Tripterygium wilfordii Hook F extract in cART-treated HIV patients with poor immune response: a pilot study to assess its immunomodulatory effects and safety

Abstract

Background:

Despite combination antiretroviral therapy (cART), 20% of HIV-infected patients are unable to achieve adequate immunologic recovery, in which immune activation plays a crucial role. We hypothesize that extract of Tripterygium wilfordii Hook F (TwHF), a Chinese medication used to treat autoimmune diseases, has immunomodulatory effects that may help CD4 cell recovery.

Methods:

Eighteen cART-treated HIV-infected patients virally suppressed for over 12 months with suboptimal CD4 cell recovery were enrolled. TwHF extract was administered at a dosage of 10 mg three times daily for 12 months. T-cell subsets and activation markers were evaluated at baseline and during follow-up. The trial was registered at Clinicaltrials.gov (NCT02002286).

Results:

TwHF extract was associated with a mean increase in CD4 cell count of 88 cells/μl (95% confidential interval [CI], 72–105 cells/μl) after one year of treatment. A significant increase in the mean rate of CD4 cell recovery (26 before vs 75 cells/μl/year after TwHF use, P < 0.001) was observed. Analysis of 13 patients with activation profiles suggested that TwHF extract was associated with a decrease in T-cell immune activation which was temporally correlated with CD4 cell recovery. No discontinuation of TwHF extract was reported.

Conclusion:

Use of TwHF extract in HIV-infected patients was associated with a reduction in T-cell activation and improved CD4 recovery with an excellent safety profile.

Keywords:

• HIV
• Immune activation
• Tripterygium wilfordii Hook F
• Immune reconstitution

Introduction

Although combination antiretroviral therapy (cART) has substantially improved patient outcomes by suppressing HIV-1 replication leading to sustained CD4 cell recovery for most HIV-infected patients,¹ approximately 20% of patients do not achieve adequate CD4 cell reconstitution despite prolonged virologic suppression. Typically, the CD4 cell recovery of these patients, called immunologic non-responders (INRs),² plateaus beyond 10 years. Factors including advanced age (>45 years), low nadir CD4 cell count, higher baseline viral load, reduced thymic output, a history of AIDS-defining events, and especially increased immune activation are the main driving force behind poor CD4 cell recovery.^2–7 An inadequate immunologic response is associated with a higher risk of opportunistic infections,^8,9 cardiovascular disease,¹⁰ renal disease,¹¹ malignancy,¹² neurological complications,¹³ as well as higher mortality.^9,12 Specific medications including hydroxychloroquine (HCQ), chloroquine and celecoxib dampen immune activation in HIV infection.^14–17 In virally suppressed INRs treated with cART and HCQ, however, only a trend toward CD4 cell count recovery was observed,¹⁴ while studies using chloroquine and celecoxib in HIV-patients not on cART failed to show a significant increase in CD4 cell count.^15–17 To date, no immunomodulants have been shown to promote CD4 cell recovery significantly.¹⁸ Finding an effective therapy that decreases the level of immune activation in HIV infection and enhances immune recovery addresses an unmet need.

The extract of Tripterygium wilfordii Hook F (TwHF), a traditional Chinese medication, has been utilized as an anti-inflammatory therapy to treat different autoimmune diseases including rheumatoid arthritis (RA)¹⁹ and Crohn's disease.²⁰ A recent multicenter, open-label, randomized controlled trial in China comparing TwHF and methotrexate showed that TwHF was not inferior to methotrexate monotherapy in controlling acute RA.²¹ Multiple in vitro and in vivo studies have demonstrated that triptolide, the major bioactive component extracted from TwHF²² (chemical structure shown in Fig. S1 see supplementary material online for this article www.maneyonline.com/doi/suppl/10.1179/1528433614Z.0000000005), can reduce lipopolysaccharide-induced inflammation,^23–26 a crucial factor for immune activation in HIV-1 infection.^24,27,28 In addition, triptolide inhibits proinflammatory markers like COX-2 and iNOS²⁶ and suppresses their proinflammatory downstream signals.²⁵

In this pilot study, we evaluated the efficacy of TwHF extract on immune activation and immune recovery.

