Efficacy of Human Monoclonal Antibody Monotherapy Against Bundibugyo Virus Infection in Nonhuman Primates

Abstract

Background

The 2013–2016 Ebola virus disease (EVD) epidemics in West Africa highlighted a need for effective therapeutics for treatment of the disease caused by filoviruses. Monoclonal antibodies (mAbs) are promising therapeutic candidates for prophylaxis or treatment of virus infections. Data about efficacy of human mAb monotherapy against filovirus infections in preclinical nonhuman primate models are limited.

Methods

Previously, we described a large panel of human mAbs derived from the circulating memory B cells from Bundibugyo virus (BDBV) infection survivors that bind to the surface glycoprotein (GP) of the virus. We tested one of these neutralizing mAbs that recognized the glycan cap of the GP, designated mAb BDBV289, as monotherapy in rhesus macaques.

Results

We found that recombinant mAb BDBV289-N could confer up to 100% protection to BDBV-infected rhesus macaques when treatment was initiated as late as 8 days after virus challenge. Protection was associated with survival and decreased viremia levels in the blood of treated animals.

Conclusions

These findings define the efficacy of monotherapy of lethal BDBV infection with a glycan cap–specific mAb and identify a candidate mAb therapeutic molecule that could be included in antibody cocktails for prevention or treatment of ebolavirus infections.

Ebolaviruses, which are members of the Filoviridae family, cause severe disease in humans, with high mortality rates and significant epidemic potential. The 2013–2016 Ebola epidemic in West Africa was the largest of the known 29 outbreaks of Ebola virus disease (EVD) reported since ebolaviruses were first identified, with 28646 cases and 11323 deaths identified [1]. There are 5 known Ebolavirus species: Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), Sudan ebolavirus (SUDV), Tai Forest ebolavirus, and Reston ebolavirus. EBOV, BDBV, and SUDV are clinically relevant viruses that are known to cause lethal disease in humans [2]. There are no licensed ebolavirus vaccines or treatments. Studies of animal challenge models for EVD showed that mAbs and their mixtures are promising therapeutic candidates that, in some cases, confer complete postexposure protection of nonhuman primates (NHPs). The experimental therapeutic mAb mixture ZMapp, comprising 3 murine-human chimeric EBOV glycoprotein (GP)–specific mAbs, of which 2G4 and 4G7 recognize the base region and c13C6 recognizes the glycan cap, fully protected NHPs from lethal EBOV challenge [3]. This cocktail also exhibited some activity when used as treatment of EVD in humans in incomplete clinical trial testing during the recent EVD epidemic [4]. The use of ZMapp is limited to EBOV infection because the mAbs in that cocktail do not recognize BDBV or SUDV. Also, experience with human mAb–mediated protection in NHPs has been limited previously to studies with EBOV infection [3, 5, 6]. The species of Ebolavirus that might cause future ebolavirus outbreaks cannot be predicted. Therefore, it is desirable to identify broader human antibodies with a pan-ebolavirus recognition pattern for use in future therapeutic cocktails. Protection studies in NHPs with other ebolavirus species, such as BDBV and SUDV, are necessary to further elucidate the efficacy of mAb-based therapeutics against these infections.

We recently described a large panel of GP-specific mAbs from survivors of natural BDBV infection. One of the mAbs in that panel, designated BDBV289, possesses neutralizing activity against both BDBV and EBOV and protected both mice and guinea pigs from lethal EBOV challenge in monotherapy experiments [7]. Here, we assessed the efficacy of postexposure treatment with recombinant mAb BDBV289-N, using a rhesus macaque model of BDBV infection.

METHODS

Ethics Statement

NHP research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the eighth edition of the Guide for the Care and Use of Laboratory Animals [8]. The facility where this research was conducted (the University of Texas Medical Branch) is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and has an approved Office of Laboratory Animal Welfare Assurance (no. A3314-01).

