Ethics Statement. All animal procedures reported herein were conducted under IACUC protocols approved by WRAIR/NMRC (#19-IDD-24) and BIOQUAL (#20-019P) in compliance with the Animal Welfare Act and in accordance with the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 2011. The studies were carried out in compliance with the ARRIVE guidelines60.
Humanized DRAGA Mice. DRAGA mice express the HLA-A2.1 and HLA-DR0401 transgenes on a Rag1KO.IL2RγcKO.NOD (NRG) background, and they have been described previously28-31 De-identified umbilical cord bloods positive for HLA-A2.1.HLA-DR0401 were commercially procured through the New York Blood Center (Long Island City, NY, USA, https://nybloodcenter.org/products-and-services/blood-products/research-products/). Mice were irradiated (350 rads) and injected intravenously with CD3+ T-cell-depleted cord blood cells (EasySep Human CD3 Positive Selection Kit, Stem Cell Technologies, Vancouver, BC, Canada) containing approximately 105 human CD34+ hematopoietic stem cells (HSC) determined by FACS using a mouse anti-human CD34 antibody (BD Biosciences, San Jose, CA, USA) as described49,52,54. The procedures for assessing human T and B cell reconstitution in peripheral blood by FACS have been described49,52,54. As documented in our previous studies, >90% of HIS-reconstituted DRAGA mice generated using these procedures reconstitute a human immune system by 3 to 4 months post-CD34+ HSC infusion. The human reconstitution status of DRAGA mice at the time of our SARS-CoV-2 infection experiments was determined based on FACS measurement of T cells and B cells in peripheral blood (Table S1). The DRAGA mice were transferred to BIOQUAL Inc for SARS-CoV-2 challenge experiments in their BSL-3 facility.
RT-PCR detection of hACE2 mRNA in HIS-DRAGA mouse lungs. RNA was extracted using a Qiagen RNA extraction kit (Qiagen, Hilden, Germany) from lungs of HIS-DRAGA and control (non-HSC-infused DRAGA) mice. Human lung mRNA (Sigma-Aldrich, St. Louis, MO, USA) served as a positive control. PCR primers specific for hACE2 were: forward, CAGGAAATGTTCAGAAAGCA and reverse, TCTTAGCAGAAAAGGTTGTG. The murine ACE2 specific primers were: forward: AGCAGATGGCCGGAAAGTTG, and reverse: TCTTAGCAGGAAAGGTTGCC. RT-PCR was performed using a One-step RT-PCR kit (Qiagen) for 45 cycles using 1 µg RNA template and 1.6 µM of each primer, following the manufacturer’s instructions. The PCR amplicons were run on a 3% agarose gel. PCR bands were purified from the agarose gels and nucleotide sequenced (Eurofins, Coralville, Iowa, USA).
RT-qPCR measurement of viral RNA copies in SARS-CoV-2 infected HIS-DRAGA mouse lungs. RNA from lungs of mice #M2-M4 and #F11-F13 was extracted using RNA-STAT 60 extraction reagent (Tel-Test, Inc., Friendswood, TX, USA) + chloroform, precipitated and resuspended in RNAse-free water. Control RNA was isolated from SARS-CoV-2 viral stocks following the same procedure and quantified by OD260. These control stocks were serially diluted and OD260 values measured to generate a standard curve. RT-qPCR of the lung RNA was carried out using the following primers : 2019-nCoV_N1-F :5’-GAC CCC AAA ATC AGC GAA AT-3’; 2019-nCoV_N1-R: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’; and probe 2019-nCoV_N1-P: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’ (Integrated DNA Technologies, Coralville, IA, USA) which were designed to bind to and amplify a conserved region of SARS-CoV-2 Nucleocapsid (N) RNA. Amplification was performed with an Applied Biosystems 7500 Sequence detector using the following program: 48oC for 30 minutes, 95oC for 10 minutes followed by 40 cycles of 95oC for 15 seconds, and 1 minute at 55oC. Reactions were carried out using a TaqMan RT-PCR kit (Meridian Bioscience, Memphis, TN, USA) in 50 µL volume containing 5 µL of template, 2 µM of each primer and 2µM of each probe. The number of viral RNA copies per mL was calculated by extrapolation from the standard curve, and values were then converted to the number of viral RNA copies per gram of lung tissue.
