SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta

Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Because of confounding factors in the human population, such as preexisting immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were up-regulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggest that, in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is under constant evolutionary pressure. The unprecedented speed and volume of whole-genome sequencing used during the pandemic has allowed for near real-time surveillance of amino acid substitutions. The close surveillance of virus genomes for such substitutions additionally led to early detection and analysis of variants of concern (VOCs) (1). A variant is deemed a VOC when it displays evidence for increased transmissibility, increased disease severity, or decreased effectiveness of available diagnostics, vaccines, and therapeutics (2). The first recognized VOC was detected in September 2020 (3) and was designated Alpha. Thus far, five VOCs have been identified: Alpha (Pango lineage B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529, which includes BA.1, BA.2, BA.3, BA.4, BA.5, and all its descendent lineages). The Delta VOC was first detected in the spring of 2021 in India. It spread very quickly on a global level, replacing the Alpha variant in the United Kingdom and the United States (3,4). Delta is characterized by a number of key substitutions, such as the L452R and P681R substitutions in the S protein (5). The Omicron VOC was then detected in November 2021 in South Africa and subsequently replaced the Delta VOC. Omicron is characterized by >30 substitutions in the S protein (5).
Studies aiming to identify the evolutionary advantages of each VOC in the human population are complex, due to populationwide confounding factors such as previous SARS-CoV-2 infections and vaccine coverage. Animal models allow us to study pathogenesis and compare viral replication kinetic in naïve animals, thereby circumventing these confounders. We previously used the rhesus macaque model to examine differences in pathogenicity between an ancestral strain (Wuhan like) with the D614G mutation, the Alpha VOC, and the Beta VOC and showed that inoculation with the Beta VOC resulted in lower clinical scores, lower lung virus titers, less severe lung lesions, and lower cytokine and chemokines in the bronchoalveolar lavage (BAL) (6). In the current study, we aim to extend this dataset to include the Delta, Omicron BA.1, and Omicron BA.2 VOCs in naïve rhesus macaques.
To determine the entry profile of the respective VOCs, we compared the entry of pseudotyped vesicular stomatitis virus (VSV) particles expressing the S protein of Wuhan1 virus to particles expressing the S protein of the Delta, Omicron BA.1, and Omicron BA.2 VOC into baby hamster kidney (BHK) cells expressing either the human or rhesus Angiotensin-converting enzyme 2 (ACE2). Entry was observed under all conditions but was significantly less efficient for the Omicron VOCs compared to the Delta VOC, both with human and rhesus ACE2 (fig. S1A). We repeated this assay using 293T cells, which are low in Transmembrane serine protease 2 (TMPRSSII) expression, which has been shown to effect entry of Omicron (7). Entry of Omicron BA.1 was particularly low, whereas entry of BA.2 was significantly lower than Delta [P < 0.001, one-way analysis of variance (ANOVA)] but significantly higher than BA.1 for both human and rhesus macaque ACE2 (P < 0.001 and 0.0037, relatively, one-way ANOVA) (fig. S1B).

Reduced clinical signs in Omicron-inoculated rhesus macaques
Three groups of six rhesus macaques were inoculated intranasally and intratracheally with a total dose of 2 × 10 6 median tissue culture infectious dose (TCID 50 ) of one of the SARS-CoV-2 VOCs. Although animals in all three groups showed mild signs of disease after challenge, inoculation with Delta resulted in noticeable higher clinical scores than inoculation with Omicron BA.1 and BA.2, a result that was only statistically significant for Omicron BA.1 (Fig. 1A). Most animals in all three groups had days with reduced appetite throughout the study. However, respiratory signs were significantly different between groups: They were observed in four animals inoculated with Delta, only one animal inoculated with Omicron BA.2, and no animals inoculated with Omicron BA.1 (Fig. 1, B and C). Radiographs collected on all exam days were analyzed for the presence of pulmonary infiltrates. Most animals in all three groups did not present with pulmonary infiltrates, except for one animal each in the Delta and Omicron BA.

