A Two-Antibody Pan-Ebolavirus Cocktail Confers Broad Therapeutic Protection in Ferrets and Nonhuman Primates

Abstract

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134^AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134^AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134^AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.

Keywords: ADI-15878; ADI-23774; BDBV; Bundibugyo; EBOV; MBP134; SUDV; Sudan; ebolavirus; pan-ebolavirus.

Figure 1.. MBP134^AF protects ferrets from lethal EBOV, SUDV and BDBV challenge.

(A) Survival curves for ferrets challenged with EBOV/Makona and treated with 15 mg of MBP134^AF on either Day 2 and 5 (orange) or Day 3 and 6 (green) p.i. **, P<0.01. (B) Quantitative RT-PCR measuring average copies of EBOV/Makona genomic equivalents per mL of whole blood (GEQ/mL) from animals treated on day 2 and 5 (orange) and day 3 and 6 (green) p.i. (C) Infectious EBOV/Makona measured via plaque assays (PFU/mL) present in the blood of animals treated on day 2 and 5 (orange) or day 3 and 6 (green). (D) Survival curves for ferrets challenged with SUDV/Gulu and treated with 15-mg (green) or 5-mg (blue) doses of MBP134^AF on day 3 and 6 post infection. (E) The average GEQ/mL of SUDV/Gulu present in the blood of animals treated with 15-mg (green) or 5-mg doses (blue). (F) SUDV/Gulu viremia present in the blood of animals treated with 15-mg (green) or 5-mg (blue) doses from panel D. (G) Survival curves for ferrets challenged with BDBV/But-811250 and treated with 15-mg (green) or 5-mg (blue) doses of MBP134^AF on day 3 and 6 post infection. (H) The average GEQ/mL of BDBV/But-811250 from animals treated with two 15-mg (green) or 5-mg doses (blue). (I) Viremia (PFU/mL) present in the blood of animals treated with two 15-mg (green) or 5-mg doses (blue) from panel G. LOD = limit of detection

Figure 2.. A single 25 mg/kg dose of MBP134^AF protects rhesus macaques challenged with EBOV/Kikwit.

(A) Survival curves for NHPs challenged with EBOV/Kikwit and treated with a single 25-mg/kg dose of MBP134^AF on day 4 (green) p.i or a more conservative two-dose regimen of 50 mg/kg on day 4 and 25 mg/kg on day 7 (orange) post infection. *, P<0.05. (B) The average GEQ/mL of EBOV/Kikwit present in the blood of animals treated with a single dose of MBP134^AF (green) or two doses of MBP134^AF (orange). All detectable EBOV/Kikwit was eliminated 10 days post treatment. (C) Infectious EBOV/Kikwit (PFU/mL) present in the blood of animals treated with either a single (green) or two-dose course of MBP134^AF (orange). Infectious EBOV/Kikwit was no longer detectable by plaque assay by the next bleed of treated animals on day 7 post infection. (D) Clinical scores of animals within the study cohort are shown. Aside from the control animals only NHP-1 and NHP-8 scored for partially or completely refusing their daily nutrition. (E) Body temperatures taken during the course of the study show nine of the ten animals registered an elevated temperature by day 7 p.i. Animals receiving MBP134^AF returned to baseline temperature by day 10 p.i. (F) The platelet counts for the cohort show severe thrombocytopenia in the control animals post-infection. By contrast, animals receiving MBP134^AF rapidly recovered and displayed little or no signs of thrombocytopenia. Data are represented as mean ± SD. (G) The graphed CRP levels show NHP-3 and NHP-7 suffered from acute inflammation as a result of EVD prior to MBP134^AF treatment. (H) The ALT levels from each animal are shown, NHP-3 and NHP-8 demonstrated signs of advanced EVD that were alleviated post-treatment. (I) AST levels for all the challenged animals showed initial signs of liver damage resultant of EVD. Samples from NHP-8 in particular show a significant spike in AST levels that returned to baseline post-treatment. Legend for graphs in top right-hand corner, LOD = limit of detection.

Figure 3.. Induction of Fc effector function by plant- and CHOKI-produced ADI-23774^AF and ADI-15878^AF.

(A-C) Markers of antibody-dependent NK cell activation utilizing NK cells from seronegative donors are displayed. The levels of NK cell activation markers CD107a (A), intracellular cytokine IFNγ (B) and MIP-1β (C) are comparatively graphed for ADI-23774 and ADI-15878 produced from transiently expressed HEK293 cells as fucosylated mAbs or as afucosylated mAbs produced from either the plant (-N) or CHOK1-AF (-CHO) cell-based expression platforms. The graphs demonstrate a marked enhancement of Fc-mediated NK cell activation from the presence of an afucosylated glycan in the Fc region of either antibody. Further, both mAbs exceed the Fc effector function potency of c13C6-N, a component of the ZMapp cocktail used here as a positive control. Data are represented as mean ± SD. (D) The phagocytic score or ability of each mAb to facilitate Fc-mediated antibody-dependent neutrophil activation/phagocytosis (ADNP) is shown. Expression in the CHOK1-AF platform greatly enhanced ADNP activity over the other expression platforms likely do to the more uniform heavy chain glycosylation propensity offered by the CHOK1-AF stable pools over transient expression of the antibodies within plants. The formula for calculating the phagocytic score is described in the materials and methods. The anti-human immunodeficiency virus GP120 mAb, 2G12, is utilized here as an irrelevant negative control mAb (Trkola et al., 1996). *, P<0.05. **, P<0.01. ***, P<0.001. ****, P<0.0001.

