ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques

Ethics Statement

Mice - Mice were used in accordance with the UK Animals (Scientific Procedures) Act under project license number P9804B4F1 granted by the UK Home Office. Animals were group housed in IVCs under SPF conditions, with constant temperature (20–24°C) and humidity (45 to 65%) with lighting on a fixed light/dark cycle (12-hours/12-hours)

NHPs - Animal experiment approval was provided by the Institutional Animal Care and Use Committee (IACUC) at Rocky Mountain Laboratories. Animal experiments were executed in an Association for Assessment and Accreditation of Laboratory Animal Care (AALAC)-approved facility by certified staff, following the basic principles and guidelines in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. Rhesus macaques were housed in individual primate cages allowing social interactions, in a climate-controlled room with a fixed light/dark cycle (12-hours/12-hours) and monitored a minimum of twice daily. Commercial monkey chow, treats, and fruit were provided by trained personnel. Water was available ad libitum. Environmental enrichment consisted of a variety of human interaction, commercial toys, videos, and music. The Institutional Biosafety Committee (IBC) approved work with infectious SARS-CoV-2 virus strains under BSL3 conditions. All sample inactivation was performed according to IBC approved standard operating procedures for removal of specimens from high containment.

Generation of vaccine ChAdOx1 nCoV-19

The spike protein of SARS-CoV-2 (GenBank accession number YP_009724390.1), the surface glycoprotein responsible for receptor binding and fusion/entry into the host cell, was codon optimised for expression in human cell lines and synthesised with the tissue plasminogen activator (tPA) leader sequence at the 5’ end by GeneArt Gene Synthesis (Thermo Fisher Scientific). The sequence, encoding SARS-CoV-2 amino acids 2–1273 and tPA leader, was cloned into a shuttle plasmid using InFusion cloning (Clontech). The shuttle plasmid encodes a modified human cytomegalovirus major immediate early promoter (IE CMV) with tetracycline operator (TetO) sites, poly adenylation signal from bovine growth hormone (BGH), between Gateway® recombination cloning sites. ChAdOx1 nCoV-19 was prepared using Gateway® recombination technology (Thermo Fisher Scientific) between the shuttle plasmid described and the ChAdOx1 destination DNA BAC vector described in⁵ resulting in the insertion of the SARS-CoV-2 expression cassette at the E1 locus. The ChAdOx1 adenovirus genome was excised from the BAC using unique PmeI sites flanking the adenovirus genome sequence. The virus was rescued and propagated in T-Rex 293 HEK cells (Invitrogen) which repress antigen expression during virus propagation. Purification was by CsCl gradient ultracentrifugation. Virus titers were determined by hexon immunostaining assay and viral particles calculated based on spectrophotometry^16,17.

Study design animal experiments

Mice – Female BALB/cOlaHsd (BALB/c) (Envigo) and outbred Crl:CD1(ICR) (CD1) (Charles River) mice of at least 6 weeks of age, were immunized IM in the musculus tibialis with 6x10⁹ VP of ChAdOx1 nCoV-19 unless otherwise stated.

NHPs - 18 adult rhesus macaques (17M, 1F) between 2–4 years old were randomly divided into three groups of six animals. Animal group size was based on initial model development. Group 1 was vaccinated with ChAdOx1 nCoV-19 at −28 DPI, group 2 was vaccinated with ChAdOx1 nCoV-19 at −56 and −28 DPI, group 3 was vaccinated with ChAdOx1 GFP at −56 and −28 DPI (1 animal) or at −28 DPI (5 animals). All vaccinations were done with 2.5 x 10¹⁰ VP/animal diluted in sterile PBS. Blood samples were obtained before vaccination and 14 days thereafter. Animals were challenged with SARS-CoV-2 strain nCoV-WA1–2020 (MN985325.1) diluted in sterile DMEM on 0 DPI; with administration of 4 mL intratracheally, 1 mL intranasally, 1 mL orally and 0.5 mL ocularly of 4 x 10⁵ TCID₅₀/mL virus suspension. Animals were scored daily by the same person who was blinded to study group allocations using a standardized scoring sheet. Scoring was based on the following criteria: general appearance, appearance skin and coat, discharge, respiration, feces and urine appearance, appetite, and activity. The scoring sheet can be found in⁷. Clinical exams were performed on −28, −14, 0, 1, 3, and 5 and 7 DPI. Nasal swabs and blood were collected at all exam dates. BAL was performed on 3, 5, and 7 DPI by insertion of an endotracheal tube and bronchoscope into the trachea, then past the 3^rd bifurcation, and subsequent installation of 10 mL of sterile saline. Manual suction was applied to retrieve the BAL sample. Serum biochemistry (albumin, AST, ALT, GGT, BUN, creatinine) was analyzed using the Vetscan VS2 Chemistry Analyzer and VetScan 2 Preventative Care 12 discs (Abaxis) (Tables S1–3). Necropsy was performed on 7 DPI and the following tissues were collected: cervical lymph node, mediastinal lymph node, conjunctiva, nasal mucosa, oropharynx, tonsil, trachea, all six lung lobes, right and left bronchus, heart, liver, spleen, kidney, stomach, duodenum, jejunum, ileum, cecum, colon, urinary bladder.