Extraction and quantification of hACE2 protein in HIS-DRAGA and non-HIS-reconstituted DRAGA mouse lungs and in a human lung control. Lungs from 10 non-infected HIS-DRAGA and 10 non-infected, non-HIS reconstituted DRAGA mice were homogenized in the presence of MPER mammalian protein extraction reagent (Fisher Scientific, Waltham, MA, USA) containing complete protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany) using tubes loaded with ceramic beads (MP Biologicals, Irvine, CA, USA) in a Fast-prep homogenizer (MP Biologicals). Pooled lung homogenates from each group of mice were sonicated on ice in a Fisher Ultrasonicator for 10 cycles of 10 seconds each, the cellular debris was removed by centrifugation at 5,000 rpm, and the protein in the clear supernatant was quantified using a BCA reagent (Thermo Fisher Scientific, Waltham, MA, USA). 9 mg of total lung protein extract from each group of mice and 2 mg of human lung total protein lysate (Zyagen, San Diego, CA, USA) were then individually incubated with gentle shaking (300 strokes per min) in an Eppendorf thermomixer for 1 h at 370C with 10 µg of the S1(RBD)-mFcγ2a protein (ACRO Biosystems, Newark, DE, USA) followed by incubation with gentle shaking for 1 h at 370C with 50 µl of rat anti-mouse IgG2a-magnetic microbeads (Miltenyi Biotech, Berdisch Gladback, Germany). The total lysate from each sample was next passed over MACS magnetic columns (Miltenyi Biotec), and the hACE2/S1(RBD)-mFcγ2a/rat anti-mouse IgG2a-magnetic beads were eluted according to the manufacturer’s instructions and concentrated to 52 µl each. The amounts of hACE2 protein in these immunoprecipitates were then quantified using the highly sensitive human ACE2 ELISA kit PicoKineTM (Boster Biological Technology, Pleasanton, CA) per the manufacturer’s protocol. The provided recombinant hACE2 protein was serially diluted in the provided sample buffer. The human lung sample was diluted 1:100 and the DRAGA and HIS-DRAGA samples were diluted 1:50 each in the provided sample buffer. OD450nm values were then read for duplicate samples (100 µl each) using a BioTEK microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). A hACE2 standard curve was constructed by applying a four-parameter logistic fit formula using the BioTEK Gen 5 software (BioTek Instruments, Inc.). The sample OD450nm mean values were then converted to hACE2 concentrations using this curve, per the manufacturer’s instructions.
Western blot analysis of hACE2 protein in HIS-DRAGA lung lysates.
Western blots were next run using aliquots from the same concentrated immunoprecipitates used for the ELISA assays above: immunoprecipitates obtained from (1) 2 mg human lung extract; (2) 9 mg HIS-DRAGA mice lungs lysate; (3) 9 mg DRAGA mice lungs lysate. One µl of each immunoprecipitate added to wells of a 4-12% Bis-Tris gradient pre-cast gel from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA) and the samples were electrophoresed under denaturing conditions and then electro-transferred onto a PVDF membrane. The membrane was blocked overnight at 40C with shaking using 5% non-fat milk plus 3% BSA in PBS, incubated with a mouse monoclonal anti-human ACE2 (Abcam, Cambridge, MA, USA, 1:750) for 2h at room temperature, and washed with PBS + 0.01% Tween 20. The membrane was then incubated with goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA, 1:3000) and SuperSignalTM West Pico PLUS chemiluminescent substrate according to the manufacturer’s instructions (Thermo Fisher Scientific). The chemiluminescent bands were then imaged using a Fluorchem E Imaging System (ProteinSimple, San Jose, CA, USA).
Infection of mice with SARS CoV-2 virus. HIS-DRAGA mice were infected intranasally (i.n.) with SARS CoV-2 strain USA-WA1/2020 (BEI Resources NR‐52281, batch #70033175), which was provided to Bioqual, Inc. by the Centers for Diseases Control and Prevention (CDC). This virus strain was originally isolated from an oropharyngeal swab of a patient with a respiratory illness who had returned to Washington State, USA, from travel to China and developed COVID-19 in January 2020. Infection of HIS-DRAGA mice, harvesting of serum and organs, and all experiments requiring BSL-3 conditions were conducted in a BSL-3 laboratory at Bioqual, Inc. (Rockville, MD, USA) in compliance with local, state, and federal regulations, under IACUC protocol #20-019P. The SARS-COV-2 stock was expanded at Bioqual in Vero E6 cells, and the challenging virus was collected at day 5 of culture when the infection reached 90% cytopathic effect. The full viral genome sequence showed 100% identity with the parent virus sequence listed in GenBank (MN985325.1). A plaque forming assay carried out with confluent layers of Vero E6 cells was used to determine the concentration of live virions, reported as plaque-forming units (pfu). HIS-DRAGA mice were infected i.n. with three different doses (2.8x103, or 2.8x104, or 1x103 pfu) of the same SARS-COV-2 virus strain (NR‐52281, batch #70033175) as summarized in Table S1.