Reduced shedding after Omicron BA.1 or BA.2 inoculation
Nasal swabs were collected at 0, 2, 4, and 6 days postinoculation (dpi) and analyzed for the presence of viral genomic RNA (gRNA) and subgenomic RNA (sgRNA). The amount of viral gRNA detected in nasal swabs from Delta animals was significantly higher than that detected in nasal swabs from Omicron BA.1 or BA.2 animals ( Fig. 2A). Similar differences were also observed in the amount of sgRNA found in nasal swabs between groups, but significance was only found 2 dpi between Delta and Omicron BA.1, and on 4 and 6 dpi between Delta and Omicron BA.2 (Fig. 2B). For each animal, the area under the curve was calculated as a measure of the total amount of viral gRNA and sgRNA shed between 2 and 6 dpi. Animals inoculated with Delta shed significantly more gRNA than Omicron BA.1-inoculated animals and more gRNA and sgRNA than Omicron BA.2-inoculated animals (Fig. 2, A and B). Oropharyngeal and rectal swabs were also obtained on each exam day. The presence of viral RNA in these samples was limited, compared to nasal swabs. The only significant difference between groups was found 2 dpi in gRNA in rectal swabs, where Delta animals shed significantly more than Omicron BA.1 or BA.2 animals. No viral RNA was detected in blood samples on any exam day ( fig. S3).
Reduced virus replication in the lower respiratory tract of rhesus macaques inoculated with Omicron BA.1 and BA.2 BAL and bronchial cytology brush (BCB) samples were collected on 2, 4, and 6 dpi (BCB only) and analyzed for the presence of gRNA and sgRNA. Viral load in BAL and BCB samples was highest on 2 dpi and declined by 4 and 6 dpi ( Fig. 2, C to F). As seen in the nasal swabs, less viral RNA was detected in BCB samples in animals inoculated with Omicron BA.1 or BA.2 compared to that with Delta ( Fig. 2, C and D). In contrast, no significant differences between groups were detected in the amount of viral RNA detected in BAL samples (Fig. 2, E and F). At 6 dpi, animals were euthanized, and tissues were collected, including tissues from the upper and lower respiratory tract and intestinal tract. Although significant differences in the amount of virus detected in nasal swabs were found, the amount of viral RNA in nasal turbinates was not significantly different between groups (Fig. 3A). In contrast, viral RNA in lung tissue was significantly lower in animals inoculated with Omicron BA.1 and BA.2 compared to Delta (Fig. 3B). Additional tissue samples were analyzed and, if positive, showed a higher gRNA

Omicron BA.1 and BA.2 inoculation caused decreased respiratory pathology
In nasal turbinates, minimal to moderate inflammation was observed and consisted of a submucosal infiltrate of neutrophils, macrophages, and lymphocytes that infiltrated the overlaying mucosa and were interspersed with individual and small clusters of necrotic cells. SARS-CoV-2 antigen in the nasal turbinates was extremely rare and was detected in three of the six Delta-challenged animals, one of the six Omicron BA.1-challenged animals, and one of the six Omicron BA.2-challenged animals within both respiratory and olfactory epithelium (Figs. 4, A and B; 5, A and B; and 6A). It is unknown as to what extent the inflammation in the turbinates may be attributable to viral challenge or is background inflammation, as SARS-CoV-2 antigen was found in both inflamed and noninflamed tissue sections. The trachea showed a milder inflammation than the nasal turbinates. Three of the six animals in the Delta group, all six Omicron BA.1 animals, and no Omicron BA.2 animals were found to have inflammation in the trachea. Unexpectedly, only two animals had SARS-CoV-2 antigen, and both were challenged with the Delta variant. This may suggest that the virus was cleared from these antigen-negative tissues or that inflammation was caused by repeated intubation of the animals (Figs. 4C and 5C).
Less inflammation was noted in the bronchi when compared to the trachea, two of the six Delta animals, three of the six Omicron BA.1 animals, and none of the Omicron BA.2 animals exhibited inflammation. Like the trachea, only two Delta-challenged macaques had bronchial mucosal immunoreactivity to SARS-CoV-2 (Figs. 4D and 5D).
Gross lung lesions associated with SARS-CoV-2 pneumonia were identified as foci of consolidation and were noted in three animals in the Delta group, one animal in the Omicron BA.1 group, and two animals in the Omicron BA.2 group (Fig. 6B). The observed features of SARS-CoV-2 pneumonia in this study included thickening of the alveolar septa with fibrin, edema and inflammatory cells, intraalveolar inflammation, type II pneumocyte hyperplasia, reactive endothelial cells in blood vessels, and perivascular inflammation. The inflammatory cells present included neutrophils, macrophages, and lymphocytes. When present, lesion severity ranged from minimal to mild in the Omicron BA.1 and BA.2 groups, and minimal to moderate in the Delta groups. Two of the six animals in the Delta group developed lesions in much higher frequency and severity than the other four animals. SARS-CoV-2 antigen was rarely detected but was present in type I pneumocytes and mononuclear cells in foci with and without features of pneumonia in all six Omicron BA.1-inoculated macaques and three of the six Omicron BA.2-challenged macaques. Comparatively, more SARS-CoV-2 antigen could be detected in all six of the Delta-challenged group, which ranged from rare to multifocal in severity (Figs. 4E, 5E, and 6C).
Overall, infection with all three VOCs resulted in lesions typical of SARS-CoV-2 pneumonia in macaques. Omicron BA.1 and BA.2 VOCs resulted in a lower number of lesions with lesser severity than observed in animals infected with the Delta VOC.

