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

Traditional Chinese Medicine TN-01 Reconstitutes T Cells in SIV–infected Rhesus Monkeys | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Traditional Chinese Medicine TN-01 Reconstitutes T Cells in SIV–infected Rhesus Monkeys Qiaoli Wang, Qiongzhen Zeng, Zhe Ren, Yuefeng Li, Rongze Wang, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-693617/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 beneficial 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 influences immune reconstruction to enhance the antiviral immune response in simian immunodeficiency virus (SIV)-infected rhesus monkeys. Methods: The SIV-infected animals were firstly 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 flow cytometry analysis revealed that treatment with TN-01 alone or combined with ART significantly 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 efficacy of ART and promote immune reconstruction, suggesting TN-01 treatment as a potent strategy in immune functional cure of HIV infection. Internal Medicine HIV/AIDS Antiretroviral therapy Traditional Chinese medicine Immune reconstruction Immune functional cure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Backgroud Acquired immune deficiency syndrome (AIDS) is a type of CD4 + T cells exhaustion mediated immunodeficiency syndrome caused by human immunodeficiency virus (HIV) infection.[ 1 , 2 ] T cell plays significant roles in suppression of virus replication,[ 3 ] and eliminates the virus infected cells.[ 4 , 5 ] Therefore, functional cure of HIV infection through modulation of T cell reconstitution becomes long-term goal. Currently, most of HIV-infected people are widely treated by antiretroviral therapy (ART).[ 6 ] Despite ART can effectively control virus replication whereas there is a substantial proportion of AIDS patients that do not exhibit any improvements in the normalization of T cell counts and T cell function under long-term ART.[ 7 – 10 ] Besides, viral load rapidly rebounds upon ART interruption in vast majority of HIV-infected individuals.[ 6 , 11 ] Long-term ART also causes some unacceptable side effects due to the drug toxicity, poor adherence to therapy, and drug resistance.[ 12 , 13 ] Therefore, it is necessary to develop new drugs or complementary methods to overcome the limitations of ART in treating AIDS and AIDS-related complications. Traditional Chinese herbal medicine (TCM) plays important roles in treating immune deficiency 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 significantly 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 SIV-infected 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. Materials And Methods Animals Adult rhesus monkeys in this study were obtained from Guangdong Landau Biotechnology Co. Ltd (Guangzhou, China). All rhesus monkeys were confirmed 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 immunodeficiency 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 flow 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 flow cytometry at week 23, 25, 27, 29, 31, 33, 38 and 40. Plasma viral loads were measured every week. Flow cytometry analysis of T cell phenotype Flow cytometry analysis of T-cell phenotype was performed according to the protocols as previously described.[21-23] Monoclonal antibodies used in this study including anti-CD3-BV605, anti-CD45-PE, anti-CD4-BV421, anti-CD8-APC-R700 anti-CD95-DX2, anti-CD28-CD28.2, anti-HLA-DR-PE-Cy7, anti-CD69-APC and anti-CCR5-PE, were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD38-FITC was obtained from StemCell Technologies (Vancouver, BC, Canada). Anti-CD25-APC was from Biolegend (San Diego, CA, USA), and anti-CD127-PE was a product of Invitrogen (Carlsbad, CA, USA). CD4 + T cell differentiation was identified in terms of CD28 and CD95 expression, as CD28 + CD95 + CD4 + T cell defined as central memory CD4 + T cell (CD4 + Tcm) and CD28 + CD95 - CD4 + T cell as effector memory CD4+ T cell (CD4 + T EM ). CD28 + CD95 + CD8 + T cell was defined as CD8 + T CM cell whereas CD28 + CD95 - CD8 + T cell was CD8 + T EM cell. In addition, expression of CD25 and CD127 were measured to evaluate regulatory T Cells (Tregs). Activation markers HLA-DR, CD38 and CD69 were measured on CD4 + and CD8 + T cells, and CD4 + CCR5 + T cells were defined as SIV-infected cells. All dates were acquired and analyzed on a flow cytometer (FACSCanto; Becton Dickinson). SIV-1 viral load measurement Plasma SIV viral load was quantified 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. Briefly, 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 significance among multiple groups and between two groups, respectively. Data are reported as the mean ± SD. P < 0.05 was considered statistically significant. Results 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 firstly 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 efficacy 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 flow 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, flow cytometry analysis revealed that CD4 + and CD8 + T cell counts were remained significantly 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 efficacy 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 influenced the different subsets of memory T cell. After 2 weeks, the proportions of CD4 + and CD8 + central memory cells (T CM )were significantly 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 significantly 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 influenced 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 confirm the fore mentioned results obtained from flow cytometry analysis of memory T cell subsets (Fig. 3), we next explored whether TN-01 could influence 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 significantly 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 SIV-infected 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 