Cells and virus

SARS-CoV-2 strain nCoV-WA1–2020 (MN985325.1) was provided by CDC, Atlanta, USA. Virus propagation was performed in VeroE6 cells in DMEM supplemented with 2% fetal bovine serum, 1 mM L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. The used virus stock was 100% identical to the initial deposited genbank sequence (MN985325.1) and no contaminants were detected. VeroE6 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1 mM L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. VeroE6 cells were provided by Dr. Ralph Baric and were not authenticated in-house; mycoplasma testing is performed at regular intervals and no mycoplasma has been detected.

Virus isolation from tissue

Tissue sections were weighed and homogenized in 1 mL of DMEM. 250 µl of homogenate was added to VeroE6 cells in a 24 well plate in duplicate. After 1 hour at 37°C and 5% CO₂, cells were washed with PBS and 500 µl of DMEM containing 2% FBS was added. Cells were incubated at 37°C and 5% CO₂. CPE was read 6 days later.

Virus neutralization assay SARS-CoV-2

Sera were heat-inactivated (30 min, 56 °C), two-fold serial dilutions were prepared in 2% DMEM and 100 TCID₅₀ of SARS-CoV-2 was added. After 1hr incubation at 37 °C and 5% CO2, virus:serum mixture was added to VeroE6 cells and incubated at 37°C and 5% CO2. At 5 dpi, cytopathic effect was scored. The virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum which still inhibited virus replication.

Virus neutralization assay ChAdOx1

Chimpanzee adenovirus ChAdOx1-specific neutralizing antibody titers were assessed using a secreted placental alkaline phosphatase (SEAP) quantitation assay as described⁶. Briefly, GripTite MSR 293 cells (Invitrogen, catalog no. R795–07) were infected with the serial diluted serum in phenol red–free DMEM (Life Technologies, catalog no. 31053028) and the ChAdOx1-SEAP reporter virus in a 1:1 mixture for 1 hour before replacing with phenol red–free 10% FBS DMEM for 24 hours. For each sample, SEAP concentration was assessed in 50 μl aliquots of culture supernatant, with CPSD as an indicator substrate (Tropix Phospha-Light Chemiluminescent Assay Kit, Life Technologies, catalog no. T1017). Luminescence intensity was measured using a Varioskan Flash luminometer (Thermo Fisher Scientific). Serum dilution neutralization titers were measured by linear interpolation of adjacent values (to 50% inhibition) to determine the serum dilution required to reduce SEAP concentration by 50% compared to wells with virus alone.

RNA extraction and quantitative reverse-transcription polymerase chain reaction

Tissues (up to 30 mg) were homogenized in RLT buffer and RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. RNA was extracted from BAL fluid and nasal swabs using the QiaAmp Viral RNA kit (Qiagen) according to the manufacturer’s instructions. Viral gRNA¹⁸ and sgRNA¹⁹ specific assays were used for the detection of viral RNA. Five μl RNA was tested with the Rotor-GeneTM probe kit (Qiagen) according to instructions of the manufacturer. Dilutions of SARS-CoV-2 standards with known genome copies were run in parallel.

