Traditional Chinese Medicine TN-01 Reconstitutes T Cells in SIV–infected Rhesus Monkeys

Background: Long-term antiretroviral therapy (ART) cannot recover the T cell counts in a substantial proportion of AIDS patients and viral loads rapidly rebound after ART suspension. Therefore, exploring novel alternative and adjuvant ART has become a priority in HIV treatment. Traditional Chinese medicine (TCM) have benecial effects in regulating T cells and our previous clinical trial data have shown that TCM TN-01 maintains good health of patients with AIDS with an unclear mechanism. Herein, we preliminary investigated whether TN-01 inuences immune reconstruction to enhance the antiviral immune response in simian immunodeciency virus (SIV)-infected rhesus monkeys. Methods: The SIV-infected animals were rstly administered with TN-01 alone (5 ml/kg; twice a day, i.g.). More than 2 months later, these 2 SIV-infected rhesus monkeys were intraperitoneally injected with PMPA (30 mg/kg; once a day) and FTC (20 mg/kg; once a day) for about 3 months. Moreover, SIV-infected rhesus monkeys were also administered with TN-01 each 2 weeks from week 23 to week 35 during ART. Flow cytometer were used to assess the phenotype of T cell and plasma was isolated from whole blood for viral load detection. Results: The ow cytometry analysis revealed that treatment with TN-01 alone or combined with ART signicantly upregulated T cells counts and the proportions of T cell subsets. TN-01 treatment also delayed viral rebound after ART suspension. Conclusions: Our data indicate that TN-01 could enhance the ecacy of ART and promote immune reconstruction, suggesting TN-01 treatment as a potent strategy in immune functional cure of HIV infection.

Traditional Chinese herbal medicine (TCM) plays important roles in treating immune de ciency diseases. [14,15] For instance, clinical data showed that TCM is effective in treating AIDS via maintaining immune function and reducing the adverse effects of ART. [15] TCM steadily increased CD4 + and CD8 + T cell counts during viral infection, [16,17] while has no effect on viral load in patients. Interestingly, combination of TCM and ART signi cantly inhibited HIV infection, [16,18] suggesting TCM as an adjuvant treatment of ART to achieve the goal of AIDS eradication. [19] Notably, our previous study showed that a unique formula of TCM (TN-01) maintained normal CD4 + T cells counts in AIDS patients. [20]. In this study, we investigated its action on the immune function reconstruction. We revealed that TN-01 upregulated CD4 + and CD8 + T cell counts and enhanced T cells differentiation and activation in SIVinfected rhesus monkeys. Treatment with TN-01 combined with ART therapy also delayed the rapidly rebound of viral load after treatment interruption. Our results highlight the potential function of TN-01 in eradicating HIV infection.

Animals
Adult rhesus monkeys in this study were obtained from Guangdong Landau Biotechnology Co. Ltd (Guangzhou, China). All rhesus monkeys were con rmed to be free of Tuberculin, B virus, D-type simian retrovirus, simian T lymphotropic virus type 1 and SIV. All rhesus monkeys were housed at the Non-Human Primate Animal Center of the Guangdong Landau Biotechnology Co. Ltd and acclimatized in a separate cage, with standard primate food and water. All animal experiments were performed in accordance with the animal experiment manual and reviewed and approved by the Institutional Animal Care and Use Committee of Guangdong Landau Biotechnology Co., Ltd (LDACU 20170410-01).

Rhesus monkey model of SIV infection
Rhesus monkey model of simian immunode ciency virus (SIVmac251, SIV) infection was established as previously described. [21] In brief, rhesus monkeys were infected intravenously (i.v.) with 300 TCID 50 of SIVmac251. 16 weeks later, the SIV-infected rhesus monkeys were ready to treat with TN-01 alone or TN-01 combined with ART therapy, as described in Fig.1.

Blood collection and preparation
Whole blood was collected from SIV-infected rhesus monkeys at week 0 (before treatment), 2, 4, 6, 8, 10, 12 and 14. Subsequently, T cell counts and the phenotype of T cell were determined using a ow cytometer (FACSCanto; Becton Dickinson). Plasma was isolated from whole blood, and was cryopreserved at -80 °C for viral load detection. In addition, whole blood was collected after combined treatment of TN-01 and ART, and T cell phenotype was then determined by ow cytometry at week 23, 25, 27, 29, 31, 33, 38 and 40. Plasma viral loads were measured every week.

SIV-1 viral load measurement
Plasma SIV viral load was quanti ed by SYBR green Real-time reverse transcription-polymerase chain reaction (RT-PCR) based on published study. [24] Primers and probes synthesized by Invitrogen (Carlsbad, CA, USA) were designed as previously study, [25] Alu1217-F, GCA GAG GAG GAA ATT ACC CAG; SIVgagA, CAA TTT TAC CCA GGC ATT TAA TGT T; and Alu1217-PFAM, TCGG GCTTAATGGCAGGTGGACA. Brie y, the viral RNA was isolated using QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the provided instructions. RNA measurement was performed using One-step qPCR Kit RNA-direct Real time PCR Master Mix (TaKaRa, Tokyo, Japan).

