key: cord-0981479-jvbucw9l
authors: Dhakal, Santosh; Ruiz-Bedoya, Camilo A.; Zhou, Ruifeng; Creisher, Patrick S.; Villano, Jason S.; Littlefield, Kirsten; Castillo, Jennie Ruelas; Marinho, Paula; Jedlicka, Anne; Ordonez, Alvaro A.; Majewski, Natalia; Betenbaugh, Michael J.; Flavahan, Kelly; Mueller, Alice L.; Looney, Monika M.; Quijada, Darla; Mota, Filipa; Beck, Sarah E.; Brockhurst, Jacqueline; Braxton, Alicia; Castell, Natalie; D’Alessio, Franco R.; Pate, Kelly A. Metcalf; Karakousis, Petros C.; Mankowski, Joseph L.; Pekosz, Andrew; Jain, Sanjay K.; Klein, Sabra L.
title: Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model
date: 2021-04-04
journal: bioRxiv
DOI: 10.1101/2021.04.02.438292
sha: d840c69c7cfdcbd78f38e048e450df460456ecab
doc_id: 981479
cord_uid: jvbucw9l

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 105 TCID50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFNβ and TNFα, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population. Importance Men experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection. One Sentence Summary Following SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.

At the start of the coronavirus disease 2019 (COVID-19) pandemic, early publications 76 from Wuhan, China (1, 2) and European countries (3) began reporting male biases in 77 hospitalization, intensive care unit (ICU) admissions, and mortality rates. Ongoing real-time 78 surveillance (4) and meta-analyses of over 3 million cases of continue to show 79 that while the incidence of COVID-19 cases are similar between the sexes, adult males are 80 almost 3-times more likely to be admitted into ICUs and twice as likely to die as females. 81

Differential exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is 82 likely associated with behaviors, occupations, comorbidities and societal and cultural norms (i.e., 83 gender differences) that impact the probability of exposure, access to testing, utilization of 84 healthcare, and risk of disease (6-8). This is distinct but also complementary to biological sex 85 differences (i.e., sex chromosome complement, reproductive tissues, and sex steroid hormone 86 concentrations) that can also impact susceptibility and outcomes from COVID-19 (9, 10). While 87 exposure to SARS-CoV-2 may differ based on gender, the increased mortality rate among males 88 in diverse countries and at diverse ages likely reflect biological sex. Studies have shown that in 89 males, mutations in X-linked genes (e.g., TLR7) resulting in reduced interferon signaling (11), 90 elevated proinflammatory cytokine production (e.g., IL-6 and CRP) (2, 12), reduced CD8+ T cell 91 activity (e.g., IFN-) (13), and greater antibody responses (i.e., anti-SARS-CoV-2 antigen-92 specific IgM, IgG, and IgA, and neutralizing antibodies) (14) are associated with more severe 93 COVID-19 outcomes as compared with females. Because COVID-19 outcomes can be impacted 94 by both gender and biological sex, consideration of the intersection of these contributors is 95 necessary in human studies (15) . 96

Animal models can mechanistically explore sex differences in the pathogenesis of SARS-97

CoV-2 independent of confounding gender-associated factors that impact exposure, testing, and 98 use of healthcare globally. Transgenic mice expressing human ACE2 (K18-hACE2) are 99 susceptible to SARS-CoV-2 and in this model, males experience greater morbidity than females, 100 despite having similar viral loads in respiratory tissues (e.g., nasal turbinates, trachea, and lungs) 101 (16, 17) . Transcriptional analyses of lung tissue revealed that inflammatory cytokine and 102 chemokine gene expression is greater in males than females early during infection, and these 103 transcriptional patterns show a stronger correlation with disease outcomes among males than 104 females (16, 17) . In addition to utilizing hACE2 mice, mouse-adapted strains of SARS-CoV-2 105 have been developed and can productively infect wild-type mice but have not yet been used to 106 evaluate sex-specific differences in the pathogenesis of disease (18) (19) (20) . 107