Figure 4.. MBP134^AF produced from CHOK1-AF cells is therapeutically equivalent to the plant produced MBP134^AF.

(A) Hartley guinea pigs were divided into eight treatment groups and given a 5-mg total dose of CHO or plant produced MBP134^AF on either Day 4, 5, 6, or 7 post infection. Animals receiving MBP134^AF treatment on Day 4 or 5 post infection showed either equivalent and/or significant levels of protection, while all animals treated on day 6 or 7 succumbed to SUDV/Boniface infection with no detectable delay to death when compared to the PBS-treated controls. (B) Weight loss curves from animals treated with CHO or plant produced MBP134^AF display equivalent weight change trends. Data are represented as mean ± SD. (C) The viral titers (PFU/mL) in blood samples taken from each animal immediately prior to treatment are graphed. The data suggests MBP134^AF produced from either platform equivalently protected animals with <10⁵ PFU/mL of infectious virus, with reduced efficacy in animals that showed viral loads >10⁵ PFU/mL. Data are represented as mean ± SD.

Figure 5.. A single 25 mg/kg or 7.5 mg/kg dose of MBP134^AF protects rhesus macaques challenged with SUDV/Boniface.

(A) Survival curves for NHPs challenged with SUDV/Boniface receiving either PBS (black), a 25-mg/kg dose of MBP134^AF (green) or a 7.5-mg/kg (purple) dose of MBP134^AF on day 5 p.i. (B) The average GEQ/mL of SUDV/Boniface present in the blood of animals receiving a 25-mg/kg dose of MBP134^AF (green), a 7.5-mg/kg dose of MBP134^AF (purple) or the PBS controls (black). In the MBP134^AF treated groups detectable levels of SUDV/Boniface were eliminated by day 8 post infection (the next bleed post treatment). (C) Infectious SUDV/Boniface (PFU/mL) present in the blood of animals receiving a 25-mg/kg dose of MBP134^AF (green) or a 7.5-mg/kg dose of MBP134^AF (purple) as well as in PBS controls (black). Infectious SUDV/Boniface was no longer detectable by plaque assay by the next bleed on day 8 p.i. (D) All of the MBP134^AF treated animals in the cohort had clinical scores on day 5 p.i. prior to receiving treatment. Animals dosed with MBP134^AF no longer registered a clinical score by day 14 p.i., while the surviving controls continued scoring out to day 21 p.i. (E) Body temperatures taken from the blinded cohort showed all the animals registered an elevated temperature by day 5 p.i., prior to receiving MBP134AF. (F) The platelet counts for the cohort show declining counts p.i. with the MBP134^AF treated animals rapidly recovering and the PBS controls displaying severe thrombocytopenia. Data are represented as mean ± SD. (G) The ALT levels from each animal are shown; the control animals all display elevated levels from baseline post infection. In contrast, all of the animals receiving MBP134^AF show little to no signs of EVD. (H) AST levels for all the MBP134^AF treated animals show little to no increase, while the control animals all show highly elevated AST levels by day 10 p.i. (I) ALP levels are graphed for each animal in the cohort, no sign of EVD induced ALP levels are present in any of the MBP134^AF treated animals. In contrast, all of the control animals that received PBS display elevated ALP levels resulting from EVD. Legend for graphs in top right-hand corner, LOD = limit of detection.

Figure 6.. A single 25 mg/kg dose of MBP134^AF protects cynomolgus macaques challenged with BDBV/But-811250.

(A) Survival curves for NHPs challenged with BDBV/But-811250 and treated with a single 25-mg/kg dose of MBP134^AF on day 7 post infection (green or pink) or PBS (black). **, P<0.01. (B) The average GEQ/mL of BDBV/But-811250 present in the blood of animals treated with a single dose of MBP134^AF (green or pink) or PBS (black). Serum samples taken from NHP-4 and NHP-6 on days 20 and 28 tested negative for viral genetic material. (C) Infectious BDBV/But-811250 (PFU/mL) present in the blood of animals treated with MBP134^AF (green or pink) or PBS (black). Infectious BDBV was no longer detectable by plaque assay by the next bleed on day 10 post infection in the surviving animals. (D) Clinical scores show NHP-5 and NHP-6 registering scores from refusing nutrition, having a hunched posture and displaying petechiation over 10% of their bodies prior to receiving MBP134^AF on day 7 p.i. While NHP-6 cleared clinical signs of infection by day 12 p.i., NHP-5 failed to recover and succumbed to infection on 13 p.i. (E) Body temperatures from all the animals showed the majority registered an elevated temperature by day 7 p.i. prior to receiving MBP134^AF. (F) The platelet counts for the cohort show thrombocytopenia occurring in all the animals by day 7 p.i. prior to receiving MBP134^AF. All the MBP134^AF treated animals (excluding NHP-5, pink) cleared signs of thrombocytopenia by day 14 p.i., seven days post treatment. (G) ALP levels are graphed for each animal in the cohort. (H) ALT levels from each animal are shown. Notably NHP-5 (pink) displayed advanced signs of EVD prior to treatment. (I) AST levels for the challenged animals show varying signs of liver damage resultant of EVD. Samples from NHP-5 (pink) in particular show a significant spike in AST levels suggesting a severe onset of disease beyond that of the other animals in the cohort, a possible explanation of NHP-5’s succumbing to EVD despite receiving the MBP134^AF treatment course. Legend for graphs in top right-hand corner, LOD = limit of detection.