Animal Challenge

Seven healthy adult rhesus macaques (Macaca mulatta) of Chinese origin (body weight, 4–6 kg) were studied. All animals were inoculated intramuscularly with a target dose of approximately 1000 plaque-forming units (PFU) of BDBV (isolate 200706291 Uganda, kindly provided by Dr Thomas Ksiazek). The back titer of the inoculum identified 825 PFU as the actual inoculation dose. Animals were randomized by random number assignment (with Microsoft Excel) into a treatment group of 6 animals and a control animal. The 6 BDBV-infected macaques in the treatment group received 35 mg/kg BDBV289-N mAb (made in tobacco, Nicotiana benthamiana, at Kentucky Bioprocessing [9]) on days 8 and 11 after virus challenge by intravenous injection. Antibody concentration was approximately 20 mg/mL, resulting in an administered volume of 1.5 mL/kg. The control animal was not treated. Historical untreated controls included 9 animals from 3 separate studies (unpublished data) that were challenged with the same target dose of BDBV and by the same route. The back titer of the inoculum identified 835, 1088, or 763 PFU as the actual inoculation dose for each of the 3 cohorts of historical controls. All animals underwent physical examinations, and blood specimens were collected at the time of and various times after BDBV infection. In addition, all animals were monitored daily and scored for disease progression with an internal filovirus scoring protocol approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee. The scoring measured from baseline and included posture/activity level, attitude/behavior, food and water intake, respiration, and disease manifestations, such as visible rash, hemorrhage, ecchymosis, or flushed skin. A score of ≥9 indicated that an animal met criteria for euthanasia. These studies were not blinded. Identification of individual NHPs is shown in Supplementary Table 1.

Detection of Virus Load by Plaque Assay or Real-Time Quantitative Polymerase Chain Reaction (qPCR) Analysis

Titration of virus in plasma samples was performed by plaque assay in Vero E6 cell culture monolayers, as previously described [10]. Briefly, increasing 10-fold dilutions of the samples were adsorbed to Vero E6 cell monolayers in duplicate wells (200 µL); the limit of detection was 25 PFU/mL. For real-time qPCR analysis, RNA was isolated from whole blood using the Viral RNA Mini-Kit (Qiagen) using 100 µL of blood into 600 µL of buffer AVL. Primers/probe targeting the VP35 intergenic region of BDBV were used for real-time qPCR with the probe sequence of 6 carboxyfluorescein–5′-CGCAACCTCCACAGTCGCCT-3′–6 carboxytetramethylrhodamine (Fisher Scientific). BDBV RNA was detected using the CFX96 detection system (BioRad Laboratories, Hercules, CA) in 1-step probe real-time qPCR kits (Qiagen) with the following cycle conditions: 50°C for 10 minutes, 95°C for 10 seconds, and 40 cycles at 95°C for 10 seconds and at 57°C for 30 seconds. Threshold cycle (CT) values representing BDBV genomes were analyzed with CFX Manager Software, and data are depicted as genome equivalents (GEq); the limit of detection was 3.7 log₁₀ GEq/mL.

Detection of Circulating BDBV289-N and Assessment of mAb Binding to GP

ELISA plates were coated overnight at 4°C with 0.1 µg/mL of mouse anti-human IgG (human CH2 domain with no cross-reactivity to rhesus macaque IgG; clone R10Z8E9; BioRad) and then blocked for 2 hours. The serum samples were assayed at 4-fold dilutions starting at a 1:100 dilution in ELISA diluent (1% heat-inactivated fetal bovine serum, 1× phosphate-buffered saline, and 0.2% Tween-20). Samples were incubated for 1 hour at ambient temperature and then removed, and plates were washed. Wells then were incubated for 1 hour with goat anti-human IgG conjugated to horseradish peroxidase (Fitzgerald Industries International) at a 1:5000 dilution. Wells were washed and then incubated with 2,2′-azino-di(3ethylbenzthiazoline-6-sulfonate) peroxidase substrate system (KPL) and read at 405 nm on a microplate reader (Molecular Devices Emax system). mAb were quantified using Prism software, version 7.04 (GraphPad), to analyze sigmoidal dose-response (variable slope), using BDBV289-N as standard. To assess whether the binding of recombinant BDBV289-N mAb protein was similar to that of the hybridoma cell-produced mAb we originally described, ELISA plates were coated with EBOV GP ΔMuc protein kindly provided by Erica Saphire. Purified mAbs were tested at different concentrations, starting at 30 μg/mL, and tested in quadruplicate.

