Journal: Molecular Therapy. Methods & Clinical Development

Article Title: HBVZ10, an AAV8 vector-based new HBV therapy candidate for cccDNA elimination

doi: 10.1016/j.omtm.2025.101646

Figure Lengend Snippet: In vivo expression levels and duration of a single dose of HBVZ10 and therapeutic effects of HBVZ10 monotherapy in uPA/SCID chimeric mice with humanized livers (A1) Kinetic serum anti-HBs antibody levels (mIU/mL) after a single injection of HBVZ10 at a dose of 2.5E10, 1.8E11 or 7E11 genomic copies on day 11, 22 or 44 post inoculation (PI) among 18 individual chimeric mice or replicates. Each of 18 replicates is labeled as T1, T2 … T18. (A2). Average anti-HBs antibody levels (mIU/mL) per group. Note: The chimeric mice lack functional T and B cell immunity and therefore do not produce anti-HBs antibodies in response to HBV infection. A pretreatment serum sample from each replicate was used as a negative control, and all pretreatment anti-HBs antibody levels were plotted as 1 mIU/mL for consistency. (B1) Kinetic serum HBV DNA levels (copies/mL log10) among 8 individual mice. Four of them were mock treated with 1.8E11 copies of AAV vector expressing malaria antibody (blue, labeled as MT1-MT4) and the remaining 4 mice with HBVZ10 monotherapy at a dose of 1.8E11 copies on day 22 PI (orange, labeled as T1-T4). (B2) Average serum HBV DNA levels (copies/mL log10) per group. (C1) Kinetic serum HBsAg levels (IU/mL) among the same 8 individual mice as described in B1. (C2) Average serum HBsAg levels (IU/mL) per group. The lower limit of quantification for serum HBV DNA and HBsAg is 100 copies/mL and 0.05IU/mL, respectively. Error bars: standard deviation (SD).

Article Snippet: Blood was collected triweekly for quantification of serum HBV DNA (qPCR), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA anti-HBs 20-Calibrator kit 25219, Bio-Rad), and human albumin (human albumin ELISA kit E−80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: In Vivo, Expressing, Injection, Labeling, Functional Assay, Infection, Negative Control, Plasmid Preparation, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: HBVZ10, an AAV8 vector-based new HBV therapy candidate for cccDNA elimination

doi: 10.1016/j.omtm.2025.101646

Figure Lengend Snippet: Different serum HBsAg responses to different levels of anti-HBs antibody monotherapy (A) Experimental procedures and time course of HBV infection and treatment evaluation using uPA/SCID chimeric mice model. (B–E) Kinetic serum HBsAg levels (IU/mL) and anti-HBs antibody levels (mIU/mL) in four infected mice (ID: 906, 909, 996, and 970) treated with triweekly infusions of 250 μg mouse anti-HBs antibody starting on day 74 PI. Note: A dynamic relationship appears to exist between serum HBsAg and anti-HBs antibody levels. For example, in both mice 906 and 909 (2A and B), serum HBsAg became undetectable when anti-HBs antibody levels reached and remained at ≥100,000 mIU/mL, but reappeared when anti-HBs antibody levels declined below 100,000 mIU/mL. (F) Kinetic serum HBsAg levels (IU/mL) in 5 mice (MT1-MT5) mock treated with 1E10 copies of vector expressing malaria antibody on day 49 PI. (G) Kinetic serum anti-HBs antibody levels (mIU/mL) expressed by a single dose (1E10 copies) of HBVZ10 administration on day 49 PI among 13 individual chimeric mice (T1-T13). (H) Average anti-HBs antibody levels (mIU/mL) per group from F. (I) Kinetic serum HBsAg levels (IU/mL) among the 13 mice (T1-T13) treated with a single 1E10 dose of HBVZ10 on day 49 PI, as described in G and H. Note. Mice with relatively lower serum HBsAg levels at baseline (day 49 PI) appeared to respond more effectively to lower levels of anti-HBs antibody treatment than those with higher baseline HBsAg levels. (J) Comparison of average serum HBsAg levels between mock-treated and low-dose HBVZ10-treated groups. A pretreatment serum sample from each mouse was used as a negative control, and all pretreatment anti-HBs antibody levels were plotted as 1 mIU/mL for consistency in (A–D) and (F). Dashed line: anti-HBs antibody level at 100,000 mIU/mL threshold. Error bars: standard deviation (SD).