Cytokines and chemokines are up-regulated in animals inoculated with Delta
The presence of nine different cytokines was analyzed in nasosorption, serum, and BAL samples. Compared to baseline, nasosorption samples obtained from animals challenged with Delta showed an elevated immune response on all days. In particular, interleukin-1 (IL-1) receptor antagonist (IL1-RA), IL-6, IL-15, and tumor necrosis factor-α (TNF-α) were increased. In comparison, IL-6, IL-15, and TNF-α in nasosorption samples from animals inoculated with Omicron BA.1 and BA.2 were only moderately elevated or decreased compared to baseline samples. IL-1RA was elevated on 0 dpi in animals that received Omicron BA.1 and did not significantly increase over time ( Fig. 7A and fig. S6). Very few changes in cytokine and chemokine levels were observed in BAL samples: IL-1RA was up-regulated in animals that were inoculated with Omicron BA.1 on 2 dpi (Fig. 7B and fig. S6). In serum samples, similar responses were seen in all groups. IL-1RA was up-regulated in all three groups on 2 dpi. TNF-α was slightly down-regulated on 4 and 6 dpi, although the absolute values showed only a minor drop. On 6 dpi, the groups diverged slightly: IL-1RA, IL-6, and IL-8 were up-regulated in the Delta group, whereas IL-6 and monocyte chemoattractant protein 1 (MCP-1) were down-regulated in the Omicron BA.1 and BA.2 groups ( Fig. 7C and fig. S6).