Hematologic and Serum Biochemical Analyses

Total white blood cell counts, white blood cell differential counts, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed in blood specimens collected in tubes containing ethylenediaminetetraacetic acid, using a laser-based hematologic analyzer (Beckman Coulter). Serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, glucose, cholesterol, total protein, blood urea nitrogen, creatinine, uric acid, and C-reactive protein by using a Piccolo point-of-care analyzer and Biochemistry Panel Plus analyzer discs (Abaxis).

Statistical Analysis

Data were plotted using Prism software. Survival curves were estimated using the Kaplan-Meier method, and the proportion surviving at day 28 after infection was compared using a 2-sided exact unconditional test of homogeneity [11]. Virus titers were compared using the Mann-Whitney U test. A P value of ≤ .05 was considered statistically significant.

RESULTS

A rhesus macaque BDBV challenge model was used to determine the efficacy of treatment with recombinant BDBV289-N antibody produced in tobacco. BDBV289-N possessed similar binding activity to EBOV GP when compared to the original mAb expressed from hybridoma cells (Supplementary Figure 1). In this experiment, rhesus macaques were assigned to 1 treatment group of 6 animals, and all treated NHPs received 2 doses of mAb BDBV289-N spaced 3 days apart (8 and 11 days after virus inoculation). An additional animal was studied as a contemporaneous control, along with 9 historical untreated controls; all 10 were inoculated by the same route with the same stock of virus. After intramuscular challenge with 825 PFU of BDBV, we treated the animals with mAb 8 and 11 days after virus inoculation. The control animals were left untreated. All 6 animals treated with mAb BDBV289-N survived infection, while 60% of 10 control animals survived (Figure 1A). The difference in survival was associated with a statistical value of (P = .1138) by an exact unconditional test of homogeneity. This survival finding was consistent with prevention of mortality by BDBV289-N treatment, although not significant.

Figure 1.

Therapeutic potency of monoclonal antibody (mAb) BDBV289-N to prevent death and reduce viremia. Nonhuman primates (NHPs) received 825 plaque-forming units (PFU) of Bundibugyo virus (BDBV) intramuscularly and a dose of mAb (35 mg/kg) intravenously 8 and 11 days after inoculation (data are for 6 treated animals and 1 untreated control this study and for 9 untreated historical controls). A, Kaplan-Meier survival plot. Arrows indicate the day of mAb dosing. Untreated groups represent 1 animal from this study, and 9 historical controls represent animals (cumulative 60% survival) inoculated by the same route with the same stock of virus. The proportion surviving at day 28 after infection was compared using a 2-sided exact unconditional test of homogeneity. A P value of ≤ .05 was considered significant. B, A comparison of blood viral RNA load was determined at 11 days after virus inoculation for treated or control animals, as described in panel A. The group of 6 untreated animals included 1 NHP from this study and 5 historical controls that also were assessed for viremia on day 11 after infection. Virus titers were compared using the Mann–Whitney U test. The median titer for each group is shown. C, Kinetics of the blood viral RNA load, as determined by real-time quantitative polymerase chain reaction, for 6 treated or 6 of 10 control animals, comprising 1 NHP assessed at the same time point and 9 historical controls. The dotted line indicates the limit of detection (LOD), which was 3.7 log₁₀ genome equivalents (GEq) per milliliter. Each measurement represented the mean value of technical duplicates. Numbers indicate individual NHPs from the respective group. Abbreviations: C, untreated, this study (gray); HC, untreated, historical control (gray curves); M, treated, this study (orange curves).

