Balb c annhsd

Eleven to 12-month-old female BALB/c mice (BALB/c AnNHsd, Envigo, stock# 047) were used for mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA) in vivo protection experiments as described previously^{37 (link)}. Ten-week-old HFH4-hACE2 transgenic mice were used for SARS-CoV-2 WT in vivo protection experiments^{37 (link),77 (link)}. For evaluating the prophylactic efficacy of single mAbs and mAb combinations, mice were injected intraperitoneally (ip) with the appropriate concentration of each mAb combination 12 hours prior to infection. Mice were infected intranasally with 1 × 10⁵ PFU SARS-CoV-2 MA or SARS-CoV-2 WT, respectively. At 48 hours post-infection, mice were euthanized, and lung tissue was harvested for viral titer as measured by plaque assays. For viral plaque assays, the caudal lobe of the right lung was homogenized in PBS, and the tissue homogenate was then serial-diluted onto confluent monolayers of Vero E6 cells, followed by agarose overlay. Plaques were visualized with overlay of Neutral Red dye on day 2 post-infection. All mouse studies were performed at the University of North Carolina (Animal Welfare Assurance #A3410-01) using protocols (19-168) approved by the UNC Institutional Animal Care and Use Committee (IACUC) and were performed in a BSL3 facility at UNC.

For immunogenicity studies, female BALB/cAnNHsd were purchased from Envigo (order code 047) at 7 weeks of age. Mice were housed in a specific-pathogen free facility within the Department of Comparative Medicine at the University of Washington, Seattle, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animal studies were conducted in accordance with the University of Washington’s Institutional Animal Care and Use Committee. For each immunization, low-endotoxin nanoparticles were diluted to 20 μg/mL in buffer and mixed with 1:1 v/v AddaVax adjuvant (InvivoGen vac-adx-10) to obtain a final dose of 1 μg of immunogen per animal, per injection. At 8 weeks of age, 10 mice per group were injected subcutaneously in the inguinal region with 100 μL of immunogen at weeks 0 and 4. Animals were bled using the submental route at weeks 0, 2, and 6. A terminal bleed was collected at week 8 via cardiac puncture. Whole blood was collected in serum separator tubes (BD #365967) and rested for 30 min at room temperature for coagulation. Tubes were then centrifuged for 10 min at 2,000 g and serum was collected and stored at −80°C until use.

All of the animal work was reviewed and approved by the University of Illinois IACUC and was performed under the protocol number 21197. The competition experiments were performed using 5 to 6-week-old mice. The BALB/cAnNHsd and C3H/HeNHsd mice were purchased from Envigo. The Salmonella strains were grown overnight in LB medium, mixed 1:1, and diluted to a target inoculum of approximately 1,000 CFU in 200 μL sterile PBS. For the Δsynth background strains, we used an inoculum of approximately 10,000 CFU. The mice were infected via the intraperitoneal route. Each inoculum was plated on LB medium to measure the total inoculum and was replica plated to the appropriate selective medium in order to calculate the input ratio for each strain. After 4 days of infection for the BALB/cAnNHsd mice or 5 days of infection for the C3H/HeNHsd mice, the animals were sacrificed via CO₂ asphyxiation, and their spleens were removed and homogenized. Serial dilutions of the spleen homogenates were plated on LB medium and were replica plated to the appropriate selective medium in order to calculate the output ratio for each competition. The competitive index (CI) was calculated as (percent strain A recovered/percent strain B recovered)/(percent strain A inoculated/percent strain B inoculated). The statistical comparisons of individual competitions were done using Student’s t tests.