Measurement of antibody serum titers to SARS-CoV-2 viral proteins. Titers of human IgM and IgG serum antibodies (1/20 serum dilution) to the recombinant S1(RBD) viral protein from mice infected i.n. with SARS-CoV-2 virions (103 pfu/mouse) were measured prior to infection and at 24 dpi using ELISA kits according to the manufacturer’s instructions (Bethyl Laboratories). In addition, titers of human IgM and IgG serum antibodies in aliquots of these same serum samples against a recombinant His-tagged S trimeric protein and against a recombinant His-tagged N protein (both from ACRO Biosystems) were determined using an in-house ELISA. Briefly, His-S trimeric protein or His-N protein, respectively, were coated on high-binding ELISA plates (Corning Costar, Fisher Scientific, Waltham, MA, USA) at 0.2 µg/well/100µL in carbonate buffer, pH 9.0. The plates were then incubated overnight at 4OC, then blocked with PBS + 1% BSA for 2h at room temperature, washed with PBS + 0.05% Tween 20, and incubated at room temperature for 1h with the serum samples diluted in PBS + 1% BSA + 0.05% Tween 20. Bound human IgM and IgG antibodies against the His-S trimer protein were then revealed by adding anti-human IgM or IgG antibody-HRP conjugates (Bethyl Laboratories) to the His-S-trimer-coated plates. Due to limited serum volumes, total IgG + IgM antibodies against the His-N protein were revealed by adding anti-human kappa plus lambda antibody-HRP conjugates (Bethyl Laboratories) to the His-N protein coated plates. The ELISA plates were then incubated with soluble HRP substrate for 15 minutes, and reactions were stopped by adding H2SO4 (0.18M, 100 µL/well). Plates were read in an ELISA reader at 450nm and 570 nm. OD450nm values were corrected by subtracting the OD570nm values (ranging from 0.045–0.067) of serum samples from the same mice prior to infection. Standard deviations (+/-SD) for each serum sample in duplicate wells were determined at 99% interval of confidence by SigmaPlot v.14 software. The positive control (anti-S1(RBD)) antibody from the kit was used as a positive control for both S1(RBD) and S1-trimer binding ELISA assays. The antibody titer against the N protein in serum from a non-infected mouse served as a negative control for the N-binding ELISA assays.
Histopathology of lungs from infected HIS-DRAGA mice. Lungs harvested from infected mice at the experimental end-points (14 dpi or 25 dpi) were fixed in 10% formalin, embedded in a paraffin block, and 5 µm sections were stained with Hematoxylin/Eosin (H&E) or Masson’s Trichrome by Histoserv, Inc. (Germantown, MD, USA). Microscopic images were captured using an Olympus BX43 microscope (Shinjuku-ku, Tokyo, Japan).
Immunofluorescence microscopy. Tissue sections (5µm) from paraffin-embedded cassettes or frozen OCT cassettes from infected and non-infected HIS-DRAGA mice, and from non-infected, non-HIS reconstituted DRAGA mice were prepared by Histoserv, Inc. Thawed OCT-frozen tissue slides were rehydrated with PBS, and paraffin-embedded sections were de-paraffinized with xylene and rehydrated with graded concentrations of ethanol. Slides were then fixed, permeabilized with fixation/permeabilization buffer (Invitrogen, Waltham, MA, USA), blocked with 3% BSA in PBS for 30 min at 370C, and stained with fluorochrome-conjugated antibodies in PBS containing 0.01% Evans Blue at 370C for 50 min. To visualize hACE2, slides were probed with the S1(RBD)-mFcγ2a protein (10 µg/ml), washed with PBS, and then incubated with a goat anti-mouse IgG-FITC conjugate (Southern Biotech, Birmingham, AL, USA). Other antibodies to detect antigens of interest were: anti-human CD3-FITC, anti-human CD4-PE, anti-human CD8-PE, anti-human CD45-FITC, anti-human granzyme-B-PE, anti-human CD103-FITC (all from BD Biosciences, San Jose, CA, USA), anti-human CD326-PE (Miltenyi Biotech), anti-human Perforin-PE (Biolegend, San Diego, CA, USA), anti-human CD103-FITC (BD PharMingen, Irvine, CA, USA), anti-mouse CD61-PE (Invitrogen), and anti-hACE2 antibody (clone# MM0073—11A3, Abcam), anti-human TMPRSS2 monoclonal IgG1 mouse antibody (#clone P5H9-A3, Sigma Aldrich), mouse IgG1/κ-Alexa Fluor 594 anti-human CD31 Antibody (clone WM59, Biolegend), goat F(ab')2 anti-mouse IgG1-PE conjugate (Southern Biotech), and goat F(ab')2 IgG anti-mouse IgG2a (Southern Biotech). After staining, the slides were washed 3X with PBS, air-dried, and mounted with 10 µl of Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA), and images were acquired with a Zeiss Axioscan Confocal microscope or an Olympus BX43 microscope.
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