant difference in time-weighted area under the curve for change from baseline through week 24 in CD41 T-cell count compared


severity using the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (December 2004 version). CD41 and CD81 T cells coexpressing CD381 and HLA-DR1 were enumerated in whole blood within 72 hours of collection by flow cytometry using a standardized protocol on either a FACSCanto II or FACSAria cell sorter. Each subject had flow measurements starting from week 0 performed in 1 lab. All assays were undertaken in 1 of 3 core laboratories, which took part in an ongoing quality assurance exercise to standardize analysis and reporting methods. To improve the consistency of gating of activation markers, a bulk data analysis was performed by 1 experienced flow cytometrist blinded to treatment allocation. Microbial translocation markers were measured from plasma samples that had been stored at 270°C following blood collection. Plasma LPS was measured using a commercial chromogenic limulus amebocyte lysate assay (Lonza). The sCD14 in plasma was measured using the Quantikine Human sCD14 kit (R&D Systems). Details of both methods are described elsewhere [19]. Plasma 16S rDNA levels were assessed by real-time quantitative polymerase chain reaction on DNA extracted from plasma (DNeasy Kit, Qiagen). Plasma HIV-1 RNA levels were measured using the Amplicor HIV-1 Monitor assay (version 1.5; Roche) or Abbott RealTime ultrasensitive assays with lower limits of detection at 50 and 40 copies/mL, respectively. In addition, levels of HIV-RNA were measured at 1 central laboratory using Amplicor HIV-1 Monitor assay test (MWP, version 1.5), with the Ultra Boosted specimen preparation protocol with a lower limit of detection of 4 copies/mL.

CORAL study participant disposition. Abbreviations: HIBC, hyperimmune bovine colostrum; RAL, raltegravir.

with placebo (mean difference, 95% confidence interval [CI]: 9.43 cells/lL, 27.81 to 26.68, P 5 .279; and 3.09 cells/lL, 214.27 to 20.45, P 5 .724, respectively)dITT (Table 2). There was no significant interaction between HIBC and raltegravir (P 5 .275). A similar result was obtained in a PP population analysis in which participants were censored when randomized therapy was stopped (data not shown). There were no significant differences in absolute changes in CD41 T-cell count or CD41 T-cell percentage at any time point between HIBC or raltegravir and placebo; neither were there any significant differences in proportions of patients with CD41 T cells .350 cells/lL at weeks 4, 8, and 24 (data not shown). For the 38 participants that completed week 48 on randomized therapy, there were no significant differences observed in proportion of participants with CD41 T cells .350 cells/lL, time weighted area under the curve for change from baseline in CD41 T-cell count, plasma microbial translocation markers, T-cell immune activation markers, or plasma HIV-RNA between participants who received HIBC or raltegravir and those who received placebo. Effect of HIBC or Raltegravir on Microbial Translocation, Immune Activation, and Plasma HIV-RNA

Compared with placebo, we observed no significant differences in changes from baseline to week 24 in plasma levels of LPS, 16S

rDNA and sCD14, proportion of CD41 and CD81 T cells expressing CD381 and HLA-DR1, and plasma HIV-RNA in participants who received either HIBC or raltegravir (Table 3). Associations Between CD41 T-Cell Count, Microbial Translocation Markers, T-Cell Activation, and Plasma HIV-RNA

LPS and 16S rDNA were significantly correlated (r 5 0.37, P , .001; and r 5 0.36, P , .001, respectively) at baseline and week 24, respectively. There was a significant negative correlation between LPS and sCD14, both at baseline and week 24 and between the proportion of CD81 T cells expressing CD381 and HLA-DR1 and CD41 T-cell counts (Table 4). There was no correlation between CD41 T-cell count and microbial translocation markers or plasma HIV-RNA; neither was there any association between microbial translocation markers, T-cell activation, or plasma HIV-RNA levels. Safety and Tolerability of HIBC and Raltegravir

Two participants stopped study medications due to grade 2 adverse symptoms thought to be related to study drugs. The following 8 serious adverse events were reported: myocardial infarction; unstable angina pectoralis; and hospitalization for cellulitis (event occurred twice in the same participant), possible influenza A, a methamphetamine-induced psychotic episode, and investigation of chest pain (event occurred in two different


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Figure 1.

