key: cord-0794482-noxt3ikd
authors: Meyer, Michelle; Wang, Yuan; Edwards, Darin; Smith, Gregory R.; Rubenstein, Aliza B.; Ramanathan, Palaniappan; Mire, Chad E.; Pietzsch, Colette; Chen, Xi; Ge, Yongchao; Cheng, Wan Sze; Henry, Carole; Woods, Angela; Ma, LingZhi; Stewart-Jones, Guillaume B. E.; Bock, Kevin W.; Minai, Mahnaz; Nagata, Bianca M.; Periasamy, Sivakumar; Shi, Pei-Yong; Graham, Barney S.; Moore, Ian N.; Ramos, Irene; Troyanskaya, Olga G.; Zaslavsky, Elena; Carfi, Andrea; Sealfon, Stuart C.; Bukreyev, Alexander
title: mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge
date: 2021-01-25
journal: bioRxiv
DOI: 10.1101/2021.01.25.428136
sha: a6c538847b813791a9c3a3162b6d768bb865182d
doc_id: 794482
cord_uid: noxt3ikd

The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

When the World Health Organization declared SARS-CoV-2 a global pandemic in March 2020, Phase 1 clinical trials on the most promising vaccine candidates had already commenced.

Nucleoside modified mRNA, a relatively new addition to the arsenal of vaccine platforms, has shown promise against numerous viral infectious diseases in preclinical trials (1, 2) , and phase 1 and 2 trials that are completed (3, 4) or currently underway (5-7). Previous preclinical work on a related betacoronavirus enabled the rapid development of mRNA-1273, a vaccine composed of a modified mRNA encoding for a stabilized prefusion form of the SARS-CoV-2 spike (S) protein encapsulated in lipid nanoparticles (1) . Vaccination with mRNA-1273 prevented infection in the lungs and upper airways of mice (1) and rhesus macaques (8). In phase 1 (9, 10) and phase 2 studies the vaccine was found to be safe and to elicit robust neutralizing antibody responses. More recently, interim analysis of Phase 3 data indicated mRNA-1273 was 94.1% efficacious in prevention of COVID-19 disease and highly efficacious in protecting against severe disease (11) . On December 18, 2020, the U.S. Food and Drug Administration authorized the emergency use of mRNA-1273.

Nonhuman primate (12) (13) (14) , ferret (15) (16) (17) and mouse (18) Here, we tested the efficacy of the mRNA-1273 vaccine with prime only and three dose levels of prime-boost regimens using the stringent golden Syrian hamster model. Serological, virological, clinico-pathological and single cell (sc) RNA-seq analyses were conducted to characterize vaccine-mediated immunity before and after challenge. We show that vaccination induced robust virus neutralizing antibody responses, attenuated virus replication, and mitigated against the influx of inflammatory innate immune cells and the relative reduction of lymphocytes in the lungs after challenge, although activated immune cell populations were observed. Our data illustrates the viral, cellular, and immune dynamics within the lungs of vaccinated hamsters which may offer a unique perspective into the events which occur within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

Three groups of outbred hamsters (n = 15) were vaccinated with 25 µg, 5 µg and 1 µg of mRNA-1273 via the intramuscular (IM) route in a prime (week 0)-boost (week 3) regimen ( Figure 1A ). One group of hamsters (n =15) received a prime-only dose of 25 µg at week 0 and another group (n =15) were mock vaccinated as the study's control. The humoral responses to vaccination were measured by enzyme-linked immunosorbent assay (ELISA) specific for the SARS-CoV-2 S protein ( Figure 1B ) and its receptor binding domain (RBD; Figure 1C ). Three weeks after the prime dose, higher S-specific IgG titers were detected in hamsters vaccinated with 25 µg and 5 µg doses compared to the 1 µg dose ( Figure 1B ). S-specific IgG titers were significantly augmented in all groups following the booster, but continued to be significantly higher in the 25 µg and 5 µg dose groups compared to 1 µg dose group. RBD-specific IgG titers were comparable among hamsters in the prime-boost regimen groups after receipt of their first dose ( Figure 1C ). After a boost dose, RBD titers had increased significantly (P<0.0001) and remained comparable among the prime-boost groups. RBD titers in the prime-boost vaccine groups were also significantly higher after the second dose when compared to the 25 µg primeonly group (P<0.0001), while titers remained unchanged for the 25 µg prime-only group between weeks 3 and 6 post-vaccination. The ability of serum to neutralize live SARS-CoV-2 reporter virus was also determined ( Figure 1D ). Most vaccinated hamsters produced neutralizing titers after the prime dose. As with RBD-binding titers, neutralizing titers significantly increased in all groups which received a booster dose, and the levels remained comparable among these hamster groups, but higher versus the prime-only vaccine group. The magnitude of neutralizing antibody titers in all booster-vaccinated groups was higher than in convalescent COVID-19 patients, while titers in the prime-only group were comparable to those seen in these subjects. Neutralizing titers significantly correlated with S-specific IgG titers (Supplemental Figure 1A ) and RBD-specific titers (Supplemental Figure 1B) , albeit the correlation was greater with RBD-specific IgG titers at both weeks 3 and 6 post-vaccination

