Heterozygous K18-hACE2 mice [strain: JAX 034,860 B6.Cg-Tg(K18-hACE2)2Prlmn/J] were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Seven-week-old male K18-hACE2 mice were administrated 2.5 × 104 TCID50/mL SARS-CoV-2 via the intranasal route. The K18-hACE2 mice were monitored for changes in weight loss, lethality, and clinical symptoms every day after inoculation. At 6 dpi, SARS-CoV-2–infected K18-hACE2 mice were sacrificed by isoflurane and autopsied to assess the clinical lesions in several organs such as the brain, heart, lungs, spleen, and kidneys. The animal experiments were approved by the Institutional Animal Committee of the Jeonbuk National University (JBNU 2020-11-001) and performed in accordance with the guidelines of the Institutional Biosafety Committee. The study was conducted in compliance with the ARRIVE guidelines.
B6 cg tg k18 hace2 2prlmn j
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B6.Cg-Tg(K18-hACE2)2Prlmn/J is a genetically modified mouse strain. The strain expresses the human angiotensin-converting enzyme 2 (hACE2) gene under the control of the keratin 18 (K18) promoter.
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6 protocols using b6 cg tg k18 hace2 2prlmn j
1
SARS-CoV-2 Infection in K18-hACE2 Mouse Model
2
SARS-CoV-2 Virus Challenge in hACE2 Mice
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For the virus challenge experiments, 14 female hACE2 knock‐in transgenic mice (B6.Cg‐Tg(K18‐hACE2)2Prlmn/J; aged 6–8 weeks; Jackson Laboratory, ME) were immunized with NSctVac (30 μg) plus alum hydroxide via the intramuscular route. Mice were boosted 2 weeks after the initial immunization. On Day 28 post‐priming, mice were anaesthetized by intra‐peritoneal injection avertin (200 mg/kg) and inoculated intranasally with 5 × 104 pfu SARS‐CoV‐2 virus (BetaCoV/Korea/KCDC03/2020). Three mice were euthanized humanly, and lungs were collected to determine the viral load in a plaque assay (Cho et al., 2020 (link)) and histopathology on Days 3, 5 and 7 post‐infection. The remaining mice (n = 5) were monitored continuously for 14 days to assess body weight changes and survival.
3
Evaluation of ChulaCov19 Vaccine in K18-hACE2 Mice
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Challenge study was conducted in ABSL-3 facility at AFRIMS, Bangkok, Thailand. Seventeen female K18-hACE2 mice (B6.Cg-Tg(K18-hACE2)2Prlmn/J), 7 weeks old (The Jackson Laboratory, Bar Harbor, ME, USA) were randomly divided into 3 groups. For group 1 and 2, there were 6 mice/group immunized intramuscularly via quadricep muscles with 2 doses, 3 weeks apart of ChulaCov19 at dose of 1 µg and 10 µg, respectively. In negative control (group 3), 5 mice were immunized with PBS instead of ChulaCov19 using the same schedule. At 2 weeks after the second immunization, mice were challenged intranasally with 2×104 pfu (in 50 µL) of SARS-CoV-2 (wild-type). Blood was collected at wk0, wk2, wk3, wk4 + 6 and wk5 + 6 days for antibody kinetic analysis (Fig. 1b ). Six-day post challenge, wk5 + 6 days, mice were sacrificed to determine virus titers in different tissues (nasal turbinate, brain, lung, and kidney) and for histopathology. Virus titers were quantified by RT-qPCR and by determined the log10TCID50 values. K18-hACE2 mice were also housing at 20–22 °C and a relative humidity of 45 ± 10% on a 12 h light/dark cycle.
4
SARS-CoV-2 Infection Model in Mice
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C57BL/6 mice and K18-hACE2 mice [B6.Cg-Tg(K18-HACE2)2Prlmn/J] (6–8 weeks old) were provided by the Jackson Laboratory. Mice challenged intranasally with 2×104 p.f.u. of SARS-CoV-2 WA1 strain 2020 diluted in a total volume of 50 µl of PBS (25 µl per nare). All mice were uniquely identified with numerical ear tags (Stoelting). Anti-SARS-CoV-2 antibody or PBS was injected by the intraperitoneal route on the indicated days in a total volume of 0.5 ml per dose. The plasma was screened for SARS-CoV-2 antibody prior to use in animals. Sick animals were supplied nutrient gel.
5
Serial Passage of SARS-CoV-2 Variants
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B6.Cg-Tg(K18-hACE2)2Prlmn/J (stock#3034860) and B6 mice were purchased from the Jackson Laboratories (Bar Harbor, ME). All mouse studies were conducted in a BSL-3 laboratory. Mice of both sexes and between the ages of 7–9 weeks were used throughout the protocol. One K18-ACE2 mouse was intranasally infected with either B.1.351 (Beta) or B.1.617.2 (Delta) SARS-CoV-2 lineages. Following 3–4 days, the animals were sacrificed and lung homogenate collected. The homogenate was used to intranasally infect sequential animals, defined as passage. This was repeated for 10 passages. After passage 10 (P10), virus in lung homogenate was sequenced. K18-ACE2 mice were then intranasally infected with Beta P10 or Delta P10 with viruses passaged in three mice at a time for an additional 10 times (total of 20 passages) with virus in lung homogenate sequenced following passages 13, 17, and 20 (Fig. 1 ). Three viral stocks of Beta P20 (1-1, 1-2, 1-3) and three viral stocks of Delta P20 (2-1, 2-2, 2-3) viruses were then made from lung homogenates. K18-ACE2 mice were infected intranasally with 50050% tissue culture infectious dose (TCID50) of P0 or P20 Beta (1-2) and Delta (2-3) viruses. Lung viral loads were determined on day 3 post-infection with weight loss and survival monitored daily for up to 9 days.
6
Murine SARS-CoV-2 Infection and Immunization
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Six- to 8-week-old female mice (Envigo) or K18-hACE2 mice [B6.Cg.Tg(K18-hACE2)2Prlmn/J, Jackson Laboratory] were used in these studies. The mice were anesthetized by intraperitoneal injection of 250 μl of 2,2,2-tribromoethanol in tert-amyl alcohol (Avertin) and intranasally inoculated with 50 μl of PBS (or DMEM) or 104, 105, or 106 PFU of CVXGA1 or 106 PFU of PIV5 vector. Twenty-eight days after immunization, the mice were euthanized, serum was collected via cardiothoracic bleeds, and spleens were harvested. The mice were housed and immunized in enhanced BSL2 facilities in HEPA-filtered isolators. All experiments were performed in accordance with protocols approved by the IACUC at the University of Georgia and at the University of Iowa. UV-inactivated SARS-CoV-2 (106 PFU) was 1:1 (v/v) mixed with alum adjuvant (Thermo Fisher Scientific, catalog no. 77161) in a volume of 200 μl and delivered to mice intramuscularly.
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