Animal Ethics and Biosafety Statement

All macaque work was performed in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, the Office of Animal Welfare and the United States Department of Agriculture. Animal procedures were carried out under anesthesia by trained personnel under the supervision of veterinary staff and all efforts were made to promote the welfare and to minimize animal suffering in accordance with the “Weatherall report for the use of non-human primates” recommendations. Animals were housed in adjoining individual primate cages allowing social interactions, under controlled conditions of humidity, temperature and light (12-hour light/12-hour dark cycles). Food and water were available ad libitum. Animals were monitored at least twice daily (pre- and post-infection) and fed commercial monkey chow, treats and fruit twice daily by trained personnel. Environmental enrichment consisted of commercial toys. Humane endpoint criteria, specified and approved by the Institutional Animal Care and Use Committee (IACUC), were applied to determine when animals should be humanely euthanized. All infectious animal work was performed in the maximum containment laboratory at the Rocky Mountain Laboratories (RML), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Montana, USA applying standard operating protocols approved by the Institutional Biosafety Committee (IBC).

Vaccine Vectors

We constructed RhCMV/EBOV-GP essentially as previously described11, by using E/T linear recombination to manipulate the parental RhCMV strain 68-1 genome cloned within a bacterial artificial chromosome (BAC)30. A codon-optimized version of GP (optZGP) from EBOV (Mayinga strain 76; Accession number AF086833) was used as the target antigen28. The optZGP open reading frame (ORF) was inserted within the RhCMV genome to replace the non-essential endogenous RhCMV Rh112 (pp65b) ORF (nucleotide positions 111,240 to 112,868 of RhCMV; see schematic Fig. 1A)56. This strategy places optZGP under control of the Rh112 promoter. The optZGP was epitope-tagged at the carboxy terminus with a V5 epitope. RhCMV/EBOV-GP BAC clones were analyzed in vitro as previously described (data not shown)11. Recombinant RhCMV/EBOV-GP viruses were reconstituted by transfection of BAC DNA into RFs30. Stability of optZGP expression in RhCMV/EBOV-GP vectors over at least 7 passages was confirmed by western analysis of infected cell lysates using monoclonal antibodies directed against EBOV GP1 and against the V5 epitope tag (Invitrogen) (Fig. 1C). Multi-step growth analysis of the RhCMV/EBOV-GP was performed as described11.

Challenge Virus

EBOV strain Mayinga (passage 5) was propagated on Vero E6 cells (mycoplasma negative), titrated on these cells and stored in liquid nitrogen. Deep sequencing confirmed the “7U phenotype” for this EBOV strain.

SDS-PAGE and western blotting

RFs infected at a multiplicity of infection (MOI) of 0.03 with either RhCMV/WT, RhCMV/EBOV-GP[2–8] or RhCMV/EBOV-GP[6–1] were solubilized in 1X Lamelli’s sample buffer, homogenized using QiaShredders (Qiagen), heated to 100 °C for 10 min, loaded onto 12% polyacrylamide gels and run in Tris-glycine buffer. Proteins were transferred to PVDF membranes (Bio-Rad) and blocked with 5% skimmed milk powder in Tris-buffered saline with 0.01% Tween-20 (TBST) overnight at 4 °C. Primary and secondary antibodies (HRP labeled; Bio-Rad) were applied at indicated dilutions: anti-V5 (1:1,500), anti-GP1 (1:10,000), anti-Rh112 (pp65b) (1:100), anti-Rh156 (IE-1) (1:500), anti-Rh70 (UL44) (1:2), anti-p38 (Santa Cruz Biotechnology, Inc.) (1:500), and secondary anti-mouse HRP (1:2,000) and anti-rabbit HRP (1:2,000). All antibodies were incubated for 1 hour at room temperature followed by three 5 minute washes with TBST. Membranes were developed using either ECL Western blotting substrate (Thermo Scientific) or SuperSignal West Femto substrate (Thermo Scientific), and imaged using a UVP ChemiDoc-It2 imaging system.

Study design

Six adult male and female NHPs of Indian genetic background were used for the study. Animals were confirmed to be RhCMV seropositive by ELISA before initiation of the study (resulting from natural RhCMV infection) (Fig. 3D). Animals were filovirus-naïve and were also free of cercopithicine herpesvirus 1, simian T-lymphotropic virus type 1, D-type simian retrovirus and simian immunodeficiency virus. Animals were assigned to either RhCMV/EBOV-GP vaccine (n = 4) or parental wild-type RhCMV/WT control (n = 2) groups with an aim of achieving a relatively equal distribution based on sex and age. On day -112, the vaccine group received a single s.c. bolus of a mixture of two independent clones of the RhCMV/EBOV-GP construct (5 × 106 pfu/construct). RhCMV/EBOV-GP and RhCMV/WT were administered subcutaneously as this has been the route used for inoculation of rhesus macaques in all earlier studies using RhCMV-based vectors (in this case, expressing SIV antigens11,14,18). The RhCMV/WT control group received a single 1 × 107 pfu s.c. inoculation of parental RhCMV/WT (clone 68-1)30. Animals were boosted with RhCMV/EBOV-GP clones or RhCMV/WT at week 12 (day -28). Blood samples were collected at times indicated over the pre-challenge 112 day period (vaccination phase) (Fig. 3A). Peripheral blood mononuclear cells (PBMCs) and plasma were prepared from blood by centrifugation on a histopaque gradient (Sigma) and assayed as detailed below. On day 0 (112 days post-vaccination), all animals were challenged with a lethal dose of 1,000 focus forming units of EBOV (strain Mayinga) by intramuscular administration at two anatomical locations (left and right caudal thigh). Animals were monitored twice daily for clinical signs of disease. Disease progression was assessed based on pre-established endpoints, and animals were humanely euthanized when clinical signs indicated onset of terminal disease. Blood samples were collected at times indicated (Fig. 3A) over the 35 day post-challenge period.