significantly 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 significantly suppression by combination therapy (Fig. 5d), indicating that combination therapy was more benefit 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 find an effective and long-lasting intervention to enhance efficacy 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 influences 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 confirming 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 significant 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 findings highlight the potential application of TN-01 as an adjuvant treatment of ART therapy in functional cure of HIV/SIV infection. Abbreviations ART: antiretroviral therapy; TCM: Traditional Chinese medicine; SIV: simian immunodeficiency virus; AIDS: Acquired immune deficiency syndrome; HIV: human immunodeficiency virus. i.v.: infected intravenously; i.g.: intragastrically Declarations Ethics approval and consent to participate All animal experiments in this study 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). 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 findings of this study are available from the corresponding author upon request. Competing interests No competing financial interests exist. Funding This work was supported by the National Natural Science Foundation of China (grants 81872908 and 820722741) and The Key Laboratory of Virology of Guangzhou (grant 201705030003) and Guangzhou Major program of the Industry-University-Research collaborative innovation (grant 201704030087). 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 final manuscript. Acknowledgements The authors would like to thank all the researchers and experts for this study. References Douek DC, Roederer M, Koup RA. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med. 2009;60:471-484. Okoye AA, Picker LJ. CD4(+) T-cell depletion in HIV infection: mechanisms of immunological failure. Immunol Rev. 2013;254(1):54-64. Xu H, Wang X, Lackner AA, et al. CD8 down-regulation and functional impairment of SIV-specific cytotoxic T lymphocytes in lymphoid and mucosal tissues during SIV infection. J Leukoc Biol. 2013;93(6):943-950. Cartwright EK, Spicer L, Smith SA, et al. CD8(+) Lymphocytes Are Required for Maintaining Viral Suppression in SIV-Infected Macaques Treated with Short-Term Antiretroviral Therapy. Immunity. 2016;45(3):656-668. Agosto LM, Henderson AJ. CD4(+) T Cell Subsets and Pathways to HIV Latency. AIDS Res Hum Retroviruses. 2018;34(9):780-789. Hatano H, Yukl SA, Ferre AL, et al. Prospective antiretroviral treatment of asymptomatic, HIV-1 infected controllers. PLoS Pathog. 2013;9(10):e1003691. Wilson EM, Sereti I. Immune restoration after antiretroviral therapy: the pitfalls of hasty or incomplete repairs. Immunol Rev. 2013;254(1):343-354. Kelley Colleen F, Kitchen Christina MR, Hunt Peter W, et al. Incomplete Peripheral CD4+Cell Count Restoration in HIV‐Infected Patients Receiving Long‐Term Antiretroviral Treatment. Clinical Infectious Diseases. 2009;48(6):787-794. Volberding P, Demeter L, Bosch RJ, et al. Antiretroviral therapy in acute and recent HIV infection: a prospective multicenter stratified trial of intentionally interrupted treatment. AIDS. 2009;23(15):1987-1995. Ndhlovu ZM, Kamya P, Mewalal N, et al. Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point. Immunity. 2015;43(3):591-604. Davey RT, Bhat N, Yoder C, et al. HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression. Proc Natl Acad Sci USA. 1999;96(26):15109-15114. Montessori V, Press N, Harris M, et al. Adverse effects of antiretroviral therapy for HIV infection. CMAJ. 2004;170(2):229-238. Wainberg MA, Zaharatos GJ, Brenner BG. Development of antiretroviral drug resistance. N Engl J Med. 2011;365(7):637-646. Wu X-f, Wang J, Li Y, et al. Thoughts on intervention in HIV/AIDS with traditional Chinese medicine. J Tradit Chin Med. 2011;31(4):265-268. Liu Z-B, Yang J-P, Xu L-R. Effectiveness and safety of traditional Chinese medicine in treating acquired immune deficiency syndrome: 2004-2014. Infectious diseases of poverty. 2015;4:59. Zou W, Wang J, Liu Y. Effect of traditional Chinese medicine for treating human immunodeficiency virus infections and acquired immune deficiency syndrome: Boosting immune and alleviating symptoms. Chin J Integr Med. 2016;22(1):3-8. Wang J, Yang FZ, Zhao M, et al. Randomized double-blinded and controlled clinical trial on treatment of HIV/AIDS by Zhongyan-4. Chin J Integr Med. 2006;12(1):6-11. Wang J, Zou W. Practices, challenges, and opportunities: HIV/AIDS treatment with traditional Chinese medicine in China. Front Med. 2011;5(2):123-126. Liu ZB, Yang JP, Xu LR. Effectiveness and safety of traditional Chinese medicine in treating acquired immune deficiency syndrome: 2004-2014. Infect Dis Poverty. 2015;4:59. Wang Y, Jin F, Wang Q, et al. Long-Term Survival of AIDS Patients Treated with Only Traditional Chinese Medicine. AIDS Res Hum Retroviruses. 2017;33(2):90-92. Zhan XY, Wang N, Liu G, et al. Plasmodium infection reduces the volume of the viral reservoir in SIV-infected rhesus macaques receiving antiretroviral therapy. Retrovirology. 2014;11:112. Liu G, Li Y, Qin L, et al. SIV infection aggravates malaria in a Chinese rhesus monkey coinfection model. BMC infectious diseases. 2019;19(1):965. Maino VC, Suni MA, Ruitenberg JJ. Rapid flow cytometric method for measuring lymphocyte subset activation. Cytometry. 1995;20(2):127-133. Zhan X-Y, Wang N, Liu G, et al. Plasmodium infection reduces the volume of the viral reservoir in SIV-infected rhesus macaques receiving antiretroviral therapy. Retrovirology. 2014;11:112. Hofmann-Lehmann R, Swenerton RK, Liska V, et al. Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one- versus two-enzyme systems. AIDS research and human retroviruses. 2000;16(13):1247-1257. Kulpa DA, Talla A, Brehm JH, et al. Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4(+) T Cells. J Virol. 