Methods

Patients

Eighteen HIV-infected patients at Peking Union Medical College Hospital Clinic receiving cART for at least two years with plasma HIV-1 viral load < 40 copies/ml and suboptimal CD4 cell recovery were enrolled after informed consent was obtained. Patients were recruited from August 1, 2011 to August 1, 2012 and were followed until August 1, 2013 (Fig. 1).

Figure 1. Patient flowchart.

Definitions

Immune non-responders were defined as patients with CD4 cell count < 200 cells/μl or < 20% increase in CD4 cell count compared with baseline after at least 12 months; inadequate responders (InaR) were defined as patients with CD4 count between 200 and 300 cells/μl after at least12 months of viral suppression.^2,3

Study design

TwHF extract was administered orally at a dosage of 10 mg three times daily for 12 months. Patients also continued on their usual cART regimen. The primary endpoints were changes in CD4 cell count and in T-cell subsets. The secondary endpoints were immune activation levels and the safety profile. T-cell subsets and activation markers were evaluated at baseline and at weeks 12, 24, and 48.

HIV-1 viral load and T-cell subsets

Measurement of plasma HIV-1 viral load has been described.²⁹ In brief, viral load was measured with COBAS Ampliprep/TaqMan48 real-time RT-PCR Test (Roche Diagnostics) with HIV-1 viral detection range of 40 to 10 000 000 copies/ml. T-cell subsets and immune activation were measured using six-color FACSCanto flow cytometer (BD Biosciences) to detect, CD4(+) T cells (CD4(+)CD3(+)), CD8+T cells (CD8(+)CD3(+)), naive CD4+T cells (CD4(+)CD45RA(+)CD62L(+)), memory CD4+T cells (CD4(+)CD45RA( − )), and activated T cells (CD38(+)HLA-DR(+)), as described previously.³

Statistical analysis

Noncategorical baseline variables were analyzed using unpaired t test (data with normal distribution) or Mann–Whitney U test (data with non-normal distribution). Categorical variables were analyzed with chi square test or Fisher's exact test. CD4 cell recovery was evaluated using one-way ANOVA with repeated measures. Generalized estimating equations (GEE) were utilized to evaluate slopes of CD4 cell recovery. We also used linear regression to calculate CD4 cell recovery rate in each patient and used paired t test to compare differences in CD4 cell recovery rate before and after TwHF use, which is a method utilized in previous studies.^30,31 Wilcoxon paired test was utilized to evaluate changes in immune activation.¹⁵ Spearman's rank-correlation was used to evaluate the correlation between immune activation and CD4 cell recovery. Normality was tested by using the Shapiro-Wilk test. For all tests, P < 0.05 was considered to be statistically significant. SPSS 20.0 statistical package was used for all analyses.

Role of the funding source

The funders had no role in study design, data collection, data analyses, preparation of the manuscript, or decision to publish.

Ethics statement

Informed consent was obtained from every participant, and the study was approved by the Institutional Review Board of Peking Union Medical College Hospital. It was in compliance with local regulations and the Declaration of Helsinki.

Results

Baseline characteristics

Baseline characteristics are summarized in Table 1. Demographic and clinical characteristics were similar between INR and InaR except for baseline and nadir CD4 count as well as duration of cART use and viral suppression. Most patients had been on cART for over two years and virally suppressed for more than one-and-a-half years.