Golden Syrian hamsters are also being used as an animal model of SARS-CoV-2 108 pathogenesis because they are susceptible to human clinical strains of viruses, without the need 109 for genetic modifications in either the host or virus. While studies have included males and 110 females in analyses of age-associated differences in the pathogenesis of SARS-CoV-2 (21), few 111 studies have specifically evaluated males vs. females to better understand sex differences in 112 disease. There are studies of golden Syrian hamsters that have included male and female 113 hamsters but did not have sufficient numbers of animals to accurately compare the sexes (22). 114

Sex differences are not reported in either viral RNA, infectious virus, or cytokine mRNA 115 expression at a single time point (i.e., 4 days post-infection) in the lungs of golden Syrian 116 hamsters (23). There is a gap in the literature of studies designed to rigorously test the hypothesis 117 that biological sex alters disease severity and immune responses after SARS-CoV-2 infection. 118

Results 120

Intranasal inoculation of human clinical isolates of SARS-CoV-2 causes productive 123 infection in golden Syrian hamsters (24) (25) (26) . To test the hypothesis that SARS-CoV-2 infection 124 results in sex differences in disease outcomes, adult male and female golden Syrian hamsters 125 were infected with 10 5 TCID 50 of virus and changes in body mass were monitored for 28 days 126 post-inoculation (dpi). Mortality was not observed in either sex, but infected hamsters 127 progressively lost body mass during the first week before starting to recover ( Figure 1A) . The 128 peak body mass loss in female hamsters was observed at 6 dpi (-12.3±1.8%), whereas peak mass 129 loss in male hamsters was observed at 7 dpi (-17.3±1.9%). The percentage of body mass loss was 130 significantly greater in male than female hamsters at 8 to 10 dpi and throughout the recovery 131 period (p<0.05; Figure 1A ). Recovery to baseline body mass after SARS-CoV-2 infection 132 occurred within 2 weeks for female and at 3 weeks for male hamsters ( Figure 1A) . 133

To evaluate pulmonary disease in SARS-CoV-2-infected males and females, chest 134 computed tomography (CT) was performed at the peak of lung disease (7 dpi). As previously 135 reported by others (26), multiple and bilateral mixed ground-glass opacities (GGO) and 136 consolidations were detected in both females and males ( Figure 1B and Supplementary Figure  137 1). In order to reduce bias in the visual assessment, we developed an unbiased approach to 138 quantify lung disease by chest CT. Volumes of interest (VOIs) were drawn to capture total and 139 diseased (pneumonic) lung volumes ( Figure 1C) . As reported in COVID-19 patients who 140 underwent CT (27, 28) , there was significantly more disease in the lung of male versus female 141 hamsters (p<0.05) ( Figure 1D) . These results indicate that infected male hamsters developed 142 more severe disease, including more extensive lung injury, than females. 143

Previous studies show that estrogens, including but not limited to estradiol (E2), are anti-144 inflammatory and can reduce pulmonary tissue damage following respiratory infections, 145 including with influenza A viruses or Streptococcus pneumoniae (29) (30) (31) . To test the hypothesis 146 that E2 could dampen inflammation and pulmonary tissue damage to improve outcomes in male 147 hamsters, males received either exogenous E2 capsules or placebo capsules prior to SARS-CoV-148 2 infection. Plasma concentrations of E2 were significantly elevated in E2-treated males 149 compared with placebo-treated males (p<0.05; Figure 2A ) and were well within the normal 150 range of plasma concentrations of E2 in cyclic female hamsters (30-700pg/mL) (32). Animals 151 were followed for 7 dpi and changes in body mass and chest CT score were quantified. There 152 was no effect of E2-treatment on morbidity as placebo-and E2-treated males had equivalent 153 percentages of body mass loss ( Figure 2B) . CT findings noted in E2-treated males were similar 154 to those noted in placebo-treated males (Supplementary Figure 1) and chest CT scans revealed 155 in CT score between groups ( Figure 2C ). Moreover, histopathology demonstrated similar cell 156 infiltration and pneumonic areas between groups ( Figure 2D ). From these data, we conclude that 157 the treatment of gonadally-intact males with E2 did not improve morbidity or pulmonary 158 outcomes from SARS-CoV-2 infection. 159