DISCUSSION
Severity of disease is an important variable when considering a public health response, more so when the infectious agent causing disease has become as widespread as SARS-CoV-2. Omicron is the first VOC that has been reported to cause less severe disease in the human population than the preceding VOC wave (8,9). Disease severity is likely to be reduced by the presence of SARS-CoV-2-specific immunity in the population, either through vaccination or previous infections (10). In South Africa, where Omicron rapidly displaced the Delta VOC, a lower proportion of reported infections ended in hospitalizations and deaths during the Omicron wave as compared to previous waves with the ancestral, Beta, and Delta variants (11). However, the seroprevalence of SARS-CoV-2 immunoglobulin G was determined to be 68.4% before the Omicron wave, compared to 19.1% after the Beta wave. The increased rates of immunity generated either by vaccine or infection likely play a crucial role in the reduction of disease severity (12). Whether disease severity would have been reduced in the absence of preexisting immunity is now not known. Studies using both hamsters and mice have shown that infection with Omicron BA.1 resulted in a lack of weight loss and lower viral burdens in the upper and lower respiratory tract compared to other SARS-CoV-2 VOCs (13). Furthermore, viral loads in nasal swabs obtained from NHPs inoculated with Omicron BA.1 appear low compared to viral loads in nasal swabs obtained from NHPs inoculated with a lineage A isolate, whereas viral load in BAL appears similar (14,15).
Here, we show that rhesus macaques infected with Omicron BA.1 or BA.2 behave very similarly. Animals inoculated with Omicron BA.1 or BA.2 shed less virus and have a lower virus load in the lower respiratory tract than rhesus macaques infected with the Delta variant. This is accompanied by a reduction in observed clinical signs of disease, inflammatory lesions in the respiratory tract, and a decrease in the innate immune response in Omicron-inoculated animals compared to Delta-inoculated animals. Whereas the detection of viral RNA was mostly limited to the respiratory tract in Omicron-inoculated animals, viral RNA in Delta-inoculated animals was found in extra-respiratory tissues. Overall, these results support the notion that Omicron infection results in less severe disease, even in the absence of preexisting immunity. It is possible that this difference is driven by the S protein of Omicron. In our entry studies, we show a reduced entry of Omicron compared to Delta for both human and rhesus macaque ACE2 in a BHK cell line. A similar difference in entry has been observed in Calu-3 and A549 cell lines, but not human embryonic kidney (HEK) cell lines, which may be driven by the TMPRSSII-independent, cathepsin-dependent endosomal entry pathway that Omicron favors compared to Delta (16). In our hands, Omicron BA.1 was extremely inefficient at entry in 293T cells, which are low in TMPRSSII, whereas this was not observed for BA.2. There thus may be a difference in TMPRSSII dependency between different Omicron variants. The reduction in shedding of viral RNA that we observed in animals inoculated with Omicron compared to Delta aligns with some of the shedding data published on vaccinated and unvaccinated individuals (10), although not all (17). Puhach et al. (17) determined viral load and infectious virus titers in nasopharyngeal samples of 384 symptomatic individuals and did not find a difference in viral load or infectious virus. Chaguza et al. (10) analyzed 37,877 nasal swab samples and showed consistently lower viral loads for samples obtained from participants infected with Omicron compared to Delta, independent of vaccination status. Because the difference in the cycle threshold (Ct) values are subtle in this study (less than one cycle), it is possible that, in humans, the Fig. 6. Scoring of pathology and SARS-CoV-2 antigen staining in nasal turbinate and lung tissue, and gross pathology in lung tissue. (A) Scoring between 0 (no pathology or staining) and 5 (severe pathology or diffuse staining) of nasal turbinates was done by a board-certified veterinary pathologist who was blinded to the study groups. (B) Gross pathology was scored per lung lobe (6 in total), dorsal and ventral side. (C) Scoring between 0 (no pathology or staining) and 5 (severe pathology or diffuse staining) of lung tissue per lung lobe was done by a board-certified veterinary pathologist who was blinded to the study groups. H&E, hematoxylin and eosin; IHC, immunohistochemistry sample numbers must be high to show significant differences in virus loads. We compared the amount of virus shed and detected in respiratory tract tissue between all six VOCs. Previously, we showed that there was no difference in the amount of viral RNA shed in nose swabs, BAL, or BCB samples for D614G, Alpha, and Beta VOCs. In nasal swabs, Omicron BA.1 and BA.2 were mostly similar to D614G, Alpha, and Beta, whereas the viral load detected in nasal swabs from animals inoculated with the Delta VOC was higher. In BCB samples, viral load was again highest for the Delta VOC, whereas no differences between the VOCs were observed in BAL samples. In lung tissue, the amount of viral RNA was significantly higher for animals inoculated with the Delta VOC when compared to all other VOCs. Thus, Delta is the VOC most efficient at replication in the naïve rhesus macaque model.
Nonetheless, the Omicron VOC has replaced the Delta VOC in the human population, and our study was not designed to address this question. Omicron is antigenically the most distant VOC (18), and recent studies suggest that Omicron variants can readily overcome immunity acquired from previous infection with earlier variants and vaccination (19)(20)(21)(22)(23). The rise in Omicron cases could be a combination of immune evasion, waning immunity, relaxation of coronavirus disease-related restrictions, and other factors that may affect transmission, such as reduced symptoms caused by Omicron, resulting in prolonged contact with other humans. None of these features were investigated in our study, and their influence can thus not be assessed.
We assessed the cytokine and chemokine response in three different samples: Nasosorption samples represent the upper respiratory tract, BAL samples represent the lower respiratory tract, and serum samples represent the systemic response. In the upper respiratory tract, cytokines and chemokines were up-regulated to higher levels in Delta-inoculated animals than in Omicron BA.1-or BA.2inoculated animals, whereas the systemic response was comparable. This is likely directly correlated to the amount of antigen: We consistently found higher viral loads in nasal swabs, BCBs, and lung tissues of animals inoculated with Delta compared to that with Omicron. This highlights the need for obtaining samples from the site of virus replication to obtain a full understanding of the innate immune response, both in animal studies and in patients.
Here, we show that, in naïve rhesus macaques, the Delta VOC replicated to higher viral loads than the D614G, Alpha, Beta, and Omicron BA.1 and BA.2 variants, resulting in more virus shed and increased replication in lung tissue. Although similar results were found in small animal models of SARS-CoV-2 infection, this study directly compares Delta, Omicron BA.1, and Omicron BA.2 in a species that share the same ACE2 receptor sequences to humans. The reduction in viral load, disease, and pathology detected following Omicron BA.1 and BA.2 infection is reflective of what is seen in the human population. Last, this study further validates the rhesus macaque model for continued evaluation and comparison of the phenotype and pathogenicity of novel emerging variants.

