Jennifer K. in SARS-CoV-2 contamination. Our results suggested that disruption of the CXCL5 and CXCL1/2 axis may be important early components of the inflammatory dysregulation that is characteristic of severe cases of COVID-19. = 10 per group) or vehicle [(phosphate-buffered saline (PBS)] control (= 7) on day 0 by intramuscular injection in a total volume of 100 L. Twenty-one days later, blood samples were collected from each animal by cheek bleed and processed in serum separator tubes by centrifugation at 1000 for 5 min at room temperature. Mice were then given a second dose of vaccine (15 or 45 g) via intramuscular injection. Blood was drawn day 36 (post-initial vaccination) and processed to collect serum and evaluate antibody response prior to challenge. Mice were then challenged with 1 105 pfu in 50 L of USA-WA1/2020 SARS-CoV-2 (2019-nCoV) by intranasal delivery forty-two days after initial vaccination with either CoV-RBD121-NP or vehicle control. Mice were evaluated twice daily for clinical symptoms and weight loss for 10 days post-challenge. Categories included in clinical scoring were (i) posture and appearance of fur (piloerection) (0C3) and (ii) development of respiratory distress (0C3). Four days following challenge, blood samples were collected from mice by cheek bleed and pooled per group. Blood samples were collected from all mice again at the conclusion of this study (day 10 post-infection) or when the animal reached end point criteria. All vaccine materials were stored at 4 C for a duration of less than two months prior to use. All mouse work was approved by the University of Louisville Institutional Animal Care and Use Committee (University of Louisville, IACUC 19625, approved 9 September 2021), and all procedures were performed in the universitys certified animal biosafety level three laboratory. 2.2. Human Convalescent Sera In mid-March of 2020, prior to availability of EUA-authorized vaccines, health care workers who had actively Diethylstilbestrol been in contact with SARS-CoV-2 patients were invited to participate in a study to examine contamination rates and immune responses to SARS-CoV-2 [26]. Our analysis of the sera in this study was approved by the Institutional Review Board at University of Louisville (IRB Approval #20.0312). 2.3. Neutralizing Titer Assay Vero E6 cells (ATCC, cat# VERO C1008; CRL-1586, Manassas, VA, USA) were seeded at a density of 2 104 cells in 96-well tissue culture plates (Corning/Costar) and incubated overnight at 37 C with 5% CO2. The following morning, cells were washed once with Diethylstilbestrol Vero incubation medium [VIM: 100 L DMEM made up of penicillin/streptomycin and 5% fetal bovine serum (FBS)] followed by two washes with 200 L of PBS prior to Rabbit Polyclonal to OR4F4 the addition of 100 L of VIM. Prior to the addition of virus, 2-fold serial dilutions of serum samples were prepared in VIM in a separate 96-well dilution plate. SARS-CoV-2 was then added to the dilution plate at a concentration of 60 pfu/well for a final multiplicity of contamination (MOI) of 0.003 and incubated with the serum at 37 C, 5% CO2. Following a 1 h incubation, medium around the cells was replaced with 100 L of the serum and virus mixture, and the cells were returned to the incubator. After four days, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet for 20 min at room temperature. Cells were then washed twice with 200 L of filtered tap water and assessed for cytopathic effects (CPE). For assessment of neutralization titer in samples from mice collected at day 4 post-challenge, the sera were pooled for evaluation due to limited volume of blood that could be collected without affecting the health of Diethylstilbestrol the mouse. Neutralizing titers were evaluated with four technical replicates per group.