Cells and viruses
Human embryonic kidney (HEK)-293T cells, and Vero cells were obtained from ATCC. SARS-CoV-2 (icSARS-CoV-2) virus was obtained from BEI resources and grown in Suthar’s laboratory at the Emory University. mNeonGreen SARS-CoV-2 (2019-nCoV/USA_WA1/2020) virus was produced by Pei Yong Shi’s laboratory at the University of Texas46. The infectious clone SARS-CoV-2 (icSARS-CoV-2) was propagated in VeroE6 cells (ATCC) and sequenced46. The titer of SARS-CoV-2 viruses (icSARS-CoV-2 and 2019-nCoV/USA_WA1/2020) using VeroE6 cells. VeroE6 cells and HEK-293T cells were cultured in complete DMEM medium consisting of 1× DMEM (Corning, Cellgro), 10% fetal bovine serum (FBS), 25 mM HEPES buffer (Corning Cellgro), 2 mM l-glutamine, 1 mM sodium pyruvate, 1× non-essential amino acids, and 1× antibiotics. Viral stocks were stored at −80 °C until further use.
Specific-pathogen-free (SPF) 6–8-week-old female BALB/c mice (00065 strain) were obtained from Jackson Laboratories (Wilmington, MA, USA) and housed in the animal facility at the Yerkes National Primate Research Center of Emory University, Atlanta, GA. Male Indian rhesus macaques (Macaca mulatta), 3–4.5 years old, were housed in pairs in standard non-human primate cages and provided with both standard primate feed (Purina monkey chow) fresh fruit, and enrichment daily, as well free access to water. Immunizations, blood draws, and other sample collections were performed under anesthesia with ketamine (5–10 mg/kg) or telazol (3–5 mg/kg) performed by trained research and veterinary staff.
Construction and characterization of DNA/RBD trimer plasmid
The plasmid containing amino acids residues 319–541 of SARS-CoV-2 spike protein fused to His tag was obtained from BEI resources (Cat# NR-52309). The plasmid expressing RBD monomer (DNA/RBD mono) was generated by cloning insert in pGA8 vector between NheI and AvrII restriction enzyme sites. Further, the plasmid expressing RBD trimer proteins (DNA/RBD tri) was generated by placing T4 trimerization sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL) at the C-terminus without His tag using In-Fusion cloning technology (Takara). Both plasmids contain the TPA signal sequence under the control of CMV promoter with intron A.
Flow staining for RBD trimer protein expression
HEK293 cells were transfected with 1 µg of plasmid (DNA/RBD tri) expressing trimerized from SARS-CoV-2 RBD. Cells were harvested, initially stained with live dead marker followed by cells were then fixed with Cytofix/cytoperm (BD Pharmingen), permeabilized with permwash (BD Pharmingen), and intracellularly stained with anti-SARS-CoV-2 RBD antibody (40592-T62, SinoBiological). Later, donkey anti-rabbit IgG coupled to PE (406421, BioLegend) secondary antibody was used to confirm the expression.
