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. In brief, rhesus monkeys were infected intravenously (i.v.) with 300 TCID50 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+ TEM). CD28+ CD95+ CD8+ T cell was defined as CD8+ TCM cell whereas CD28+ CD95- CD8+ T cell was CD8+ TEM 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. Primers and probes synthesized by Invitrogen (Carlsbad, CA, USA) were designed as previously study, 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 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.