Cells and Viruses
Vero E6 cell line (Cat # CRL-1586) was purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin and L-glutamine. Calu-3 cell line (Cat # HTB-55) was obtained from ATCC and maintained in EMEM + 20%FBS. H1299-hACE2 is a human lung carcinoma cell line stably expressing human ACE2. It was made by S. Liu in our group from the NCI-1299 human lung carcinoma cell line (ATCC CRL-5803). H1299-hACE2 cells were maintained in DMEM supplemented with 5% penicillin and streptomycin, and 10% fetal bovine serum (FBS) at 37°C with 5% CO2.
Production of Nsp1-K164A/H165A was described elsewhere23. The SARS-CoV-2 isolate WA1/2020 (NR-52281, lot 70033175) was obtained from BEI Resources, NIAID, NIH, and had been passed three times on Vero cells and 1 time on Vero E6 cells prior to acquisition. It was further passed once on Vero E6 cells in our lab. SARS-CoV-2 hCoV-19/USA/MD-HP05647/2021 (Delta variant, Pango lineage B.1.617.2) was obtained from BEI resources, NIAID, NIH (NR-55672, Lot 70046635) and had been passaged once in Vero E6-TMPRSS2 and once in Calu-3 cells prior to acquisition. It was passaged once more in H1299-hACE2 cells in our lab to generate viral stocks. Passaged viruses were deep sequenced to confirm identity. SARS-CoV-2 isolates hCoV-19/USA/HI-CDC-4359259-001/2021 (B.1.1.529 Omicron, NR-56475), hCoV-19/USA/NY-MSHSPSP-PV56475/2022 (BA.2.12.1 Omicron, NR-56782), USA/MD-HP30386/2022 (BA.4, Omicron, NR-56802), and hCoV-19/USA/COR-22-063113/2022 (BA.5 Omicron, NR-58620) were obtained from BEI resources and used directly in experiments.
Hamster Challenge Experiments
Adult male and female outbred Syrian hamsters were previously purchased from Envigo and held at FDA vivarium. All experiments were performed within the biosafety level 3 (BSL-3) suite on the White Oak campus of the U.S. Food and Drug Administration. The animals were implanted subcutaneously with IPTT-300 transponders (BMDS), randomized, and housed 2 per cage in sealed, individually ventilated rat cages (Allentown). Hamsters were fed irradiated 5P76 (Lab Diet) ad lib, housed on autoclaved aspen chip bedding with reverse osmosis-treated water provided in bottles, and all animals were acclimatized at the BSL3 facility for 4–6 days or more prior to the experiments. The study protocol details were approved by the White Oak Consolidated Animal Care and Use Committee and carried out in accordance with the PHS Policy on Humane Care & Use of Laboratory Animals.
Adult male (5–6 months old) Syrian hamsters (Mesocricetus auratus) were anesthetized with (3–4% v/v) isoflurane and oxygen following procedures as described previously 35,41,42. Intranasal inoculation was done by pipetting 102 PFU or 104 PFU SARS-CoV-2 in 50 µl volume dropwise into the nostrils of the hamster under anesthesia. Following infection, hamsters were monitored daily for clinical signs and weight loss. Nasal washes were collected by pipetting ~ 200 µl sterile phosphate buffered saline into one nostril when hamsters were anesthetized by 3–5% isoflurane. Nasal swabs were done as described previously 32.
For airborne transmission, a subset (n = 7) of hamsters inoculated with 102 PFU WA1/2020 or Nsp1-K164A/H165A were paired in divided cages to prevent direct contact to measure transmission to naive sentinels 27. One hamster (WH363), paired with an actively shedding Nsp1-K164A/H165A vaccinated animal, did not show evidence of productive infection or seroconvert at 14 DPE and remained seronegative until just prior to BA.2.12.1 challenge 4.5 months later. For these reasons, WH363 was removed from the challenge datasets.