Table 1. Population characteristics

	INR (n = 9)	InaR (n = 9)	P value
Age, mean ± SEM (years)	41 ± 3	44 ± 2	0.378
Sex, n (%)			0.471
Male	7 (77.8)	9 (100.0)
Female	1 (11.1)	0 (0.0)
Transgender	1 (11.1)	0 (0.0)
Route of transmission, n (%)			0.294
Sex	8 (88.9)	5 (55.6)
Blood	1 (11.1)	4 (44.4)
CD4+T cell count before TwHF use, mean ± SEM (cells/μl)	134 ± 9	241 ± 10	< 0.001
Nadir CD4+T cell count, median, interquartile range (cells/μl)	21 (17, 46)	99 (21, 157)	0.058
Duration of viral suppression#, mean ± SEM (years)	1.4 ± 0.1	2.6 ± 0.5	0.004
Duration of current cART use before TwHF extract administration, mean ± SEM (years)	2.0 ± 0.2	3.6 ± 0.7	0.038
Viral load before cART, mean ± SEM (log copies/ml)	5.05 ± 0.70	4.65 ± 0.57	0.248
NRTI, n (%)			>0.999
3TC+AZT	5 (55.6)	5 (55.6)
3TC+TDF	4 (44.4)	4 (44.4)
Third antiretroviral therapy, n (%)			0.576
NNRTI (NVP or EFV)	8 (88.9)	6 (66.7)
PI (LPV/r)	1 (11.1)	3 (33.3)
HBsAg positive, n (%)^※	0 (0.0)	0 (0.0)	N/A
HCV antibody positive, n (%)^※	2 (22.2)	2 (25.0)	>0.999

Note: INR, immune nonresponders; InaR, inadequate responders; SEM, standard error of mean; cART, combined antiretroviral therapy

* xscalculated by using Fisher's exact test

# in INR group, viral load of patient 16 before TwHF extract use was missing, but this patient received three years of cART before TwHF extract use

‡ patient 16 in INR group and patient 11, 14 in InaR group did not have baseline viral load since their viral load was measured at other hospitals and was not available

※ baseline HBV and HCV profiles in patient 11 (InaR group) were missing. P values were calculated by using Fisher's exact test.

Total and memory CD4 cells increased during TwHF treatment

Immunologic response before and after TwHF extract administration is summarized in Fig. 2. After one year of TwHF extract use, CD4 cell count increased by a mean of 88 cells/μl (95% confidential interval [CI] 72–105 cells/μl). In both INR and InaR, a significant increase in CD4 cell count was observed (Fig. 2A and Supplementary Table 1 in Supplementary Material). Importantly, a rise in memory CD4 cells accounted for this increase rather than naive cells (Fig. 2B and C and Supplementary Table 1 see supplementary material online for this article www.maneyonline.com/doi/suppl/10.1179/1528433614Z.0000000005). CD4 cell recovery for each patient is shown in Fig. 3.

Figure 2. Association between TwHF extract and CD4 cell recovery. (A–C) an increase in total and memory CD4 cell count was observed during TwHF use. In B and C, data from 14 patients were available for T-cell subgroup analysis (n = 7 in INR group and n = 7 in InaR group). In Fig. 2A–C, gray asterisks indicate a significant difference between T-cell subsets before TwHF use and at TwHF initiation; black asterisks indicate a significant difference between T-cell subsets after TwHF use and at TwHF initiation. Dashed lines indicate TwHF initiation. (D, E) increase in total and memory CD4 T cells calculated by generalized estimating equation (GEE). Rates of CD4 cell recovery and 95% confidential intervals (CI) before and after TwHF extract use are labeled in these figures. (F–H) increase rates of CD4 cell and subgroups before and after TwHF extract use. The rate of increase in each patient was derived from linear regression, and the comparison was made using paired t-test, with changes in mean (95% CI) rates of increase labeled in these figures. *, 0.01 < P < 0.05; * *, 0.001 < P ≤ 0.01; * * *, P ≤ 0.001.

Figure 3. CD4 cell recovery for each patient. Horizontal lines with arrows indicate TwHF extract use. cART was administered at the beginning of each graph.

