Fully Human Immunoglobulin G From Transchromosomic Bovines Treats Nonhuman Primates Infected With Ebola Virus Makona Isolate

Abstract

Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic.

Figure 1.

Ebola virus (EBOV)-specific transchromosomic bovine (Tc-bovine)-derived human immunoglobulin G (hIgG) potently recruits multiple innate immune effector functions. (A and B) Induction of antibody-mediated monocyte phagocytosis (ADMP) (A) or antibody-mediated neutrophil phagocytosis (ADNP) (B) using antibodies from SAB-139/V3–V4 (blue), SAB-139/V6–V8 (purple), sera from Tc-bovines (2314 [black squares] and 2316 [white circles]), anti-EBOV monoclonal antibodies c13C6 (green) and KZ52 (orange), no antibody control (white), and irrelevant Tc IgG control (red). Uptake of EBOV glycoprotein (GP) onto coated fluorescein isothiocyanate (FITC) beads was determined by flow cytometry. A phagocytic score was determined using the percentage of FITC⁺ cells and the mean fluorescent intensity (MFI) of the FITC⁺ cells. The average phagocytic score of 2 replicates is shown. (C) The percentage of natural killer (NK) cells expressing CD107a protein and production of the cytokine, interferon (IFN)-γ, and the chemokine macrophage inflammatory protein (MIP)-1b was determined by flow cytometry (average of 2 replicates). (D) EBOV-specific IgG subclass MFI was determined via Luminex EBOV GP-coupled beads followed by detection with a subclass-specific secondary antibody (average of 2 replicates). (E) Correlation analyses using Spearman correlation coefficients between ADMP, ADNP, CD107a, IFNγ, MIP-1β, and EBOV GP-specific subclass titers (total IgG, IgG1–4) indicated a significant correlation between indicated function and/or titer, as marked by an asterisk. The r_s values are represented by a heat map (range, −1 to 1).

Figure 2.

Study design. Three sequential Ebola virus (EBOV) challenge studies using Chinese-origin rhesus macaques were conducted to assess the postinfection efficacy of SAB-139 in early intervention ([EI] starting on 1 day [d] postinfection), delayed intervention ([DI] on 3 d postinfection) and late intervention ([LI] on 5 d postinfection). Collection of blood and cerebrospinal fluid samples was preplanned for each experiment, but collection differed by d based on group assignment and the experiment. (A) Experiment 1 evaluated 4 57 mg/kg intravenous (IV) doses of SAB-139/V3–V4 in an EI group (n = 6), an irrelevant transchromosomic human immunoglobulin G (Tc-hIgG) control group (n = 6), or a saline control group (n = 2). (B) Experiment 2 evaluated three 150-mg/kg doses then one 125-mg/kg IV dose of SAB-139/V6–V8 in EI (n = 6) and DI (n = 6) groups or an irrelevant Tc-hIgG in the control group (n = 2). Surviving animals were back-challenged at d 77 after initial challenge to assess the development of an adaptive immune response postinfection and treatment. (C) Experiment 3 evaluated three 150-mg/kg doses then one 125-mg/kg dose of SAB-139/V6–V8 in a LI group (n = 6), an irrelevant Tc-hIgG in a control group (n = 4), or saline in a negative control group (n = 2). The * indicates that the fourth administration could be administered on d 11, 12, 13, or 14 postinfection based upon clinical and laboratory criteria. Abbreviation: EOS, end of study.

Figure 3.

SAB-139 can reduce or eliminate morbidity and mortality in rhesus macaques. (A) Experiment 1: Of the early-intervention (EI) nonhuman primates (NHPs) receiving SAB-139/V3–V4 intravenous (IV), 33% survived (2 of 6) compared with 0% (0 of 6) after administration irrelevant transchromosomic human immunoglobulin G (Tc-hIgG) or normal saline (0 of 2) controls. Surviving animals had mild clinical signs but no detectable viremia (10⁵ genome equivalents [GE]/mL limit of quantification [LOQ]). The NHPs had low anti-Ebola virus (EBOV) glycoprotein (GP) enzyme-linked immunosorbent assay (ELISA) titers after infusion (day [d] 3, 2908–7042 ELISA units [EU]/mL). One surviving NHP abruptly developed high anti-EBOV GP ELISA titers near end of study (EOS). (B) Experiment 2: Of EI and delayed intervention (DI) NHPs treated with SAB-139/V6–V8 IV, 100% survived (6 of 6 in both groups) compared with 0% of irrelevant Tc-hIgG controls (0 of 2). All SAB-treated NHPs were asymptomatic, but 2 EI animals had low sporadic viremia. All EI and DI NHPs had anti-EBOV GP ELISA titers after infusion that peaked at d 14 postinfection before falling at EOS. (C) Experiment 3: Of late intervention NHPs treated with SAB-139/V6–V8 IV, 0% survived (0 of 6) as did controls. All NHPs had viremia ranging from 10⁸ to 10¹⁰ GE/mL at d 5 postinfection. All animals died before or on d 9 postinfection. All NHPs had a low anti-EBOV GP ELISA titer at necropsy (1688–3629 EU/mL). Abbreviation: GGT, gamma-glutamyl transferase.

Figure 4.

Back-challenge survival, blood chemistries, and viremia. Early intervention (EI) and delayed intervention (DI) nonhuman primates (NHPs) from experiment 2 were back-challenged with Ebola virus (target dose 1000 plaque-forming units) on day (d) 77 after initial exposure. Of the total NHPs in both groups (n = 10), 12.67% (1 of 6) of the EI group and 100% (4 of 4) of the DI group survived, respectively. Nonsurvivors had high viremia (8.3 × 10⁸ to 3.6 × 10¹⁰ genome equivalents [GE]/mL). Survivors had no detectable viremia or changes in blood chemistries. Abbreviations: GGT, gamma-glutamyl transferase; LOQ, limit of quantification.