Table 1. Patient Characteristics at Baseline HIBC 1 RAL (n 5 19) Gender (male), no.

HIBC 1 Placebo (n 5 19)

17

Age (y), mean (SD) Duration of infection (y),mean (SD) CDC category C, no. (%) Duration from start of ART (y), median (IQR)

Placebo 1 RAL (n 5 18)

Placebo 1 Placebo (n 5 17)

All (N 5 75)

18

18

16

69

52 (11)

56 (9)

50 (10)

55 (10)

53 (10)

11.5 (6.9) 8 (42)

12.0 (8.2) 8 (42)

11.4 (7.2) 7 (39)

11.6 (8.1) 7 (41)

11.6 (7.5) 30 (41)

6 (3–7)

4 (2–8)

5 (3–7)

4 (2–6)

5 (2–7)

Current ART, no. (%) PI

4 (21)

7 (37)

6 (33)

6 (35)

23 (32)

NRTI

19 (100)

19 (100)

18 (100)

17 (100)

73 (100)

NNRTI

15 (79)

13 (68)

12 (67)

12 (71)

52 (71)

Ultrasensitive plasma viral load Single-copy assay mean (SD), log10 copies/mL Immunology, mean (SD) Nadir CD41 T-cell count (cells/lL)

20.01 (0.22)

0.04 (0.38)

20.04 (0.30)

20.05 (0.18)

0.54 (0.29)

100 (73)

68 (52)

62 (48)

79 (60)

230 (68)

208 (73)

201 (63)

194 (56)

209 (66)

CD41 T-cell %

17.3 (8.6)

15.7 (4.9)

14.4 (4.6)

14.5 (4.9)

15.5 (6.0)

CD81 T-cell count (cells/lL)

811 (496)

773 (306)

887 (465)

807 (428)

819 (422)

CD81 T-cell %

50.1 (16.3)

55.7 (10.8)

57.3 (11.4)

54.0 (12.7)

54.3 (13.0)

12 (63)

10 (53)

8 (44)

9 (53)

39 (53)

0.7 (1.2)

0.8 (0.9)

1.5 (2.4)

0.4 (0.4)

0.9 (1.4)

1.7 (0.9)

3.3 (2.9)

3.3 (2.8)

2.2 (1.6)

2.6 (2.3)

Proportion with CD41 T-cell count $200, no (%) T-cell activation markers (%), mean (SD) CD4 CD381 HLA-DR1 CD8 CD381 HLA-DR1 Microbial translocation markers, mean (SD) LPS (pg/mL)

79.6 (48.0)

74.6 (31.7)

75.0 (22.7)

85.0 (25.3)

78.4 (33.3)

16S rDNA (copies/mL)

6933 (8140)

4562 (2117)

7113 (6616)

6035 (3253)

6151 (5606)

2.1 (0.7)

2.2 (0.7)

2.0 (0.6)

1.9 (0.5)

2.1 (0.6)

Monocyte activation, mean (SD) sCD14 (3 106 pg/mL)

Abbreviations: ART, antiretroviral therapy; CDC, Centers for Disease Control and Prevention; HIBC, hyperimmune bovine colostrum; IQR, interquartile range; LPS, lipopolysaccharide; NNRTI, nonnucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; RAL, raltegravir; sCD14, soluble CD14; SD, standard deviation.

participants). All serious adverse events resolved, none were related to the study drugs, and all participants continued study medications. Adherence to study drugs was approximately 100% for all 4 treatment arms.