Protein expression and purification
Monomeric and trimeric form of RBD proteins of SARS-CoV-2 was produced by transfecting FreeStyle 293-F cells using plasmids DNA/RBD mono and DNA/RBD tri, respectively. Transfections were performed according to manufacturer’s instructions (Thermo Fisher). Briefly, FreeStyle 293-F cells were seeded at a density of 2 × 106 cells/ml in Expi293 expression medium and incubated in an orbital shaking incubator at 37 °C and 127 rpm with 8% CO2 overnight. Next day, 2.5 × 106 cells/ml were transfected using ExpiFectamineTM 293 transfection reagent (ThermoFisher, cat. no. A14524) as per manufacturer’s protocol. The cells were grown for 72 h at 37 °C,127 rpm, 8% CO2. The cells were removed by centrifugation at 2000×g for 10 minutes at 4 °C, the supernatant was collected and filtered using a 0.22 µm stericup filter (ThermoFisher, cat. no. 290-4520) and loaded onto pre-equilibrated affinity column for protein purification. The SARS-CoV-2 RBD monomer (with His tag) and RBD trimer proteins were purified using Ni-NTA resin (ThermoFisher, cat. no. 88221) and Agarose bound conA (Vector Labs, cat. no. AL-1243-5) respectively. Briefly, His-Pur Ni-NTA resin was washed with PBS by centrifugation at 2000×g for 10 min. The resin was resuspended with the supernatant and incubated for 2 h on a shaker at RT. Polypropylene column was loaded on the supernatant–resin mixture and washed with wash buffer (25 mM Imidazole, 6.7 mM NaH2PO4·H2O, and 300 mM NaCl in PBS) four times, after which the protein was eluted in elution buffer (235 mM Imidazole, 6.7 mM NaH2PO4·H2O and 300 mM NaCl in PBS). RBD trimer protein supernatants were mixed with ConA Agarose-resin overnight on rocker at 4 °C. The supernatant-resin mix was loaded onto the column and washed three times with PBS and eluted using 1 M methyl-α-d mannopyranoside (pH 7.4). Eluted proteins were dialyzed against PBS using Slide-A-lyzer Dialysis Cassette (ThermoScientific, Cat# 66030) and concentrated using either 10 kDa Amicon Centrifugal Filter Units (for RBD-mono) or 50 kDa Amicon Centrifugal Filter Units (for RBD-tri) at 2000×g at 4 °C. The concentrated protein elutes were run on a Superdex 200 Increase 10/300 GL (GE Healthcare) column on an AktaTMPure (GE Healthcare) system and collected the peak that is matching to corresponding protein. The quantity of the proteins was estimated by BCA Protein Assay Kit (Pierce) and quality by BN-PAGE (NuPAGE™, 4–12% Bis–Tris Protein Gels, ThermoScientific), SDS–PAGE, and Western blot. Image Lab 5.2 version software was used to acquire images.
HEK293-T cells were transfected with 1 µg of plasmid (DNA/RBD-monomer and DNA/RBD-trimer) expressing monomer and trimerized forms of SARS-CoV-2 RBD. Cells were harvested and lysed in ice-cold RIPA buffer and supernatants were collected. Lysates were kept on ice for 10 min, centrifuged, and resolved by SDS–PAGE using precast 4–15% SDS polyacrylamide gels (BioRad). Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat# 1610782 BioRad), and incubated for 2 h at room temperature with primary RBD antibody (Cat# 40592-T62, Sino Biologicals) diluted 1:4000 in blocking buffer. The membranes were washed in PBS containing Tween-20 (0.05%) and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Cat #4030-05, Southern Biotech). The membranes were washed, and proteins were visualized using the ECL select chemiluminescence substrate (Cat# RPN2235, GE Healthcare).
BALB/c mice of 6–8-week-old female were immunized with 1–10 μg SARS-CoV-2 RBD trimer or monomer protein, intramuscularly, at weeks 0 and 4 with either aluminum hydroxide (100 µg) or Alum- 3M-052 (100 µg alum, 1 µg 3M-052). Antigen was mixed with the adjuvant and incubated at 4 °C for 15 min on a rocker before immunization. The blood samples were collected at two weeks following each immunization by facial vein puncture in BD Microtainer® Tube for analyzing SARS-CoV-2 RBD-specific serum antibody responses.