2019;93(24). Hunt PW. HIV and inflammation: mechanisms and consequences. Curr HIV/AIDS Rep. 2012;9(2):139-147. Pace M, Frater J. A cure for HIV: is it in sight? Expert review of anti-infective therapy. 2014;12(7):783-791. Davenport MP, Khoury DS, Cromer D, et al. Functional cure of HIV: the scale of the challenge. Nature reviews. Immunology. 2019;19(1):45-54. Xu LR, Guo HJ, Liu ZB, et al. Unified-planning, graded-administration, and centralized-controlling: a management modality for treating acquired immune deficiency syndrome with Chinese medicine in Henan Province of China. Chin J Integr Med. 2015;21(4):243-248. Jiang F, Zhang R, Gu Z, et al. Fuzhengpaidu granule regulates immune activation molecules CD38 and human leukocyte antigen-D related on CD4+ and CD8+ T cells in patients with acquired immunodeficiency syndrome/human immunodeficiency virus. J Tradit Chin Med. 2013;33(4):439-443. Groot F, van Capel TM, Schuitemaker J, et al. Differential susceptibility of naive, central memory and effector memory T cells to dendritic cell-mediated HIV-1 transmission. Retrovirology. 2006;3:52. Holmes D, Jiang Q, Zhang L, et al. Foxp3 and Treg cells in HIV-1 infection and immuno-pathogenesis. Immunol Res. 2008;41(3):248-266. Pereira LE, Villinger F, Onlamoon N, et al. Simian immunodeficiency virus (SIV) infection influences the level and function of regulatory T cells in SIV-infected rhesus macaques but not SIV-infected sooty mangabeys. J Virol. 2007;81(9):4445-4456. Aandahl EM, Michaelsson J, Moretto WJ, et al. Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol. 2004;78(5):2454-2459. Xia H, Jiang W, Zhang X, et al. Elevated Level of CD4+ T Cell Immune Activation in Acutely HIV-1-Infected Stage Associates With Increased IL-2 Production and Cycling Expression, and Subsequent CD4+ T Cell Preservation. Front Immunol. 2018;9. McBrien JB, Kumar NA, Silvestri G. Mechanisms of CD8(+) T cell-mediated suppression of HIV/SIV replication. Eur J Immunol. 2018;48(6):898-914. Pitsios C, Dimitrakopoulou A, Tsalimalma K, et al. Expression of CD69 on T-cell subsets in HIV-1 disease. Scandinavian journal of clinical and laboratory investigation. 2008;68(3):233-241. Vallejo A, Molina-Pinelo S, De Felipe B, et al. Toll-like receptor 9 1635A/G polymorphism is associated with HIV-1 rebound after four weeks of interruption of antiretroviral therapy. J Acquir Immune Defic Syndr. 2020. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-693617","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":38713842,"identity":"d0a4ed54-9f0e-4405-adb9-06f3c4f47769","order_by":0,"name":"Qiaoli Wang","email":"","orcid":"","institution":"Jinan University College of Life Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiaoli","middleName":"","lastName":"Wang","suffix":""},{"id":38713843,"identity":"b677ae8a-1269-47dc-940d-a99694359d70","order_by":1,"name":"Qiongzhen Zeng","email":"","orcid":"","institution":"Jinan University College 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Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yexuan","middleName":"","lastName":"Zhu","suffix":""},{"id":38713853,"identity":"10d8d6aa-971f-4fa7-aeba-7e8ac7bcd0bb","order_by":11,"name":"Feng Liang","email":"","orcid":"","institution":"Jinan University College of Information Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Liang","suffix":""},{"id":38713854,"identity":"5799fabe-b745-41dc-a1ee-ab0705a33b0f","order_by":12,"name":"Ziyao Li","email":"","orcid":"","institution":"Jinan University College of Life Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ziyao","middleName":"","lastName":"Li","suffix":""},{"id":38713855,"identity":"7bd1ff6e-02a3-43ef-ad3b-65dfcd57e227","order_by":13,"name":"Hemei Qi","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hemei","middleName":"","lastName":"Qi","suffix":""},{"id":38713856,"identity":"f9e496ef-3907-4f0e-9f46-4537ff1137a3","order_by":14,"name":"Li Qin","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Qin","suffix":""},{"id":38713857,"identity":"e571c4ba-c02e-4f01-b2a5-61fce799ac04","order_by":15,"name":"Fujun Jin","email":"","orcid":"","institution":"Jinan University College of Life Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fujun","middleName":"","lastName":"Jin","suffix":""},{"id":38713858,"identity":"aa77631a-003e-4ddc-96e9-09681d26845a","order_by":16,"name":"Yifei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYPACOTnGBhDNRoRaHghlbEy6lkSwDqK02EskP3v4tc0gvXl2jwHDh7LDDPyzGwjYIpFmbizbZpDbOOeMAeOMc4cZJO4cIKBFOsFMWrLtT27jjBwDZt62wwwGEgmEtKR/A2oxSGcEaflLnJYcM8mPbQYJYC2MRGm5/6ZMmuGcgWHjjLSCgz3n0nkkbhDQwt5zfJvkjzIDecMZyRsf/CizluOfQUALCDDzAqPDsIGB4QADPKIIAMYffxgY5IlSOgpGwSgYBSMSAAAOqj7u6xR1KQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9918-2865","institution":"Jinan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2021-07-07 17:21:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-693617/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-693617/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":11420388,"identity":"cae07cec-bc71-40a2-b328-ad7739bcac58","added_by":"auto","created_at":"2021-07-13 16:36:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101768,"visible":true,"origin":"","legend":"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.","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-693617/v1/518acb8b0fcce92696a6f7c5.jpg"},{"id":11420390,"identity":"80280c0a-dbdb-4e61-8075-9889eb453d98","added_by":"auto","created_at":"2021-07-13 16:36:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282736,"visible":true,"origin":"","legend":"TN-01 increased T cell counts. SIV-infected rhesus monkeys were treated with TN-01 alone (a) or combined with ART therapy (b) for indicated time periods. (a) Flow cytometry was used to assess CD4+ or CD8+ T cell counts on 8 time points (week 0, 2, 4, 6, 8, 10, 12 and 14 in the course). Histograms show the total CD4+ or