Enzyme-linked immunosorbent assay for mouse sera

MaxiSorp plates (Nunc) were coated with S1 (monomeric, AA 1–674) or S2 (monomeric, AA 685–1211 The Native Antigen Company; 50 ng/well) or with prefusion stabilized SARS-CoV-2 spike protein (250 ng/well) in PBS for overnight adsorption at 4°C. Plates were washed in PBS/Tween (0.05% v/v) and wells blocked using casein (ThermoFisher Scientific) for 1hr at RT. Serially diluted mouse serum samples were added and incubated overnight at 4°C or 37°C for 2hr for specific IgM detection. Plates were washed and Alkaline Phosphatase-conjugated goat anti-mouse IgG (Sigma) or IgM (Abcam) was added to all wells for 1hr at RT or 2hr at 37°C, respectivelly. After washing pNPP substrate (Sigma) was added. Optical density (OD) values for each well were measured at 405 nm. Endpoint titers were calculated as follows: the log₁₀ OD against log₁₀ sample dilution was plotted and a regression analysis of the linear part of this curve allowed calculation of the endpoint titer with an OD of three times the background. The same calculation was used for diluting the sera to the same amounts of total IgG for further testing on different IgG subclasses with anti-mouse IgG subclass-specific antibodies (Abcam). The results of the IgG subclass ELISA are presented using OD values.

Enzyme-linked immunosorbent assay for NHP sera

Prefusion stabilized SARS-CoV-2 spike protein with a T4 fibritin trimerization motif²⁰ was obtained from the Vaccine Research Centre, Bethesda, USA. Maxisorp plates (Nunc) were coated overnight at 4°C with 100 or 250 ng/well spike protein in PBS for IgG and IgM detection, respectively. Plates were blocked with 100 µl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum serially diluted 2x in casein in PBS was incubated at RT for 1hr or 37°C for 2hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, 074–11-021) in casein and TMB 2-component peroxidase substrate (Seracare, 5120–0047), developed for 5–10 min, and reaction was stopped using stop solution (Seracare, 5150–0021) and read at 450 nm or anti-monkey IgM – AP labelled after adding the pNPP substrate and measuring OD values at 405 nm. All wells were washed 4x with PBST 0.1% tween in between steps. Threshold for positivity was set at 3x OD value of negative control (serum obtained from non-human primates prior to start of the experiment) or 0.2, whichever one was higher, or OD of three times the background for the calculation of the IgM endpoint values.

ELISpot assay and ICS analysis

Single cell suspension of murine splenocytes were prepared by passing cells through 70μM cell strainers and ACK lysis prior to resuspension in complete media. Rhesus macaque PBMCs were isolated from ethylene diamine tetraaceticacid (EDTA) whole blood using LeucosepTM tubes (Greiner Bio-one International GmbH) and Histopaque®−1077 density gradient cell separation medium (Sigma-Aldrich) according to the manufacturers’ instructions.

Mice - For analysis of IFN-γ production by ELISpot, cells were stimulated with pools of S1 or S2 peptides (final concentration of 2μg/ml) on IPVH-membrane plates (Millipore) coated with 5μg/ml anti-mouse IFN-γ (AN18). After 18–20 hours of stimulation, IFN-γ spot forming cells (SFC) were detected by staining membranes with anti-mouse IFN-γ biotin (1μg/ml) (R46A2) followed by streptavidin-Alkaline Phosphatase (1μg/ml) and development with AP conjugate substrate kit (BioRad, UK).

For analysis of intracellular cytokine production, cells were stimulated at 37°C for 6 hours with 2μg/ml S1 or S2 pools of peptide, media or cell stimulation cocktail (containing PMA-Ionomycin, Biolegend), together with 1μg/ml Golgi-plug (BD) with the addition of 2μl/ml CD107a-Alexa647. Cell supernatant was collected and frozen at –20̊C for subsequent analysis by MesoScale Discovery (MSD) assay (see below). Following surface staining with CD4-BUV496, CD8-PerCPCy5.5, CD62L-BV711, CD127-BV650 and live-dead Aqua, cells were fixed with 4% paraformaldehyde and stained intracellularly with TNF-α-A488, IL-2-PECy7, IL-4-BV605, IL-10-PE and IFN-γ-e450 diluted in Perm-Wash buffer (BD).

