EBOV (Zaire Kikwit strain) stocks were developed at the USAMRIID with virus originally isolated from an infected patient during the 1995 outbreak and passaged in Vero E6 cells.
The stock material for the study was obtained in four passages from the original isolate. This stock has been deemed the national stock for preclinical studies for advanced products.
The stock was made by the Department of Defense Critical Reagents Program under the Joint Program Executive Office for Chemical and Biological Defense.
The virus and future viruses generated under this program have been developed under the Filovirus Animal Non-Clinical Group (FANG), a multiple-agency group including the U.S. Food and Drug Administration, to prepare for validated/regulated efficacy studies.
NHP challenge and care
Adult male and female rhesus macaque NHPs were caged individually. NHPs underwent surgery for TA10TA-D70 telemetry implantation (DSI). After the NHPs were placed in training jackets (Lomir Biomedical) for acclimatization, which lasted for 6 days.
Surgeries to place central venous catheters (CVCs) (Groshong 7F; Bard) were performed, and adequate recovery time was provided before transfer into biosafety level 4 (BSL4) containment.
After CVC placement, custom jackets (Lomir Biomedical) were used to contain and protect the CVC lines connected to a mounted swivel system (Instech Solomon). These lines were locked with taurolidine-citrate catheter solution (UNO B.V.)
After the NHPs were moved into containment, acclimated, and connected to the swivel system, the lines were flushed at least once daily with PBS (BD) and locked with heparin (BD) to maintain line functionality.
NHPs were given monkey chow (Harlan), primate treats, fruits, and vegetables for the duration of the study. Before the challenge, NHPs were anesthetized and given a physical examination.
Animals were challenged intramuscularly with a target dose of 1000 PFU/ml diluted from the stock concentration with minimum essential medium (MEM). After the challenge, the animals were returned to their cages and observed until reasonable mobility was recovered.
Verification of target dose and viremia
Target challenge doses and viremia were verified by agarose-based plaque assay. Dilution points were serially diluted 10-fold in Eagle’s MEM and adsorbed onto Vero E6 cell monolayers in six-well plates.
The plates were incubated for 1 hour at 37°C/5% CO2 with rocking about every 15 min, and 2 ml of a 1:1 mixture of medium [2× Eagle’s basal medium with Earle’s salts, 10% (v/v) fetal bovine serum] and 1% agarose (Lonza) was added.
Plates were then incubated for 7 days at 37°C/5% CO2. After incubation, 2 ml of a 1:1 mixture of the same medium and 4% neutral red stain was added to each well.
After an additional 24 hours of incubation at 37°C/5% CO2, the plaques were counted, and viremia titers were calculated. Viremia analysis was performed on plasma samples with the same methodology.
MB-003 preparation and treatment Treatments consisted of a mixture of three mAbs, and each mAb was equally represented in the treatment mixture. Treatment for each group was set at 16.7 mg/kg per mAb.
The treatment for each specific NHP was determined according to the animal’s weight before the challenge. NHPs were treated via intravenous infusion through the CVC with 60-ml syringes (BD) and syringe pumps (Lomir Biomedical) over about 280 min.
NHPs received MB-003 (50 mg/kg). After the initial positive triggers, all NHPs were treated every 3 days. In total, three in- fusions were given to each NHP.
Animal monitoring and sample collection
NHPs were monitored for changes in health, diet, behavior, and appearance. Samples used for RT-PCR analysis were collected twice per day at 12-hour increments. If anesthesia was required for the collection samples were only collected once daily in the morning.
Samples used for CBCs and chemistry analyses were collected on days 0, 3, 5, 7, 10,14, 21, and 28. CBCs were performed with the Hemavet 950 (Drew Scientific), and chemistry analyses were performed with Piccolo 13 general chemistries (Abaxis).
All blood collections were performed with 1-ml mini collect serum and K3 EDTA plasma tubes (Greiner Bio-One). All sample tubes were centrifuged at 1800g for 10 min, and the resulting material was used or frozen at −80°C for further analysis.
NHP temperatures were monitored remotely via DSI telemetry every 10 min with the corresponding software (DSI). Experiments were conducted under BSL4 containment conditions.
Enzyme-linked immunosorbent assay
ELISAs were performed with a recombinant EBOV glycoprotein (National Cancer Institute), and plasma samples were serially diluted at half-log increments. Goat anti-human IgG (heavy + light) (KPL) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Millipore) were used as secondary antibodies and substrate, respectively. Assays were evaluated at an absorbance at 405 nm (SpectraMax M5; Molecular Devices).
RNA isolation and analysis
Qualitative real-time RT-PCR (rRT-PCR) was used for the detection of Ebola Zaire virus in plasma samples from exposed NHPs. Samples from the NHPs were inactivated with TRI reagent LS (at a ratio of three parts of TRI Reagent LS to one part of plasma).
RNA in each sample was purified with the Qiagen QIAamp Viral RNA Mini Kit. A volume of 70 ml of inactivated plasma was placed onto the column, and samples were eluted in 70 ml of AVE buffer provided by Qiagen.
The eluted RNA was run in triplicate with the USAMRIID DSD EZ1 rRT-PCR pre-EUA Assay Kit on the ABI 7500 Fast Dx instrument. This assay kit uses SuperScript II One-Step rRT-PCR sequence-specific primers and a hydrolyzable probe (TaqMan) against a conserved region of the Ebola Zaire virus.
The PCR cycling conditions were as follows: reverse transcription for 15 min at 50°C, Taq activation for 5 min at 95°C, and 45 cycles of amplification with a denaturation step for 1 s at 95°C and an anneal/extension step for 26 s at 60°C.
Samples were run immediately for qualitative detection at two-time points on days 3 to 5 after infection, with a.m. and p.m. time points where applicable. The sample was considered positive if one of the three replicate reaction was detected with a Ct value or negative if all three replicate reactions were undetected.
The time required from sample receipt to reportable detection was about 3 hours.
Quantitative RNA isolation and RT-PCR
RNeasy kits (Qiagen) were used for RNA extraction. One-step quantitative real-time RT-PCRs were performed with a Light Cycler 480 (Roche) in 20-ml volumes with 5 ml of purified RNA using the SuperScript III One-Step RT-PCR System (Invitrogen).
Primers (forward, 5′-CGGA- CCTGGTTTGGTTGTG-3′; reverse, 5′-GCTGCAGTGTCGCATCTGA-3′) and TaqMan probe (6-carboxyfluorescein-5′-CCCTTGCCACAATCT- minor groove binder nonfluorescent quencher-3′) (Applied Biosystems) specific for the Ebola Zaire glycoprotein gene were used.
Program conditions consisted of reverse transcription at 50°C for 20 min and initial denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 5 s; annealing, synthesis, and signal acquisition at 60°C for 20 s; and a final cooling step at 40°C for 30 s. Measurements of viral gene expression were performed with a viral RNA standard.
Survival curves were analyzed with the log-rank Mantel-Cox test. For each test, P < 0.05 was considered statistically significant. Survival data are consistent with proportional hazards.
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