Rhesus Macaques. Eight colony-bred Indian origin rhesus macaques (Macaca mulatta) were housed at the California National Primate Research Center and maintained in accordance with American Association for Accreditation of Laboratory Animal Care guidelines and Animal Welfare Act/Guide. As described by us 37, strict social distancing measures were implemented at the CNPRC at the start of the pandemic in March to reduce risk of human-to-rhesus SARS-CoV-2 transmission. Animals were screened for SARS-CoV-2 and housed in barrier rooms with increased PPE requirements prior to study assignment. Animals were four to five years of age with a median weight of 8.6 kg (range 5.4-10.7 kg), were SIV- STLV- SRV-. Animals were seronegative for SARS-CoV-2 at study initiation. Sex distribution within experimental groups was as follows; Infected (n=3 females, n=1 male); Infected + Convalescent Plasma (n=2 males); Infected + Normal Plasma (n=2 males). Table S1 provides details of the animals in the study. For blood collection, animals were anesthetized with 10 mg/kg ketamine hydrochloride injected i.m. For virus inoculation and nasal secretion sample collection, animals were additionally anesthetized with 15-30 ug/kg dexmedetomidine HCl inject i.m. and anesthesia was reversed with 0.07-0.15 mg/kg atipamezole HCl injected i.m.
Virus and inoculations. SARS-CoV-2 virus was isolated from the nasal swab of a COVID-19 patient with acute respiratory distress syndrome admitted to University of California, Davis Medical Center, Sacramento 38. Vero cells (ATCC CCL-81) were used for viral isolation and stock expansion. The passage 2 viral stock (SARS-CoV-2/human/USA/CA-CZB-59X002/2020) used for animal inoculations had a titer of 1.2x106 PFU/ml (corresponding to 2x109 vRNA) (Genbank accession number: MT394528). To recapitulate relevant transmission routes of SARS-CoV-2, animals were inoculated with 1 ml stock instilled into the trachea, 1 ml dripped intranasally, and a drop of virus stock in each conjunctiva.
Convalescent plasma and infusions. Convalescent plasma was sourced from Vitalant and represented a pool from up to four donors. Plasma was pooled prior to infusion into monkeys. Pooled plasma had a nAb titer of 1:1149, binding antibody titers for SARS-CoV-2 antigen were as follows; anti-S1-IgG, 24.5 µg/ml; anti-S2 IgG, 2.9 µg/ml; anti-NC IgG, 10.7 µg/ml. Normal plasma was collected prior to the COVID-19 pandemic and was negative for SARS-Cov2 antibody. Concentrations were as follows; anti-S1-IgG, 0.00004µg/ml; anti-S2 IgG, 0.003 µg/ml; anti-N IgG, 0.001 µg/ml. Twenty fours following virus inoculation, animals were infused with plasma at 4 ml/kg volume (total volume infused was 33-39 ml) at an infusion rate of 1 ml/minute. Control animals (n=4) were not infused.
Specimen collection and processing. On days 2, 4, 5, 7, 8, and 10, a 5-french feeding tube was inserted into the nasal cavity. 2 ml PBS were instilled through each nostril and the maximum volume was aspirated, secretions were spun at 800 g for 10 min and 1 ml of supernatant and cell pellet were lysed in 3 ml Trizol LS for RNA isolation. EDTA-anticoagulated blood was collected on Days 0, 2, 4, 7, and 10 for immunophenotyping. PBMCs were isolated from whole blood collected in CPT vacutainer tubes, sampled at Day 7 and necropsy, by density gradient centrifugation as previously described 39. For serum, coagulated blood was centrifuged at 800 g for 10 min to pellet clotted cells, followed by extraction of supernatant and storage at -80°C. Lymph nodes, spleen, and lung tissue were obtained at necropsy and digested enzymatically using collagenase followed by manual disruption to obtain single cell suspensions for flow cytometry based assays.
Activation induced Marker (AIM) assay. Cells were stimulated with overlapping peptide pools representing SARS-CoV-2 and responding cells were identified by upregulation of activation markers, as described previously 39,40. All antigens were used at a final concentration of 2 mg/mL in a stimulation cocktail made with using 0.2 mg of CD28 and 0.2 mg CD49d costimulatory antibodies per test. Unstimulated controls were treated with volume-controlled DMSO (Sigma-Aldrich). Tubes were incubated in 5% CO2 at 37°C overnight. Following an 18 h stimulation, the cells were stained, fixed, and acquired the same day. AIM assays on splenocytes and mediastinal lymph nodes were performed on cryopreserved cells (Table S2). AIM assay on day 7 PBMCs were performed on fresh cells. Phenotype panel on LNs and PBMCs was performed using standard flow cytometry assays.
vRNA quantitation by quantitative real time polymerase chain reaction (qRT-PCR)
Trizol lysed nasal samples were processed using a modified Qiagen RNeasy Lipid Tissue Mini Kit protocol. Briefly, 5.6ul polyacryl carrier was added to trizol lysate, followed by 1/10 volume BCP and phase separated as described in Qiagen protocol. 8ul of eluted RNA was DNase treated with ezDNase per kit instructions and converted to cDNA with Superscript IV using random primers in a 20ul reaction and quantified in quadruplicate by qPCR on an Applied Biosystems QuantStudio 12K Flex Real-Time PCR System using Qiagen QuantiTect Probe PCR Mastermix with primers and probe that target the SARS-CoV-2 Nucleocapsid (forward 5’-GTTTGGTGGACCCTCAGATT-3’, reverse 5’-GGTGAACCAAGACGCAGTAT-3’, probe 5’-/5-FAM/TAACCAGAA/ZEN/TGGAGAACGCAGTGGG/3IABkFQ/-3’).
