Combination therapy of vaccinia virus infection with human anti-H3 and anti-B5 monoclonal antibodies in a small animal model

Abstract

Background: Treatment of rare severe side effects of vaccinia virus (VACV) immunization in humans is currently very challenging. VACV possesses two immunologically distinct virion forms in vivo - intracellular mature virion (MV, IMV) and extracellular virion (EV, EEV).

Methods: Antibody-mediated therapeutic efficacy was determined against VACV infection in a small animal model of progressive vaccinia. The model consisted of severe combined immunodeficiency mice infected with VACV New York City Board of Health vaccine strain and treated with monoclonal antibodies (mAbs).

Results: Here, we show that combination therapy with two fully human mAbs against an immunodominant MV antigen, H3 (H3L), and an EV antigen, B5 (B5R), provides significantly better protection against disease and death than either single human monoclonal or human vaccinia immune globulin, the currently licensed therapeutic for side effects of smallpox vaccination.

Conclusions: The preclinical studies validate that this combination of mAbs against H3 and B5 is a promising approach as a poxvirus infection treatment for use in humans.

Figure 1. In vitro neutralization of murine anti-H3 mAb #41

A) Neutralization of VACV_WR by murine anti-H3 mAb #41 in a standard overnight neutralization assay. “VACV” = no mAb added. B) Neutralization of VACV_WR by murine anti-H3 mAb #41 in a 60 minute neutralization assay. C) Neutralization of VACV_WR by murine anti-H3 mAb #41 in a 60 minute neutralization assay in the presence of 1% complement. Anti-H3 mAb #41 was used at 10 μg/mL in all conditions. Data are representative of three or more experiments.

Figure 2. In vivo protection of murine anti-H3 mAb vs. VIG

A) SCID model VACV_NYCBOH titration. Adult SCID mice were infected intravenously (i.v.) with 1 × 10³ PFU, 1 × 10⁴ PFU, or 1 × 10⁵ PFU VACV_NYCBOH. Body weight was tracked. Dotted lines indicate starting body weight (upper) and terminal body weight (bottom). (B–C) SCID mice were treated with a single dose of 1.25 mg VIG, 200 μg anti-H3 murine mAb #41, or PBS at day -1 and then infected with 1 × 10⁴ PFU VACV_NYCBOH i.v. Body weight, clinical disease (pox), and survival were tracked. N = 5/group. Data are representative of four independent experiments. B) Body weight kinetics. C) Clinical scores. D) Efficacy of murine anti-H3 mAb against virulent VACV_WR in SCID mice. SCID mice were treated with 200 μg anti-H3 murine mAb #41, 600 μg VIG, or PBS at day -1 and then infected with 1 × 10³ PFU VACV_WR i.v. Body weight was tracked. N = 6/group. Data are representative of two experiments. Error bars are +/− SEM.

Figure 3. In vivo protection of human anti-H3 mAb vs. VIG

(A–B) In vitro neutralization of fully human anti-H3 mAbs. A) Neutralization of VACV_WR by human anti-H3 mAb hV26 in a standard overnight neutralization assay. Dose titration showed a 50% neutralization (PRNT₅₀) at ~100 ng/mL. Dashed lines indicate VACV plaque number in the absence of mAb (top line) and the 50% plaque number (middle). B) Neutralization of VACV_WR by hV26. Three neutralization assays were used. (left) Overnight neutralization assay. **, P = 0.006. (middle) one hour neutralization in absence of complement. **, P = 0.0012. (right) one hour neutralization in the presence of 1% complement (1hr + C′). **, P = 0.005. “VACV” = no mAb added. hV25 = an anti-H3 mAb exhibiting no neutralization. Data are representative of three or more independent experiments. (C–E) SCID mice were treated with a single dose of 100 μg human anti-H3 mAb hV26, 1.25 mg VIG, or PBS at day -1 and then infected with 1 × 10⁴ PFU VACV_WR i.v. Body weight, clinical disease (pox), and survival were tracked. N = 6/group respectively. All data are representative of three or more experiments. C) Body weights, averages per group. Anti-H3 hV26 vs. PBS, P < 0.003. VIG vs. PBS, P < 0.0002. D) Survival curves, per treatment group. Anti-H3 hV26 vs. PBS, P < 0.0006. VIG vs. PBS, P < 0.004. E) Clinical scores, per treatment group. Anti-H3 hV26 vs. PBS, P < 0.0003. VIG vs. PBS, P < 0.0005. Error bars are +/− SEM.

Figure 4. Titration of human anti-H3 mAb protection

SCID mice were treated with a single dose of human anti-H3 mAb hV26 (10 μg, 20 μg, 50 μg, 100 μg, or 200 μg), 1.25 mg VIG, or PBS at day -1 and then infected with 5 × 10⁴ PFU VACV ACAM 2000 strain i.v. N = 5/group, except PBS group N = 4. A) Body weights. B) Clinical score. Data are representative of two independent experiments. Errors bars are +/− SEM.

Figure 5. In vivo protection of human anti-B5 mAb vs. VIG

SCID mice were treated with a single dose of human anti-B5 mAb h101 (50 μg), 1.25 mg VIG, or PBS at day −1 and then infected with 1 × 10⁴ PFU VACV_NYCBOH i.v. N = 6 mice/group for VIG and PBS, 10/group for h101. A) Body weights. B) Time (days) to 5% weight loss. ** P < 0.01. C) Clinical scores. P < 0.0001, h101 vs. PBS. D) Survival. P < 0.0001, h101 vs. PBS. Data are representative of three or more experiments. Error bars are +/− SEM.

Figure 6. Titration of human anti-B5 mAb protection

SCID mice were treated with a single dose of human anti-B5 mAb h101 (25 μg, 50 μg, or 100 μg), 1.25 mg VIG, control human mAb (IgG1 anti-DNP hapten), or PBS at day -1 and then infected with 5 × 10⁴ PFU VACV (ACAM 2000 strain) i.v. N = 5/group. A) Body weights. B) Time to 5% weight loss. C) Clinical score. Error bars are +/− SEM. D) Survival curves.

Figure 7. In vivo protection of anti-H3 plus anti-B5 vs VIG

SCID mice were treated with a single dose of human anti-H3 mAb hV26 (50 μg), human anti-B5 mAb h101 (50 μg), 25 μg combination of human anti-H3 mAb hV26 and anti-B5 mAb h101 (25 μg or each), 50 μg combination of human anti-H3 mAb hV26 and anti-B5 mAb h101 (50 μg or each) 1.25 mg VIG, or control human anti-DNP mAb (50 μg) at day -1 and then infected with 5 × 10⁴ PFU ACAM2000 clone of VACV_NYCBOH i.v. N = 9–10/group. A) Body weights. B) Time to 5% weight loss. * P < 0.05, ** P < 0.01, *** P < 0.001. C) Clinical score. D) Survival. Data are representative of two to four independent experiments. Experiment was ended at day 81, with 50% of h101 + hV26 50 μg combination group still healthy (> 100% initial body weight). Error bars are +/− SEM.