markers, plasma HIV-RNA levels, or T-cell activation. Neither plasma HIV-RNA nor markers of microbial translocation were associated with CD41 T-cell count. Markers of T-cell activation were inversely correlated with CD41 T-cell count, consistent with the notion that immune activation is a determinant of CD41 T-cell recovery during cART. However, no correlation was found between immune activation and microbial translocation or plasma HIV viremia. An earlier study investigating the use of bovine colostrum in patients with HIV-associated diarrhea showed a CD41 T-cell

DISCUSSION Addition of HIBC or raltegravir intensification of a suppressive cART regimen did not result in an increase in CD41 T-cell count or demonstrable changes in plasma microbial translocation

Table 2. Time-Weighted Area Under the Curve for Change From Baseline Through Week 24 in CD41 T-Cell Count (cells/mL)dIntention to Treat HIBCa 95% CI

Main effects

No.

Mean

Placebo

35

15.09

1.31, 28.87

Active

38

5.65

25.43, 16.73

Difference

.

9.43

27.81, 26.68

P value

Mean

.

36

11.74

2.49, 23.98

.

37

8.65

24.07, 21.38

.

.279

.

3.09

214.27, 20.45

.724

Abbreviations: CI, confidence interval; HIBC, hyperimmune bovine colostrum. a

P value for interaction between HIBC and raltegravir was .275.

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Raltegravira 95% CI

No.

P value .


83 (59)

CD41 T-cell count (cells/lL)

Table 3. Mean Change From Baseline Through Week 24 in CD381/HLA-DR1 Expression, Plasma Microbial Translocation Markers, and Plasma HIV-RNA for Individuals Who Received Raltegravir or HIBC (n 5 73, Intention to Treat) Mean 1

1

95% CI

P value

1

CD4 CD38 HLA-DR (%) HIBC vs placebo RAL vs placebo

20.03

2.22, .15

.713

0.08

2.10, .27

.370

0.16 0.31

2.72, 1.04 2.57, 1.20

.719 .479

CD81 CD381 HLA-DR1 (%) HIBC vs placebo RAL vs placebo sCD14 (3 106 pg/mL) HIBC 1 placebo

0.02

2.23, .28

.859

RAL vs placebo

0.07

2.19, .32

.596

LPS (pg/mL) HIBC vs placebo

2.40 211.9, 16.71

.739

RAL vs placebo

21.31 215.61, 13.00

.856

RAL vs placebo

2437.00 1977.20

22928, 2053.80 2469.60, 4423.90

.728 .112

Ultrasensitive HIV-RNA (log10 copies/mL)a HIBC vs placebo RAL vs placebo

2.19, .07

.350

0.02 211.00, .16

.725

20.06

Abbreviations: CI, confidence interval; HIBC, hyperimmune bovine colostrum; HIV, human immunodeficiency virus; LPS, lipopolysaccharide; RAL, raltegravir; sCD14, soluble CD14. a Ultrasensitive assay with lowest level of detection of 4 copies/mL used for measurement of HIV-RNA.

gain of up to 125 6 75% cells/lL after 7 weeks [35]. Similarly, a randomized placebo-controlled study using NR100157, a multicomponent nutritional supplement containing bovine colostrum, showed significantly slowed CD41 T-cell count decline (228 vs 268 cells/lL, respectively; difference: 40 cells/lL; P 5 .030) at week 52 independent of viral load changes [36]. Notably, unlike participants enrolled in our study, patients enrolled in these 2 studies were ART naive. In addition, NR100157 is different in content to HIBC and contains prebiotic oligosaccharides, N-acetyl cysteine, omega-3 longchain polyunsaturated fatty acids, and micronutrients as well as bovine colostrum [36]. In this study, HIBC did not affect plasma levels of markers of microbial translocation. Possible explanations are that HIBC did not reach the mucosal sites at which translocation occurs; HIBC reached the sites but did not impact microbial translocation; and we were unable to detect bacterial translocation by measurement of plasma biomarkers. We were unable to draw any conclusions about the role of microbial translocation in CD41 T-cell recovery during cART in this study, given that no significant changes in CD41 T-cell count were observed in participants who received HIBC.


