A yeast-expressed RBD-based SARS-CoV-2 vaccine formulated with 3M-052-alum adjuvant promotes protective efficacy in non-human primates

Description


ELISpot assays to quantify ASCs in blood, LN and BM:
Briefly, 96 well multi-screen HTS filter plates (Millipore; MSHAN4B50) were coated overnight at 4C with 10g/mL of anti-monkey IgG, IgA, or IgM (H&L) goat antibody (Rockland) or with 4g/mL of recombinant RBD protein for enumeration of total or antigen-specific antibody secreting cells (ASCs) respectively. Wells were washed 4 times with PBS 0.05% Tween 20 (PBS-T) and 4 times with PBS, and blocked with complete RPMI medium with L-glutamine (supplemented with 10% FBS, 10mM HEPES, 100mM sodium pyruvate, non-essential amino acids, 2-mercaptoethanol and Penicillin/Streptomycin with fungicide) for 2 hours in a 5% CO 2 incubator at 37°C. Whole PBMC, lymph node or bone marrow preparations were diluted in complete RPMI medium, plated in serial 3-fold dilutions starting with 5 million cells/ml and incubated overnight in a 5% CO 2 incubator at 37°C. Wells were washed 4 times with PBS and 4 times with PBS-T, followed by incubation with either anti-monkey IgG, or IgA, -biotin conjugated antibodies (Rockland), diluted 1:1000 in PBS-0.05% Tween 20 1% FBS solution (PBS-T-F), for 2 hours at room temperature. Wells were again washed 4 times with PBS-T before adding Avidin D-HRP (Vector labs) diluted 1:1000 in PBS-T-F. After a 2-3 hour incubation at room temperature, wells were washed 4 times with PBS-T and 4 times with PBS. Spots were developed with filtered 3-amino 9-ethylcarbazole (AEC) substrate (0.3mg/mL AEC diluted in 0.1M of sodium acetate buffer (pH 5.0), containing a 1:1000 dilution of 3% hydrogen peroxide).
To stop the reaction, wells were washed with water. Spots were documented and counted using the Immunospot CTL counter and Image Acquisition 4.5 software (Cellular Technology). Once counted, the number of spots specific for each immunoglobulin isotype was reported as the number of either total or antigen-specific ASCs per million PBMCs.
Stained cells were acquired using a Fortessa Flow Cytometer (BD Biosciences, CA). Flow cytometry data was analyzed using Flow Jo (TreeStar, Or).

MSD cytokine assay.
Briefly, 25L microliters of plasma from each samples were combined with the biotinylated antibody plus the assigned linker and the SULFO-TAG™ conjugated detection antibody; in parallel a multi-analyte calibrator standard was prepared by doing 4-fold serial dilutions. Read buffer was added to both samples and calibrators and loaded in a 10-spot U-PLEX plate, which was read by the MESO QuickPlex SQ 120. The plasma cytokines values (pg/mL) were extrapolated from the standard curve of each specific analyte. Cytokine clustering was performed using independent methods: gap statistic method to identify and characterize optimal number of k-means clusters, and hierarchical clustering ward clustering; Euclidean distance.

Viral stock
Vero E6 cell line (African Green Monkey Kidney cell line; CRL-1586, ATCC) was cultured and maintained in MEM (Sigma) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco) and 1 mM L-glutamine (Gibco), 50U/ml penicillin and 50μg/ml streptomycin (Gibco). The cells were kept at 37°C in the presence 5% CO 2 . At the time of virus inoculation and propagation, the concentration of FBS was reduced to 2%. SARS-CoV-2 (NR-52281: BEI Resources, Manassas, VA; USA-WA/2020, Lot no. 70033175) was passaged on Vero E6 cells at a MOI of 0.01 to produce the infectious viral stock. SARS-CoV-2 has been propagated and titrated by TCID 50 method followed by storage of aliquots at -80°C until further use in the experiments. Back titration of viral stocks via plaque assay was used to determine the infectious dose delivered to the RMs. The virus stock was also directly sequenced via metagenomic methods prior to inoculation to confirm the presence of the furin cleavage motif, which has been shown to be lost upon sequential passage of SARS-CoV-2 in culture(73). Our stock contained fewer than 6% of viral genomes with a mutation that could potentially abrogate furin-mediated cleavage of S.

Quantifying viral load RNA:
Quantitative PCR (qPCR) was performed on total viral RNA using the N2 primer and probe set   designed  by  the  CDC  for  their  diagnostic  algorithm:  CoV2-N2-F:  5'-TTACAAACATTGGCCGCAAA-3

Histopathology:
For each animal, all the lung lobes were used for analysis and affected microscopic fields were scored semi-quantitatively as Grade 0 (None); Grade 1 (Mild); Grade 2 (Moderate) and Grade 3 (Severe). Scoring was performed based on these criteria: number of lung lobes affected, type 2 pneumocyte hyperplasia, alveolar septal thickening, fibrosis, perivascular cuffing, peribronchiolar hyperplasia, and inflammatory infiltrates. A total pathological score was calculated by the accumulative scores from each criterion of all lobes affected, and an average pathological score was calculated by combining scores from each criterion taking into account the number of lobes affected. Digital images of H&E stained slides were captured at 20x and 10x magnification with an Olympus BX43 microscope equipped with a digital camera (DP27, Olympus) using Cellsens® Standard 2.3 digital imaging software (Olympus).

Statistics:
The repeated measures linear mixed model included three predictors (study group, time on study (categorical) and the statistical interaction between study group and time on study). A compound-symmetric variance-covariance form in repeated measurements was used for total viral RNA (and sqRNA) and robust estimates of the standard errors of parameters were used to perform statistical tests and construct 95% confidence intervals. A P-value ≤0.05 was considered statistically significant for the main effects (study group and time on study) and for the study group by time on study interaction effect from the repeated-measures analysis. The statistical test for interaction between time on study and study group was the overall hypothesis test to determine whether total viral RNA (and sgRNA) in the three study groups changed in significantly different ways during follow-up (i.e., different temporal patterns by study group). If the statistical interaction was not significant, then the main effect test for study group was used as the primary hypothesis test to compare the 3 study groups. If a significant interaction was detected, then t-tests were used to compare the differences between the model-based study group means at each time point and to compare differences over time within each study group.
Specific statistical tests will be done within the framework of the mixed linear model. All  The size reduced after PNGase-F treatment suggested that RBD was N-glycosylated. (C) The purity of SARS-CoV-2 RBD was evaluated using SE-HPLC. Briefly, 50μg of RBD was injected into a Yarra SEC-3000 column (300mm X 7.8mm; catalog # 00H-4513-K0), and was eluted in 20mM Tris, 150mM NaCl, pH 7.5 at the flow rate of 0.6 ml/min. The % purity was estimated as ~99%.       Contribution of each variable from high to low is presented as gradient color from light red to cyan color. C) Determining the optimal number of clusters. The x and y axes represent the

A B C
RBD+alum RBD+3M-052-alum two tailed Mann-Whitney test was used to compare the significance of the difference. * represents a P value = 0.0476.