To test the hypothesis that male-biased disease outcomes were caused by increased virus 162 load or faster replication kinetics, subsets of infected male and female hamsters were euthanized 163 at 2, 4, or 7 dpi and infectious virus titers were measured in the respiratory tissue homogenates. 164

The peak infectious virus titers in the nasal turbinates ( Figure 3A) , trachea (Figure 3B) , and 165 lungs ( Figure 3C ) were detected at 2 dpi, decreased at 4 dpi, and was cleared at 7 dpi. There 166 were no sex differences in either peak virus titers or clearance of SARS-CoV-2 from any of the 167 respiratory tissues tested (Figure 3A-C) . Although the infectious virus was cleared from the 168 respiratory tract of most of the hamsters by 7 dpi (Figure 3A-C) , viral RNA was still detectable 169 in the lungs at 14 dpi in all of the SARS-CoV-2 infected hamsters, with no differences between 170 the sexes ( Figure 3D) . These data illustrate that sex differences in the disease phenotype are not 171 due to differences in infectious virus loads or persistence of viral RNA. 172 173

To test whether local or systemic cytokine activity differed between the sexes, 175

concentrations of cytokines were measured in lung and spleen homogenates at 2, 4, or 7 dpi. Sex 

To evaluate whether females developed greater antiviral antibody responses than males, 197 as is observed in response to influenza A viruses (33), we measured virus-specific 198 immunoglobulins as well as neutralizing antibody (nAb) titers in plasma and respiratory samples 199 collected throughout the course of infection. To begin our evaluation, we inactivated SARS-200

CoV-2 virions to analyze plasma IgG that recognize diverse virus antigens. Anti-SARS-CoV-2 201

IgG titers were detected within a week post-infection, with females developing greater antibody 202 titers than males at 21 and 28 dpi (p<0.05; Figure 5A ). Using live SARS-CoV-2, we measured 203 nAb titers in plasma, which were detectable 7-28 dpi, with females having or trending towards 204 significantly greater titers than males at 14-28 dpi (p<0.05; Figure 5B) . S-RBD mutants N501Y, Y453F, N439K, and E484K were significantly greater in female than 219 male hamsters (p<0.05 in each case; Figure 5F ). Overall, IgG responses to the E484K, but not 220 the N501Y variant, were significantly lower in both sexes as compared with responses to the 221 wild-type S-RBD (p<0.05 for main effect of variant; Figure 5F ). 222

Local antibody responses at the site of infection are critical for SARS-CoV-2 control and 223 recovery (39, 40). Anti-S-RBD-IgM titers were greatest in the lungs at 7 dpi, being significantly 224 greater in female than male hamsters (p<0.05; Figure 6A ). A cornerstone of mucosal humoral 225 immunity is IgA and anti-S-RBD IgA titers peaked at 7 dpi, with a trend for higher titers in 226 females than males (p=0.07; Figure 6B ). By 28 dpi, females still had detectable anti-S-RBD IgA 227 titers in their lungs, whereas males did not (p<0.05; Figure 6B ). Anti-S-RBD IgG titers in the 228 lungs were elevated 7-28 dpi with a higher trend observed at 28dpi in females than males 229 (p=0.09; Figure 6C ). In the trachea, but not in nasal turbinate or lung homogenates, females had 230 significantly greater anti-S-RBD IgG titers than males (p<0.05; Figure 6D ). In summary, these 231 data demonstrate that female hamsters develop greater systemic and local antiviral antibody 232 responses compared with male hamsters during SARS-CoV-2 infection.

Sex differences in COVID-19 outcomes are well documented (9, 13). There is a critical 236 need to develop accurate animal models that reflect the male-bias in disease outcomes to better 237 understand the underlying mechanisms. We show that male hamsters suffer more systemic (body 238 mass loss) and local (pulmonary pathology) symptoms of SARS-CoV-2 infection than females. 239