Statistical analysis
Statistical analysis was performed using GraphPad Prism7.0 (GraphPad Software Inc., San Diego, CA, United States). One-way analysis of variance (ANOVA) followed by Turkey post hoc test and unpaired.
Student's t-test were used to analyze the statistical signi cance among multiple groups and between two groups, respectively. Data are reported as the mean ± SD. P < 0.05 was considered statistically signi cant.

Study design
Rhesus monkeys infected SIV before TN-01 treatment (see Methods). We used 2 rhesus monkeys successfully infected with SIV in this study of TN-01 treatment on immune reconstitution, and we named these 2 monkeys as S1 and S3, respectively. The SIV-infected animals were rstly administered with TN-01 alone (5 ml/kg; twice a day, i.g.) from week 0 to week 2, and from week 4 to week 6, respectively (Fig. 1a). Plasma viral loads and immunological e cacy of TN-01 were detected at week 0 (without therapy) and the indicated weeks (up to week 14) (Fig.1a). More than 2 months later, these 2 SIV-infected rhesus monkeys were intraperitoneally injected with PMPA (30 mg/kg; once a day) and FTC (20 mg/kg; once a day) for about 3 months. Moreover, SIV-infected rhesus monkeys were also administered with TN-01 each 2 weeks from week 23 to week 35 during ART. T cell phenotype and plasma viral loads were detected by ow cytometry at the indicated time points (see materials and methods).

Effects of TN-01 on total T-cell counts
Immune cells are essential for AIDS patients, [16] we therefore assessed the immunomodulatory effects of TN-01 in SIV-infected rhesus monkeys (named S1 or S3). As expected, ow cytometry analysis revealed that CD4 + and CD8 + T cell counts were remained signi cantly higher than week 0 (Fig.   2a). Similarly, combinatory treatment with ART and TN-01 also increased CD4 + and CD8 + T cell counts after therapy interruption (Fig. 2b). These changes were sustained for 14 weeks, suggesting that TN-01 promoted the e cacy of ART. Together, these results indicated that total CD4 + and CD8 + T cell counts were elevated during TN-01 therapy and these changes were sustained at therapy discontinuation.
Effects of TN-01 on memory T cell Effector memory T cell plays a key role in immune response against virus infection.
[26] We next explored whether TN-01 in uenced the different subsets of memory T cell. After 2 weeks, the proportions of CD4 + and CD8 + central memory cells (T CM )were signi cantly decreased ∼20% in monkeys treated with TN-01 alone (Fig. 3a). In addition, the proportions of CD4 + and CD8 + effector memory cells (T EM )were signi cantly different from those T CM cells at week 2 and had a steady increment up to week 14 after TN-01discontinuation (Fig. 3b). These changes were sustained at 14 weeks, corroborating that TN-01 in uenced proportion of memory T cell. Interestingly, T CM cells showed a steady increase during TN-01 and ART combinatory treatment and remained high level up to 15 weeks after treatment discontinuation (Fig. 3c), which were not observed in T EM cells (Fig. 3d). Together, these results indicated that TN-01 therapy increased the proportions of T CM cells duringSIV-infection, and continuously enhanced the differentiation of T CM cells into T EM cellsto against SIV during persistent infection.

Effects of TN-01 on T cells immune response
To con rm the fore mentioned results obtained from ow cytometry analysis of memory T cell subsets (Fig. 3), we next explored whether TN-01 could in uence T cell immune response in SIV-infected rhesus monkeys. The result showed that the percentages of CD4 + and CD8 + Tregs were markedly suppressed after secondary TN-01 discontinuation (Fig. 4a). Moreover, the proportion of CD4 + and CD8 + Tregs were slightly lower than pre 2 weeks by combined with ART therapy (Fig. 4c), suggesting that TN-01 may more effective to recover T cell immune response during SIV-infection. Next, we investigated the effects of TN-01 on the activation of different subsets of CD4 + and CD8 + T cells. At week 6, TN-01 therapy decreased the proportions of CD38 + CD4 + and CD38 + CD8 + T cells (Fig. 4b). The proportions of CD69 expression signi cantly increased after secondary TN-01 discontinuation and HLA-DR expression slightly upregulated for 14 weeks (Fig. 4b), suggesting that TN-01 could achieve T cell reconstruction for SIVinfected rhesus monkeys. Interestingly, combination therapy did not change the expression levels of CD69, CD38 and HLA-DR (Fig. 4d). All these results indicated that TN-01 enhanced T cell activation.