Six weeks after prime vaccination (3 weeks after the boost vaccination), all treatment group hamsters were challenged intranasally with 10 5 plaque forming units (PFU) of SARS-CoV-2.

Hamsters were monitored daily for changes in body weight. At 2 and 4 days post infection (dpi), 5 animals from each group were serially euthanized, and the viral load in the right lung and nasal turbinates was determined. The remaining hamsters were observed until the study's endpoint at day 14. Mock vaccinated hamsters lost an average maximum body weight of 12% by day 6 (Figure 2A and Supplemental Figure 2 , A and B). The prime-boost regimen prevented significant weight loss for all but one hamster in the 5 µg prime-boost group; excluding animals necropsied at 2 and 4 dpi, the average maximum weight loss for the combined prime-boost dose vaccine groups was 2.25% over the 14 day infection period (Supplemental Figure 2B) . The prime-only vaccinated group lost a maximal mean weight of 6.2%. Moderate inverse correlations were observed between maximum percent weight loss and S-binding IgG and neutralizing antibody titers at week 6 (Supplemental Figure 3A ). At 2 dpi, high viral loads were detected by plaque assay in the lungs and nasal turbinates of the mock vaccinated group with a peak mean of 6.8 log 10 PFU/g and 6.9 log 10 PFU/g, respectively ( Figure 2B ). Markedly lower levels of virus were detected in the lungs of all vaccinated groups, while the 1 µg prime-boost dose group had no detectable virus in the lungs. The mean peak virus load in the nasal turbinates were similar among the prime-boost groups and 4.3 log 10 lower compared to the mock (P<0.0001). At 4 dpi, there was no detectable virus in the lungs and nasal turbinates of all prime-boost recipients. Hamsters that received the prime-only vaccine dose had 4.8 log 10 and 3.0 log 10 reductions of peak virus load means, respectively, in the lungs and nasal turbinates compared to the control group.

We measured viral subgenomic RNA (sgRNA) in these tissues by qRT-PCR as a potential gauge for replicating virus. Peak sgRNA were detected in the mock group at 2 dpi, with geometric means of 7.4 log 10 (95% CI range 7.0 log 10 -7.9 log 10 ) copies/g in the lungs and 7.3 log 10 (95% CI range 7.1 log 10 -7.5 log 10 ) copies/g in nasal turbinates (Supplemental Figure 2C ).

All prime-boost groups showed similar lower sgRNA levels compared to the mock vaccine group with peak geometric means of 2.1 log 10 (95% CI range 0.86 log 10 -3.3 log 10 ) sgRNA copies/g in lungs and 6.3 log 10 (95% CI range 6.4 log 10 -6.1 log 10 ) sgRNA copies/g in nasal turbinates. In the prime-only vaccine group, sgRNA levels were not significantly reduced in either of these tissues. In close agreement with viral load, no sgRNA was detected in the lungs of prime-boost groups by 4 dpi, except for one hamster in the 1 µg dose group. While viral load was not detected in the nasal turbinates of prime-boost recipients, sgRNA was detected, albeit at markedly reduced levels compared to the mock-vaccinated group. SARS-CoV-2 transmission has been shown to correlate with levels of infectious virus and not sgRNA (22). Therefore, the likelihood of onward transmission was highly reduced by mRNA-1273. The prime-only dose afforded a less robust protection according to sgRNA levels, particularly in the nasal turbinates.

