Ethical statement

Wageningen Bioveterinary Research is authorized to perform animal experiments according to the Dutch Law on Animal Experiments (WoD) and in accordance with European legislations and guidelines. The current study was performed under project license no. AVD4010020209446 of the Dutch Central Authority for Scientific procedures on Animals (CCD). The experimental plan was approved by the Animal Welfare Body of Wageningen University and Research prior to the start of the in-life phase. Protocols were prepared in compliance with 3 R policies, and the study reports are presented in compliance with the ARRIVE guidelines⁵⁰.

Vaccine preparation

For production of the virus stock, Vero/hSLAM cells (Sigma-Aldrich, Merck, Darmstadt, Germany, Cat# 04091501-1VL) were used, cultured in minimum essential medium (Life Technologies, Carlsbad, CA, USA) supplemented with 4% heat-inactivated FBS (Sigma-Aldrich), 25 mM Hepes (Life Technologies) and geneticin (0.4 µg/ml) (Gibco, Thermo Fischer Scientific; Waltham, MA, USA). Production of the stock, preparation of the formalin-inactivated whole virus (FIWV) SARS-CoV-2 vaccine, and the subsequent purification and characterization of the FIWV have been described previously¹⁹. Prior to administration, frozen formalin-inactivated whole virus was thawed, diluted with phosphate buffered saline (PBS), and mixed 1:1 with 2% Alhydrogel (InvivoGen, Toulouse, France) to a final concentration of 0.5 µg vaccine antigen per dose (low dose) or 5 µg vaccine antigen per dose (high dose) for the first study, or to 5 µg vaccine antigen per dose for the second study. The same vaccine preparation was used for both studies described in this manuscript.

Viruses and cells

The virus used for inoculation in both studies was SARS-CoV-2 D614G, strain SARS-CoV-2/human/NL/Lelystad/2020⁵¹. In the first study, an undiluted virus stock prepared as previously described⁵¹ was used at a dose 10^4.22 TCID₅₀ per hamster. For the second study, the virus stock was obtained following a second passage of the original isolate on Vero/hSLAM cells at MOI 0.0001. The culture medium consisted of MEM (Gibco, Thermo Fischer Scientific; Waltham, MA, USA, Cat# 21090;), supplemented with 2% FCS, 1% antibiotic/antimycotic, 1% L-glutamine, 1% Minimal Essential Medium Non-Essential Amino Acids (MEM-NEAA) (all from Gibco). Inoculations of hamsters were performed with undiluted virus stock at a dose of 10^4.13 TCID₅₀ per hamster. Sequences of both virus stocks were determined by next generation sequencing and were identical to the original isolate, with no deletions or mutations in the furin cleavage site. For virus neutralization test, VERO-E6 cells (ATCC® CRL-1586™; Manassas, VA, USA) were used, cultured in MEM (Gibco, Cat# 21090;), supplemented with 5% FCS, 1% antibiotic/antimycotic, 1% L-glutamine, 1% Minimal Essential Medium Non-Essential Amino Acids (MEM-NEAA) (all from Gibco).

Experimental design

For both studies, Syrian hamsters (Mesocricetus auratus), strain RjHan:AURA were obtained from Janvier, France. All hamsters were housed solitary in cages with open grids in one animal room under human BSL3 containment level. Water and food were provided ad libitum. Hamsters were monitored daily for their general health from the day of arrival until the end of the study and were allowed to acclimatize for at least 7 days before subjected to any study-related handlings. Vaccines and mock-treatment (phosphate buffered saline (PBS)) were administered intramuscularly (IM) by injection of 100 µL in the left hind leg. Where relevant, booster vaccination was administered IM in the right hind leg. Body weights of all hamsters were measured approximately twice per week during the acclimatization and vaccination period. Blood drawn from the retroorbital sinus and challenge infection with SARS-CoV-2 via the intranasal (IN) route were performed under general anesthesia with 0.15 mg/kg medetomidine (Sedastart, ASTfarma; Oudewater, The Netherlands) and 100 mg/kg ketamine (Narketan, Vetoquinol; Breda, The Netherlands). The anesthesia was antagonized with atipamezole (Sedastop, ASTfarma; Oudewater, The Netherlands). Animals were euthanized by anesthesia with 0.25 mg/kg medetomidine and 200 mg/kg ketamine, followed by exsanguination. Definition of humane endpoints (HEPs) were described previously⁵¹.

