This DATSETSTAATSreadme.txt file was generated on 2021-06-25 by Brandi Johnson-Weaver GENERAL INFORMATION 1. Title of Dataset: Nasal Immunization with Small Molecule Mast Cell Activators Enhance Immunity to Co-administered with Subunit Immunogens 2. Author Information A. Principal Investigator Contact Information Name: Herman Staats, PhD Institution: Duke University Address: Email: Herman.staats@duke.edu B. Associate or Co-investigator Contact Information Name: Soman Abraham, PhD Institution: Duke University Address: Email: Soman.abraham@duke.edu C. Alternate Contact Information Name: Brandi Johnson-Weaver, PhD Institution: Duke University Address: Email: brandi.t.johnson@duke.edu 3. Date of data collection : 2014-09-30 - 2020-12-1 4. Geographic location of data collection : Durham, NC, United States 5. Information about funding sources that supported the collection of the data: National Institute of Allergy and Infectious Diseases (NIAID) Adjuvant Discovery Contract #HHSN272201400054C SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: 2. Links to publications that cite or use the data: 3. Links to other publicly accessible locations of the data: https://gitlab.oit.duke.edu/hy140/staats_adjuvant. 4. Was data derived from another source? yes/no NO DATA & FILE OVERVIEW 1. File List: Mast cell acitvator_Data Summary_Submitted (available as XML and XLSX) Data Dictionary Positive Control Adjuvants: MPL- TLR4 Ligand C48/80- polymeric mast cell activator M7- mast cell activating peptide Experimental Mast Cell Activators: L147192 L201863 R127655 R529877 R606278 ST026567 ST027688 ST029248 ST029279 ST045940 ST048871 ST081379 ST086136 ST099914 ST101036 Vaccine antigen: West Nile Virus Envelope Domain III (EDIII) MC/9: Mouse Mast Cell line JAWSII: Mouse monocyte/dendritic cell line MATERIALS AND METHODS: Mice Female BALB/cJ (Jax stock # 000651) and C57BL/6J (Jax stock # 000664) mice (6-8 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed under specific pathogen-free conditions on a twelve-hour light cycle. All experimental procedures were conducted with the approval of the Duke University’s Institutional Animal Care and Use Committee. Mast cell activating compounds Small molecule mast cell activating compounds were purchased from TimTec (Newark, DE) or synthesized by Duke University’s small molecule synthesis facility (SMSF). All compounds were prepared in high concentration stocks (20-40 mM) using DMSO (Spectrum Chemical, Gardena, CA) as the solvent for in vitro assays and PEG400 (Spectrum Chemical) for in vivo assays. Compound solutions were stored at -20°C until used. The mast cell activating peptide mastoparan 7 (M7; amino acid sequence INLKALAALAKALL-NH2) was synthesized by CPC Scientific (San Jose, CA). In vitro compound-induced cytokine and cytotoxicity Mouse MC/9 mast cells (cat #CRL-8306), JAWSII dendritic cells (cat #CRL-11904), J774A.1 macrophages (cat #TIB-67), and LA-4 lung epithelial cells (cat #CCL-196) were purchased from ATCC (Manassas, VA) and cultured in media according to the manufacturer’s instructions. MC/9 media was prepared as previously described with the minor modification that used rat T-stim (Cat# 354115 Corning; Corning NY) as a source of Con A(26). JAWSII cells were cultured in MEM Alpha Modification (HyClone Cat#SH30265.01) with 20% FBS. J774A.1 (27) and LA-4 cells (28) were cultured in media as previously described by others. For cytokine induction, 100 μL of cells were plated in 48-well plates at 5 x106 cells/ml in the presence of the 15 hit mast cell activating compounds or M7 at a final concentration of 100 μM. Cells were incubated at 37°C for 24 hours. Supernatants were collected and measured for cytokine content using a 32-cytokine/chemokine multiplex assay from Millipore (Burlington, MA) according to the manufacturer’s instructions. Compound-induced cytotoxicity was also measured 24-hours after stimulation with the mast cell activating compounds tested at 100, 50, 25, and 12.5 μM. Celltiter96™ MTS (Promega; Madison, WI) was added to the cells and incubated for one hour at 37°C before reading absorbance at 490 nm. In vivo mast cell activation BALB/cJ mice were injected subcutaneously with temperature transponders (BMDS; Seaford, DE) one week before in vivo mast cell activation evaluation. Baseline temperature was recorded for each mouse before exposure to mast cell activators. Mast cell activating compounds were prepared in a 50% PEG400 solution and M7 was prepared in saline. MCA compounds (20 μmoles) or M7 (200 nmoles) were injected into the mouse peritoneal cavity in 100 μL and evaluated for their ability to activate mast cells as monitored by a drop in body