Article Snippet: Blood was collected triweekly for quantification of serum HBV DNA (qPCR), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA anti-HBs 20-Calibrator kit 25219, Bio-Rad), and human albumin (human albumin ELISA kit E−80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: Infection, Plasmid Preparation, Expressing, Comparison, Negative Control, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: HBVZ10, an AAV8 vector-based new HBV therapy candidate for cccDNA elimination

doi: 10.1016/j.omtm.2025.101646

Figure Lengend Snippet: Significant reduction of cccDNA levels in infected chimeric mice upon blocking cccDNA replenishment (A) Average cccDNA levels (copies/cell) among 440 cccDNA samples isolated from 15 mice, 13 of which received a single administration of HBVZ10 at a dose of 1E10 copies on day 49 PI and the remaining 2 (mice 909 and 996) received mouse anti-HBs antibody infusions at a dose of 250 μg/injection triweekly started on day 74 PI. Each of the 15 livers underwent an initial round of 20 samplings, and a second round of 20 samplings was conducted for 7 of the 15 livers. The second-round samples were labeled as 972.21, 964.21, and so on. (B) Comparison of the numbers of cccDNA samples with average cccDNA levels >1 copies/cell between mock-treated and anti-HBs antibody-treated groups. The difference in percentages between the two groups were significant (1% vs. 13% p = 4.07E−10 by chi-square test). (C) Average cccDNA levels per liver or per 20 samplings in two groups, the 1st group (15 mice, orange) treated with anti-HBs antibody only (anti-HBs antibody levels <100,000 mIU/mL, resulting in HBsAg+/anti-HBs antibody+ profile, reflecting partial blocking of de novo infection), and 2nd group (green) with complete blocking of de novo infection, resulting in anti-HBs antibody+/HBsAg-) in 17 mice either under a combination of HBVZ10 with ETV in 15 mice or anti-HBs antibody monotherapy in 2 mice (mouse 819 with a single injection of HBVZ10 at a dose of 1.8E11 and mouse 970 with mouse anti-HBs antibody at a dose of 250 μg/injection triweekly for 9 consecutive times). Anti-HBs antibody levels in group 2 were maintained at ≥100,000 mIU/mL. # anti-HBs antibody monotherapy. ∗ HBVZ10/anti-HBs antibody and 9 weeks of ETV, and remaining mice with HBVZ10 + 12 weeks of ETV. (D) Comparison of average cccDNA levels among three groups: mock-treated (averaged from 220 cccDNA samples, blue), partial blockade of de novo infection (averaged from 440 samples, orange), and complete blockade of de novo infection (averaged from 380 samples, green). The differences in average cccDNA levels among the three groups were statistically significant (Student’s t test). (E and F) Average rcDNA levels (copies/cell) among 15 mice with partial blocking of de novo infection, harvested either on day 204 PI (E) or day 253 PI (F). Blue: mock treated with malaria antibody. Green: HBVZ10. Red: mouse anti-HBs antibody in both E and F. (G) Average cccDNA levels among 380 cccDNA samples isolated from 17 mice with complete blocking of de novo infection. p value >0.05 is considered significant. Error bars: standard deviation (SD). 909, 968 … or M905, M908 … etc. are mice ID.

Article Snippet: Blood was collected triweekly for quantification of serum HBV DNA (qPCR), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA anti-HBs 20-Calibrator kit 25219, Bio-Rad), and human albumin (human albumin ELISA kit E−80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: Infection, Blocking Assay, Isolation, Injection, Labeling, Comparison, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: HBVZ10, an AAV8 vector-based new HBV therapy candidate for cccDNA elimination