VAED model development: establishing vaccination conditions that result in VAED (Fig. 1, upper panel)

A total of 60 golden Syrian hamsters, (30 male and 30 female), 6–8 weeks of age at arrival, were used for the study. Hamsters from the same sex were assigned to one of 5 treatment groups (n = 6 males and n = 6 females per group) based on equal distribution of body weights across groups and subgroups (per day of euthanasia). One group of hamsters was immunized with a low (0.5 µg) dose, and one with a high (5 µg) dose of FIWV vaccine 33 days prior to challenge (−33 DPI) and then at −14 DPI (2x low dose and 2x high dose groups, respectively). Two other groups received either a single low or high dose of the same vaccine at −14 DPI only (1x low dose and 1x high dose, respectively). The fifth group received a vaccination with PBS at −14 DPI and served as mock-vaccinated control. All vaccinations were applied in a volume of 100 µl via the intramuscular (IM) route. Thirteen days post last vaccination (−1 DPI), n = 4 hamsters of each group were sacrificed for necropsy and served as unchallenged controls. The rest of the hamsters (n = 8 hamsters per group) were inoculated with SARS-CoV-2 at 0 DPI. From the inoculated hamsters, n = 4 per group were euthanized at 5 days post inoculation (5 DPI) and the other n = 4 animals per group were sacrificed at 13 DPI. During the challenge phase, body weights were measured daily. In addition, activity of the hamsters to be sacrificed on 13 DPI (n = 4 per group) was monitored daily by means of individual activity tracking wheels (Tecnilab BMI, Someren, The Netherlands)⁵¹. The running wheels were connected to an automatic rotation counter. Counts were recorded once per 24 h at approximately the same time of day and the counters were reset to 0. One complete wheel rotation corresponded to 4 counts. Serum samples were collected on the day prior to each vaccination (−34 and −15 DPI), two days prior to challenge (-2 DPI) and on both necropsy days (5 and 13 DPI) by retroorbital puncture under general anesthesia. From the hamsters that were euthanized at -1 DPI, serum samples were taken during necropsy. At necropsy, the left lung lobe was weighed and collected for pathological investigation, while cranial, medial and caudal right lung lobes were collected for viral load. The cardiac lung lobe was collected for cytokine measurements.

One hamster of the 1x high dose vaccine group reached a HEP on 7 DPI, showing depression, abdominal breathing and 19% body weight loss, and it was euthanized. This same animal also exhibited nonspecific symptoms (ruffled fur, hunched posture, and reduced alertness) on the day of challenge infection, prior to administration of the anesthesia. Furthermore, it was also one of the smallest in the study and consistently had the lowest body weight from −5 DPI until reaching HEP at 7 DPI, except for two measurements (0 DPI and 3 DPI). We cannot rule out the possibility that an unknown underlying condition contributed to the deterioration of the this one hamster post-challenge. Therefore, data from this hamster was excluded from the study.

VAED kinetics: investigation of the kinetics of VAED during infection (Fig. 1, lower panel)

A total of 45 female golden Syrian hamsters, 8 weeks of age at arrival, were assigned to one of 3 treatment groups (n = 15 animals per group in total) based on equal distribution of body weights across groups and subgroups (per day of euthanasia). One group of hamsters was inoculated IN with SARS-CoV-2 (re-challenge group) at −22 DPI. To confirm successful infection in this group, oropharyngeal swabs (MW100, Medical Wire, Corsham, UK) were collected every 2 to 3 days, starting 3 days prior to inoculation until 10 days post inoculation. Hamsters from the other two groups were injected IM at −14 DPI with either vaccine (5 µg vaccine antigen) or with PBS (1x vaccinated and mock-vaccinated groups respectively), both in a volume of 100 µl. Two weeks post vaccination (vaccinated and mock-vaccinated groups) and approximately 3 weeks post first challenge (re-challenge group), all hamsters were inoculated IN with SARS-CoV-2 (0 DPI). At 2, 4, 6, 8 and 10 DPI, n = 3 hamsters per group were euthanized to perform necropsies and to collect lung tissue. The left lung lobe was collected for pathological analysis and the right caudal and cardiac lobes for virological and mRNA expression analysis (Nanostring® nCounter technology, Seattle, WA, USA). During the challenge phase, body weights were measured once daily starting 5 days prior to challenge and ending on the day of necropsy. Serum samples were collected before vaccination, prior to challenge and during necropsy by retroorbital puncture under general anesthesia.