We calculated the slope of CD4 cell increase before and after TwHF extract initiation. The rate of increase before TwHF extract use was calculated by linear regression of CD4 cell recovery from three months after cART (second phase of CD4 cell increase³) to initiation of TwHF extract. In comparison with the rates of CD4 cell count increase in the slow increase phase, rates of increase were significantly higher after TwHF extract administration (Fig. 2D); in addition, memory CD4 cell recovery accounted for total CD4 cell recovery (Fig. 2E). This was further confirmed by comparing slopes of CD4 total T-cell and memory T-cell increase (Fig. 2F–H), in which significant increases in mean CD4 cell recovery rates (26 before vs 75 cells/μl/year after TwHF extract use, P < 0.001) and mean memory CD4 cell recovery rates (17 before vs 65 cells/μl/year after TwHF extract use, P < 0.001) were observed after TwHF extract administration. We also observed changes in CD8 cells and the CD4/CD8 ratio (see Fig. S2 in supplementary material online for this article www.maneyonline.com/doi/suppl/10.1179/1528433614Z.0000000005).

Association between TwHF use and decrease in immune activation

We next explored the potential mechanisms by which TwHF extract promotes CD4 cell recovery. The median percentage of CD38(+)HLA-DR(+)CD8(+) T cells decreased significantly during the 12-month treatment period (Fig. 4A) Importantly, the decrease in immune activation was most prominent in the first three months (Fig. 4A), which preceded the recovery of CD4 cells and memory CD4 cells in the InaR and INR groups (Fig. 2A and B). Similarly, the percentage of CD38(+)HLA-DR(+)CD4(+) cells also decreased (Fig. 4B).

Figure 4. Association between TwHF extract use and immune activation in patients with poor immune response. (A, B) CD8 and CD4 cell activation profiles during TwHF extract treatment (n = 13 in month 0 and 3, n = 10 in month 6, n = 11 in month 9, n = 5 in month 12). P values were derived by using Wilcoxon paired test. Median level of immune activation is also labeled in figures. (C–F) correlation between CD38(+)HLA-DR(+) double positive CD8 or CD4 T-cell percentage and total or memory CD4 cell count. *, 0.01 < P < 0.05; * *, 0.001 < P ≤ 0.01; * * *, P ≤ 0.001.

Association between immune activation and CD4 cell recovery

We last assessed the association between CD8(+) T-cell immune activation and CD4 cell recovery after TwHF extract treatment. Total CD4 cell count was negatively correlated with CD38(+)HLA-DR(+)/CD8(+) percentage (Fig. 4C) Similar results were also observed in memory CD4 cells (Fig. 4D). Interestingly, CD4 cell activation was negatively correlated with CD4 T memory cell counts, but not with total CD4 cell counts (Fig. 4E and F).

Safety profile

Throughout the 12-month treatment period, TwHF extract was well tolerated. No discontinuation of TwHF extract was reported. Two patients developed hypercholesterolemia and one patient developed transient thrombocytopenia (Grade II or higher per the Division of AIDS grading of severity of adult and pediatric adverse events, http://www.hptn.org/web%20documents/hptn046/ssp/appendices/appendixe-toxicitytables_daids_ae_gradingtable_finaldec2004.pdf) after TwHF administration. No other TwHF-associated adverse effects including hematologic complications, infection, hepatic impairment, gastrointestinal events greater than Grade II were reported. Participants remained virally suppressed throughout the trial, although occasional isolated blips (defined as a transiently detectable low viral load, typically less than 400 copies/ml, at no more than one visit) were observed.

Discussion

This is the first study to demonstrate the association of TwHF extract, a well-tolerated traditional Chinese medication, with immune recovery and decreased immune activation.

We showed an association between low dose TwHF extract and reduced T-cell activation in the setting of viral suppression on cART. In addition, this immune modulatory effect was temporally correlated to immune recovery: decreased CD8 cell activation correlated to total CD4 cell and memory CD4 cell recovery, while decreased CD4 cell activation correlated in particular to memory CD4 cell recovery. Taken together, these results are consistent with previous findings that HIV infection and immunologic failure are related to preferential depletion of memory CD4 cells, which is correlated with immune activation.^32–34 Thus, as immune activation decreases with TwHF extract use, memory CD4 cells are able to recover. Notably, the CD4/CD8 ratio did not normalize with TwHF use, reflecting the expansion of effector CD8 cells which are associated with aging and accelerated disease progression. Interventions aimed at fully restoring this ratio should be investigated.³⁵