CD8+ T cell counts. (b) Histograms show total CD4+ or CD8+ at indicated time periods (week 23, 25, 27, 29, 31, 33, 38 and 40 in the course). ","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-693617/v1/804e9a61925a0d712b4c11d1.jpg"},{"id":11420389,"identity":"b7b6c91d-68cd-4351-8a78-635d2c98d099","added_by":"auto","created_at":"2021-07-13 16:36:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431017,"visible":true,"origin":"","legend":"Increasing T cells differentiation after TN-01 treatment. SIV-infected rhesus monkeys were treated as FIG.1. Histograms show the percentages of several CD4+ or CD8+ TCM (a) and CD4+ or CD8+ TEM (b) with TN-01 treatment. (c-d) The percentages of indicated CD4+ or CD8+ T cells subsets by TN-01 combined with ART therapy. ","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-693617/v1/d147e8987ae488c7ff84e1db.jpg"},{"id":11420392,"identity":"ed6d8910-46e2-4991-8138-41ca8ebf26af","added_by":"auto","created_at":"2021-07-13 16:36:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":849194,"visible":true,"origin":"","legend":"High levels of activation phenotype expression on CD4+ and CD8+ T cells after TN-01 therapy. TN-01 alone (a-b) or TN-01 combined with ART therapy (c-d) treated as Fig. 1. Flow cytometry analysis of indicated phenotype on CD4+ and CD8+ T cells. (a-c) Histograms show the levels of Treg. (b-d) Histograms show the percentages of CD4+ and CD8+ T cells activation marks CD38, HLA-DR and CD69. ","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-693617/v1/c7293d4502d79b0c519519fe.jpg"},{"id":11420391,"identity":"a79117d2-f695-4a7f-8e75-728a73d1a390","added_by":"auto","created_at":"2021-07-13 16:36:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":372570,"visible":true,"origin":"","legend":"TN-01 combined with ART therapy restrain viral loads rebound. SIV-infected rhesus monkeys were treated with TN-01 alone (a-b) or TN-01 combined with ART therapy (c-d) for indicated time periods. (a-c) CCR5 expression on CD4+ T cells were analyzed by flow cytometry. Histograms show the percentages of CD4+ CCR5+ T cells. (b-d) Histograms show the levels of viral load on indicated time points.","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-693617/v1/d176fdc2be6e6eae0a708276.jpg"},{"id":13703740,"identity":"1ca93bd8-2ead-4a6f-8988-b7f1823c0441","added_by":"auto","created_at":"2021-09-17 13:43:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":912243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-693617/v1/058a368f-49dc-4698-a576-a96ee6df88e8.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTraditional Chinese Medicine TN-01 Reconstitutes T Cells in SIV–infected Rhesus Monkeys\u003c/p\u003e","fulltext":[{"header":"Backgroud","content":"\u003cp\u003eAcquired immune deficiency syndrome (AIDS) is a type of CD4\u003csup\u003e+\u003c/sup\u003e T cells exhaustion mediated immunodeficiency syndrome caused by human immunodeficiency virus (HIV) infection.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] T cell plays significant roles in suppression of virus replication,[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and eliminates the virus infected cells.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Therefore, functional cure of HIV infection through modulation of T cell reconstitution becomes long-term goal.\u003c/p\u003e \u003cp\u003eCurrently, most of HIV-infected people are widely treated by antiretroviral therapy (ART).[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Despite ART can effectively control virus replication whereas there is a substantial proportion of AIDS patients that do not exhibit any improvements in the normalization of T cell counts and T cell function under long-term ART.[\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Besides, viral load rapidly rebounds upon ART interruption in vast majority of HIV-infected individuals.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Long-term ART also causes some unacceptable side effects due to the drug toxicity, poor adherence to therapy, and drug resistance.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Therefore, it is necessary to develop new drugs or complementary methods to overcome the limitations of ART in treating AIDS and AIDS-related complications.\u003c/p\u003e \u003cp\u003eTraditional Chinese herbal medicine (TCM) plays important roles in treating immune deficiency diseases.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] For instance, clinical data showed that TCM is effective in treating AIDS via maintaining immune function and reducing the adverse effects of ART.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] TCM steadily increased CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell counts during viral infection, [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] while has no effect on viral load in patients. Interestingly, combination of TCM and ART significantly inhibited HIV infection,[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] suggesting TCM as an adjuvant treatment of ART to achieve the goal of AIDS eradication.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Notably, our previous study showed that a unique formula of TCM (TN-01) maintained normal CD4\u003csup\u003e+\u003c/sup\u003e T cells counts in AIDS patients.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we investigated its action on the immune function reconstruction. We revealed that TN-01 upregulated CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell counts and enhanced T cells differentiation and activation in SIV-infected 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.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult rhesus monkeys in this study were obtained from Guangdong Landau Biotechnology Co. Ltd (Guangzhou, China). All rhesus monkeys were confirmed 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRhesus monkey model of SIV infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRhesus monkey\u0026nbsp;model of simian immunodeficiency virus (SIVmac251, SIV) infection was established as previously described.