Pathological evaluation of lung tissue and immunohistochemistry

The left lung lobe was removed and weighed, and the weight was expressed as percentage of the body weight measured on the day of challenge (relative lung weight). Subsequently, the left lung lobe was gently inflated and immersed in 10% neutral buffered formalin, fixed for 14 days and embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin (H&E) for histological examination. The percentage of the total extent of the left lung lobe that was microscopically affected by SARS-CoV-2-related lesions, was estimated in a blinded fashion by a board-certified veterinary pathologist. Three blinded estimates with different order of slides were performed and averaged for a final score. For the model development study, a section from the lung of each hamster from both 5 and 13 DPI was evaluated by a second, independent board-certified veterinary pathologist in a blinded fashion. Percentage of affected lung tissue was calculated using digital image analysis (Nikon NIS-Ar software, Tokyo, Japan). The agreement between the scores assigned by both pathologists was assessed by correlation analysis (GraphPad (San Diego, CA, USA) software Prism v. 9.4.0). The severity and characteristics of the (histo)pathological lesions were scored semi-quantitatively as described previously⁵¹ with slight modifications as shown in Table 1a. To account for the different scales of the 6 individual parameters that were scored, all scores were normalized by multiplying by a correction factor (4/highest score of each scale). Normalized scores were used to calculate cumulative (“sum”) scores for visual representation and for statistical analysis. The severity and characteristics of the lung lesions were also analyzed by the second pathologist in a blinded fashion and in a descriptive way.

SARS-CoV antigen expression was evaluated with immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded (FFPE) lung tissue sections. Heat-induced epitope retrieval (HIER) method was used to prepare slides for IHC stain as previously described⁵¹. Briefly, after routinely dewaxing and endogenous peroxidase quenching (methanol/0.3%H₂O₂), the sections were heated for 15 min at 100 °C (Pascal, Dako, Agilent, Santa Clara, CA, USA pressure cooker) in 10 mmol citrate buffer pH 6,0 (Dako, Cat# S1699). The slides were blocked with 10% goat serum (Dako) and stained with primary polyclonal rabbit anti-SARS-CoV NucleoProtein (Sino Biological, Beijing, China, Cat# 40163-T62) and secondary HRP-conjugated anti-rabbit reagent (Envision + Single Reagent, Dako, Cat# K4003). For visualization, the 3,3’-diaminobenzidine (DAB) (Dako, Cat# K3468) substrate was used. Slides were counterstained with haematoxylin. The semiquantitative scoring system for the level of SARS-CoV-2 virus antigen expression in the lungs is shown in Table 1b (Level of antigen expression).

Oropharyngeal swab sampling and analysis

Following sampling, the swabs were directly submerged in 2 ml of complete culture medium and kept on melting ice until transport to the lab and freezing at ≤−70 °C. Upon thawing, swab samples were vortexed, and 200 µl of the sample was mixed with 211 µl lysis master mix, consisting of 200 µl lysis buffer supplemented with 1 µl Poly-A RNA and 10 µl Proteinase K solution (Molgen Pureprep Pathogens Kit, Utrecht, The Netherlands). Lysis buffer-inactivated samples were stored at ≤−15 °C until RNA isolation and PCR analysis. RNA from oropharyngeal swabs was isolated by an automated robot system (PurePrep 96, Molgen), using the Pureprep Pathogens RNA isolation kit (Molgen) according to manufacturer instructions.

