Fig 1.
DiABZI activates a robust innate immune response in mice.
(A-B) C57BL/6J male mice were treated with diABZI (0.625, 1.25 and 2.5 mg/kg) or vehicle by either intraperitoneal (IP) injection or tail intravenous (IV) injection (A). C57BL/6J mice were treated with GS-9620 (15 mg/kg) by gavage and DMXAA (12.5 and 25 mg/kg) or vehicle by intraperitoneal (IP) injection (B). At 4 h after treatment, the serum levels of IFNβ were determined by ELISA (n = 4-5/group). ***P < 0.001,**P < 0.01, *P < 0.05 by One-way ANOVA. (C-O) C57BL/6J male mice were treated with diABZI (0.625, 1.25 and 2.5 mg/kg), DMXAA (20 mg/kg) or vehicle by IP injection, or GS-9620 (15 mg/kg) by gavage and sacrificed at 2 h after treatment. The mRNA levels of IFNβ, IFNα, TNFα, IL-6, CXCL10, IFIT1, IFITM3, ISG15, Mx1, Mx2, OAS1, OAS3 and STAT1 in the livers were determined by qRT-PCR (normalized to GAPDH) (n = 3-4/group). Fold induction of gene expression relative to that in vehicle-treated controls was presented as mean values ± standard deviations (SD). With the exception of IFITM3, the others were subjected to a natural logarithm transformation (ln transformation), and then a One-way ANOVA analysis was performed. ***P < 0.001,**P < 0.01, *P < 0.05.
Fig 2.
Effect of diABZI treatment on HBV replication in rAAV-HBV transduced STING humanized (hSTING) mice.
(A) The STING protein level in the spleen of hSTING mice was detected by a Western blotting assay with an anti-STING antibody which can detect both human and mouse STING. (B-C) Effect of DMXAA and diABZI on the expression of CXCL10, IFIT1, ISG15, Mx1 and Mx2 in splenocytes and PBMCs from hSTING mice. The amounts of mRNAs specifying the specific cytokines and ISGs were quantified by qRT-PCR assay. Data are expressed as fold induction of gene expression relative to that in negative control (NC) treated with DMSO. ***P < 0.001,**P < 0.01, *P < 0.05 by Student’s t-test. (D-I) Experimental setup for in vivo efficacy study in humanized STING mice (D). All mice received a single i.v. injection of 8 × 1010 vg of rAAV8-HBV1.3. On day 52 after inoculation, mice were grouped and treated with 2 mg/kg of diABZI or 20 mg/kg of DMXAA. Mice treated with vehicles served as control. Images of the mouse, syringe, and liver were sourced from https://openclipart.org/17558, 282069, and 37315, respectively. (E) Body weight of mice before and after drug treatment is presented. (F-H) Serum levels of HBV DNA, HBsAg and HBeAg were determined by qPCR or ELISA on days 39 (before treatment), 54, and 61 after rAAV-HBV injection. ***P < 0.001 by Two-way ANOVA. (I) Liver HBV DNA levels were determined by qPCR (normalized per 1 μg of total liver DNA).***P < 0.001 by One-way ANOVA. (J) Effect of DMXAA and ETV on serum levels of HBV DNA in the rAAV-HBV transduced WT mice. C57BL6/J mice injected with 8 × 1010 vg of rAAV8-HBV1.3 genotype D by the tail vein for 8 weeks were divided into three groups (n = 6/group). Mice were then administered with a single dose of vehicle or DMXAA (25 mg/kg) via IP. Mice were also given ETV (0.1 mg/kg) by gavage daily. After one week posttreatment, the levels of serum HBV DNA in three groups of mice were measured by qPCR assay.***P < 0.001,*P < 0.05 by One-way ANOVA.
Fig 3.
Effects of diABZI treatment on HBV replication and humoral immune response in the rAAV-HBV infected mice.
(A) Schematic illustration of experimental schedule. Briefly, 6-week-old male C57BL6/J mice were transduced with 8 × 1010 vg of rAAV8-HBV1.3 genotype D by tail vein injection. Eight weeks later, the transduced mice were divided into four groups (n = 8-9/group) and dosed with vehicle (Group 1) or diABZI by IP injection at the indicated times, alone (Group 2 and Group 3) or in combination with ETV (0.1 mg/kg, once daily) (Group 4) by gavage. Mice were euthanized on day 29 after treatment (n = 4/group) or at end of treatment (next day after last dose) (n = 4-5/group). Body weight and virological markers were measured at the indicated time points. Day 0 corresponds to the start of dosing. Images of the mouse, syringe, and liver were sourced from https://openclipart.org/17558, 282069, and 37315, respectively. (B-E) The mice body weight, serum levels of HBV DNA, HBsAg and HBeAg in the four groups were determined. Mean values ± SD are plotted for each group. *P < 0.05,**P < 0.01,***P < 0.001, Group 2 vs Group 1; ##P < 0.01,###P < 0.001, Group 3 vs Group 1; +++P < 0.001, Group 4 vs Group 1 by Two-way ANOVA. (F-H) Dynamic levels of HBV DNA, HBsAg, and HBeAg from day -7 to the end of the experiment for each of mice in Group 1 and Group 2 are presented. (I) Dynamic levels of HBsAb during day 29-64 for each of the mice in Group 1 and Group 2 are presented. (J-K) Intrahepatic HBV DNA load was determined by qPCR and presented as log copies per 1 μg of total liver DNA or by Southern blot hybridization, respectively. ss, single-stranded DNA. (L-M) The levels of pgRNA in the liver tissues were quantified by qRT-PCR (normalized to GAPDH) or by Northern blot hybridization, respectively. (N) The serum levels of HBV pgRNA were quantified by qRT-PCR. (O) The levels of AAV vector DNA were quantified by PCR (normalized to GAPDH). (P) The levels of HBV core protein (HBc) in liver tissues were measured by Western blotting. β-actin served as a loading control.