[21]\u0026nbsp;In brief, rhesus monkeys were infected intravenously (i.v.) with 300 TCID\u003csub\u003e50\u003c/sub\u003e of SIVmac251. 16 weeks later, the SIV-infected rhesus monkeys\u0026nbsp;were ready to treat with TN-01 alone or TN-01 combined with ART therapy, as described in Fig.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood collection and preparation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood\u0026nbsp;was collected from SIV-infected rhesus monkeys at week 0 (before treatment), 2, 4, 6, 8, 10, 12 and 14.\u0026nbsp;Subsequently, T cell counts and the phenotype of T cell were determined using a flow cytometer (FACSCanto; Becton Dickinson). Plasma was isolated from whole blood, and was cryopreserved at -80 \u0026deg;C for viral load detection. In addition, whole blood was collected after combined treatment of TN-01 and ART, and T cell phenotype\u0026nbsp;was then determined by flow cytometry\u0026nbsp;at week 23, 25, 27, 29, 31, 33, 38 and 40. Plasma viral loads were measured every week.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis of T cell phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis of T-cell phenotype was performed according to the protocols as previously described.[21-23]\u0026nbsp;Monoclonal antibodies used in this study including anti-CD3-BV605, anti-CD45-PE, anti-CD4-BV421, anti-CD8-APC-R700 anti-CD95-DX2, anti-CD28-CD28.2, anti-HLA-DR-PE-Cy7, anti-CD69-APC and anti-CCR5-PE, were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD38-FITC was obtained from StemCell Technologies (Vancouver, BC, Canada). Anti-CD25-APC was from Biolegend (San Diego, CA, USA), and anti-CD127-PE was a product of Invitrogen (Carlsbad, CA, USA). CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation was identified in terms of CD28 and CD95 expression, as CD28\u003csup\u003e+\u003c/sup\u003e CD95\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cell defined as central memory CD4\u003csup\u003e+\u003c/sup\u003e T cell (CD4\u003csup\u003e+\u003c/sup\u003e Tcm) and CD28\u003csup\u003e+\u003c/sup\u003e CD95\u003csup\u003e-\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cell as effector memory CD4+ T cell (CD4\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eEM\u003c/sub\u003e). CD28\u003csup\u003e+\u003c/sup\u003e CD95\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cell was defined as CD8\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eCM\u003c/sub\u003e cell whereas CD28\u003csup\u003e+\u003c/sup\u003e CD95\u003csup\u003e-\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cell was CD8\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eEM\u0026nbsp;\u003c/sub\u003ecell. In addition, expression of CD25 and CD127 were measured to evaluate regulatory T Cells (Tregs). Activation markers HLA-DR, CD38 and CD69 were measured on CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells, and CD4\u003csup\u003e+\u003c/sup\u003e CCR5\u003csup\u003e+\u003c/sup\u003e T cells were defined as SIV-infected cells. All dates were acquired and analyzed on a flow cytometer (FACSCanto; Becton Dickinson).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSIV-1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eviral load measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma SIV viral load was quantified 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. Briefly, 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical 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\u0026rsquo;s t-test were used to analyze the statistical significance among multiple groups and between two groups, respectively. Data are reported as the mean \u0026plusmn; SD. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRhesus monkeys infected SIV before TN-01 treatment (see\u0026ensp;Methods). We used 2 rhesus monkeys successfully infected with SIV in\u0026ensp;this study\u0026ensp;of TN-01 treatment on immune reconstitution, and we named these 2 monkeys as S1 and S3, respectively. The SIV-infected animals were firstly 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 efficacy 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 flow cytometry at the indicated time points (see materials and methods).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TN-01 on total T-cell counts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmune cells are essential for AIDS\u0026nbsp;patients,[16]\u0026nbsp;we therefore assessed the immunomodulatory effects of TN-01 in SIV-infected rhesus monkeys (named S1 or S3). As expected, flow cytometry analysis\u0026nbsp;revealed that\u0026nbsp;CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell counts were remained significantly higher than week 0 (Fig. 2a). Similarly, combinatory treatment with ART and TN-01 also increased CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eand CD8\u003csup\u003e+\u003c/sup\u003e T cell counts after therapy interruption (Fig. 2b). These changes were sustained for 14 weeks, suggesting that TN-01 promoted the efficacy of ART. Together, these results indicated that total CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cell counts were elevated during TN-01 therapy\u0026nbsp;and these changes were sustained at therapy discontinuation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TN-01 on memory T cell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEffector\u0026nbsp;memory T cell\u0026nbsp;plays a key role in immune response against virus infection.