Assessment of viral genome loads

In the VAED model development study, the collected cranial, medial and caudal right lung lobes were kept on melting ice until initial storage at ≤−70 °C. Upon further processing, tissue samples were weighed, thawed and homogenized in 6 mL Earl’s MEM (Gibco), supplemented with 1% antibiotic/antimycotic (Gibco), using DT-20 Tubes with Rotor Stator Element (Thermo Fisher Scientific) and an Ultra-Turrax® homogenizer (IKA; Staufen, Germany). The tissue homogenates were centrifuged for 15 min at 3400 × g at 4 °C and 85 µl of the supernatant was mixed with 255 µl Trizol-LS and stored at ≤−15 °C. In the VAED kinetics study, the right cranial and middle lung lobes were directly snap-frozen in liquid nitrogen and kept on dry ice until initial storage at ≤−70 °C. Upon further processing, snap-frozen lung tissue samples were weighed and homogenized in 5 mL Trizol by an Ultra-Turrax® homogenizer. The tissue homogenate was centrifuged for 15 min at 3400 × g at 4 °C and supernatants were aliquoted and stored at ≤−15 °C. RNA extraction from the lung suspensions in Trizol was performed manually with individual columns of the Direct-zol Miniprep Kit (Zymogen, Irvine, CA, USA), following manufacturer instructions. The isolated RNA samples were stored at ≤−70 °C until further use for analysis by SARS-CoV-2 qPCR to assess total viral RNA levels and subgenomic RNA levels. Viral RNA loads were measured by qPCR essentially as previously described⁵¹, using primer/probe sets for both total viral E-gene PCR⁵² and subgenomic PCR (sgPCR)²⁵. The former PCR detects all positive RNA species (genomic and subgenomic) containing the E-gene sequence, while the latter detects only the subgenomic species generated during active virus replication.

Cytokine PCR

Lung tissue samples were used for analyses with RT-qPCR to assess mRNA expression levels of Th1 (Ifng, Tnf, Il2, Il6) and Th2 (Il4, Il13) cytokines. To that end, in the model development study the accessory lung lobe was submerged in 1 mL Trizol in a vial prefilled with lysing beads (matrix D, MP Biomedicals, Irvine, CA, USA). Subsequently the lobe was grinded using a Fastprep-24 instrument (MP Biomedicals) and then stored at ≤−70 °C until further processing. In kinetics study, the right cranial and middle lung lobes were used and were processed as described above (“Assessment of viral genome loads”). RNA extraction in both studies was performed as described above, using individual columns of the Direct-zol Miniprep Kit (Zymogen), according to manufacturer instruction, including a DNAse step performed on the columns to degrade host DNA. To prepare cDNA, 200 ng RNA per sample (model development study) or 100 ng RNA (kinetics study) was used as an input for a reverse transcription reaction, using the Superscript IV First-strand synthesis system Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. The cDNA reaction was performed on an Applied Biosystems 7500 Instrument with the following temperature conditions: 10 min at 23 °C (annealing step); 10 min at 50 °C (reverse transcription step); 10 min at 80 °C (denaturation); 4 °C (termination). For normalization, the reference genes RPL18 and Ywhaz were selected, following an initial screening of 6 reference genes (Rpl18, Rpl13, Pp1a, Ywhaz, B-actin, B2m) and determining the two most stable ones with the GeNex Software v.7 (MultiD Analyses AB, Götenburg, Sweden). For the screening, half of the samples were used in the first study and selection was confirmed on one third of the samples in the second study. Primer sequences and cycling conditions are provided in Tables 2 and 3. Primers were ordered at Biolegio (Nijmegen, The Netherlands). All PCRs were performed using Power SYBR Green PCR Master mix (Thermo Fisher Scientific) according to manufacturer’s instructions with 1:10 diluted sample cDNA and primer concentrations as indicated in Table 2 in a total reaction volume of 20 µl or 50 µl in the first or second study, respectively. All PCRs were performed on an Applied Biosystems 7500 Instrument.