Serum cytokines. Serum cytokines. Luminex® (NHP Cytokine Luminex Performance Pre-mixed kit, R&D, FCSTM21) was performed to evaluate cytokines in rhesus macaque sera. The assay was performed according to the manufacturer’s protocol. The beads for each sample, control, and standard curve point were interrogated in a Luminex® 200 dual laser instrument (Luminex, Austin, TX) which monitors the spectral properties of the beads and amount of associated phycoerythrin (PE) fluorescence for each sample. xPONENT® software was used to calculate the median fluorescent index and calculate the concentration for each cytokine in
Flow cytometry. Cell staining was performed as previously described. Whole blood and single cell suspensions from the lung and lymph nodes were stained fresh and acquired the same day. Staining on spleen was performed on cryopreserved samples. Fluorescence was measured using a BD Biosciences FACSymphony™ with FACSDiva™ version 8.0.1 software. Compensation, gating and analysis were performed using FlowJo (Version 10). Reagents used for flow cytometry are listed in Table S2.
BAMA for IgG and IgM antibodies to S1, S2 and N proteins A customized BAMA was developed to simultaneously measure antibodies to the following recombinant SARS CoV-2 proteins (all from SinoBiologicals, Wayne, PA): S1 (#40591-V08H), S2 extracellular domain (#40590-V08B) and nucleocapsid (N; #40588-V08B). Briefly, proteins were dialyzed in PBS and conjugated to Bioplex Pro carboxylated magnetic beads (BioRad, Hercules, CA) as previously described 39. Standard and serum samples treated with 1% TritonX-100 detergent were serially diluted in PBS containing 1% BSA, 0.05% azide, and 0.05% Tween-20 and mixed with beads overnight at 1100rpm and 4˚C. The following day, the beads were washed and treated with biotinylated antibody followed by neutralite avidin-phycoerythrin (Southern Biotechnology Associates: SBA, Birmingham, AL) as described (Phillips 2017). A BioRad Bioplex 200 and BioManager software were used to measure fluorescent intensity and construct standard curves for interpolation of antibody concentrations in test samples.
The standard was pooled serum from macaques infected for 11-14 days with SARS CoV-2. The following humanized (IgG1) monoclonal antibodies were used to estimate concentrations of IgG, IgM, and IgA antibodies in the rhesus serum standard: anti-S1 RBD (Genscript #HC2001), anti-S2 (SinoBiologicals #40590-D001) and anti-NC (Genscript #HC2003). Human and rhesus IgG antibodies were both detected in these calibration assays using biotinylated affinity-purified goat anti-human IgG γ chain polyclonal antibody (SBA #2048-08). In subsequent BAMA assays for SARS CoV-2-specific rhesus macaque IgG antibodies, biotinylated mouse anti-monkey IgG γ chain monoclonal antibody (SBA cat#4700-08) was used as the secondary antibody. IgM antibodies were detected using biotinylated affinity-purified goat anti-human IgM µ chain polyclonal antibody (SBA#2020-08) which cross-reacts well with macaque IgM. Results obtained for IgM in the rhesus standard were multiplied by 3.3 to account for under-estimation by the monomeric IgG monoclonal antibody standard. Macaque IgA antibodies were detected with the antibodies described below. IgG, IgM or IgA antibodies had to be increased 3-fold over the day 0 value to be considered significant
ELISA for SARS-specific IgA and antibodies to RBD
These assays were done using methods similar to those described 41 and Immulon 4 microtiter plates (VWR, Radnor, PA) coated with 100ng per well of S1, S2, N or RBD protein (SinoBiological #40592-VNAH). For IgA assays, a pooled rhesus serum collected day 14 after infection with SARS CoV-2 was used as standard after depletion of IgG using GE Healthcare Protein G Sepharose (Sigma, St. Louis, MO) as described 41. Test sera were also depleted of IgG to facilitate detection of low levels of IgA antibodies. Macaque IgA was detected using a mixture of biotinylated clone 10F12 (NHP Reagent Resource) and biotinylated clone 40.15.5 (Ward et al 1995) anti-rhesus IgA monoclonal antibodies, which do not cross-react with human IgA and, when combined, appear to recognize all allotypes of rhesus macaque IgA (Kozlowski, personal observation). For RBD IgG assays, the above pooled rhesus serum standard and secondary monoclonal antibody specific for macaque IgG were used.
Neutralizing assay Pseudovirus neutralization assay was performed as described 42.
Statistics Statistical analyses were performed using GraphPad Prism 8.4.2. Within group comparisons, such as immune responses and antibody levels at different time points, were done using the two-tailed Wilcoxon matched-pairs signed rank test. For correlation analysis, the two-tailed Spearman rank correlation test was used.
Materials Availability. The study generated SARS-CoV-2/human/USA/CA-CZB-59X002/2020 and is available upon request from CJM (Genbank accession number: MT394528).