SARS-CoV-2 (MA10) challenge
All SARS-CoV-2 (MA10) challenge experiments were carried out at the University of North Carolina (UNC) at Chapel Hill. Briefly, mice were anesthetized using ketamine/xylazine and infected intranasally with 105 PFU SARS-CoV-2 MA10 strain diluted in PBS28. Briefly, the mouse-adapted SARS-CoV-2 virus (MA10) was generated from an infectious clone of SARS-CoV-2 MA stock and further genetically engineered to introduce Q498Y/P499T substitutions into the spike protein. SARS-CoV-2 MA10 stock was generated from a P10-infected mouse lung homogenate via inoculation of Vero E6 cells. Clinical signs of disease (weight loss and body score) were monitored daily. The mice were euthanized by isoflurane overdose at indicated time points when samples for titer (caudal right lung lobe) were collected. Plaque assay was used to define Lung viral titers. Briefly, right caudal lung lobes were homogenized in 1 ml PBS using glass beads and serial dilutions of the clarified lung homogenates were added to a monolayer of Vero E6 cells. After 3 days plaques were visualized via staining with Neutral Red dye and counted.
Rhesus macaque study
A total of 16 Indian origin rhesus macaques (Macaca mulatta; male) from 4 to 6 years of age were included in this study. The animal study was conducted at Yerkes National Primate Research Center, Emory University, and was approved by the Emory IACUC. Nine macaques were randomly allocated into two vaccine groups. Group 1 (n = 5) received 30 µg RBD trimer antigen adjuvanted with 1 mg amounts of Aluminum Hydroxide. Group 2 (n = 4) received 30 µg RBD trimer antigen adjuvanted with Alum (1 mg)-3M-052 (10 µg) formulations. Macaques were immunized intramuscularly with indicated RBD trimer-adjuvanted vaccines, at week 0 and 4, respectively with a total volume of 1 ml in the thigh. The serum samples and PBMCs were collected at the indicated time points and subjected to immunological assays. The remaining 7 animals were used as controls. Of the 7, 5 animals received wild-type modified vaccinia Ankara (MVA) vaccine as reported previously47 and 2 did not receive any vaccine.
4 weeks after final immunization the macaques were challenged with a total of 5 × 104 pfu (2.5 × 104 pfu/ml) of SARS-CoV-2 (2019-nCoV/USA_WA1/2020). Virus was administered as 1 ml by IT and 1 ml by intranasal (IN) route (0.5 ml in each nostril). Nasal swabs and BAL samples were collected, stored immediately in the viral transport media, and processed for viral RNA extraction on the same day. Starting from the day of challenge, the nasal swabs and BAL fluid were collected on day 2, 4, 7, and 10 and subjected to viral load measurements. At Day 10 after SARS-CoV-2 challenge, all vaccinated and non-vaccinated macaques were euthanized. Necropsy samples were collected (lung tissues) and were subjected to Hematoxylin and Eosin staining.
Binding antibody responses using ELISA
SARS-CoV-2 S (RBD, S1, and S)-specific IgG in serum and BAL was quantified by enzyme-linked immunosorbent assay (ELISA)48. Briefly, Nunc high-binding ELISA plates were coated with 2 µg/ml of recombinant SARS-CoV-2 proteins (RBD, S1, and S) proteins in Dulbecco’s phosphate-buffered saline (DPBS) and incubated overnight at 4 °C. SARS-CoV-2 RBD and S1 proteins were produced in the lab whereas, S1 and S (S1 + S2 ECD) proteins were purchased. Plates were then blocked with 5% blotting-grade milk powder and 4% whey powder in DPBS with 0.05% Tween 20 for 2 h at room temperature (RT). Plates were then incubated with serially diluted serum samples (starting from 100, 3-fold, 8x) and incubated for 2 h at RT followed by six washes. Total SARS-CoV-2 S (RBD, S1, and S)-specific mouse IgG and monkey IgG antibodies were detected using HRP-conjugated anti-mouse (1:6000) (Southern Biotech; AL, USA) and goat anti-monkey IgG secondary antibody (1:10,000), respectively for 1 h at RT. The plates were washed and developed using TMB (2-Component Microwell Peroxidase Substrate Kit) and the reaction was stopped using 1 N phosphoric acid solution. Plates were read at 450 nm wavelength within 30 min using a plate reader (Molecular Devices, San Jose, CA, USA). ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >2-fold over background values.