[26]\u0026nbsp;We next explored whether\u0026nbsp;TN-01 influenced the different subsets of\u0026nbsp;memory\u0026nbsp;T cell. After 2 weeks, the proportions of\u0026nbsp;CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e central memory cells (T\u003csub\u003eCM\u003c/sub\u003e)were significantly decreased\u0026nbsp;\u0026sim;20% in monkeys treated with TN-01 alone\u0026nbsp;(Fig. 3a). In addition, the proportions of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e effector memory cells (T\u003csub\u003eEM\u003c/sub\u003e)were significantly different from those T\u003csub\u003eCM\u003c/sub\u003e 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 influenced proportion of memory T cell. Interestingly, T\u003csub\u003eCM\u003c/sub\u003e 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\u003csub\u003eEM\u0026nbsp;\u003c/sub\u003ecells (Fig. 3d). Together, these results indicated that TN-01 therapy increased\u0026nbsp;the proportions of T\u003csub\u003eCM\u0026nbsp;\u003c/sub\u003ecells duringSIV-infection, and continuously enhanced the differentiation of T\u003csub\u003eCM\u003c/sub\u003e cells into T\u003csub\u003eEM\u0026nbsp;\u003c/sub\u003ecellsto against SIV during persistent infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TN-01 on T cells immune response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the fore mentioned results obtained from flow cytometry analysis of memory T cell subsets (Fig. 3), we next explored whether TN-01 could influence T cell immune response in SIV-infected rhesus monkeys. The result showed that the percentages of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e Tregs were markedly suppressed after secondary TN-01 discontinuation (Fig. 4a). Moreover, the proportion of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eTregs 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\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells. At week 6, TN-01 therapy decreased the proportions of CD38\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e and CD38\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig. 4b). The proportions of CD69 expression significantly 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 SIV-infected 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TN-01 on viral load\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we assayed the effects\u0026nbsp;of TN-01 on viral load. Flow cytometry analysis showed that the number of SIV-infected T cells (CD4\u003csup\u003e+\u003c/sup\u003e CCR5\u003csup\u003e+\u003c/sup\u003e) was significantly 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\u003csup\u003e+\u003c/sup\u003e CCR5\u003csup\u003e+\u003c/sup\u003e cells as compared to TN-01 alone therapy (Fig. 5c). Furthermore, viral load also showed significantly suppression by combination therapy (Fig. 5d), indicating that combination therapy was more benefit 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\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLong-term ART treatment just controls HIV replication and does not restore normal T cell function to eliminate HIV.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Therefore, it is necessary to find an effective and long-lasting intervention to enhance efficacy of ART and eliminate HIV completely by immune reconstruction[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It has been demonstrated that TCM can safely and effectively treat AIDS,[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] as well as control HIV-infected patients.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] For instance, clinical data showed that Zhongyan-4 has an immuno-protective effect in HIV-infected patients for 6 months by elevating CD4\u003csup\u003e+\u003c/sup\u003e T cells counts and reducing viral load.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] 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\u003csup\u003e+\u003c/sup\u003e counts in nine living AIDS patients, suggesting that TN-01 is more conducive for HIV-infected patients to maintain health for long time.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] In the present works, we examined whether TN-01 influences 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.\u003c/p\u003e \u003cp\u003eOne 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.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Besides, TN-01 combined with ART further increased T cell counts. It is known that T\u003csub\u003eCM\u003c/sub\u003e can differentiate into T\u003csub\u003eEM\u003c/sub\u003e when encounters the pathogen again,[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and T\u003csub\u003eEM\u003c/sub\u003e activation plays crucial role in immune response process against HIV infection. Consistently, we showed that TN-01 treatment alone increased the proportion of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eEM\u003c/sub\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Interestingly, combined treatment with TN-01 and ART elevated the proportion of T\u003csub\u003eCM\u003c/sub\u003e, but did not change the proportion of T\u003csub\u003eEM\u003c/sub\u003e, 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\u003csub\u003eCM\u003c/sub\u003e into T\u003csub\u003eEM\u003c/sub\u003e. In addition, Tregs can balance immune activation in the immune system,[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and several studies showed that Tregs exerted a negative role in HIV defense by inhibiting the response of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in HIV-infected patients or SIV-infected rhesus monkeys.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] We also found that TN-01 reduced the proportions of Tregs, furthering confirming the negative function of Tregs in immune response against HIV infection.\u003c/p\u003e \u003cp\u003eActivation of CD4\u003csup\u003e+\u003c/sup\u003e T cell is enhanced in acute HIV infection,[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell activation plays significant role for suppression of HIV replication.