For tissue collection, a subset of hamsters was humanely euthanized by intraperitoneal injection of pentobarbital at 200mg/kg at 4 and 7 DPC. Lungs, trachea, and nasal turbinates were dissected for histopathology or homogenized for RNA extraction or titration in cell culture. Blood collection was performed under anesthesia (3–5% isoflurane) through gingival vein puncture or cardiac puncture when animals were euthanized. The left lobes of hamster lungs (~ 0.2 gram) were diced, divided, and resuspended in 1 milliliter MEM or TriZol reagent (RNA extraction) and homogenized on a Precellys Evolution tissue homogenizer with a Cooling Unit (Bertin). Trachea and nasal turbinates were homogenized the same way in TriZol Reagent. Splenocytes were extracted at 14 DPI from vaccinated and naive hamsters and IFNγ-secreting cells were identified after stimulation with spike and nucleocapsid antigen pools (Genscript) by ELISpot (MABTECH, 3102-2H).
RNA isolation and qRT-PCR
Procedures as described previously35,41. In brief, RNA was extracted from 0.1-gram tissue homogenates using QIAamp vRNA mini kit or the RNeasy 96 kit (QIAGEN) and eluted with 60 µl of water. 5 µL RNA was used for each reaction in real-time RT-PCR. When graphing the results in Prism 9, values below the limit of quantification (LoD) were arbitrarily set to half of the LoD values. Unless otherwise specified, the unit for RNA copies are as presented as Log10 RNA copes/µg tissue RNA.
Histopathology Analyses
Procedures as described previously35,41. Tissues (lungs, trachea, and nasal turbinates) were fixed in 10% neutral buffered formalin overnight and then processed for paraffin embedding. The 5-µm sections were stained with hematoxylin and eosin for histopathological examinations. Images were scanned using an Aperio ImageScope. Blinded samples were graded by a licensed pathologist for the following twelve categories: consolidation, alveolar wall thickening, alveolar airway infiltrates, perivascular infiltrates, perivascular edema, type II pneumocyte hyperplasia, atypical pneumocyte hyperplasia, bronchiole mucosal hyperplasia, bronchiole airway infiltrates, proteinaceous fluid, hemorrhage, and vasculitis. Grading: 0 = none, 1 = mild, 2 = moderate, 3 = severe. A graph was prepared by summing up the score in each category.
Virus titration
Tissue culture infectious dose 50% (TCID50) assays were done described previously 35,41 for initial nasal wash titrations post-inoculation. In brief, Vero E6 cells were plated the day before infection into 96 well plates at 1.5 × 104 cells/well. On the day of the experiment, serial dilutions of 20 µl nasal wash samples were made in media and a total of six to eight wells were infected with each serial dilution of the virus. After 48 h incubation, cells were fixed in 4% PFA followed by staining with 0.1% crystal violet. The TCID50 was then calculated using the formula: log (TCID50) = log(do) + log (R) (f + 1). Where do represents the dilution giving a positive well, f is a number derived from the number of positive wells calculated by a moving average, and R is the dilution factor.
For focus-forming assay, nasal wash, BALF, and lung homogenate samples were 10-fold serially diluted in 96-well plates and dilutions added to 96-well black-well plates for fluorescent focus forming assays in H1299-hACE2 cells43. After 1 h the Tragacanth gum overlay (final concentration 0.3%) was added. Cells were incubated at 37°C and 5% CO2 for 1 day, then fixed with 4% paraformaldehyde, followed by staining of cells with primary rabbit anti-N Wuhan-1 antibody (Genscript) overnight followed by secondary anti-rabbit Alexa-488 conjugated antibody and DAPI staining. The infectious titers were then counted using Gen5 software on a Cytation7 machine and calculated and plotted as focus forming units per milliliter (FFU/ml).