Neutralizing antibody titers at week 6 inversely correlated with viral load and sgRNA in both the lungs and nasal turbinates at 2 and 4 dpi (Supplemental Figure 3B and C).

Fourteen days post challenge, the mock vaccinated hamsters had measurable serum S-(Supplemental Figure 4A ) and nucleocapsid (NP)-specific (Supplemental Figure 4B ) IgG titers and neutralizing titers (Supplemental Figure 4C ). An anamnestic response was observed as soon as 4 dpi with the S-specific IgG increasing in recipients of the prime only vaccine regimen.

At 14 dpi, all vaccinated hamsters displayed an anamnestic S-specific IgG antibody response.

This result contrasts with the unwavering virus-specific IgG levels observed post challenge in vaccinated NHPs (8). Furthermore, NP-specific IgG titers were detected in all groups at 14 dpi (Supplemental Figure 4B ) confirming, together with the detection of sgRNA in the upper and lower respiratory tract (Supplemental Figure 2C) , replication of the challenge virus before clearance.

The lungs of hamsters were evaluated histologically following challenge with SARS-CoV-2 at 2-, 4-and 14-day timepoints ( Figure 2C , Supplemental Figure 5 and Supplemental Figure 6) . A naïve group of hamsters (n = 4), intranasally administered media to mimic virus inoculum and euthanized 4 days later, was included as a control and presented with a moderately prominent alveolar interstitial hypercellularity. While these hypercellular areas may represent regions of atelectasis, the presence of Pasteurella multocida, a common respiratory commensal, was also detected by metagenomics analysis of all lung samples used in scRNA-seq (Supplemental Table 1 ). At 2 dpi, SARS-CoV-2 infection in mock vaccinated animals caused mild interstitial inflammation in the lungs with some animals exhibiting a largely polymorphonuclear cellular infiltrate, comprised predominantly of neutrophils/heterophils, in and around the lung airways (Supplemental Figure 6 and Supplemental Table 2 ). By 4 dpi, inflammation was largely associated with perivascular and peribronchiolar regions in both a focally diffuse or multifocal distribution affecting, on average, 30-50% of the lung that persisted until the study's endpoint Table 2 ). In the 1 µg prime-boost group, the predominant inflammatory phenotype was mild to moderate interstitial and rarely perivascularperibronchiolar (4 dpi) inflammation. However, one animal exhibited a more severe pulmonary inflammatory response at 2 dpi, characterized by mild to moderate edema and rare foci of hemorrhage and vascular congestion that was previously described in SARS-CoV-2 infected hamsters (19). Prime-only vaccinated animals generally exhibited reduced inflammation compared to mock-vaccinated animals at 4 dpi; one outlier hamster presented mild pulmonary edema and scant fibrin deposition which was not observed in the rest of the group. Irrespective Table 2 ). Large amounts of viral antigen were found throughout the lungs of mock-vaccinated hamsters. Prime-boost vaccination limited the amount of detectable total viral antigen more so than prime-only vaccination; SARS-CoV-2 antigen was minimal to absent in the 25 µg and 5 µg prime-boost groups.

Notably, the three outlier hamsters with more severe histopathological phenotypes identified in the 25 µg prime-only group at 4 dpi and in the 1 µg prime-boost group at 2 and 4 dpi, did not exhibit drastic weight loss. Their levels of infectious virus or sgRNA in the lungs or nasal turbinates were also comparable with other group members, with the exception of the single 1 µg recipient at 4 dpi that had detectable sgRNA in the lungs that were not detected in other 1 0 prime-boost recipients at the same time point. Both 4 dpi outliers had the highest level of viral antigen among all vaccinated hamster samples. Interestingly, all three outliers did not produce neutralizing titers after prime vaccination, a finding that suggests the importance of vaccine dose level and regimen, and the evolving immune response following administration of a second dose.