temperature secondary to mast cell degranulation. Temperatures were recorded 15, 30, and 45 minutes after compound exposure. Animals were immediately euthanized by CO2 exposure if they displayed a temperature decrease greater than 10°C as a humane endpoint or 45 minutes post-exposure as the experimental endpoint. Change in body temperature was determined by subtracting the baseline temperature from the temperatures recorded 15, 30, and 45 minutes post-exposure. In vivo RNA Sequence Analysis Female C57BL/6J mice (5 mice per group) were nasally instilled with the mast cell activating compounds (200 nmoles), M7 (20 nmoles), MPL (10 μg), or saline in 10 μL. Mice were euthanized six hours post compound exposure. The upper pallet and nasal septum were harvested from each mouse. Harvested tissues were homogenized and total RNA was purified from the homogenized tissues according to the Qiagen® Rneasy kit (Cat.#74106 Hilden, Germany). Total RNA was sequenced utilizing RNA-seq technology performed by BGI® (Cambridge, MA). All genomic analyses used build GRCm38 of the Mus musculus genome. The genome sequence and annotation were downloaded from Ensembl release 98 (29). Analysis was performed using scripts written in the R programming language, Bash, and publicly available software detailed below. Custom Jupyter notebooks, which used the following R and Bioconductor packages: dedexted, DESeq2, EsDb.Mmusculus.v79, foreach, fs, gage, gageData, limma, pathview, pheatmap, plotly, RColorBrewer, Rtse, tidyverse. Basic assessments of sequence data quality were performed using FastQC v0.11.9 (30) and MultiQC v1.9 (31). Raw sequencing reads were trimmed and filtered using fastq-mcf v1.04.807 (32) using the BGISEQ-500 AD1_Long, AD1_Short, AD2_Long, and AD2_Short adapter sequences and their reverse complements (33). Reads were then mapped to the reference genome and read counts were generated using STAR v2.5.4b (34). For quantification of reads mapped to genes, we use the second column of the STAR count output because the libraries were unstranded. Comparative analysis of the resulting count matrices was performed using DESeq2 (35), and the top 20 genes that were under- (negative log-fold change) or over-expressed (positive log-fold change) relative to vehicle only reported for each adjuvant, together with p-values adjusted for multiple comparisons by the Benjamini-Hochberg method (36). Reproducible scripts are maintained under version control at https://gitlab.oit.duke.edu/hy140/staats_adjuvant. Mouse immunization to evaluate adjuvant activity BALB/cJ mice (n=5 mice per group) received three doses of West Nile Virus envelope domain III (EDIII; GenScript Biotech; Piscataway, NJ) alone (15 μg) or combined with M7 (20 nmoles) or MPL (10 μg) as controls, or the mast cell activating compounds (R127655, R529877, ST101036, ST027688, and ST048871; 200 nmoles) on days 0, 7, and 21 by nasal delivery. For mouse WNV infection studies, BALB/cJ mice (n = 9-13 mice per group) were nasally immunized on days 0, 7, 21, and 35. The first vaccine dose contained 15 µg of EDIII and the subsequent doses contained 30 µg of EDIII on days 7 and 21 alone or co-administered with M7, MPL, or the mast cell activators. Because the MCAs display hydrophobic properties that were not suitable for co-administration with the aqueous antigen solution, we utilized an immunization method that administered the adjuvant dissolved in a 50%PEG400:water formulation in 15 µL 15-minutes before administering the EDIII antigen in saline (15 µL). On day 35, all vaccine groups were immunized with 30 µg of EDIII alone to boost anti-EDIII antibody responses before a lethal WNV infectious challenge. Mouse West Nile virus infection Mice were anesthetized with isoflurane. West Nile Virus, strain NY99-35262-11 (BEI Resources NR-677) was diluted to 1.6 x105 PFU/mL. Each mouse was injected with 0.1 mL of the virus via the intraperitoneal route. After mice fully recovered from anesthesia, the animals were returned to their home cages and provided water and chow ad libitum. Changes in body weight, temperature, and activity were monitored daily for 14 days after challenge. Animals that displayed a 20% weight loss versus pre-challenge weight or developed hind limb paralysis were humanely euthanized(37). All work with live virus and infected animals was completed in an animal biosafety level (ABSL)-3 suite in the Regional Biocontainment Laboratory (RBL) at Duke University. Serum collection for ELISAs Blood was obtained from immunized mice using the submandibular lancet method. The whole blood was centrifuged for 10 minutes at 4°C. Serum was collected by removing the supernatant from the clotted blood and stored at -20°C until analysis. ELISA ELISAs were performed as previously described (21) with modifications indicated below. 