We tested several potential mechanisms that could mediate male-biased outcomes from 240 infection, including: 1) lack of estrogenic protection, 2) greater virus replication, 3) exacerbated 241 cytokine responses, and 4) reduced humoral immunity. Our data reveal that females produce 242 greater antibody responses, both locally in the respiratory tract as well as systemically in plasma, 243 but if this causes female hamsters to suffer less severe outcomes from SARS-CoV-2 infection 244 remains to be determined. shown that hamsters lose body mass after infection, reaching peak loss at 5 to 7 dpi, followed by 248 recovery (24-26). Body mass loss in hamsters, regardless of age, has been associated with the 249 dose of virus inoculum, with higher dose resulting in greater body mass loss (26, 41). Body mass 250 loss also is influenced by age; older hamsters (i.e., 7 to 9 months old) had greater mass loss than 251 younger animals (i.e., 4-6 weeks old) (21, 26). Sex is another factor impacting body mass loss, as 252 a reliable clinical sign of disease in hamsters following SARS-CoV-2 infection. As reported in 253 humans, older age and male sex are clinical variables associated with greater clinical 254 manifestations of disease in hamsters. 255

A novel determinant of clinical disease that was utilized in the current study was 256 unbiased, quantitative chest CT-imaging analysis. Previous reports describe chest CT findings in 257 female SARS-CoV-2 infected hamsters only and show lung abnormalities, including 258 multilobular ground-glass opacities (GGO) and consolidation (26), as observed in patients with 259 COVID-19 (42). In the current study, CT-imaging revealed that multilobular GGO and 260

consolidations were observed to a greater extent in male than female SARS-CoV-2-infected 261 hamsters at 7 dpi. Whether the sexes differ in the recovery of pulmonary damage following 262 infection requires greater consideration. There are a number of registered clinical trials of 263 therapeutic E2 administration (NCT04359329 and NCT04539626) in COVID-19, which raised 264 the question as to whether disease outcomes in male hamsters could be improved through 265 administration of E2. In this study, pre-treatment of male hamsters with E2 prior to SARS-CoV-266 2 infection did not reduce either weight loss, observed histological damage to lung tissue or the 267 observed multilobular GGO and consolidations. 268 SARS-CoV-2 replicates in the nasal turbinates, trachea, and lungs of infected golden 269

Syrian hamsters (24, 25). Virus replication peaks in respiratory tissue within 2-4 dpi, with virus 270 clearance typically occurring within one week (21, 24, 25) . Viral RNA, however, is present in 271 the lungs of infected hamsters beyond 7 dpi (21, 22, 25 Ifnα, and Ifnγ in the nasal turbinates and lungs, is triggered at 2 dpi, peaks at 4 dpi, and returns to 280 baseline by 7 dpi, but comparisons between males and females have not performed (24, 41, 43) . Studies have reported that both IgG and virus neutralizing antibodies are detected in 292 serum from SARS-CoV-2-infected golden Syrian hamsters as early as 7 dpi and persist through 293 43 dpi (24, 35, 44) . In the present study, females developed greater IgG responses against both 294 SARS-CoV-2 wild type and variant S-RBD as well as antiviral nAb titers in both plasma and 295 respiratory tissue homogenates than males. We also showed that mucosal IgA titers are greater 296 in the lungs of female than male hamsters and are detectable as early as 7 dpi. Passive transfer of 297 convalescent sera from infected to naïve hamsters as well as reinfection of previously infected 298 hamsters carrying high antibody titers, have both been shown to provide protection by reducing 299 virus titers in the respiratory tissues (24, 26). Likewise, hamster models of SARS-CoV-2 300 immunization have shown an inverse correlation between antibody responses and either virus 301 titers in the respiratory tissues or body mass loss (45). These studies highlight the possible 302 protective role of antibodies during SARS-CoV-2 infection, which may contribute to faster 303 recovery in female than male hamsters. 304