We performed scRNA-seq on the cranial lung lobe tissue samples from three of the study's hamster groups that were euthanized at 4 dpi: the naive hamsters mock-infected with media (Naive, N, n = 4) and the 5 µg prime-boost vaccinated (Vaccinated + Infected, VI, n = 5) and mock-vaccinated (Mock-Vaccinated + Infected, MI, n = 5) groups infected with SARS-CoV-2.

Lung samples from the N group served as a baseline control for analysis. The scRNA-seq data were aligned against the hamster and SARS-CoV-2 genomes and integrated using the Seurat scRNA-seq analysis pipeline (24). The expression profile of one VI animal that showed excessive weight loss at 4 dpi (Supplemental Figure 2A and Supplemental Figure 7A Visualizing these 13 datasets by group on the integrated UMAP coordinates showed an overall change in cell type proportion across MI and VI groups when compared to the N group. Most noticeably, there was a more than 30% increase in the presence of interstitial macrophages in the MI hamsters compared to a negligible fraction in N hamsters ( Figure 3 , B and C).

Importantly, this increase in macrophages was not seen in VI hamsters. Significant increases in proportion of alveolar macrophages, DC subtypes (plasmacytoid DC (pDC), conventional DC (cDC) and activated DC (DCα)), and granulocytes were also found in MI hamsters and not VI hamsters. We also detected decreases in proportion of CD4 + , CD8 + and CD8 + activated T cells in MI hamsters and not VI hamsters. The MI and VI hamsters both showed increases in the proportion of the dividing immune cells and monocytes when compared to N hamsters. The moderate changes in the lung cell composition of VI hamsters and infiltration of inflammatory cells in lungs of MI hamsters are consistent with histology results described in the previous section.

Overlaying the data from the outlying VI hamster onto the UMAP coordinates, together with the other 4 VI samples, showed different cell type composition in this sample ( Figure 3B Figure 7D ). The abnormal cellular composition in the VI outlier was likely due to sampling of heterophilic cellular lesions constrained to the cranial lobe given histopathological analysis on the remaining left lung showed drastic reduction of inflammation.

The activity of heterophils concentrated at a lesion is expected to be different to those patrolling the lung parenchyma, adopting a functional state at the site of a foreign antigen.

The scRNA-seq reads were mapped to the SARS-CoV-2 genome to identify which cell types 

To determine whether immune cells in the lungs showed differences in their transcriptional states in the three groups studied, we focused on two lymphoid cell types critical for viral clearance during respiratory infections, CD8 + T cells and NK cells, which were relatively abundant across all samples. We also examined myeloid cells, including pDCs, cDCs, and monocytes, which bridge innate sensing with adaptive responses. We performed differential expression analysis for each cell type (see Methods), comparing the MI to N samples and the VI to N samples. This analysis in CD8 + T cells showed that less transcripts were differentially expressed in the CD8 + T cells from VI lungs compared to N than CD8 + T cells from MI lungs compared to N, with the DEGs in VI lungs being predominantly a subset of those regulated in the cells from MI samples ( Figure 5A ). A scatter plot comparing the log fold-changes of the set of transcripts significantly regulated (FDR <0.05) in either MI or VI comparisons to N showed that changes of these transcripts were highly correlated (p<2.2e^-16). The slope of the linear regression fit was less than 1, indicating that the gene expression changes were relatively lower in the VI samples. These results suggested that similar transcriptional programs were modulated in these immune cells in the MI and VI hamsters, although the regulation was to a lesser extent in the VI hamsters. Similar analyses of the NK cells, pDC, cDC, and monocytes revealed that the expression of DEGs for both MI and VI conditions compared to N was also highly significantly correlated ( Figure 5B , Figure 6 , A and B and Supplemental Figure 12A ). The slopes of the regression lines showed that the changes were also of lower magnitude in the VIderived cells. Overall, these results indicated that across all cell types analyzed, the transcriptome regulation in the VI group was similar to that of MI group, but the changes were of a lower magnitude in the VI group.

To elucidate the common pathways regulated in the MI and VI groups, we performed a modulebased functional enrichment analysis. Among the modules within the set of upregulated DEGs, immune activation and viral response were noted across CD8 + T cells, NK cells, cDC, and monocytes ( Figure 5 , C and D, Figure 6C and Supplemental Figure 12B ). Common pathway enrichment analysis could not be performed for the pDCs due to limited DEGs identified in the VI group.