Relative expression of the genes of interest (GOI) was determined with the ΔΔCT method⁵³, using Rpl18 and Ywhaz as reference genes for normalization. This method generates a unit-free number indicating expression of the GOI relative to a reference gene, taking variations among PCR plates into consideration. Before normalizing the GOI, the ΔCt values obtained for the two reference genes were averaged. Standard curves for each gene were prepared by 5-fold serial dilutions of a sample pool, including pre-selected samples mixed in equal amounts (Rpl18, Ywhaz and Tnf) or the sample pool was spiked with 5-fold serial dilutions of a synthetic DNA fragment (synthesized by GenScript Biotech, Piscataway, NJ, USA), encompassing the generated by PCR amplicon (Il2, Il4, Il6, Il13 and Ifng). A standard curve was included on each PCR test plate.

Virus neutralization test

Serial 3-fold dilutions of sera were prepared in duplicates, starting at 1:10 initial dilution. A standard dose of ~100 TCID₅₀ of SARS-CoV-2/human/NL/Lelystad/2020 was mixed with the serum dilutions and incubated for 1 h. Subsequently, VERO-E6 cells were added to the mixture, at a density of 2 × 10⁴ cells/well. The plates were incubated for 4 days at 37 °C and 5% CO₂, after which cell monolayers were fixed with 4% formaldehyde, followed by fixation with pure methanol (kept at −20 °C) for cell permeabilization. Cell monolayers were then washed 3 times with PBS and stored at 4 °C until staining with an immunoperoxidase monolayer assay to visualize viral antigen, as described previously⁵¹. Briefly, cell monolayers were stained using primary anti-SARS-CoV-2 S1 (Wuhan strain) polyclonal antibody (custom-made by Davids Biotechnologie GmbH and obtained by immunization of rabbits with S1 protein; kindly provided by Dr. Berend Jan Bosch and Dr. Wentao Li, University of Utrecht, The Netherlands) and secondary HRP-conjugated anti-rabbit antibody (DAKO Cat# P0448). Both antibodies were diluted in PBS supplemented with 5% horse serum and incubated with the cell monolayer for 1 h at RT. Between the incubation steps, cell monolayers were washed 3 times with PBS, supplemented with 0.5% Tween-80. Staining was visualized following 30–40 min incubation with AEC substrate (4 mg/ml AEC in DMSO), freshly dissolved in substrate buffer (0.05 M NaAc buffer, pH adjusted to 5.0 using 0.05 M HAc) in the following manner: 19 mL substrate buffer + 1 mL 4 mg/mL AEC + 50 µL 3% H₂O₂. Evaluation of staining was performed using a standard light microscope.

The titer of each duplicate was determined as the average of the reciprocal value of the last dilution that showed ≥50% neutralization (lack of staining) in the well, evaluated by eye. The detection limit of the assay in this experimental setting was a titer of 5.7. All samples with undetectable titer were attributed a titer of 3.3.

Enzyme-linked immunosorbent assay (ELISA)

Anti-N and anti-S total IgG titers were determined by end-point dilutions of serum samples using a custom ELISA test. ELISA plates (medium bind, Greiner Bio-One, Krensmünster, Austria) were coated with 50 ng/well of either N or S protein (40588-V08B and 40589-V08B1 respectively, both purchased from Sino Biological, Beijing, China), dissolved in coating buffer (0.05 M Na2CO3 and 0.05 M NaHCO3, pH 9.6) overnight at 4 °C. Blocking was performed with StabilBlock (Surmodics, Eden Prairie, MN, USA) blocking buffer for 1 h at 37 °C. Test sera and the secondary antibody were diluted in dilution buffer (PBS, supplemented with 0.05% Tween 20 and 5% bovine serum albumin, sterile filtered with 0.45 µm filter) and incubated on the plates for 1 h at 37 °C with gentle agitation. Serum samples were serially diluted (2-fold dilution step), starting at dilution 1:50 (low positive sera), 1:200 (mid positive sera) or 1:1600 (high positive sera), based on a pilot test. As secondary antibody, anti-hamster IgG (Thermo Fischer Scientific Cat# HA6007) was used. For color development, TMB substrate (ImmunoChemistry Technologies, Davis, CA, USA) was added to the plates and incubated for 15 min at room temperature. Reaction was stopped with 0.5 M sulfuric acid and OD values were measured at 450 nm on a SpectraMax ABS Plus spectrophotometer (VWR; Radnor, PA, USA). All reagents were added to the ELISA plate wells in a volume of 100 ul (except the blocking solution, which was added in a volume of 300 ul). Between all incubation steps, the plates were washed 3 times with wash buffer (distilled water, supplemented with 0.05% Tween 20).