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] We therefore evaluated the effect of TN-01 on T cell activation. CD69 is the earliest activation marker during T cells activation,[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and CD69 expression becomes a reliable and rapid assessment for the activation and antiviral functions of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003eT cells.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] Indeed, we found that TN-01 treatment greatly increased the proportion of CD69\u0026thinsp;+\u0026thinsp;T cell, implying enhanced T cell immune response by TN-01.\u003c/p\u003e \u003cp\u003eVirus 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.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] 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.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur 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 findings highlight the potential application of TN-01 as an adjuvant treatment of ART therapy in functional cure of HIV/SIV infection.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eART: antiretroviral therapy; TCM: Traditional Chinese medicine; SIV: simian immunodeficiency virus; AIDS: Acquired immune deficiency syndrome; HIV: human immunodeficiency virus. i.v.: infected intravenously; i.g.: intragastrically\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments in this study 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetails of data mining, selection, extraction and assessment carried out to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo competing financial interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grants 81872908 and 820722741) and The Key Laboratory of Virology of Guangzhou (grant 201705030003) and Guangzhou Major program of the Industry-University-Research collaborative innovation (grant 201704030087).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQZZ\u0026nbsp;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 final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all the researchers and experts for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDouek DC, Roederer M, Koup RA. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med. 2009;60:471-484.\u003c/li\u003e\n \u003cli\u003eOkoye AA, Picker LJ. CD4(+) T-cell depletion in HIV infection: mechanisms of immunological failure. Immunol Rev. 2013;254(1):54-64.\u003c/li\u003e\n \u003cli\u003eXu H, Wang X, Lackner AA, et al. CD8 down-regulation and functional impairment of SIV-specific cytotoxic T lymphocytes in lymphoid and mucosal tissues during SIV infection. J Leukoc Biol. 2013;93(6):943-950.\u003c/li\u003e\n \u003cli\u003eCartwright EK, Spicer L, Smith SA, et al. CD8(+) Lymphocytes Are Required for Maintaining Viral Suppression in SIV-Infected Macaques Treated with Short-Term Antiretroviral Therapy. Immunity. 2016;45(3):656-668.\u003c/li\u003e\n \u003cli\u003eAgosto LM, Henderson AJ. CD4(+) T Cell Subsets and Pathways to HIV Latency. AIDS Res Hum Retroviruses. 2018;34(9):780-789.\u003c/li\u003e\n \u003cli\u003eHatano H, Yukl SA, Ferre AL, et al. Prospective antiretroviral treatment of asymptomatic, HIV-1 infected controllers. PLoS Pathog. 2013;9(10):e1003691.\u003c/li\u003e\n \u003cli\u003eWilson EM, Sereti I. Immune restoration after antiretroviral therapy: the pitfalls of hasty or incomplete repairs. Immunol Rev. 2013;254(1):343-354.\u003c/li\u003e\n \u003cli\u003eKelley Colleen\u0026nbsp;F, Kitchen Christina\u0026nbsp;MR, Hunt Peter\u0026nbsp;W, et al. Incomplete Peripheral CD4+Cell Count Restoration in HIV‐Infected Patients Receiving Long‐Term Antiretroviral Treatment. Clinical Infectious Diseases. 2009;48(6):787-794.\u003c/li\u003e\n \u003cli\u003eVolberding P, Demeter L, Bosch RJ, et al. Antiretroviral therapy in acute and recent HIV infection: a prospective multicenter stratified trial of intentionally interrupted treatment. AIDS. 2009;23(15):1987-1995.\u003c/li\u003e\n \u003cli\u003eNdhlovu ZM, Kamya P, Mewalal N, et al. Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point. Immunity. 2015;43(3):591-604.\u003c/li\u003e\n \u003cli\u003eDavey RT, Bhat N, Yoder C, et al. HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression. Proc Natl Acad Sci USA. 1999;96(26):15109-15114.\u003c/li\u003e\n \u003cli\u003eMontessori V, Press N, Harris M, et al. Adverse effects of antiretroviral therapy for HIV infection. CMAJ. 2004;170(2):229-238.\u003c/li\u003e\n \u003cli\u003eWainberg MA, Zaharatos GJ, Brenner BG. Development of antiretroviral drug resistance. N Engl J Med. 2011;365(7):637-646.\u003c/li\u003e\n \u003cli\u003eWu X-f, Wang J, Li Y, et al. Thoughts on intervention in HIV/AIDS with traditional Chinese medicine. J Tradit Chin Med. 2011;31(4):265-268.\u003c/li\u003e\n \u003cli\u003eLiu Z-B, Yang J-P, Xu L-R. Effectiveness and safety of traditional Chinese medicine in treating acquired immune deficiency syndrome: 2004-2014. Infectious diseases of poverty. 2015;4:59.\u003c/li\u003e\n \u003cli\u003eZou W, Wang J, Liu Y. Effect of traditional Chinese medicine for treating human immunodeficiency virus infections and acquired immune deficiency syndrome: Boosting immune and alleviating symptoms. Chin J Integr Med. 2016;22(1):3-8.\u003c/li\u003e\n \u003cli\u003eWang J, Yang FZ, Zhao M, et al. Randomized double-blinded and controlled clinical trial on treatment of HIV/AIDS by Zhongyan-4. Chin J Integr Med. 2006;12(1):6-11.\u003c/li\u003e\n \u003cli\u003eWang J, Zou W. Practices, challenges, and opportunities: HIV/AIDS treatment with traditional Chinese medicine in China. Front Med. 2011;5(2):123-126.\u003c/li\u003e\n \u003cli\u003eLiu ZB, Yang JP, Xu LR. Effectiveness and safety of traditional Chinese medicine in treating acquired immune deficiency syndrome: 2004-2014. Infect Dis Poverty. 2015;4:59.\u003c/li\u003e\n \u003cli\u003eWang Y, Jin F, Wang Q, et al. Long-Term Survival of AIDS Patients Treated with Only Traditional Chinese Medicine. AIDS Res Hum Retroviruses. 2017;33(2):90-92.