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16S rDNA (copies/mL) HIBC vs placebo

A causal relationship between biomarkers of microbial translocation or immune activation and CD41 T-cell count during cART is not established. Similar to previous reports, we found an association between higher CD81 T-cell activation (measured by CD381 and HLA-DR1 expression), and lower CD41 T-cell counts [14, 37]. Increased susceptibility of activated T cells to apoptosis and turnover have been described as possible mechanisms [13, 14]. We also found a positive correlation between plasma levels of LPS and 16S rDNA as reported in earlier studies [17, 19]. However, unlike previous reports, we did not find an association between 16S rDNA and CD81 T-cell activation or CD41 T-cell count [17], nor an association between plasma HIV-RNA and CD81 T-cell activation or CD41 T-cell count [21]. Interestingly, we found plasma LPS levels were negatively correlated with sCD14 levels. LPS can be cleared by multiple mechanisms and has a complex relationship with sCD14. LPS activates the release of sCD14 [38] but then LPS is cleared via high-density lipoprotein [39], formation of lipopolysaccharide binding protein (LBP)–LPS complexes [39], scavenging by endotoxin core antibody (EndoCAb) [40], and by sCD14 itself [41]. LBP, EndoCAb, and high-density lipoprotein were not measured in this study. The interactions between each of these factors are important for the clearance of LPS, and may be different in the presence and absence of cART. Although plasma levels of LPS and sCD14 have been shown to decrease with initiation of cART [16, 42], there are no extensive data sets available describing the relationship between sCD14 and LPS in cART-treated individuals. In a recent report, a positive correlation was observed between LPS and sCD14 in the absence of opportunistic infection, and a significant negative correlation in the presence of opportunistic infections in ART-naive patients. However, there was no correlation seen in cART-treated individuals [42]. In this study, we also examined the effect of raltegravir intensification on CD41 T-cell count recovery. Our findings are consistent with those from a small single-arm study [43] that found no significant increase in CD41 T-cell counts following 4 weeks of raltegravir intensification [43]. Similarly, in a retrospective study of 9 patients with poor CD41 T-cell recovery despite suppressive cART, no significant change in CD41 T-cell counts was observed after 25 weeks of intensification with maraviroc [44]. By contrast, in a recently published trial of 53 patients randomized to receive raltegravir or matched placebo, a trend toward increased CD41 T-cell counts was observed during 12 weeks of raltegravir intensification. However, participants in this study had CD41 T-cell counts .200/lL, and increased CD41 T-cell counts were not associated with an effect of raltegravir on T-cell activation [45]. Our results are also consistent with those from other studies showing no effect of raltegravir intensification on low-level viremia and immune activation [43, 45–47]. Of particular note is

Table 4. Correlation of CD41 T-Cell Counts, Microbial Translocation Markers, Immune Activation, and HIV-RNA Levelsa CD41 T cell

sCD14

LPS

CD4 CD381 HLA-DR1

CD8 CD381 HLA-DR1

16S rDNA

Week 0 CD41 T-cell count (cells/lL) sCD14 (3 106 pg/mL)

.

.

.

.

.

.

20.01, .918

.

.

.

.

.

.

.

.

.

0.08, .518

.

.

.

. 20.03, .782

. .

LPS (pg/mL)

0.03, .828

20.25, .038

CD41 CD381 HLA-DR1 (%)

0.04, .736

0.11, .348

CD81 CD381 HLA-DR1 (%) 16S rDNA (copies/mL)

20.25, .037 20.00, 1.000

0.14, .268 20.19, .123

0.01, .927 0.37, ,.001

0.52, ,.001 20.08, .512

0.02, .866

20.06, .607

0.09, .445

20.20, .102

HIV-RNA (log10 copies/mL)

0.04, .726

0.03, .809

Week 24 CD41 T-cell count (cells/lL) sCD14 (3 106 pg/mL) LPS (pg/mL)

.