Live-virus SARS-CoV-2 neutralization antibodies were assessed using a full-length mNeonGreen SARS-CoV-2 (2019-nCoV/USA_WA1/2020), generated as previously described46. Vaccinated mice, NHP, and post-challenge sera were incubated at 56 °C for 30 min and manually diluted in duplicate in serum-free Dulbecco’s modified Eagle medium (DMEM) and incubated with 750–1000 focus-forming units (FFU) of infectious clone-derived SARS-CoV-2-mNG virus46 at 37 °C for 1 h. The virus/serum mixture was added to VeroE6 cell (C1008, ATCC, #CRL-1586) monolayers, seeded in 96-well blackout plates, and incubated at 37 °C for 1 h. Post incubation, the inoculum was removed and replaced with pre-warmed complete DMEM containing 0.85% methylcellulose. Plates were incubated at 37 °C for 24 h. After 24 h, the methylcellulose overlay was removed, cells were washed three times with phosphate-buffered saline (PBS), and fixed with 2% paraformaldehyde (PFA) in PBS for 30 min at room temperature. PFA is then removed and washed twice with PBS. The foci were visualized using an ELISPOT reader (CTL ImmunoSpot S6 Universal Analyzer) under a FITC channel and enumerated using Viridot49. Viridot has only one version but, was written to work with versions up to R 3.4.1. This was mentioned in the details. The neutralization titers were calculated as follows: 1—ratio of the (mean number of foci in the presence of sera: foci at the highest dilution of respective sera sample). Each specimen is tested in two independent assays performed at different times. The focus-reduction neutralization mNeonGreen live-virus 50% titers (FRNT-mNG50) were interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3. Samples that did not neutralize at the limit of detection at 50% were plotted at 10 and were used for geometric mean calculations.
Antibody isotype, IgG subclass, and FcR binding of mouse sera
For relative quantification of antigen-specific antibody titers, a customized multiplexed approach was applied, as previously described50. Therefore, magnetic microspheres with a unique fluorescent signature (Luminex) were coupled with SARS-CoV-2 antigens including spike protein (S) (provided by Eric Fischer, Dana Farber), RBD, and CoV HKU1 RBD (provided by Aaron Schmidt, Ragon Institute), CoV-2 S1 and S2 (Sino Biologicals) as well as influenza as control (Immune Tech). Coupling was performed using EDC (Thermo Scientific) and Sulfo-NHS (Thermo Scientific) to covalently couple antigens to the beads. 1.2 × 103 beads per region/ antigen were added to a 384-well plate (Greiner) and incubated with diluted plasma samples (1:90 for all readouts) for 16 h while shaking at 900 rpm at 4 °C, to facilitate immune complex formation. The next day, immune complexed microspheres were washed three times in 0.1% BSA and 0.05% Tween-20 using an automated magnetic plate washer (Tecan). Anti-mouse IgG-, IgG2a-, IgG3-, IgA- and IgM-PE coupled (Southern Biotech) detection antibodies were diluted in Luminex assay buffer to 0.65 μg/ml. Beads and detection antibodies were incubated for 1 h at RT while shaking at 900 rpm. Following washing of stained immune complexes, a tertiary goat anti-mouse IgG-PE antibody (Southern Biotech) was added and incubated for 1 h at RT on a shaker. To assess Fc-receptor binding, mouse Fc-receptor FcγR2, FcγR3, FcγR4 (Duke Protein Production facility) were biotinylated (Thermo Scientific) and conjugated to Streptavidin-PE for 10 min (Southern Biotech) before adding to immune complexes and processed as described above. Finally, beads were washed and acquired on a flow cytometer, iQue (Intellicyt) with a robot arm (PAA). Events were gated on each bead region, median fluorescence of PE for bead positive events were reported. Samples were run in duplicate for each secondary detection agent.