Study design
Three groups of six rhesus macaques were inoculated with either SARS-CoV-2 VOC Delta AY.106 (hCoV-19/USA/MD-HP05647/ 2021, EPI_ISL_2331496), SARS-CoV-2 VOC Omicron BA.1 (hCoV-19/USA/GA-EHC-2811C/2021, EPI_ISL_7171744), or SARS-CoV-2 VOC Omicron BA.2 (hCoV-19/Japan/UT-NCD1288-2 N/2022, EPI_ISL_9595604). Eighteen rhesus macaques between the ages of 2 and 22 were randomly divided into groups of six animals consisting of three females and three males. The age range of each group was as follows: Delta was 3 to 22 years, BA.1 was 6 to 19 years, and BA.2 was 2 to 4 years. Each group of animals was housed in a separate room. The animals were inoculated as previously described (1). Briefly, NHPs were inoculated intranasally (0.5 ml) and intratracheally (4 ml) with a total dose of 2 × 10 6 TCID 50 virus dilution in sterile Dulbecco's modified Eagle's medium (DMEM). The inoculum dose was confirmed by titration on Vero E6 cells. The same person, blinded to the study groups, assessed the animals throughout the study using a standardized scoring sheet (24) and based on the evaluation of the following criteria: general appearance and activity, appearance of skin and coat, discharge, respiration, feces and urine output, and appetite. Area-under-the-curve analysis was performed using GraphPad Prism 9.3.1 to obtain a single clinical score value per animal. Clinical exams were performed on 0, 2, 4, and 6 dpi. Swabs (nose, throat, and rectal), nasosorption samples, BCB samples, and blood were collected at all exam dates. Nasosorption samples were collected as previously described (25). On −10, 2, and 4 dpi, animals were intubated, and BALs were performed using 10 ml of sterile saline. Ventrodorsal and right/left lateral thoracic radiographs were taken before any other procedures. Two board-certified clinical veterinarians blinded to study groups scored the radiographs for the presence of pulmonary infiltrates according to a standard scoring system as previously described (1). Scores may range from 0 to 18 for each animal on each exam day. On 6 dpi, all animals were euthanized; after euthanasia, necropsies were performed, and 27 tissue samples were collected.

Ethics and biosafety
The Institutional Animal Care and Use Committee of Rocky Mountain Laboratories, National Institutes of Health (NIH) approved all animal experiments. Experiments are carried out in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility, according to the institution's guidelines for animal use, following the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, U.S. Department of Agriculture, and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Rhesus macaques were single-housed in adjacent primate cages, which allow social interactions. The animal room was climate-controlled with a fixed light-dark cycle (12-hour light/12-hour dark). Commercial monkey chow was provided twice daily. Water was available ad libitum. The diet was supplemented with treats, vegetables, or fruit at least once a day. Environmental enrichment consisted of a variety of human interaction, manipulanda, commercial toys, videos, and music. Animals were monitored at least twice daily throughout the experiment. The Institutional Biosafety Committee approved work with SARS-CoV-2 under the biosafety level 3 conditions and subsequent sample inactivation for removal of specimens from high containment (26).

SARS-CoV-2 entry in BHK and HEK 293T cells using human and rhesus macaque ACE2 receptors
BHK or HEK 293T cells were seeded in black 96-well plates at 6.0 × 10 5 cells/ml 1 day before transfection (n = 8 wells per variant, experiment repeated twice for BHK cells). The next day, cells were transfected with 100 ng of human or rhesus ACE2 receptor plasmid DNA using polyethylenimine (Polysciences). After 24 hours, cells were inoculated with 100 μl of pseudotype stocks at a 1:10 dilution. Plates were then centrifuged at 1200g at 4°C for 1 hour and incubated overnight at 37°C. Approximately 16 to 20 hours after infection, Bright-Glo luciferase reagent (Promega) was added to each well, at a 1:1 dilution, and luciferase was measured. Relative entry was calculated by normalizing the relative light unit for variant S pseudotypes to the plate relative light unit average for the lineage A spike pseudotype.