doi: 10.1016/j.omtm.2025.101646

Figure Lengend Snippet: Progressive reduction of both serum HBeAg and HBsAg upon blocking of cccDNA replenishment (A) Experimental procedures and time course of HBV infection and treatment evaluation using uPA/SCID chimeric mice model. (B1) The expressed anti-HBs antibody levels were >100,000 mIU/mL and sustained at steady levels for 200 days after a single injection of HBVZ10 in 8 chimeric mice (T1 and CT1-CT7) infected with HBV. A pretreatment serum sample from each replicate was used as a negative control, and all pretreatment anti-HBs antibody levels were plotted as 1 mIU/mL for consistency. (B2) Average anti-HBs antibody levels per group. HBVZ10 was administered on day 11 with a dose of 2.5E10 copies for mice CT1-CT4 on day 22 with 1.8E11 copies for mice T1 and CT5-CT6, or on day 44pi with 7E11 copies of HBV10 for mouse CT7. All 7 mice (CT1-CT7) also received 12-week ETV 10 days after HBVZ10 injection. T. HBVZ10 monotherapy. CT: Combination of HBVZ10 with ETV. (C1) Serum HBeAg kinetics among 7 mock-treated mice (MT1-MT7 blue) and 8 treated mice (T1 and CT1-CT7 green). Serum HBeAg levels are expressed as the OD ratio of the tested sample to the mean OD of three negative controls. A ratio greater than 2.1 is considered positive. (C2) Average serum HBeAg levels per group between mock-treated (blue) and treated (green) from B1. (D1) Kinetic serum HBsAg levels (IU/mL) among mock-treated 7 mice (blue) and 8 treated mice (green) from B1. (D2) Average serum HBsAg levels per group between mock-treated (blue) and treated group (green) from C1. (E) Comparison of average cccDNA levels between mock-treated and combination treated groups (cccDNA was undetectable in mice CT2 and CT4 and not plotted). (F) Comparison of average rcDNA levels between mock-treated and combination-treated groups. Statistical significance was determined using Student’s t test; p < 0.05 was considered significant. Error bars: standard deviation (SD).

Article Snippet: Blood was collected triweekly for quantification of serum HBV DNA (qPCR), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA anti-HBs 20-Calibrator kit 25219, Bio-Rad), and human albumin (human albumin ELISA kit E−80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: Blocking Assay, Infection, Injection, Negative Control, Comparison, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: HBVZ10, an AAV8 vector-based new HBV therapy candidate for cccDNA elimination

doi: 10.1016/j.omtm.2025.101646

Figure Lengend Snippet: Add-on of HBVZ10/anti-HBs antibodies to 9-week ETV treatment prevented HBV relapse, leading to progressive reduction of both serum HBeAg and HBsAg levels (A) Experimental procedures and time course of HBV infection and treatment evaluation using uPA/SCID chimeric mice model. (B) Serum HBV DNA (copies/mL log10) kinetics in mock-treated (MT1-MT8 blue) and ETV monotherapy (ET1-ET4 orange) group. (C) Serum HBV DNA kinetics in 8 ETV-treated mice (CT1-CT8) with add-on with a single dose of 1E10 copies of HBVZ10 on day 70pi, then anti-HBs levels were further boosted with administration of mouse anti-HBs at a dose of 250 μg/injection triweekly started on day 99 or later. (D) Average serum HBV DNA levels (copies/mL log10) among 3 groups. (E1) Serum kinetic anti-HBs antibody levels (mIU/mL) expressed by HBVZ10 and boosted by infusions of mouse anti-HBs antibody among the 8 mice (CT1-CT8). Dashed line: 100,000 mIU/mL. Note: Anti-HBs antibody levels exhibited significant fluctuations in some mice receiving triweekly infusions of mouse anti-HBs antibody, contrasting the steady and consistent antibody levels expressed by sufficient HBVZ10 doses ( A). A pretreatment serum sample from each replicate was used as a negative control, and all pretreatment anti-HBs antibody levels were plotted as 1 mIU/mL for consistency. (E2) Average anti-HBs antibody levels (mIU/mL) per group. (F1) Kinetic serum HBsAg levels (IU/mL) among 2 mice with mock-treated (MT1 and MT2 blue), 2 mice with 9-week of ETV therapy (ET1 and ET2 yellow), and 8 mice with 9-week of ETV and anti-HBs antibodies add-on (CT1-CT8 green). (F2) Average serum HBsAg levels (IU/mL) per group. (G1) Kinetic serum HBeAg levels among 2 mice with mock-treated (MT1 and MT2 blue), 2 mice with 9-week of ETV therapy (ET1 and ET2 yellow), and 8 mice with 9-week of ETV and anti-HBs antibodies add-on (CT1-CT8 green). Note. Mouse CT5 had relatively higher baseline HBeAg level and HBeAg remained detectable on termination day (day 218 PI) despite progressive reduction. Serum HBeAg levels are expressed as the OD ratio of the tested sample to the mean OD of three negative controls. A ratio greater than 2.1 is considered positive. (G2) Average serum HBeAg levels per group. (H and I) Comparison of intracellular cccDNA and rcDNA levels among 2 livers with 9-week of ETV therapy (ET1 and ET2 yellow), 2 livers with mock-treated (MT1 and MT2 blue), and 6 livers with 9-week of ETV and anti-HBs antibodies add-on, 5 (CT1-CT3, CT7 and CT8) of them achieved progressive reduction of both serum HBeAg and HBsAg to undetectable levels and the remaining one with detectable serum HBeAg on the termination day (CT5). The differences in cccDNA and rcDNA levels were analyzed by Student t test, p < 0.05 considered significant. Error bars: standard deviations (SD). The limit of detection for HBV DNA is 100 copies/mL and 0.05 IU/mL for serum HBsAg.MT: mock-treated. ET: ETV Monotherapy. CT: 9-week of ETV and anti-HBs antibodies add-on.