Since we generally saw a lower degree of gene activation in the VI group, less genes passed the statistical threshold for differential expression in that group as compared to the MI group.

Given the general conservation of the transcriptional programs between the MI and VI groups, a substantial number of DEGs were common to the two comparisons while another large number of DEGs were specific to the MI group, and relatively few DEGs were specific to the VI group (see Venn diagrams in Figure 5 , A and B, Figure 6A and Supplemental Figure 12A ). We performed module-based functional enrichment analysis on the DEGs that were specific to either MI or VI groups for each cell type analyzed.

Similar pathways were modulated in MI-specific NK DEGs and CD8 + T cell DEGs; viral response, migration, regulation of apoptotic signaling, and cellular responses to interferon-γ and oxidative stress were upregulated, while homeostasis and cellular maintenance were downregulated (Supplemental Figure 10A and Supplemental Figure 11A ). However, the proliferation process was upregulated in CD8 + T cells and negatively regulated in NK cells. Type I interferon production and viral response pathways were upregulated, and cellular maintenance pathways were downregulated in MI-specific cDC DEGs and monocyte DEGs (Supplemental Figure 12C and Supplemental Figure 13A ). Adaptive immune response, including T-cell activation, was modulated in both MI-specific pDC DEGs and MI-specific cDC DEGs ( Figure 6D and Supplemental Figure 13A ). Finally, additional viral response pathways were upregulated in MI-specific pDC DEGs ( Figure 6D ). Since fewer DEGs were identified as VI-specific, there were few regulated pathway modules detected (Supplemental Figure 10B , Supplemental Figure 11B and Supplemental Figure 13B ).

Overall, these data show that SARS-CoV-2 infection induced largely similar transcriptional programs in immune cells for both VI and MI animals, including many viral response and immune activation pathways. In MI-specific transcriptional programs, positive regulation of cytokine signaling stood out as enriched across all cell types studied, while cellular maintenance functions were typically downregulated. On the other hand, in the VI animals, the magnitude of gene expression was lower and fewer differential genes were detected, resulting in few regulated pathway modules.

In humans, COVID-19 can progress to severe clinical disease which manifests as pneumonia. mRNA-1273 was previously shown to be efficacious in NHPs (8), mice (1) and recently, in a large Phase 3 trial (11) . Hamsters consistently exhibit the hallmarks of severe COVID-19 disease and are therefore an important model for preclinical vaccine efficacy studies. We show that two doses of mRNA-1273 reduced viral load in the upper and lower airways of hamsters and protected against weight loss while a prime-only vaccination provided partial protection.

Two doses of mRNA-1273 were required to induce neutralizing titers comparable with the higher titers seen in convalescent COVID-19 patients. Although neutralizing titers were not dependent on dosage in a prime-boost schedule, the highest prime-boost dose of 25 µg provided better protection against lung injury and weight loss. Two hamsters which experienced more severe weight loss were identified in the low prime-boost dose or prime-only vaccine groups despite having a high binding and neutralizing antibody titers. Moreover, despite a strong inverse correlation between neutralizing antibody titers and virus load in the respiratory tract, the inverse correlation between neutralizing or binding antibody titers and weight loss was only moderate. Other antibody-and cell-mediated mechanisms may therefore be required for complete protection against SARS-CoV-2 disease in this model. This emphasizes the importance of a qualitative, not just quantitative, immune response, which may depend on the vaccine dose and regimen. The efficacy of mRNA-1273 in hamsters is distinguished from efficacy studies with NHP and mouse models, as protection was afforded against more severe pathological phenotypes and clinical disease, and the immunity was non-sterilizing. Patients with severe COVID-19 display aberrant T cell activation and differentiation, lymphopenia (31, 32), and generally have more proliferative T cells but less CD8 + T cell proportions with limited clonal diversity in BALF (25). Conversely, non-hospitalized, recovered individuals have virus-specific T cell memory (33, 34). In the lungs of MI hamsters, T cell proportions declined indicating that extravasation to affected tissues, one of the suggested causes of lymphopenia in severe patients, was not occurring. Instead, macroautophagy, a process which supports cell survival against environmental stresses or upon the activation of naïve T cells (35, 36) , was downregulated in CD8 + T cells of MI hamsters despite their responsiveness to oxidative stress and cytokine stimulus. Together with an extended lifespan, reduced telomerase maintenance and upregulated proapoptotic pathways, activated CD8 + T cells in MI hamsters may develop an effector phenotype but are replicative senescent, susceptible to depletion or unable to form functional memory T cells (37, 38) . CD8 + T cell exhaustion from hyperactivation or augmented expression of pro-apoptotic molecules has been linked to their depletion in severe COVID-19 cases (39, 40) . Vaccination prevented a reduction in T-cell frequencies in the lungs. Moreover, CD8 + T cells in VI hamsters were less enriched for activation and proliferative pathways compared to MI hamsters, but sustained effector functions.