On each plate, the same negative and positive control were taken along in duplicates. These controls consisted of pooled sera obtained from previous experiments involving naive hamsters or hamsters recovered from SARS-CoV-2 infection, respectively. The negative control was diluted 1:50 and the positive control was diluted 1:250 (N-ELISA) or 1:500 (S-ELISA). The positive and negative controls were used to determine an S/P (sample/positive control) ratio for each sample according to the formula:

SP = (OD_SAMPLE – OD_NEG)/(OD_POS – OD_NEG)

For determination of the cut-off, nine pools were prepared from the serum samples obtained prior to vaccination of the hamsters from the respective experiment. These pools were diluted 1:50 and taken along on each plate. The cut-off of the ELISA was defined as the average S/P ratio of these pools plus 3x the standard deviation (SD). The ELISA titers of the tested sera were determined as the log₂ value of the last serum dilution that showed an S/P ratio above the detection limit. Samples for which the starting dilution of 1:50 was negative were assigned the log₂ value of one (theoretical) reciprocal dilution step lower (1:25).

Gene expression analysis in lung samples on the nCounter® platform

Digital nCounter® technology (NanoString Technologies) was used to assess mRNA expression levels of selected genes (n = 128) associated with Th1, Th2, Th17, and Treg pathways, and with genes associated with different immune cell types in hamster lungs. Custom nCounter panel selection was based on human and/or mouse genes from relevant pathway annotations in the human CAR-T (Chimeric antigen receptor T-cell) Characterization Panel (NanoString Technologies, Seattle, WA, USA), as well as on genes related to different immune cell types based on available literature for mice. In addition, n = 3 reference genes identified as stable/suitable based on previous qRT-PCR analyses were included for normalization purposes (Rpl13, Rpl18, and Ywhaz). Hamster-specific probes for transcripts homologous to the selected genes were designed and manufactured by NanoString Technologies, resulting in a custom hamster Code Set.

Total RNA was isolated from hamster lungs as described under “Assessment of viral genome loads”. RNA concentrations were quantified with a Qubit™ RNA HS Assay Kit on a Qubit® 2.0 Fluorometer (Invitrogen) and the percentage of RNA fragments >200 nucleotides (“DV200”) was determined with an RNA screen tape on an Agilent Technologies 220 TapeStation. Except for two samples from the mock-vaccinated group at 4 DPI (22.8% and 29.7%), the DV200 was >35% (range 35.1%–91.2%).

Multiplexed hybridization reactions were performed by Prof. Stephen Gordon and Dr. John A. Browne on an SFI-funded nCounter®MAX Analysis System (NanoString Technologies) at the UCD Veterinary Sciences Centre (Dublin, Ireland), according to manufacturer’s guidelines and as originally described by Geiss et al.⁵⁴. Hybridization reactions contained 300 ng of total RNA in a 5 µl volume (except for all DPI 2 samples and DPI 4 samples of the re-challenge group, which contained 100 ng), as well as fluorescent barcode-labeled (reporter and capture) probes for endogenous and reference genes, six pairs of positive control probes, and eight pairs of negative control probes. Hybridized probes were loaded onto cartridges (n = 12 per run) and imaged on an nCounter® MAX instrument.