Antibody isotype, IgG subclass, and FcR binding of monkey sera
A Luminex assay was used to detect and quantify antigen-specific subclass, isotype, and Fc-receptor (binding) factors50. With this assay, we measured the antibody concentration against SARS-CoV-2 RBD (kindly provided by Aaron Schmidt, Ragon Institute) and SARS-CoV-2 S (kindly provided by Erica Ollmann Saphire, La Jolla Institute). Carboxylate-modified microspheres (Luminex) were activated using EDC and Sulfo-NHS and antigens were covalently bound to the beads via NHS-ester linkages. Antigen-coupled beads were washed and blocked. Immune complexes were formed by mixing appropriately diluted plasma (1:100 for IgG1, IgG2, IgG3, IgG4, IgA, IgM, and 1:1000 for FcγRs) to antigen-coupled beads and incubating the complexes overnight at 4 °C. Immune complexes were then washed with 0.1% BSA 0.02% Tween-20. PE-coupled secondary antibodies for each antibody isotype or subclass (Southern Biotech) were used to detect antigen-specific antibody titer. For FcRs, biotinylated FcRs were labeled with streptavidin-PE before addition to immune complexes. Fluorescence was measured with an iQue (Intellicyt) and analyzed using Forecyt (v 8.1) software. Data are reported as median fluorescence intensity (MFI).
ADCP, ADNP, and ADCD assays for monkey sera
ADCP, ADNP, and ADCD were measured as previously described51,52,53. For ADCP and ADNP, yellow-green fluorescent neutravidin beads were coupled to biotinylated SARS-CoV-2 S or RBD. For ADCD, red fluorescent neutravidin beads were coupled to biotinylated SARS-CoV-2 S or RBD. Antigen-coupled beads were then incubated with appropriately diluted plasma (ADCP 1:100, ADNP 1:50, ADCD 1:10) for 2 h at 37 °C to form immune complexes. For ADCP, THP-1s (ATCC) were added at 1.25 × 105 cells/ml and incubated for 16 h at 37 °C. For ADNP, leukocytes were isolated from fresh peripheral whole blood by lysing erythrocytes using ammonium-chloride potassium lysis. Leukocytes were added to immune complexes at 2.5 × 105 cells/ml and incubated for 1 h at 37 °C. Neutrophils were detected using anti-human CD66b Pacblue (Biolegend). For ADCD, lyophilized guinea pig complement (Cedarlane) was resuspended, diluted in gelatin veronal buffer with calcium and magnesium (GVB++, Boston BioProducts), and added to immune complexes. The deposition of C3 was detected using an anti-C3 FITC antibody (Mpbio).
All functional assays were acquired with an iQue (Inellicyt) and analyzed using Forecyt software. For ADCP, events were gated on singlets and fluorescent cells. For ADNP, bead-positive neutrophils were defined as CD66b positive, fluorescent cells. For both ADCP and ADNP, a phagocytic score was defined as (percentage of bead-positive cells) × (MFI of bead-positive cells) divided by 10,000. For ADCD, data were reported as median fluorescence of C3 deposition (MFI).
For mouse studies, spleens and lungs of vaccinated and control animals were removed and placed on ice in cold RPMI 1640 (1×) with 5% FBS (Company, state, USA). 1× β-Mercaptoethanol (Invitrogen, State, USA) was added to the complete medium to isolate splenocytes. Whereas lungs were cut into small pieces and incubated at 37 °C in RPMI (1×) medium containing Collagenase type IV and DNase I with gentle shaking for 30 min. After incubation, cells were isolated by forcing tissue suspensions through a 70 µM cell strainer. RBCs were removed by ACK lysis buffer and live cells counted by trypan blue exclusion. For macaques, PBMC from blood collected in sodium citrate CPT tubes was isolated using standard procedures. Post SARS-CoV-2 challenge, samples were processed and stained in BSL-3 facility.
