Protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies

Abstract

Smallpox (variola virus) is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs) against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV) primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs) against vaccinia H3 (H3L) and B5 (B5R), targeting both the mature virion (MV) and extracellular enveloped virion (EV) forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP) studies examining severe combined immunodeficiency mice (SCID) given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG). Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV). In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.

Figure 1. Pre-exposure hmAb protection of SCID mice.

Severe combined immunodeficiency (SCID) mice were treated with a single dose of human anti-H3 hV26, anti-B5 h101, a combination of the two, 2.0 mg of vaccinia immune globulin (VIG), or negative control anti-DNP mAb at day -1 and then infected intravenously via tail vein with 5×104 PFU of vaccinia virus, ACAM2000 lab stock strain; n = 12/group, except for the naïve control [n = 2]. (A) Weights. (B) Survival. (C) Clinical scores. (D) 5% weight loss.

Figure 2. Post-exposure hmAb protection of SCID mice.

(A) SCID mice were treated with a 500 µg dose of human anti-H3 hV26 and anti-B5 h101 in a split dose of 200 µL retro-orbital and 200 µL intraperitoneal on day 1 post-infection. A booster dose of 500 µg of combination therapy was provided day 14 intraperitoneal. All mice were infected with 5×104 PFU of vaccinia virus, ACAM2000. Half of the mice in the combination group were given virus intravenously through the tail vein T.V. (n = 8), while the other half received virus intravenously R.O. (n = 8). All anti-DNP control mice received virus T.V. (n = 4). (B) Days to 5% weight loss. (C) Day 25 clinical scores. (D) Survival.

Figure 3. Post-exposure hmAb protection with single animal housing.

(A) SCID mice were treated with a 500 µg dose of human anti-H3 hV26 and anti-B5 h101 retro-orbitally on day 1 post-infection. A booster dose of 500 µg of the combination therapy was provided to the mice on day 11 intraperitoneal. All mice were infected with 5×104 PFU of vaccinia virus, ACAM2000 retro-orbitally on day 0. For both groups n = 12. 4 mice in each group were sacrificed at day 28 for other analyses not shown. (B) Individual mice weights. (C) Clinical scores day 25. (D) Survival. (E) Clinical scores of the combination anti-H3+B5 group on day 25 versus day 88.

Figure 4. Post-exposure anti-H3+B5 hmAb protection in rabbits.

(A) VACV neutralization in vitro. (B–F) Post-exposure protection of rabbits against rabbitpox. 32 New Zealand White (NZW) rabbits were challenged with 1x105 PFU of RPXV intranasally. Group 1 (n = 10) received no treatment, Group 2 (n = 6) and group 4 (n = 10) received two I.V. injections of antibody cocktail at 9 mg/kg and 1.25 mg/kg respectively, one hour and 72 hours post-challenge. Group 3 (n = 6) received a single I.V. injection of antibody cocktail at 9 mg/kg 2 days post-challenge. (B) Body Weights. (C) Percent change from initial body temperature. (D) Percent change from initial respiration rate. (E) Clinical scores. (F) Percent survival.

Figure 5. Post-exposure anti-H3+B5 hmAb protection in rabbits.

(A) Peak percent weight loss amongst groups. (B) Peak percent temperature increase. (C) Peak clinical score. (D) RPXV copies in 1 mL of whole blood. (E) Viremia at peak disease (RPXV copies/mL).

Figure 6. RPXV in Lung, Liver, Spleen, and Blood.

RPXV in the liver, spleen, and blood of infected NWZ rabbits.

Figure 7. Post-exposure protection in rabbits with single hmAb.

(A) Body Weights. 14 New Zealand White (NZW) rabbits were challenged with 1×105 PFU of RPXV intranasally. Group 1 (n = 6) received no treatment, Group 2 (n = 6) received two I.V. injections of anti-B5 h101 alone at 9 mg/kg, one hour and 72 hours post-challenge. Group 3 (n = 2) received two I.V. injections with the antibody cocktail at 9 mg/kg, one hour and 72 hours post-challenge. (B) Percent change in initial body temperature. (C) Clinical scores. (D) Percent survival.

Figure 8. Post-exposure protection in rabbits with single hmAb.

(A) Peak percent weight loss amongst groups. (B) Peak percent temperature increase. (C) Peak clinical score.