Analysis of raw RCC (Reporter Code Count) files was performed using nSolver Analysis Software (version 4.0) and nCounter Advanced Analysis Plugin (version 2.0.134). The analysis was run with standard settings, and included normalization, differential expression, and pathway scoring modules. Low count data were not excluded from the analysis. Automatic normalization of raw counts based on housekeeping (reference) gene abundance was performed by the software, leading to exclusion of Rpl13 as normalization probe. A probe annotation file for pathway enrichment analysis was provided by NanoString Technologies and was based on the nCounter® CAR-T characterization and host response panels. During basic analysis, all samples passed standard QC requirements regarding imaging, binding density, and positive control linearity.

To analyze differential expression (DE), samples from all three treatment groups were analyzed separately per necropsy day (n = 9) with the same settings described above. Either mock-vaccinated or re-challenge groups were used as categorial reference (baseline) in the ‘fast/recommended’ DE analysis module which fits all genes with a negative binomial model and fold changes as well as univariate p values were calculated by the software.

Normalized count data and pathway scores were visualized with GraphPad Prism. The web app ClustVis (https://biit.cs.ut.ee/clustvis/)⁵⁵ was used to for Principal Component Analysis (PCA) and generation of heatmaps. For heatmaps, normalized expression counts were ln(x)-transformed, centered around the mean per gene, and unit variance scaling was applied to rows. Rows were clustered using Manhattan distance and average linkage.

Statistics

Analysis of the relative body weight losses for both studies was performed on the data between 5 DPI and 7 DPI for the hamsters present in the study up to 7 DPI (model development study: n = 4, subgroup sacrificed on 13 DPI; kinetic study: n = 6, subgroups sacrificed on 8 and 10 DPI). This time window was chosen because it encompasses the period with the largest body weight loss in the control mock-vaccinated group. Each hamster’s relative body weight was quantified by calculating the area under the curve (AUC). Between-group comparisons of the AUCs were made with one-way analysis of variance (ANOVA).

Activity wheel count analysis (first study) was performed on the data between 7 DPI and 10 DPI. This time window was chosen because it encompasses the period of activity recovery following the challenge-induced activity reduction. Data from 9 DPI was excluded from the analysis, because of an unexpected dip of activity in all groups, which was attributed to technical issue of unknown origin in the animal facility. Activity was analyzed by estimating the activity AUC and between groups comparisons were made by one-way ANOVA. The analysis was performed on log-transformed counts.

Group means of the VNT titers, lung pathology parameters (relative lung weights, extent of lung histopathology lesions, severity of histopathology lung lesions sum scores), viral loads (total E gene PCR) and cytokine data for the model development study were compared using two-way ANOVA, where DPI, group and interaction between DPI and group were assessed. Group means of the lung viral loads, as measured by subgenomic RNA PCR at 5 DPI were compared with one-way ANOVA, since all values at 13 DPI were “0”. ELISA antibody titers for the kinetics study on 0 DPI of the vaccinated and the re-challenged group were compared with a (non-parametric) Mann–Whitney test.

In all cases where ANOVA was used, multiple comparisons were performed following a significant ANOVA test. ANOVA was applied with relaxed rule for data normality and variance equality. One-way ANOVA was followed by a Dunnett’s multiple comparison test between the control group (mock-vaccinated) and each of the treatment groups (first study) or between the vaccinated group and the two control groups (re-challenge and Mock-vaccinated, second study). Two-way ANOVA was followed by a Dunnett’s multiple comparison test (to determine differences between the control group (mock-vaccinated) and each of the treatment groups per necropsy day) or by Sidak’s multiple comparison’s test (to test for change over time within a group).

To compare changes in kinetics of serology titers, pathological parameters and Th-pathways over time among groups in the kinetics study, a multivariate linear regression model (interaction Group:time) was used. To account for non-linear changes in time, we introduced natural spline terms to the time variable. Pairwise comparisons between the groups were corrected using the Tukey method.

As a standard, an alpha of 0.05 and two-tailed tests were used. For all cases when multiple comparisons were made, the p value was corrected for multiple comparisons.

The ANOVA and the non-parametric tests were performed with GraphPad (San Diego, CA, USA) software Prism v. 9.4.0, and the multivariate linear regression modeling was performed with software R version 4.2.2 (Team R.C. R: A Language and Environment for Statistical Computing. Available online: https://www.R-project.org/).