.

.

.

.

.

20.02, .876

.

.

.

.

.

.

.

.

.

. .

. .

20.25, .034

0.03, .798 20.26, .032

0.12, .318 0.14, .243

16S rDNA (copies/mL)

20.00, .983

20.18, .133

0.36, ,.001

20.06, .643

20.01, .923

0.02, .846

20.07, .561

0.10, .429

20.21, .086

0.03, .826

HIV-RNA (log10 copies/mL)

0.07, .588 20.00, .991

. 0.54, ,.001

. 0.02, .869

Abbreviations: HIV, human immunodeficiency virus; LPS, lipopolysaccharide; sCD14, soluble CD14. a Ultrasensitive assay with lowest level of detection of 4 copies/mL used for measurement of HIV-RNA. All data are expressed as Spearman’s correlation coefficient, P value.

a study in which 30 patients receiving suppressive ART with CD41 T-cell count ,350 cells/lL for at least 1 year were randomized to receive raltegravir intensification or placebo [47]: raltegravir had no effect on plasma HIV-RNA level or immune activation as measured by the proportion of CD81 T cells expressing CD381 and HLA-DR1. These findings together suggest that raltegravir intensification does not further suppress lowlevel HIV replication and that residual viremia in these fully suppressed patients does not arise from ongoing cycles of HIV-1 replication [47]. In this study, we were unable to draw any conclusions about the role of low-level HIV-RNA on CD41 T-cell recovery. In a recent study of raltegravir intensification, a significant transient increase in 2-long terminal repeat (2-LTR) circles was observed at weeks 2 and 4 [48]. Further, increased immune activation at baseline was associated with 2-LTR circle detection, and this normalized after raltegravir intensification. The authors interpreted these data to imply that active HIV replication persists in some HIV-infected patients despite suppressive cART and that viral replication drives immune activation. Similar to results from our study, there was no significant change in residual low-level viremia. We were unable to measure rapid changes in 2-LTR circles and therefore could not analyze our data according to similar parameters as they did with respect to residual immune activation. Patients in our study were older than those of previous cohorts of ART-treated patients, possibly affecting CD41 T-cell recovery [14, 15, 47]. An inverse correlation between age and the magnitude of CD41 T-cell recovery is consistently reported 1538

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[8, 9], possibly arising from decreased thymic function and other regenerative mechanisms with older age. Moreover, the number of circulating naive CD41 T cells observed in HIVinfected patients is positively correlated to thymic mass and function after both short-term [49] and long-term ART [50]. Very low levels of CD41 and CD81 expression of CD381 and HLA-DR1 were observed in our study compared with other cohorts of ART-treated patients with poor CD41 T-cell gain [14, 15, 47]. We can only speculate that persistent T-cell activation is unlikely to be the major cause of the low CD41 T-cell recovery in this group of patients. There is no consensus on the definition of poor immunological response to cART with regard to the duration of evaluation of the CD41 T-cell response, the threshold of CD41 T-cell gain from pretherapy levels, or the extent of CD41 T-cell recovery to a specific threshold. Therefore, findings from this study may not be generalizable. Another limitation of this study is that we did not investigate the effect of the addition of HIBC or raltegravir at the level of different anatomical sites, such as the gut and central nervous system. In conclusion, in this patient group, the addition of HIBC or raltegravir intensification of suppressive cART regimens was not associated with an increase in CD41 T-cell count or changes in immune activation, plasma HIV viremia, or microbial translocation. The absence of CD41 T-cell increases does not exclude the possibility that either microbial translocation or low-level plasma viremia plays a role in poor CD41 T-cell recovery, but makes it unlikely in this group with a relatively long duration since the start of ART. The determinants of CD41 T-cell


0.03, .780

CD41 CD381 HLA-DR1 (%) CD81 CD381 HLA-DR1 (%)

recovery on cART require more investigation. Further research is required to delineate causal relationships between biomarkers of microbial translocation and CD41 T-cell count responses to cART. Notes

References 1. Smith C, Sabin C. Lampe F. The potential for CD4 cell increases in HIV-positive individuals who control viraemia with highly active antiretroviral therapy. AIDS 2003; 17:963–9. 2. Kaufmann G, Perrin L, Pantaleo G. et al. CD4 T-lymphocyte recovery in individuals with advanced HIV-1 infection receiving potent antiretroviral therapy for 4 years: the Swiss HIV Cohort Study. Arch Intern Med 2003; 163:2187–95.