Sample acquisition was performed on a Fortessa (BD) and data analyzed in FlowJo V10 (TreeStar). An acquisition threshold was set at a minimum of 5000 events in the live CD3⁺ gate. Antigen specific T cells were identified by gating on LIVE/DEAD negative, doublet negative (FSC-H vs FSC-A), size (FSC-H vs SSC), CD3⁺, CD4⁺ or CD8⁺ cells and cytokine positive (Gating strategy detailed in Supplementary Figure 1). Cytokine positive responses are presented after subtraction of the background response detected in the corresponding unstimulated sample (media containing CD107a and Golgi-plug) of each individual spleen sample. To determine whether there was a statistically significant differences in the level of cytokines produced by CD4⁺ or CD8⁺ T cells, data in each graph was analysed with a 2-way ANOVA (repeated measure) and post-hoc Tukey’s multiple comparison test.

NHPs - IFN-γ ELISpot assay of PBMCs was performed using the ImmunoSpot® Human IFN- γ Single-Color Enzymatic ELISpot Assay Kit according to the manufacturer’s protocol (Cellular Technology Limited). PBMCs were plated at a concentration of 100,000 cells per well and were stimulated with four contiguous peptide pools spanning the length of the SARS-CoV-2 spike protein sequence at a concentration of 2 µg/mL per peptide (Mimotopes). ELISpot plates were subjected to overnight formalin inactivation prior to removal from BSL4 for reading. Analysis was performed using the CTL ImmunoSpot® Analyzer and ImmunoSpot® Software (Cellular Technology Limited). Spot forming units (SFU) per 1.0x10⁶ PBMCs were summed across the 4 peptide pools for each animal.

Measurement of cytokines and chemokines

Mouse samples were assayed using MSD Technology V-PLEX Mouse Cytokine 29-Plex kit according to the manufacturer’s instructions. Non-human primate samples were inactivated with γ-radiation (2 MRad) according to standard operating procedures and assayed on a Bio-Plex 200 instrument (Bio-Rad) using the Non-Human Primate Cytokine MILLIPLEX map 23-plex kit (Millipore) according to the manufacturer’s instructions. LLOD was used for all undetectable and extrapolated values. Only data for cytokines consistently above the lower limit of quantification were included in further analyses.

Log₁₀ Fold Change (Log₁₀FC) for mouse samples was calculated as follows:

Log₁₀FC = Log₁₀((Stimulated (pg/ml) + 1) / (Unstimulated (pg/ml) + 1))

Fold change for NHP samples was calculated as follows:

FC = Concentration (pg/mL) on DX (1, 3, 5, or 7)/Concentration (pg/mL) on D0

Histology and immunohistochemistry

Necropsies and tissue sampling were performed according to IBC-approved protocols. Lungs were perfused with 10% formalin and processed for histologic review. Harvested tissues were fixed for eight days in 10% neutral-buffered formalin, embedded in paraffin, processed using a VIP-6 Tissue Tek (Sakura Finetek, USA) tissue processor, and embedded in Ultraffin paraffin polymer (Cancer Diagnostics, Durham, NC). Samples were sectioned at 5 µm, and resulting slides were stained with hematoxylin and eosin. Specific anti-CoV immunoreactivity was detected using an in-house SARS-CoV-2 nucleocapsid protein rabbit antibody (Genscript) at a 1:1000 dilution. The IHC assay was carried out on a Discovery ULTRA automated staining instrument (Roche Tissue Diagnostics) with a Discovery ChromoMap DAB (Ventana Medical Systems) kit. All tissue slides were evaluated by a board-certified veterinary anatomic pathologist blinded to study group allocations. 18 sections, taken from 6 different lung lobes are evaluated for each animal; a representative lesion from each group was selected for the figure.

Statistical analyses

Two-tailed Mann-Whitney’s rank or Wilcoxon tests were conducted to compare differences between groups using Graphpad Prism version 8.3.0. A Bonferroni correction was used to control for type I error rate where required.