SARS-CoV-2 neutralization assay
Samples were serially diluted 2-fold in 5% FBS DMEM and mixed with 100 PFU of SARS-CoV-2 in a 96-well plate at 37°C for 1 hour. Sample:virus mixtures were then added to confluent H1299-hACE2 cells in 96-well plates. Cells were infected for 1 hour before the inoculum was removed and washed three times with DPBS. A second overlay containing 1.2% Tragacanth gum, 2X MEM, 5% FBS, and DMEM was added to the plate. Cells were incubated at 37°C for 1 day, then fixed with 4% paraformaldehyde, followed by staining of cells with primary rabbit anti-SARS-CoV-2 N antibody (Genscript U739BGB150-5) overnight followed by secondary anti-rabbit Alexa-488 conjugated antibody and 4′,6-diamidino-2-phenylindole (DAPI) staining. Plates were imaged on a Cytation7 (Agilent), and foci were counted using Gen5 software. For the neutralization assays, recombinant LY-CoV555 (Bamlanivimab) mixed with WA1/2020 44 was included as a positive control. The 50% endpoint neutralization titers were determined as the reciprocal of the highest dilution providing ≤ half of the number of foci obtained from the negative control well (plain DMEM mixed with 100 PFU virus).
Measurement of antibody by ELISA
The preparation of SARS-CoV-2 RBD antigen in a baculovirus expression system and its use in ELISA were previously described45. ELISAs were performed with slight modifications. Briefly, Immulon 2HB plates were coated with recombinant RBD protein at 1 µg/mL overnight at 4°C. Test serum samples were pre-diluted in assay diluent (PBS containing 0.05% Tween-20 [PBST] and 10% fetal bovine serum), followed by serial two-fold dilutions of each sample in duplicates across the plate. A starting dilution of 1:160, 1:80, and 1:20 was used for serum (IgA and IgG), BALF (IgG) and nasal wash and BALF (IgA) samples, respectively. Plates were incubated with the test serum samples for 2 h at 37°C. After rigorous plate washes in a microplate washer, plates were incubated with anti-hamster antibodies. For IgG ELISA, a 1:4000 dilution of an HRP-conjugated goat anti-hamster IgG (6060-05, Southern Biotech, Birmingham, Alabama) was added to assay wells. For IgA ELISA, a rabbit anti-hamster IgA antibody [sandwich antibody; (cat. #sab 3001a) Brookwood Biomedical, Jemison, Alabama] was added to assay wells at 1:4000 dilution and plates incubated for 1 hour at 37°C. Unbound sandwich antibody was washed off and a 1:4000 dilution of an HRP-conjugated goat anti-rabbit IgG (4030-05, Southern Biotech, Birmingham, Alabama) was added to assay plates. In both IgG and IgA ELISAs, incubation with HRP-conjugated secondary antibodies lasted 1 hour after which plates were rigorously washed to remove unbound antibodies. The ABTS/H2O2 peroxidase substrate (SeraCare, Gaithersburg, Maryland) was added to assay wells and plates left at room temperature for 20 to 30 minutes. Color development was stopped by adding 1% SDS and OD405 values were captured on the VersaMax microplate reader with Softmax Pro 7 software (Molecular Devices). In the IgG ELISA, the mean OD405 values of PBS treatment groups were subtracted from the mean OD405 values from other treatment groups and the assay endpoint was a mean OD405 value 0.05 (i.e., after background subtraction). In the IgA ELISA, the assay endpoint was a mean OD405 value 0.02 of duplicate wells. Antibody titer was defined as the reciprocal of the highest dilution of a sample at which the mean OD405 value for duplicate wells was 0.02 (IgA) or 0.05 after background subtraction (IgG).