Fig 4.
Effects of diABZI treatment on the liver expressed genes revealed by bulk RNA-sequencing analysis in rAAV-HBV transduced mice.
(A) Volcano plot with the comparison between Group 2 and Group 1 on day 29 of treatment. Genes shown represent p-value≤0.05 and FDR ≤ 0.05 (upregulated genes marked in red and downregulated genes marked in green). (B) Histograms with transcripts per million (TPM) of the representatives with differentially expressed genes between Group 2 and Group 1. (C-D) KEGG or GO functional enrichment analysis with the comparison between Group 2 and Group 1 based on the differentially expressed marker genes are presented.
Fig 5.
Effects of diABZI treatment on the levels of intrahepatic Kupffer cells/infiltrating monocytes (CD68+) and T cells (CD3+) in rAAV-HBV transduced mice.
(A) The levels of Kupffer cells and infiltrating monocytes (CD68+) in the liver sections of representative mice from the experiment presented in Fig 3 were determined by immunohistochemical (IHC) staining. Representative images at 20 × magnification are presented. (B) Multicolor immunofluorescent (IF) staining of HBsAg (white, yellow arrow), HBcAg (red) and Kupffer cells and infiltrating monocytes (CD68+, green) in liver sections. One representative image for each group of mice from the experiment presented in Fig 3 is shown. (C) Multicolor IF staining of HBcAg (red), Kupffer cells and infiltrating monocytes (CD68+, green), and T cells (CD3+, white) in liver sections. One representative image for each group of mice from the experiment presented in Fig 3 is shown. HC: Healthy control.
Fig 6.
Effects of diABZI treatment on the hepatic immune cell profiles revealed by single-cell transcriptome analysis in rAAV-HBV transduced mice.
Hepatic cells were collected at the end of vehicle or diABZI treatment and subjected to cell barcoding. The cDNA libraries of 5’-mRNA expression, TCR, and BCR were constructed independently, followed by high-throughput sequencing and downstream analyses. (A) The t-distributed stochastic neighbor embedding (t-SNE) plots of the 44337 single cells from 4 mice, including 1 healthy control mouse without rAAV-HBV transduction and diABZI treatment (HC, 11626 cells), 1 vehicle-treated AAV-HBV transduced mouse (G1, 12294 cells), 2 diABZI-treated mice (G2, #7, 9023 cells; G2, #8, 11394 cells). These cells contain 16 major clusters. (B) The tSNE plots for the sample-specific distribution of immune cells. (C) Gene expression heatmap in each cell cluster. Normalized mean expressions are shown (score). (D) Histogram representing the proportion of clusters among total liver cells in each sample. (E) Histogram representing the proportion of clusters among hepatic immune cells in each sample. (F) Heatmap depicting the results of Gene Set Enrichment Analysis (GSEA) using Hallmark gene sets.
Fig 7.
Features of hepatic T-cell subsets.
(A) The t-SNE plots of T-cell subsets from hepatic cells of 4 mice as indicated in Fig 6. (B) The t-SNE plots for the treatment sample-specific distribution of T cell subsets. (C) Histogram representing the proportion of T cell subclusters in each sample. (D) Histogram representing the proportion of CD8+T cells among the entire T cell population in each sample. (E) Violin plots showing the effector scores of different CD8+T subclusters across different samples. (F) Violin plots showing the effector scores, cytotoxicity scores and chemokine scores of CD8+T subclusters across different samples. (G) RNA velocity analysis showing the transition potential among T-cell subsets. (H) Violin plots show the exhaustion scores of Nr4a2+, Trgv2+, Nkg7+ and Ccl4+CD8+T cells across different samples. (I) Violin plots showing the regulatory scores of Icos+CD4+Treg cells. Statistical analysis was performed by a one-sided unpaired Wilcoxon test. *P < 0.05,**P < 0.01,***P < 0.001.