Golden Syrian hamsters have already been successfully used in SARS-CoV-2 305 transmission studies (24, 25, 46) , to compare routes of SARS-CoV-2 infection (41, 47), to 306 evaluate convalescent plasma and monoclonal antibody therapy (24, 26, (48) (49) (50) , and to test 307 therapeutics and vaccines (23, 45) . This model provides a unique opportunity to understand the 308 kinetics of SARS-CoV-2 immunopathology not only systemically but also at the site of infection, 309 infection. Virus was inactivated by the addition of 0.05% beta-propiolactone (51) followed by 327 incubation at 4C for 18 hours. The beta-propiolactone was inactivated by incubation at 37C for 2 328 hours and the inactivated virions were pelleted by ultracentrifugation at 25000g for 1h at 4 0 C and 329 protein concentration was determined by BCA assay (Thermo Fisher Scientific). animals (8-10 weeks of age) were inoculated with 10 5 TCID 50 (50% tissue culture infectious 339 dose) of SARS-CoV-2 USA-WA1/2020) in 100μL DMEM (50μl/naris) through intranasal route 340 under ketamine (60-80mg/kg) and xylazine (4-5mg/kg) anesthesia administered 341 intraperitoneally. Control animals received equivalent volume of DMEM. Animals were 342 randomly assigned to be euthanized at 2, 4, 7, 14, or 28-days post infection (dpi). Body mass was 343 measured at the day of inoculation (baseline) and endpoint, with daily measurements up to 10 dpi 344 and on 14, 21, and 28 dpi, when applicable per group. Blood samples were collected pre-345 inoculation (baseline) and at days 7, 14, 21, and 28 dpi, when applicable per group. Survival 346 blood collection was performed on the sublingual vein, whereas terminal bleeding was done by 347 cardiac puncture under isoflurane (500μl drop jar; Fluriso™, VetOne ® , Boise, ID) anesthesia. 348

Plasma was separated by blood centrifugation at 3500rpm, 15min at 4 0 C. After cardiac puncture, 350 animals were humanely euthanized using a euthanasia solution (Euthasol ® , Virbac, Fort Worth, 351 TX). Nasal turbinates, trachea, and lung samples for antibody/cytokine assays and virus titration 352 were snap frozen in liquid nitrogen and stored at -80 0 C. 353 354

To 355 obtain tissue homogenates, DMEM with 100unit/mL penicillin and 100 μg/mL streptomycin was 356 added (10% w/v) to tubes containing hamster nasal turbinate, lungs, and tracheal tissue samples. 357

Lysing Matrix D beads were added to each tube and the samples were homogenized in a 358

FastPrep-24 bench top bead beating system (MPBio) for 40sec at 6.0m/s, followed by 359 centrifugation for 5min at 10,000g at room temperature. Samples were returned to ice and the 360 supernatant was distributed equally into 2 tubes. To inactivate SARS-CoV-2, TritonX100 was 361 added to one of the tubes to a final concentration of 0.5% and incubated at room temperature for 362 30 minutes. The homogenates were stored at -70 0 C. 363

Infectious virus titers in respiratory tissue homogenates were determined by TCID 50 364 assay (14, 51). Briefly, tissue homogenates were 10-fold serially diluted in infection media (CM 365 with 2.5% instead of 10% FBS), transferred in sextuplicate into the 96-well plates confluent with 366

Vero-E6-TMPRSS2 cells, incubated at 37 0 C for 4 days, and stained with naphthol blue-black 367 solution for visualization. The infectious virus titers in TCID 50 /mL were determined by Reed and 368

Muench method. For detection of SARS-CoV-2 genome copies, RNA was extracted from lungs 369 using the Qiagen viral RNA extraction kit (Qiagen) and reverse transcriptase PCR (qPCR) was 370 performed as described (52). antibody ELISA protocol described previously (14). ELISA plates (96-well plates, Immunol 4HBX, Thermo Fisher Scientific) were coated with either spike receptor binding domain (S-395 RBD) or whole inactivated SARS-CoV-2 proteins (2 μg/mL, 50μl/well) in 1X PBS and 396 incubated at 4 0 C overnight. Coated plates were washed thrice with wash buffer (1X PBS + 0.1% 397 Tween-20), blocked with 3% nonfat milk solution in wash buffer and incubated at room 398 temperature for 1 hour. After incubation, blocking buffer was discarded and two-fold serially 399 diluted plasma (starting at 1:100 dilution) or tissue homogenates (starting at 1:10 dilution) were 400 added and plates were incubated at room temperature for 2 hours. After washing plates 3 times, were removed, fresh infection media was added, and plates were incubated at 37 0 C for 2 days. Cells were fixed with 4% formaldehyde, stained with Napthol blue black solution and 418 neutralizing antibody titer was calculated as described (14). were compared using two-way repeated measures ANOVA followed by Bonferroni's multiple 430 comparison test. Chest CT scores were compared by unpaired Mann-Whitney test. E2 431 concentration were compared by two-tailed unpaired t-test. Virus titers and antibody responses 432 were log transformed and compared using two-way ANOVA or mixed-effects analysis followed 433 by Bonferroni's multiple comparison test. Cytokine concentrations were normalized to total 434 protein content in lung homogenates and compared using two-Way ANOVA. Associations 435 between cytokines and virus titers in lungs were conducted using Spearman correlational 436 analyses. Differences were considered to be significant at p<0.05. 437 438 Data availability: All data will be made publicly available upon publication and upon request for 439 peer review. Estradiol resolves pneumonia via ERβ in regulatory T cells. JCI insight 6. 585 turbinates (A), trachea (B) , and lungs (C), were determined by TCID 50 assay on 2, 4, and 7 dpi. 737