For BAL fluid processing and single-cell isolation, up to 50 ml physiological saline was delivered through the trachea into the lungs of anesthetized animals using a camera-enabled fiberoptic bronchoscope. The flushed saline was re-aspirated five times before pulling out the bronchoscope. This collection was filtered through 70 μm cell strainer and centrifuged at 1126×g for 5 min. Pelleted cells were suspended in 1 ml R10 medium (RPMI(1×), 10% FBS) and stained as described in the sections below.
For processing lymph-node, lymph-node biopsies were dissociated using 70 μm cell strainer. The cell suspension was washed twice with R-10 media.
Intracellular cytokine staining (ICS)
Functional responses of SARS-CoV-2 RBD, S1 and S2 specific CD8+ and CD4+ T cells in vaccinated animals were measured using peptide pools and intracellular cytokine staining (ICS) assay54. Overlapping peptides (13 or 17 mers overlapping by 10 amino acids) were obtained from BEI resources (NR-52402 for spike and NR-52419 for NC) and different pools (S1, S2, RBD, and NC) were made. The S1 pool contained peptides mixed from 1 to 97, S2 pool contained peptides mixed from 98 to 181, RBD pool contained peptides 46–76 and NC pool contained 57 peptides. Each peptide was used at 1 μg/ml concentration in the stimulation reaction. Two million cells suspended in 200 μl of RPMI 1640 medium with 10% FBS were stimulated with 1 μg/ml CD28 (BD Biosciences), 1 μg/ml CD49d (BD Biosciences) co-stimulatory antibodies, and different peptide pools. These stimulated cells were incubated at 37 °C in 5% CO2-conditioned incubator. After 2 h of incubation, 1 μl Golgi-plug and 1 μl Golgi-stop/ml (both from BD Biosciences) were added and incubated for 4 more hours. After total 6 h of incubation, cells were transferred to 4 °C overnight and were stained the next day. Cells were washed once with FACS wash (1XPBS, 2% FBS, and 0.05% sodium azide) and surface stained with Live/Dead-APC-Cy7, anti-CD3, anti-CD4, and anti-CD8, each conjugated to a different fluorochrome for 30 min at RT. The stained cells were washed once with FACS wash and permeabilized with 200 μl of cytofix/cytoperm for 30 min at 4 °C. Cells were washed once with perm wash and incubated with anti-cytokine antibodies for 30 min at 4 °C. Finally, the samples were washed once with perm wash and once with FACS wash, and fixed in 4% paraformaldehyde solution for 20 min before acquiring on BD LSR Fortessa flow cytometer (v8.0.1). Data were analyzed using FlowJo software.
For histopathologic examination in macaques, the animals were euthanized due to the study endpoint, and a complete necropsy was performed. For histopathologic examination, various tissue samples including lung, nasal turbinates, trachea, tonsils, hilar lymph nodes, spleen, heart, brain, gastrointestinal tract (stomach, jejunum, ileum, colon, and rectum), testes were fixed in 10% neutral-buffered formalin for 24 h at room temperature, routinely processed, paraffin-embedded, sectioned at 4 µm, and stained with hematoxylin and eosin (H&E). The H&E slides from all tissues were examined by two board-certified veterinary pathologists. For each animal, all the lung lobes were used for analysis, and affected microscopic fields were scored semiquantitatively as Grade 0 (None); Grade 1 (Mild); Grade 2 (Moderate); and Grade 3 (Severe). Scoring was performed based on these criteria: number of lung lobes affected, type 2 pneumocyte hyperplasia, alveolar septal thickening, fibrosis, perivascular cuffing, peribronchiolar hyperplasia, inflammatory infiltrates, hyaline membrane formation. An average lung lobe score was calculated by combining scores from each criterion. For animals with multiple affected lung lobes, each lung lobe was assessed individually and then the scores for each category were averaged. The total score was then determined for each animal. Digital images of H&E stained slides were captured at ×100 and ×200 magnification with an Olympus BX43 microscope equipped with a digital camera (DP27, Olympus) using Cellsens® Standard 2.3 digital imaging software (Olympus).