Article Snippet: Blood was collected triweekly for quantification of serum HBV DNA (qPCR), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA anti-HBs 20-Calibrator kit 25219, Bio-Rad), and human albumin (human albumin ELISA kit E−80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: Infection, Injection, Negative Control, Comparison

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: HBVZ10, an AAV8 vector-based new HBV therapy candidate for cccDNA elimination

doi: 10.1016/j.omtm.2025.101646

Figure Lengend Snippet: Progressive reduction in serum HBsAg detected with ELISA was corroborated with western blot results and supported by intracellular HBsAg reduction (A) Progressive reduction of serum HBsAg detected by ELISA in mouse 838 (A1 orange) who received a single dose of HBVZ10 combined with 12-week ETV and was corroborated by western blot (A2). Mouse 831 was a mock-treated control. 100 × Hep: Positive control with concentrated viral particles from the HepAD38 cell medium. (B) Progressive reduction of serum HBsAg detected by ELISA in mouse 970 (B1 orange) who received mouse anti-HBs antibody monotherapy at a dose of 250 μg/injection triweekly for 9 consecutive times and was corroborated by western blot (B2). Mouse 907 was a mock-treated control. ADR and ADW: HBV positive human sera. 100xHepAD, Positive control with concentrated viral particles from HepAD38 cell medium. 35526 and 34477, negative human sera. X, no sample lane. Intrahepatic HBsAg was undetectable by ELISA (C1) in lysates (30 μL each) from seven mice that achieved an HBsAg − /anti-HBs + serum status. Consistently, western blot analysis (C2) of 40 μL liver lysates showed either undetectable or markedly reduced HBsAg levels in these seven mice compared with three untreated controls (mice 907, 987, and 471). (D) Intrahepatic HBc protein analyzed using western blot. 100xHepAD, Positive control with concentrated viral particles from HepAD38 cell medium. 100× GFP-HepG2 cell lysate, negative control. BHK-020, Uninfected liver lysate as negative control. X, no sample lane. Mice 812, 813, 814, 824, and 838 in green were the five mice with progressive reduction of serum HBsAg level upon blocking cccDNA replenishment. Mice 831 and 805 in blue were mock treated. Mouse 833 in purple received a single dose of HBVZ10 monotherapy on day 44pi. Four liver lysates from each liver were analyzed, and numbered as 1, 2, 3, and 4 or 5, 6, 7, and 8 or 11, 12, 13, and 14, respectively after mouse ID number.

Article Snippet: Blood was collected triweekly for quantification of serum HBV DNA (qPCR), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA anti-HBs 20-Calibrator kit 25219, Bio-Rad), and human albumin (human albumin ELISA kit E−80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Positive Control, Injection, Negative Control, Blocking Assay