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Acknowledgements. We extend our grateful thanks to all the participants, and thank Immuron (formerly Anadis) and Merck Sharp & Dohme for the provision of hyperimmune bovine colostrum/matched placebo and raltegravir/matched placebo, respectively. CORAL Study Group Protocol Steering Committee. Sean Emery, Mark Kelly, Damian Purcell, Sharon Lewin, Roger Garsia, Janaki Amin, Helen Byakwaga, Mark Boyd, Anthony Kelleher, Martyn French, and David Cooper. Site Investigators and Staff. East Sydney Doctors, Sydney: David Baker and Robyn Vale; Albion Street Centre, Sydney: Don Smith, Jega Sarangapany, and Jason Gao; Dr Doong’s Surgery, Burwood, Sydney: Nicholas Doong and Jeff Hudson; Royal North Shore Hospital, Sydney: George Kotsiou and Joanne Holahan; Holdsworth House Medical Practice, Sydney: Mark Bloch, Shikha Agrawal, and Teo Franic; Lismore Sexual Health Service, Lismore: David Smith and Cherie Mincham; Liverpool Hospital, Sydney: John Quin, Catherine Magill, and Ken Murray; Prince of Wales Hospital: Jeffery Post, Kristine Millar, and Suzanne Ryan; St Vincent’s Hospital: David Cooper, Karen Macrae, Gary Keogh, and Richard Norris; Royal Prince Alfred Hospital: Roger Garsia and Geoff Turnham; Northside Clinic: Richard Moore, Sally Carson, Sian Edwards, and Rachel Liddle; Centre Clinic: B. K. Tee and Helen Lau; Melbourne Sexual Health Centre: Tim Read and Julie Silvers; Prahran Market Clinic: Norman Roth and Helen Lau; The Alfred Hospital: Julian Elliott, Michelle Boglis, and Julie Moyer; AIDS Medical Unit: Mark Kelly and Abby Gibson; Gold Coast Sexual Health Clinic: John Chuah and May Ngieng; Nambour Hospital: David Sowden, Colleen Johnston, Karen McGill, and Alan Walker; Fremantle Hospital: John Dyer, Jacqueline Kerth, and Samantha Libertino; Royal Perth Hospital: Martyn French, Jenny Skett, and Allison Cain. CORAL Study Team. Hila Haskelberg, Janaki Amin, Kymme Courtney-Vega, Maja Berilazic, Absar Noorul, Simone Jacobs, Kat Marks, Kate Merlin, Helen Byakwaga, Mark Boyd, and Sean Emery. Laboratory Groups. Melbourne University, Melbourne: Marit Kramski; Monash University, Melbourne: Pushparaj Velayudham, Ajantha Solomon, Reena Rajasuriar, and Paul Cameron; Royal Perth Hospital, Perth: Rom Krueger and Leanne Langan; Sypath, Sydney: Sandy Smith; Centre of Applied Medical Research, Sydney: Julie Yeung, Maria Piperias, Bertha Fsadni, and Kersten Koelsch. Financial support. This work was supported by the National Health and Medical Research Council NHMRC Program Grant (number 510448). The Kirby Institute (formerly the National Centre in HIV Epidemiology and Clinical Research) is funded by the Australian Government Department of Health & Ageing and is affiliated with the Faculty of Medicine, The University of New South Wales. Potential conflicts of interest. D. F. J. P. owns stock in Immuron, and is a scientific advisor and consultant with Immuron. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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