Hematology and serum chemistry

A HemaVet® 950FS laser-based hematology analyzer (Drew Scientific) was used to analyze the following blood parameters in 20 μl volumes of EDTA-treated blood: i) total white blood cell count, ii) lymphocyte, platelet, reticulocyte and red blood cell counts, iii) hemoglobin, iv) hematocrit values, and v) mean corpuscular volume and hemoglobin concentrations. Serum chemistry was analyzed using a Piccolo Xpress Chemistry Analyzer using Piccolo General Chemistry 13 Panel discs (Abaxis).

Viral loads

Levels of infectious EBOV were measured by using standard virus titration33, followed by calculation of 50% tissue culture infectious dose (TCID 50 ) using the method of Reed and Muench57. Tissues were homogenized prior to analysis.

Intracellular cytokine staining analysis of T cells

Frequencies of CD4+ and CD8+ T cells directed against the EBOV (Mayinga) GP target antigen, as well as RhCMV IE-1 and Rh112 proteins were determined during the vaccine phase by intracellular cytokine staining (ICS) as previously described33. For stimulation, PBMC (1–2 × 106 cells/well) were incubated in vitro with peptide pools (1 μg/ml final concentration) of overlapping peptides (11-mer with 5 amino acid overlap) representing each of the target ORFs. Incubation without antigen served as a background control. After 1 hour, brefeldin A (10 μg/ml) was added and cells were incubated for an additional 14 hours. Cells were surface stained using the following mAbs in indicated combinations: CD3, CD4 (eBioscience) and CD8β (Beckman Coulter). Cells were fixed and permeabilized according to manufacturer’s recommendations (BioLegend) prior to staining for intracellular staining using mAbs against Ki67 (BD) and IFNγ and TNFα. Polychromatic flow cytometric analysis was performed on a LSR II (BD Biosciences), and data was analyzed by using FlowJo software (version 10; Tree Star, Inc.). Response frequencies were determined by subtracting background and then averaging background subtracted responses.

Enzyme-linked immunosorbent assay (ELISA)

Total IgG antibody responses to RhCMV/EBOV-GP were measured by ELISA using either EBOV GPΔTM or RhCMV/WT infected cell lysate as a source of antigen, as previously described58. The end-point dilution titer (using a 4-fold dilution series) is shown. Post-challenge plasma samples were inactivated by γ-irradiation (5Mrad) before removal from BSL-4 containment under standard RML operating procedures as approved by the RML Institutional Biosafety Committee (IBC). Samples were deemed positive for EBOV GP-specific IgG when the OD value was higher than the mean plus 3 standard deviations of negative (RhCMV WT) sera59.

Neutralization assay

Neutralizing antibody titers were determined by performing focus reduction neutralization titration assays. Briefly, Vero E6 cells (mycoplasma negative) were seeded into 96-well plates to generate a confluent monolayer on the day of infection. Two-fold serum dilutions were prepared in triplicate in plain DMEM and 25 μl were incubated with 200 pfu EBOV (strain Mayinga) in a total volume of 50 μl. After 60 min at 37 °C the media was removed from cells, the serum-virus mixture was added and samples were incubated for 60 min at 37 °C. The mixture was then removed and 100 μl of 1.2% carboxymethyl cellulose in MEM (2% FBS) was added per well followed by incubation for 4 days at 37 °C. Cells were fixed in 10% neutral buffered formalin and removed from the maximum containment laboratory according to approved standard operating procedures (SOPs). Foci based either on GFP or staining using an anti-EBOV VP40 polyclonal rabbit serum and a secondary anti-rabbit FITC antibody (Sigma). Foci were counted and the neutralizing activity was determined as percent reduction of EBOV infection compared to control infected cells without plasma. Negative control: EBOV naïve rhesus macaque plasma. Positive control: NHP plasma of EBOV survivor.

Statistical analysis

Kaplan-Meier survival curves were performed to visualize survival rates between groups in EBOV challenge studies. Analysis was performed using Prism GraphPad Software (Version 5.0d).