Immunophenotyping of BAL and LN cells
Briefly, the cells were stained with a surface antibody cocktail and incubated at RT for 30 min. The stained cells were given a FACS wash and permeabilized with 1 ml perm buffer (Invitrogen) for 30 min at RT. These cells were given a perm wash (Invitrogen) and stained with an intracellular antibody cocktail for 30 min at RT. Finally, the cells were washed once with perm wash and a FACS wash and fixed in 4% paraformaldehyde solution for 20 min before acquiring on BD LSR-II flow cytometer (v8.0.1). Samples prior to the challenge were acquired without 20-min 4% paraformaldehyde fixation.
BAL innate cell surface antibody cocktail: live/dead stain-APC-Cy7, anti-CD3-BV605, anti-CD20-BV605, anti-NKG2A-APC, anti-HLADR-PERCP, anti-CD11b-PE/Dazzle 594, anti-163-eFlour-450, anti-CD123-PE-Cy7, anti-CD11c-BV655, and anti-BDCA1-BV711. BAL innate cell intracellular antibody: anti-Ki67-BV786. T-cell phenotype surface antibody cocktail: live/dead stain-APC-Cy7, anti-CD3-PerCP, anti-CD4-BV655, anti-CD8-BV711, anti-PD1-BV421, anti-CXCR5-PE, and anti-CXCR3-BV605. T-cell phenotype intracellular antibody: anti-Ki67-BV786. B-Cell phenotype surface antibody cocktail: live/dead stain-APC-Cy7, anti-CD3-AF700, and anti-CD20-BV605, B-cell phenotype intracellular antibody: anti-BCL6-PE-CF594 and anti-Ki67-PE-Cy7.
Viral RNA extraction and quantification
SARS-CoV-2 genomic and subgenomic RNA was quantified in naso-pharyngeal (NP) swabs, and brocho-alveolar lavages (BAL). Swabs were placed in 1 ml of Viral Transport Medium (VTM; Labscoop (VR2019-1L)). Viral RNA was extracted from NP swabs, throat swabs, and BAL on fresh specimens using the QiaAmp Viral RNA mini kit according to the manufacturer’s protocol. Quantitative PCR (qPCR) was performed on subgenomic using primer and probe align the subgenomic mRNA transcript of the E gene55 are SGMRNA-E-F: 5′-CGATCTCTTGTAGATCTGTTCTC-3′, SGMRNA-E-R: 5′-ATATTGCAGCAGTACGCACACA-3′, and SGMRNA-E-Pr: 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-3′ (Table S2). qPCR reactions were performed in duplicate with the Thermo-Fisher 1-Step Fast virus master mix using the manufacturer’s cycling conditions, 200 nM of each primer, and 125 nM of the probe. The limit of detection in this assay was about 128 copies per ml of VTM/BAL depending on the volume of extracted RNA available for each assay. To verify sample quality the CDC RNase P p30 subunit qPCR was modified to account for rhesus macaque specific polymorphisms. The primer and probe sequences are RM-RPP30-F 5′-AGACTTGGACGTGCGAGCG-3′, RM-RPP30-R 5′- GAGCCGCTGTCTCCACAAGT-3′, and RPP30-Pr 5′-FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3′56. A single well from each extraction was run as described above to verify RNA integrity and sample quality via detectable and consistent cycle threshold values (Ct between 25 and 32).
Quantification and statistical analysis
GraphPad Prism version 8.4.3 (471) (GraphPad Software) was used to perform data analysis and statistics. The difference between any two groups at a time point was measured either using a two-tailed nonparametric Mann–Whitney rank-sum test or unpaired parametric t-test depending on the distribution of the data. Comparisons between different time points within a group used paired parametric t-test. P-value of <0.05 was considered significant. The correlation analysis was performed using the Spearman rank test.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.