Previously, several approaches targeting immune activation have been attempted.^18,36 HCQ has been added to cART in the setting of viral suppression.¹⁴ It had significant immunomodulatory effects and reduced inflammation and immune activation; however, only a trend toward CD4 cell count recovery was observed.¹⁴ This may be partially explained by differences in immune modulation between TwHF and HCQ, as HCQ did not show a significant decrease in CD38 expression,¹⁴ while in our study, TwHF did. In addition, whether the multiple immune activation and proinflammatory markers utilized in the HCQ study are predictive of clinical outcomes needs further assessment.

Notably, the observed immunomodulatory effect is useful only when virologic suppression has been achieved, given that its target is ‘residual’ immune activation³ due to low level plasma viremia³⁷ and microbial translocation.²⁷ In another study using HCQ in cART-naive patients, HCQ failed to decrease CD8 cell activation and was associated with poorer clinical outcomes compared with placebo.¹⁷ Other small-scale studies with chloroquine,¹⁵ celecoxib,¹⁶ and leflunomide³⁸ showed suppression of immune activation in the absence of cART and viral suppression but failure to boost CD4 recovery. These results suggest that immunomodulants exert optimal effects only after viral suppression.

Low dose TwHF extract is sufficient to suppress immune activation without causing significant adverse effects greater than Grade II. Since TwHF extract has been widely used in China for the treatment of RA and other autoimmune diseases including Crohn's disease and certain types of nephrotic syndrome at a dosage of 20 mg three times daily,²¹ its safety profile is well-established. However, because it has never been used in HIV-infected patients for this purpose, we reduced the dosage in an attempt to minimize adverse effects including hematologic complications or infections.

While this study is the first to identify a medication associated with a decrease in immune activation, and most importantly, CD4 cell recovery in HIV-infected patients, there are some limitations to our study. First, this is a pilot study with a limited sample size and, thus, limited power. The study was also not a randomized, placebo-controlled trial. As such, we were only able to demonstrate an association between TwHF and CD4 cell recovery and cannot draw any conclusions regarding causality. Based on our results, however, a randomized, placebo-controlled, multicenter study is warranted, in addition to further investigations regarding the mechanism of TwHF.

In conclusion, this pilot study establishes TwHF extract as an effective immunomodulant, and provides new strategies for targeting immune activation, inadequate immune response, and, potentially, for associated comorbidities. Further randomized placebo-controlled studies are warranted to evaluate the immunomodulatory effects of TwHF extract on CD4 cell recovery.

Acknowledgements

We thank Felicia C. Chow from University of California, San Francisco for her help in revising the manuscript. We are grateful for all the healthcare providers and all the patients who have participated in these studies.

Disclaimer Statements

Contributors

T. Li and J.-P. Routy managed and designed the research, coordinated the patient cohort, and provided critical review of the manuscript. J. Xie, Y. Han, Z. Qiu, M. Sun and X. Zhang performed primary study assays. Y.J. Li, J. Xie and T. Li analyzed data and wrote the manuscript. Y.L. Li and X. Song participated in patient management, implemented the clinical protocol and data collection. W. Lv, F. Wang and H. Jiang participated in data analyses.

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 81071372 to T. Li) and National Key Technologies R&D Program for the 12th Five-year Plan (grant no. 2012ZX10001003-001 to T. Li). J. Xie was supported by the grant of Chinese Ministry of Human Resources and Social Security and Peking Union Medical College Hospital Research Fund for Young Investigators. This study received partial support from Canadian Institute of Health, Clinical HIV trials network, to J.-P. Routy. In addition, J.-P. Routy holds the McGill University Louis Lowenstein Chair of Hematology & Oncology, Montreal, QC, Canada.

Conflicts of Interests

The authors declare no conflicts of interest.

Ethics Approval

This study was approved by the Institutional Review Board of Peking Union Medical College Hospital. It was in compliance with local regulations and the Declaration of Helsinki.