Plasmids
Plasmids of the human and rhesus macaque ACE2 receptors and S coding sequences for SARS-CoV-2 lineage A, Delta, Omicron BA.1, and Omicron BA.2 were developed. All plasmids used the pcDNA3.1 + vector (GenScript) and were verified by Sanger sequencing (ACGT). Because coronavirus S proteins with a 19amino acid deletion at the C terminus have previously been found to have an increase in incorporation for virions of VSV (27), all S sequences in the plasmids included the 19-amino acid truncation. In addition, the S sequences were codon-optimized for human cells as well as appended with a 5′ Kozak expression sequence (GCCACC) and 3′ tetra-glycine linker, followed by nucleotides encoding a FLAG-tag sequence (DYKDDDDK).

Pseudotype production
Pseudotype production followed a previously established protocol (28). Briefly, plates precoated with poly-L-lysine (Sigma-Aldrich) were seeded with 293T cells and transfected the following day with 1200 ng of empty plasmid and 400 ng of plasmid encoding coronavirus S or no-S plasmid control (green fluorescent protein). After 24 hours, transfected cells were infected with VSVΔG seed particles pseudotyped with VSV-G. After an hour of incubating with intermittent shaking at 37°C, cells were washed four times and incubated in 2 ml of DMEM2 for 48 hours. Supernatants were collected, centrifuged at 500g for 5 min, aliquoted, and stored at −80°C.
Virus RNA extraction and quantitative polymerase chain reaction RNA was extracted from liquid samples using a QiaAmp Viral RNA kit (Qiagen) according to the manufacturer's instructions, whereas tissue was homogenized and extracted using an RNeasy kit (Qiagen) according to the manufacturer's instructions. Viral gRNA (29) and sgRNA (30) were detected using specific assays: RNA (5 μl) was tested with the QuantStudio (Thermo Fisher Scientific) according to instructions of the manufacturer. SARS-CoV-2 standards with known genome copies were run in parallel to allow for quantification.

Histopathology
Tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and embedded in paraffin, followed by staining with hematoxylin and eosin or using a custom-made rabbit antiserum against SARS-CoV-2 N at a 1:1000 dilution. Stained slides were analyzed by a board-certified veterinary pathologist who was blinded to the study groups. Histologic lesion severity was scored per lung lobe according to a standardized scoring system evaluating the presence of interstitial pneumonia, type II pneumocyte hyperplasia, edema and fibrin, and perivascular lymphoid cuffing as follows: 0, no lesions; 1, minimal (1 to 10% of lobe affected); 2, mild (11 to 25%); 3, moderate (26 to 50%); 4, marked (51 to 75%); and 5, severe (76 to 100%). The presence of viral antigen was scored per lung lobe according to a standardized scoring system: 0, none; 1, rare/few; 2, scattered; 3, moderate; 4, numerous; and 5, diffuse.

Cytokine and chemokine analysis
The U-PLEX Biomarker Group 1 (NHP) Assay kit (MSD, K15068L-2) from MSD was used to test the presence of nine cytokines (granulocyte-macrophage colony-stimulating factor, interferon-γ, IL-1β, IL-1RA, IL-6, IL-8, IL-15, MCP-1, and TNF-α) in nasosorption, serum, and BAL NHP samples. The plates were immediately read using the Meso Quickplex instrument (MSD, K15203D). The data were extracted from the plates using the MSD Workbench 4.0 software. The fold change compared to prechallenge samples and log 2 values for the BAL, serum, and nasosorption samples were calculated using Microsoft Excel and graphed using GraphPad Prism 9.1.1 (225) software.

Statistical analysis
Statistical analyses were performed using GraphPad Prism software version 8.2.1. For all analyses, a P value of 0.05 was used as cutoff for statistical significance.

Supplementary Materials
This PDF file includes: Table S1 Figs. S1 to S6 View/request a protocol for this paper from Bio-protocol.