\u003c/li\u003e\n \u003cli\u003eZhan XY, Wang N, Liu G, et al. Plasmodium infection reduces the volume of the viral reservoir in SIV-infected rhesus macaques receiving antiretroviral therapy. Retrovirology. 2014;11:112.\u003c/li\u003e\n \u003cli\u003eLiu G, Li Y, Qin L, et al. SIV infection aggravates malaria in a Chinese rhesus monkey coinfection model. BMC infectious diseases. 2019;19(1):965.\u003c/li\u003e\n \u003cli\u003eMaino VC, Suni MA, Ruitenberg JJ. Rapid flow cytometric method for measuring lymphocyte subset activation. Cytometry. 1995;20(2):127-133.\u003c/li\u003e\n \u003cli\u003eZhan X-Y, Wang N, Liu G, et al. Plasmodium infection reduces the volume of the viral reservoir in SIV-infected rhesus macaques receiving antiretroviral therapy. Retrovirology. 2014;11:112.\u003c/li\u003e\n \u003cli\u003eHofmann-Lehmann R, Swenerton RK, Liska V, et al. Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one- versus two-enzyme systems. AIDS research and human retroviruses. 2000;16(13):1247-1257.\u003c/li\u003e\n \u003cli\u003eKulpa DA, Talla A, Brehm JH, et al. Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4(+) T Cells. J Virol. 2019;93(24).\u003c/li\u003e\n \u003cli\u003eHunt PW. HIV and inflammation: mechanisms and consequences. Curr HIV/AIDS Rep. 2012;9(2):139-147.\u003c/li\u003e\n \u003cli\u003ePace M, Frater J. A cure for HIV: is it in sight? Expert review of anti-infective therapy. 2014;12(7):783-791.\u003c/li\u003e\n \u003cli\u003eDavenport MP, Khoury DS, Cromer D, et al. Functional cure of HIV: the scale of the challenge. Nature reviews. Immunology. 2019;19(1):45-54.\u003c/li\u003e\n \u003cli\u003eXu LR, Guo HJ, Liu ZB, et al. Unified-planning, graded-administration, and centralized-controlling: a management modality for treating acquired immune deficiency syndrome with Chinese medicine in Henan Province of China. Chin J Integr Med. 2015;21(4):243-248.\u003c/li\u003e\n \u003cli\u003eJiang F, Zhang R, Gu Z, et al. Fuzhengpaidu granule regulates immune activation molecules CD38 and human leukocyte antigen-D related on CD4+ and CD8+ T cells in patients with acquired immunodeficiency syndrome/human immunodeficiency virus. J Tradit Chin Med. 2013;33(4):439-443.\u003c/li\u003e\n \u003cli\u003eGroot F, van Capel TM, Schuitemaker J, et al. Differential susceptibility of naive, central memory and effector memory T cells to dendritic cell-mediated HIV-1 transmission. Retrovirology. 2006;3:52.\u003c/li\u003e\n \u003cli\u003eHolmes D, Jiang Q, Zhang L, et al. Foxp3 and Treg cells in HIV-1 infection and immuno-pathogenesis. Immunol Res. 2008;41(3):248-266.\u003c/li\u003e\n \u003cli\u003ePereira LE, Villinger F, Onlamoon N, et al. Simian immunodeficiency virus (SIV) infection influences the level and function of regulatory T cells in SIV-infected rhesus macaques but not SIV-infected sooty mangabeys. J Virol. 2007;81(9):4445-4456.\u003c/li\u003e\n \u003cli\u003eAandahl EM, Michaelsson J, Moretto WJ, et al. Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol. 2004;78(5):2454-2459.\u003c/li\u003e\n \u003cli\u003eXia H, Jiang W, Zhang X, et al. Elevated Level of CD4+ T Cell Immune Activation in Acutely HIV-1-Infected Stage Associates With Increased IL-2 Production and Cycling Expression, and Subsequent CD4+ T Cell Preservation. Front Immunol. 2018;9.\u003c/li\u003e\n \u003cli\u003eMcBrien JB, Kumar NA, Silvestri G. Mechanisms of CD8(+) T cell-mediated suppression of HIV/SIV replication. Eur J Immunol. 2018;48(6):898-914.\u003c/li\u003e\n \u003cli\u003ePitsios C, Dimitrakopoulou A, Tsalimalma K, et al. Expression of CD69 on T-cell subsets in HIV-1 disease. Scandinavian journal of clinical and laboratory investigation. 2008;68(3):233-241.\u003c/li\u003e\n \u003cli\u003eVallejo A, Molina-Pinelo S, De Felipe B, et al. Toll-like receptor 9 1635A/G polymorphism is associated with HIV-1 rebound after four weeks of interruption of antiretroviral therapy. J Acquir Immune Defic Syndr. 2020.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HIV/AIDS, Antiretroviral therapy, Traditional Chinese medicine, Immune reconstruction, Immune functional cure","lastPublishedDoi":"10.21203/rs.3.rs-693617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-693617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e 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 beneficial 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 influences immune reconstruction to enhance the antiviral immune response in simian immunodeficiency virus (SIV)-infected rhesus monkeys. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The SIV-infected animals were firstly 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. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The flow cytometry analysis revealed that treatment with TN-01 alone or combined with ART significantly upregulated T cells counts and the proportions of T cell subsets. TN-01 treatment also delayed viral rebound after ART suspension. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Our data indicate that TN-01 could enhance the efficacy of ART and promote immune reconstruction, suggesting TN-01 treatment as a potent strategy in immune functional cure of HIV infection. \u003c/p\u003e","manuscriptTitle":"Traditional Chinese Medicine TN-01 Reconstitutes T Cells in SIV–infected Rhesus Monkeys","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-07-13 16:36:33","doi":"10.21203/rs.3.rs-693617/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56c4d707-a501-439e-a9db-cf18e0de5368","owner":[],"postedDate":"July 13th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":5676606,"name":"Internal Medicine"}],"tags":[],"updatedAt":"2021-07-19T07:47:13+00:00","versionOfRecord":[],"versionCreatedAt":"2021-07-13 16:36:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-693617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-693617","identity":"rs-693617","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}