Before (ie, 6–8 days after virus inoculation) the first treatment with mAb (ie, 8 days after inoculation), 5 of 6 NHPs from the treatment-designated group and all control NHPs, including 1 from this study and 9 historical controls, developed high viremia levels with a viral load that ranged from 10⁶ to 10¹⁰ GEq/mL of blood, as measured by real-time qPCR (Figure 1B and 1C). Viremia also was confirmed with a plaque assay (Supplementary Figure 2A). One animal in the treatment group showed no detectable virus in blood specimens at any time point of the analysis. An effect of the antibody treatment was observed in the rapid decline in viral RNA load by day 15 after infection in 4 of 5 treated animals that were initially viremic, and all 5 treated animals who experienced viremia had undetectable virus levels in blood by day 21 after infection (limit of detection, 3.7 log₁₀ GEq/mL). In contrast, the viral load in 8 of 10 control NHPs continued to increase or persist at the same level >11 days after virus inoculation (Figure 1C). Concordant with improved virus clearance, treated animals had significantly reduced viral loads in blood specimens on day 11 after infection (median, 6.8 log₁₀ GEq/mL), before treatment with the second mAb dose, compared with untreated controls at the same time point (median, 7.7 log₁₀ GEq/mL; P = .0087, by the Mann-Whitney U test; Figure 1B). A plaque assay that detected only infectious virus showed more-rapid clearance of BDBV infection from circulation than the time point estimated by a viral RNA real-time qPCR detection assay. All 6 surviving animals from the treated group and all 6 untreated control NHPs that survived by the end of this study had no detectable live virus in plasma by day 14 after infection (Supplementary Figure 2A).

Reduction of virus RNA load in blood on day 11 after infection and rapid virus clearance at the later time points after treatment were associated with a high concentration of circulating BDBV289-N antibody in all 6 animals from the treated group, as measured by human IgG capture ELISA of serum samples. The BDBV289-N concentration in serum was much higher than the previously determined half-maximal inhibitory concentration (IC₅₀) of this mAb for BDBV, using an in vitro neutralization assay (IC₅₀, 32 ng/mL) [7]. The mAb concentration in serum ranged from 0.3 to 0.8 mg/mL and from 0.4 to 1.3 mg/mL when assessed on days 11 and 28 after infection, respectively. The 2 control animals tested (1 from this study and 1 historical control) had no detectable human IgG in the same assay (Supplementary Figure 2B).

Treatment also was associated with protection from weight loss and illness, as each animal showed a <5% change in initial body weight before (11 days after virus inoculation) and after the last mAb treatment, and none experienced severe illness. Six of 10 untreated control animals demonstrated more-profound weight loss and illness (Figure 2A and 2B), and 4 animals met clinical euthanasia criteria. Before the first treatment, all animals had an elevated body temperature, and after the first treatment no clear difference in body temperature change was observed between the treated and untreated animals when the 2 groups were compared (Figure 2C). These findings demonstrate a beneficial effect of the 2-dose treatment with BDBV289-N to prevent mortality, weight loss, and disease, which was associated with control of viremia mediated by a high concentration of circulating neutralizing mAb.

Figure 2.

Therapeutic potency of monoclonal antibody (mAb) BDBV289-N to prevent illness. Nonhuman primates were treated and challenged as described in Figure 1. A, Body weight change. B, Clinical score. Dashed line indicates clinical score threshold for euthanasia. C, Change in body temperature. Each curve shows the data for an individual animal. Historical controls are included for comparative purposes. Arrows indicate the day of mAb dosing. Orange curves indicate treated animals, and gray curves indicate untreated animals.

We next assessed changes in blood chemistry findings and hematologic parameters to characterize the efficacy of mAb treatment further. Increases in the level of the liver enzymes alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase, which are indicators of liver damage by filovirus infections [3], were observed in both treated and untreated animals (Figure 3). We did not observe a clear difference between treated and untreated groups for the other assessed blood chemistry parameters (data not shown). Counts of platelets, white blood cells, and red blood cells were mostly reduced by day 8 after infection in animals from both groups, but no obvious difference was observed for the other assessed hematologic parameters (data not shown; Figure 4). BDBV289-N–treated NHPs experienced changes in hematologic and blood chemistry findings indicative of ebolavirus infection, such as reduced lymphocyte counts and elevated liver enzyme levels.

Figure 3.

Levels of blood biochemistry parameters in monoclonal antibody (mAb)–treated or –untreated animals. Nonhuman primates were treated and challenged as described in Figure 1, and blood specimens were collected and assessed at indicated time points. Arrows indicate the days of mAb dosing. Each curve shows data for an individual animal. Orange curves indicate treated animals, and gray curves indicate untreated animals. Historical controls are shown for comparative purposes.

Figure 4.