384-well black plates (Cat. # 460518 Thermo Scientific; Watham, MA) were coated with WNV EDIII as the coating antigen (2 µg/ml) diluted in carbonate/bicarbonate buffer (CBC, pH 9.5). Serum was diluted two-fold beginning at 1:32 in sample diluent (PBS, 1% BSA, 1% NFDM, 5% goat serum, 0.05% Tween 20, and 0.1% 2-Chloroacetamide). Goat anti-mouse IgG, IgG1, and IgG2a, alkaline phosphatase-conjugated antibodies (Southern Biotech; Birmingham, AL) were diluted 1:8,000 in secondary antibody diluent (PBS, 1% BSA, 5% goat serum, 0.05% Tween 20, and 0.1% 2-Chloroacetamide). The fluorescent Attophos substrate (Promega; Madison, WI) was added to each well and incubated for 15 minutes before reading plates in a BioTek Synergy 2 plate reader using 440/30 nm excitation and 560/40 nm emission filters to detect the light signal produced by the Attophos substrate after enzymatic activation by the alkaline-phosphatase-conjugated detection antibodies. The fluorescent signal was reported as relative light units (RLU). Endpoint titers, defined as the last log2 immune sample dilution with an RLU signal 3-fold greater than a naïve reference sample at the same dilution, were used for statistical analysis. Graphs presented in the figures were prepared using geometric mean titer antilog values. Splenocyte antigen restimulation cytokine assay BALB/cJ mice (5 mice per group) in the immunogenicity study were euthanized three weeks after the final immunization and spleens from each mouse were harvested and processed into single-cell suspensions. Single cell suspensions were prepared from whole spleens by grinding through a 70 µM filter. Cells were pelleted by centrifugation and red blood cells were lysed using RBL Lysis Buffer (Cat# R7757 Sigma; St. Louis, MO) according to the manufacturer’s recommendation. Splenocytes were washed twice in RPMI-1640 containing 5% FBS (Cat#25-514 Genesee; San Diego, CA) and 1% penicillian/streptomycin (Cat#15140122 ThermoFisher; Waltham, MA) before counting and plating. Splenocytes were plated in 48-well plates (2.5 x 106 cells/well) and cultured in media alone or media containing EDIII (25 μg/ml) in a total volume of 500 µl for 72 hours at 37°C and 5% CO2. Supernatants were collected and measured for IL-4, IL-5, IL-17, and IFN-γ using the Bioplex Multiplex (Biorad; Hercules, CA) according to the manufacturer’s instructions. EDIII-induced cytokine responses were determined by subtracting the cytokine value from cells cultured in media from the cytokine values measured in cells stimulated with EDIII. Flow cytometry to monitor in vivo cellular infiltration C57BL/6J mice (5-7 mice per group) were nasally instilled with one of the five lead mast cell activating compounds (200 nmoles) or M7 (20 nmoles). Twenty-four hours after compound exposure, draining cervical lymph nodes were harvested from mice and digested with a digestion buffer containing 10% collagenase (Cat. #C2674, Sigma (St. Louis, MO)), 1% deoxyribonuclease I (Cat. #04716728001, Roche (Basel, Switzerland)), 10 mM HEPES, and 1.5% FBS in HBSS to make single-cell suspensions. Cells were washed and pre-stained with Live/dead staining solution (Zombie Violet™ Fixable Viability Kit, Biolegend) for 10 minutes at room temperature. After washing, cells were stained with fluorescently labeled antibodies against B220 (clone RA3-6B2), CD11b (clone M1/70), CD11c (clone N418), CD45 (clone 30-F11), CD86 (clone GL01), IA/IE (clone M5/114.15.2), and isotype controls (BioLegend, San Diego, CA) for 30 minutes at 4°C. To examine the expression levels of individual markers, an LSR II flow cytometer (BD; Franklin Lakes, NJ) was utilized to analyze the samples. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). Statistical Analyses GraphPad Prism Version 9 (San Diego, CA) was used to identify statistical differences between groups in each experiment. One-way analysis of variance (ANOVA) determined if mast cell activating compounds or the positive controls statistically enhanced responses in the in vivo mast cell degranulation, in vivo cellular infiltrate, and in vivo immunogenicity assays using the Dunnett’s multiple comparison test compared to the negative controls. In the WNV infection study, one-way ANOVA determined if any adjuvant provided superior adjuvant activity by comparing antibody responses induced by each adjuvant to the other adjuvants using Tukey’s multiple comparison post-test. Survival curves were analyzed using the Gehan-Breslow-Wilcoxon test * p< 0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001.