Effects of TN-01 on viral load
Next, we assayed the effects of TN-01 on viral load. Flow cytometry analysis showed that the number of SIV-infected T cells (CD4 + CCR5 + ) was signi cantly higher after TN-01 therapy (Fig. 5a), whereas the viral load was not obviously reduced (Fig. 5b). Interestingly, TN-01 combined with ART therapy could reduce the proportions of CD4 + CCR5 + cells as compared to TN-01 alone therapy (Fig. 5c). Furthermore, viral load also showed signi cantly suppression by combination therapy (Fig. 5d), indicating that combination therapy was more bene t to eliminate SIV-infected cells and to delay viral rebound once treatment discontinued. Together, these results indicated that TN-01 combined with ART therapy has the potential to become a functional cure for SIV-infected rhesus monkeys Discussion Long-term ART treatment just controls HIV replication and does not restore normal T cell function to eliminate HIV. [27] Therefore, it is necessary to nd an effective and long-lasting intervention to enhance e cacy of ART and eliminate HIV completely by immune reconstruction [28,29]. It has been demonstrated that TCM can safely and effectively treat AIDS, [30,31] as well as control HIV-infected patients. [16,18,19] For instance, clinical data showed that Zhongyan-4 has an immuno-protective effect in HIV-infected patients for 6 months by elevating CD4 + T cells counts and reducing viral load. [17] Our previous clinical trial reports also suggested that TN-01 improves the long-term survival of HIV-infected patients. TN-01 decreased viral loads from 2003 to 2006 and maintained normal CD4 + counts in nine living AIDS patients, suggesting that TN-01 is more conducive for HIV-infected patients to maintain health for long time. [20] In the present works, we examined whether TN-01 in uences T cell function. Although only 2 SIV-infected rhesus monkeys were used in this experiment, T cell counts and several T cells subsets were increased after TN-01 treatment. Moreover, combined with ART administration inhibited the rapid rebound of viral load after treatment interruption. Therefore, TN-01 therapy is suggested to be a potential application in treating HIV/SIV infection.
One major concern of this study is the underlying mechanism for TN-01 treatment. Considering that T cell reconstruction does not recover completely during ART treatment, we hypothesized that TN-01 might have been involved in T cell immune function reconstruction during infection. In support of this notion, we found that TN-01 elevated the levels of T cell counts in SIV-infected rhesus monkeys, consistent with our previous study. [20] Besides, TN-01 combined with ART further increased T cell counts. It is known that T CM can differentiate into T EM when encounters the pathogen again, [32] and T EM activation plays crucial role in immune response process against HIV infection. Consistently, we showed that TN-01 treatment alone increased the proportion of CD4 + and CD8 + T EM (see Fig. 1). Interestingly, combined treatment with TN-01 and ART elevated the proportion of T CM , but did not change the proportion of T EM , suggesting that TN-01 was able to reshape the proportion of memory T cell subsets. This was likely due to that ART effectively suppressed early stage of virus infection and TN-01 did not need to further promote the differentiation of T CM into T EM . In addition, Tregs can balance immune activation in the immune system, [33] and several studies showed that Tregs exerted a negative role in HIV defense by inhibiting the response of CD4 + and CD8 + T cells in HIV-infected patients or SIV-infected rhesus monkeys. [34,35] We also found that TN-01 reduced the proportions of Tregs, furthering con rming the negative function of Tregs in immune response against HIV infection.
Activation of CD4 + T cell is enhanced in acute HIV infection,[36] and CD4 + and CD8 + T cell activation plays signi cant role for suppression of HIV replication. [9,10,37] We therefore evaluated the effect of TN-01 on T cell activation. CD69 is the earliest activation marker during T cells activation,[38] and CD69 expression becomes a reliable and rapid assessment for the activation and antiviral functions of CD4 + and CD8 + T cells.
[38] Indeed, we found that TN-01 treatment greatly increased the proportion of CD69 + T cell, implying enhanced T cell immune response by TN-01.
Virus reservoir is the main reason for the virus rebound of HIV patients, and viral load will rapid rebound for 4 weeks or several months after ART interruption. [39] Although virus reservoir was not detected in the present study, our experiments showed that viral load was maintained at a low level after ART treatment interruption in our observations for 8 weeks (from week 36 to week 43). Accordingly, additional investigation is needed to explore virus reservoir and to evaluate an extended observation time after treatment interruption.

Conclusion
Our study showed that TN-01 potentiates immune reconstruction to antagonize SIV infection. Moreover, TN-01 combined with ART therapy was effective in delaying the rapid viral rebound after ART treatment interruption. Therefore, our ndings highlight the potential application of TN-01 as an adjuvant treatment of ART therapy in functional cure of HIV/SIV infection.

Consent for publication
All authors consent to the publication of this manuscript.

Availability of data and materials
Details of data mining, selection, extraction and assessment carried out to support the ndings of this study are available from the corresponding author upon request.

Competing interests
No competing financial interests exist. Authors' contribution QZZ drafted the manuscript. QLW, QZZ and ZR searched the literature and conducted the assessment. FJJ and QLW gave suggestions and revised the manuscript. FJJ designed the study. FJJ and YFW gave advice and revised the manuscript. All authors read and approved the nal manuscript. Figure 1 Experimental schedule of SIV-infected rhesus monkey. After rhesus monkeys were infected with SIVmac251 (green arrows) for 16 weeks, then 2 rhesus monkeys were treated with TN-01 (blue arrows) for indicated time periods. Subsequently, SIV-infected rhesus monkeys were treated with ART (yellow arrow) from week 23 to week 35 in the presence or absence of TN-01 (blue arrows) for indicated time period. Flow cytometry detected T cell phenotype (red arrows) and viral loads.