Levels of hematologic parameters in monoclonal antibody (mAb)–treated or –untreated animals. Nonhuman primates were treated and challenged as in Figure 1, and blood specimens were collected and assessed at indicated time points. Arrows indicate the days of mAb dosing. Each curve show data for an individual animal. Orange curves indicate treated animals, and gray curves indicate untreated animals. Historical controls are shown for comparative purposes.

We next assessed the virus burden in various organs and tissues that were collected on day 28 after infection (the study end point) from 6 BDBV289-N–treated and 1 untreated control NHP. Real-time qPCR analysis revealed a variation in virus load between individual animals and tissues, ranging from below the detection limit (ie, 3.7 log₁₀ GEq/g of tissue) to 8.8 log₁₀ GEq/g of tissue (Table 1). Real-time qPCR detects both infectious and noninfectious neutralized particles that have not yet been cleared. Nevertheless, the presence of viral RNA in treated NHPs on day 28 after infection may indicate that circulating glycan cap GP–specific mAb BDBV289-N has a limited capacity to clear systemically disseminated BDBV when used as monotherapy in the setting of BDBV infection. In summary, these findings define the efficacy of 2-dose treatment of NHPs with mAb BDBV289-N to prevent mortality, disease, and viremia caused by BDBV infection.

Table 1.

Viral Loads in Various Organs Obtained 28 Days After Virus Inoculation From Nonhuman Primates (NHPs) Treated With BDBV289 Antibody

Organ	NHP Identifiera
	11-1775R	T126021R	T126209R	11080312	11100592	12-1658R
Axillary LN	ND	6.8 ± 0.0	6.7 ± 0.2	5.5 ± 0.3	5.8 ± 0.4	ND
Inguinal LN	5.2 ± 0.5	6.7 ± 0.0	7.4 ± 0.0	6.7 ± 0.1	6.1 ± 0.0	5.0 ± 0.2
Liver	ND	5.2 ± 0.3	5.9 ± 0.2	5.3 ± 0.2	6.1 ± 0.3	ND
Spleen	5.6 ± 0.1	7.8 ± 0.0	6.3 ± 0.0	6.3 ± 0.2	7.5 ± 0.0	6.2 ± 0.0
Kidney	ND	ND	6.1 ± 0.0	ND	6.3 ± 0.1	ND
Adrenal	ND	6.1 ± 0.1	7.1 ± 0.1	6.0 ± 0.1	7.1 ± 0.1	ND
Lung	ND	7.0 ± 0.1	5.7 ± 0.2	5.6 ± 0.1	5.2 ± 0.4	ND
Brain front	ND	ND	6.1 ± 0.3	5.3 ± 0.2	5.4 ± 0.5	ND
Brain stem	ND	5.9 ± 0.1	5.7 ± 0.1	5.5 ± 0.2	7.4 ± 0.1	ND
Cervical spinal cord	ND	5.2 ± 0.2	5.8 ± 0.1	5.7 ± 0.4	7.7 ± 0.2	ND
Pancreas	ND	ND	5.4 ± 0.1	ND	6.2 ± 0.1	ND
Urinary bladder	ND	5.2 ± 0.2	5.9 ± 0.1	6.2 ± 0.1	5.6 ± 0.3	ND
Gonad	ND	6.0 ± 0.0	7.9 ± 0.0	5.7 ± 0.1	7.0 ± 0.1	5.0 ± 0.2
Uterus/prostate	ND	6.6 ± 0.0	ND	6.3 ± 0.1	6.0 ± 0.1	ND
Conjunctiva	ND	5.8 ± 0.2	5.6 ± 0.1	5.2 ± 0.0	ND	ND
Eye	ND	5.4 ± 0.5	8.8 ± 0.1	ND	8.2 ± 0.0	ND
Aqueous humor	ND	ND	ND	ND	9.0 ± 0.1	ND

Data were determined by real-time quantitative polymerase chain reaction analysis and are mean (±SD) log₁₀ genome equivalents per gram of tissue from technical duplicates. The limit of detection was 3.7 log₁₀ genome equivalents per gram of tissue.

Abbreviations: LN, lymph node; ND, not detected.

^aAnimal identifiers are as described in Supplementary Table 1.