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Weights are represented as 712 mean ± standard error of the mean from two independent replications (n = 9-10/group), and 713 significant differences between groups are denoted by asterisks (*p<0.05) based on two-way 714 repeated measures ANOVA followed by Bonferroni's multiple comparison (A). Chest CT data is 715 represented as median ± interquartile range from two independent replication (13-14/group) and 716 significant differences between groups are

Histopathology 724 (H&E) in a representative SARS-CoV-2-infected placebo-treated male and E2-treated male 725 hamster lungs at 4X magnification are shown (D). The dashed yellow lines indicate lung lesions 726 (GGO, consolidations and air bronchogram). E2 concentrations represented as mean ± standard 727 error of the mean of two independent experiments (n=11-12/group) and significant differences 728 between groups are denoted in asterisk (*p<0.05) based on two-tailed unpaired t-test (A)

Chest CT data represented as median ± interquartile range (IQ) from two independent 731 experiments (n = 13/group) (C)

Considering similar antibody responses at 6 and 7 dpi, values 758 were presented together as 7 dpi. Data represent mean ± standard error of the mean from two 759 independent experiments (n = 4-14/group/sex) and significant differences between groups are 760 denoted by asterisks

Bonferroni's multiple comparison test

Antibody responses in the respiratory system of SARS-CoV-2 infected female 764 hamsters were greater than males. Lung homogenates were prepared at different dpi and S

Data represent mean ± standard error of the mean from one or two independent 768 experiment(s) (n = 3-10/group) and significant differences between groups are denoted by 769 asterisks

Bonferroni's multiple comparison test

Supplementary Figure 1: Representative transverse chest CT of five females, placebo-treated 773 males, and E2-treated male hamsters at 7 dpi. Multiple bilateral and peripheric ground-glass

opacities (GGO) and mixed GGO with consolidations are the hallmarks findings at the peak of 775 lung disease

Supplementary Figure 2: Kinetics of cytokine concentrations (pg/mg total protein)in the lungs 778 of SARS-CoV-2 infected hamsters. Male and female golden Syrian hamsters were infected with 10 5 TCID 50 of SARS-CoV-2

IFN-β (E), and IFN-γ (F) concentrations were determined in the 781 lungs by ELISA. Mock-infected animal samples from 2-, 4-, or 7-days post infection (dpi) were 782 not statistically different and were combined and presented together as 0 dpi. Data represent 783 mean ± standard error of the mean from one or two independent experiments (n = 6-12/group) 784 with significant differences between groups denoted by asterisks

ANOVA followed by Dunnett's multiple comparisons test

Supplementary Figure 3: Associations between concentrations (pg/mg total protein) of IL-1β

IFN-β (E), and IFN-γ (F) and virus titers in lungs collected 789 2 days post infection (dpi). Data were analyzed with Spearman correlation analyses with 790 significant associations represented with the R statistic

Supplementary Figure 4: Associations between concentrations (pg/mg total protein) of IL-1β

IFN-β (E), and IFN-γ (F) and virus titers in lungs collected 794 4 days post infection (dpi). Data were analyzed with Spearman correlation analyses with 795 significant associations represented with the R statistic

Mock-infected animal 799 samples from different dpi were pooled and used as 0 dpi. Data are presented as the 800 mean ± standard error of the mean from one or two independent experiments