IFN-gamma ELISpot
Hamster interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) analysis was performed using the Hamster IFN-γ ELISpotBASIC (MABTECH Mabtech 3102-2H, Nacka Strand, Sweden) kit according to the manufacturer’s instructions. MSIP Plates (Millipore) were washed 5 times with sterile water, coated with mAb (MTH21) and incubated overnight at 4°C. Coated plates were washed 5 times with 1X PBS, blocked for 30 minutes (room temperatures) with supplemented RPMI 1640 (GibcoBRL) containing 10% heat inactivated FBS, 1% 100x penicillin, streptomycin, and L-Glutamine solution (GibcoBRL). 2.5x105 freshly isolated splenocytes were seeded in each well and stimulated for 45–48 hours at 37°C with SARS CoV-2 Spike proteins peptide pools (2 µg/ml each peptide) (BEI Catalog No. NR-52418) or Nucleocapsid proteins peptide pools (2 µg/ml each peptide) (BEI Catalog No. NR-52419) prepared in serum free RPMI 1640. Negative and positive plate controls were medium or 2µg/ml concanavalin A (ConA, Sigma-Aldrich), respectively. Plates were incubated with 1µg/ml mAb (MTH29-biotin) for 2 hours, and then 1 hour with Streptavidin-HRP, and finally developed after adding TMB substrate (product No. 3651-10). Distinct spots typically emerge within 20 minutes. After drying, spots were counted using a BioTek Cytation 7 imaging reader (Agilent) and analysis software Gen5 Version No. 3.11. ELISpot data was analyzed in Microsoft excel. The average number of spots from two negative wells (unstimulated cells) was subtracted from peptide pools stimulated wells for each plate. Results were expressed as difference in spots forming cells (SFC)/106 PBMC between negative control and peptide pools stimulations conditions. Results were plotted using GraphPad Prism 9.
Lung immunofluorescence analyses
Formalin-fixed paraffin-embedded (FFPE) lung sections 4 µm thick were dewaxed, rehydrated, and heat-treated in a microwave oven for 15 minutes in 10 mM Tris/1 mM EDTA buffer (pH 9.0). After cooling for 30 min at room temperature, heat-retrieved sections were blocked in PBST with 2.5% bovine serum albumin (BSA) for 30 min at RT followed by overnight incubation at 4°C with primary antibodies in 1% BSA. Primary antibodies used included SARS nucleocapsid protein (NP) (1:800, Sino Biologicals, 40143-MM05), MX1 (Proteintech, 13750-1-AP), prosurfactant protein C (ProSPC) (1:200, EMD Millipore, AB3786), Iba1 (1:100, Abcam, ab5076), RAGE (1:400, Abcam, ab216329), and E-cadherin (ECAD) (Abcam, ab219332). Sections were rinsed and incubated with Alexa Fluor 488 (A-21206) and Alexa Fluor 647-conjugated secondary antibodies (A-31571, A-21447) for 1 hour at RT (ThermoFisher, Waltham, MA). Nuclei were counterstained with Hoechst 33342. For double labeling experiments, primary antibodies were mixed and incubated overnight at 4°C. For negative controls, sections were incubated without the primary antibody or mouse and rabbit isotype antibody controls. Sections stained with conjugated secondary antibodies alone showed no specific staining. Whole slide fluorescence imaging was performed using a Hamamatsu NanoZoomer 2.0-RS whole-slide digital scanner equipped with a 20x objective and a fluorescence module #L11600. Analysis software NDP.view2 was used for image processing (Hamamatsu Photonics, Japan). Immunofluorescence and differential interference images were also captured using an Axio Observer Z1 inverted microscope (Carl Zeiss, Thornwood, NY) equipped with an Axiocam 506 monochrome camera, an ApoTome.2 optical sectioning system, and a Plan-Apochromat 63x/1.4NA oil immersion with WD = 0.19 and Plan-Apochromat 20x/0.8 objective lens. Digital image post-processing and analysis were performed using the ZEN 2 ver. 2.0 imaging software. Images were constructed from Z-stack slices collected at 0.48 µm intervals (5 µm thickness in total) and visualized as maximum intensity projections in orthogonal mode. For semiquantitative analysis of NP staining, high resolution whole-slide digital images of each lung section were acquired and the NDP.view2 software was used to measure the NP-stained area as a percentage of total area of the section. For TUNEL staining, sections were deparaffinized, hydrated, and pretreated with Proteinase K, followed by EDTA, distilled H₂O wash, and BSA blocking. Sections were then incubated in a reaction mixture (TdT, dUTP, and buffer), washed, and incubated with anti-digoxigenin antibody. Sections were then visualized with alkaline phosphatase-ImmPACT Vector Red and counterstained with hematoxylin.
Statistical analysis
One-way ANOVA or Student t-test was used to calculate statistical significance through GraphPad Prism (9.1.2) software for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.
DATA and MATERIALS AVAILABILITY
All data are available in the main text or the supplementary materials. All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.