DISCUSSION

Here we report the efficacy of human mAb monotherapy of BDBV infection in a rhesus macaque challenge model. The data show that treatment with 2 doses of BDBV289-N reduced viremia levels and protected challenged animals from BDBV-associated death. NHPs remain the most stringent preclinical model for determining the likely efficacy of filovirus therapy in humans [12, 13]. Efficacy against an otherwise lethal challenge in NHPs is a necessary scientific and regulatory threshold for identifying human antibodies for testing in humans.

A large number of ebolavirus-specific mAbs have been described that recognize diverse antigenic sites on GP, including epitopes on the glycan cap, the internal fusion loop, the GP1 head, the GP1/GP2 interface, the receptor-binding site (RBS), and the stalk [5, 7, 14–22]. Many of these mAbs showed protective capacity in smaller-animal models of filovirus infection, which included mice (for EBOV or SUDV infection) [7, 14, 16, 17, 22, 23], guinea pigs (for EBOV or SUDV infection) [7, 17], or the recently established ferret model for BDBV infection [20–22, 24]. The therapeutic efficacy in NHPs with EBOV infection of the antibody mixture ZMapp containing 3 mAb specificities is well documented [3]. However, there is limited information on the efficacy of mAb monotherapy in NHPs with ebolavirus infection.

It is of interest to better understand the major antigenic sites on filovirus GPs that can be recognized by antibodies that confer a high level of protection, and monotherapy in NHPs with lethal infection is probably the best measure of efficacy of mAbs. Stringent studies of mAb monotherapy also inform the selection of therapeutic mAb candidates that might be optimal candidates for inclusion in improved cocktails with broader and more-potent activity than ZMapp. The first human mAb, KZ52, which was isolated from a phage display library, recognizes the GP base region and neutralizes virus in vitro but failed to protect NHPs against EBOV or even to reduce plasma titers at the doses and treatment schedule tested [6]. Similarly, the glycan cap–specific mAb 13C6, which also is a part of the ZMapp cocktail, exhibits weak neutralizing and Fc-mediated functional activity, and it also failed to protect NHPs when used as monotherapy [3]. We recently studied the efficacy of mAb MR191, which neutralizes the related filovirus Marburg virus (MARV), as monotherapy in NHPs at late time points during infection [25]. This mAb, which binds to the RBS of MARV GP and blocks binding to the human endosomal protein Niemann-Pick C1 receptor [15, 26, 27], demonstrated a high level of efficacy as monotherapy. MR191 conferred a survival benefit of up to 100% to MARV- or Ravn virus–infected rhesus macaques when treatment was initiated up to 5 days after virus challenge.

RBS-targeting mAbs are of high promise for treatment of MARV infection, owing to their high capacity to neutralize the virus by targeting the most vulnerable antigenic site on the MARV GP. However, in contrast to MARV GP, EBOV GP is heavily shielded by the mucin-like domain, glycan cap, and other GP glycans and requires proteolytic priming in the endosomal compartment to expose the RBS after virus entry into the host cell [15, 26]. The reduced accessibility of the EBOV GP RBS on the intact trimer means that it is much less likely that RBS-targeting mAbs with the same neutralizing potency can be identified for ebolavirus infections. Two ebolavirus-specific mAbs have been described, designated FVM04 and mAb114, that appeared to target a partially exposed hydrophilic region of the RBS on GP and possessed protective capacity [5, 17]. mAb114 neutralizes EBOV only and conferred complete protection from EBOV-associated death when treatment was initiated after virus exposure and evaluated alone in NHPs [5], similar to the pattern we observed with the MR191 treatment studies with MARV [25]. However, although animals survived EVD after mAb114 monotherapy, they had viremia and showed clinical signs of disease, whereas a combination of mAb114 and a second GP mAb (the base-specific mAb 100) prevented viremia and clinical signs [5, 28]. A second RBS-reactive mAb, FVM04, which potently neutralized EBOV and SUDV, was tested for protection in mice and guinea pigs. We and others recently described broadly reactive and potently neutralizing human mAbs that are specific to stalk epitopes in the region between the heptad repeat 2 and the membrane proximal external region of ebolavirus GP [14, 21, 22]. These mAbs were efficient in treatment of EBOV and BDBV infections in small-animal models. Recently, investigators identified the internal fusion loop as a site of broad vulnerability on GP, and they reported the isolation of several broadly neutralizing mAbs that also possessed protective capacity against EBOV, BDBV, and SUDV in various small-animal models [20, 21]. Further studies should determine the efficacy of monotherapy with stalk- or internal fusion loop–specific mAbs.

Here we focused on the level of protection that could be conferred by the glycan cap–specific and neutralizing mAb BDBV289-N, which was isolated from a human survivor following BDBV infection. Our previous studies of this BDBV- and EBOV-reactive mAb revealed complete protection of mice and guinea pigs against heterologous EBOV infection after monotherapy, when mAb BDBV289-N was administered in 2 doses (on days 1 and 3 after infection) after lethal challenge. Delayed treatment on day 3 after infection resulted in no protection in mice, which died of disease on days 5–6 after infection, similar to untreated animals. One-dose treatment on day 1 after infection provided incomplete protection of guinea pigs [7]. This finding suggested that earlier treatment and a 2-dose regimen are beneficial for effective suppression of viremia and protection. In this study, we evaluated the therapeutic potential of a 2-dose regimen of BDBV289-N in NHPs when treatment was delayed until 8 days after BDBV challenge. By day 8 after infection, most animals become highly viremic but did not develop clinical signs of the disease. All animals in the treatment group survived and remained free of signs of disease after 2 doses of mAbs, with the second dose provided on day 11 after infection.

A significant limitation of the rhesus macaque model of BDBV infection for these studies is the incomplete lethality observed (40% in this study), compared with the high-susceptibility NHP EBOV challenge model [13]. Protection was associated with a high level of circulating BBDV289-N mAb and decreased viremia levels in treated animals that survived. Similar to previous monotherapy studies with ebolavirus mAbs [3, 5, 6], the treated animals here exhibited viremia. One of the reasons for this incomplete therapeutic effect could be that mAb BDBV289-N not only recognizes the glycan cap on GP but also cross-reacts with the secreted form of the glycoprotein. This secreted form is present at high levels in patients with EVD [29] and may deplete mAbs that recognize it from circulation and infected tissues [30], potentially reducing their capacity to target and neutralize infectious virions. However, the partial therapeutic efficacy in NHPs we observed is consistent with monotherapy outcomes reported with other mAbs.

The historical high efficacy of ZMapp, despite the relatively low potency of the individual mAbs, may suggest that complete protection from ebolavirus infections may require a cocktail of several mAbs of differing epitope specificity [31–33]. The evidence presented here suggests that BDBV289-N is an effective mAb candidate for inclusion in future therapeutic mixtures that could be tested as treatment of BDBV infection.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Acknowledgments. L. Z., T. W. G., and J. E. C. planned the studies. J. B. G., K. N. A., D. J. D., R. W. C., C. E. M., A. I. F., M. H. P., J. V., and K. J. W. conducted experiments. P. G., J. C. S., T. W. G., and J. E. C. interpreted the studies. P. G. and J. E. C. wrote the first draft of the manuscript. L. Z., T. W. G., and J. E. C. obtained funding. All authors reviewed, edited, and approved the manuscript.

Financial support. This work was supported by U.S. N.I.H. grants U19 AI109711 (JEC and TWG) and U19 AI109762 (LZ), Defense Threat Reduction Agency grant HDTRA1-13-1-0034 (JEC), HHS contract HHSN272201400058C (JEC). Work in BSL-4/ABSL-4 containment of the Galveston National Laboratory was supported by NIH grant 5UC7AI094660-07. Animal studies were supported by the Animal Resource Center of the Galveston National Laboratory.

Potential conflicts of interest. J. E. C. is a consultant for Sanofi; is on the scientific advisory boards of PaxVax, CompuVax, GigaGen, and Meissa Vaccines; is a recipient of previous unrelated research grants from Moderna and Sanofi; and is founder of IDBiologics. J. E. C. and A. I. F. are coinventors of technology that includes the BDBV289 antibody and for which a patent application has been submitted. M. H. P., J. V., K. J. W., and L. Z. are employees of Mapp Biopharmaceutical. K. J. W. and L. Z. are co-owners of Mapp. All other authors report no potential conflicts of interest.