This suggests the CD8 + T cell response in VI hamsters was either from memory effectors established after vaccination, to facilitate viral clearance, or from clonally diverse, tissueresident cells with an effector phenotype after SARS-CoV-2 challenge, as seen in the BALF from moderate but not severe COVID-19 patients (25).

DCs that are impaired in maturation and T cell activation (41) . DC subsets infiltrated the lungs of MI hamsters but remained unaltered in VI hamsters. Similar to acute phase coronavirus infections (42) , infiltrating pDCs may provide rapid antiviral responses through the upregulation of cytokine production pathways. However, pDCs were not necessary for influenza A virus clearance and were shown to have a deleterious role on CD8 + T cells during lethal infection of mice (43, 44) . In MI hamsters, the lymphoid organ development pathway of pDCs was downregulated suggesting their role in T cell differentiation was compromised. The low numbers of pDCs in VI hamsters and their limited DEGs suggests they were not involved in vaccinemediated responses to infection. Dysfunctional T cell activation in COVID-19 patients has been associated with the downregulation of MHC and costimulatory molecules on antigen presenting cells (41, 45, 46) . While immune effector pathways and chemotactic cues from IL-1 to migrate to the lung-draining lymph nodes were upregulated in cDCs from both MI and VI hamsters, DEGs involved in the activation of T cells via MHC molecules were paradoxically downregulated.

Despite this remarkable similarity in MI and VI-induced cDC pathways, disparities in DEG expression magnitude, cDC numbers and external stimulus from the lung milieu may account for different cDC-mediated outcomes. Diminished MHC-dependent T cell activation by cDCs from MI hamsters likely impaired the adaptive immune response, while in VI hamsters, the controlled cell contact dependent activation by cDCs may be sufficient for recall responses or sustaining the effector functions of CD8 + T cells (47) . mRNA-1273 promoted a controlled response to SARS-CoV-2 infection that prevented desynchrony between the innate and adaptive immune arms which can exacerbate inflammation and disease severity.

The incomplete annotation of the hamster genome prevented identification of highly specific subtypes within immune cell populations. Cell type identification partially based on mouse data and the pathway analysis based on human gene ontology terms did permit detailed profiling of cellular responses. While this study examined the response to infection directly in the lungs of VI and MI hamsters rather than BALF, scRNA-seq analysis was limited by the fact that one region of lung was studied whereas responses to an infection within the lungs may be spatially distinct. As a result, we captured a unique perspective on a potentially focal inflammatory lesion comprised of activated neutrophils/heterophils formed after infection of a low vaccine-dose VI hamster. Nevertheless, we excluded this scRNA-seq outlier to effectively illustrate the cellular response dynamics at the site of infection in vaccinated and mock-vaccinated hamsters exposed to SARS-CoV-2. 

Pre-clinical mRNA-1273 is a purified mRNA transcript encoding the prefusion-stabilized SARS-CoV-2 S-2P protein and encapsulated by a lipid nanoparticle. The process for mRNA synthesis, purification, and encapsulation was described previously (1).

Human convalescent sera (n = 6) were obtained from adults between 18 and 55 years old with 2 0 mild (n = 2), medium (n = 2), and severe (n = 2) COVID-19 and a history of laboratory-confirmed SARS-CoV-2 infection 1 to 2 months before providing specimens. In addition, SARS-CoV-2 naïve sera samples (n = 3) were also included in analyses. These specimens were obtained from Aalto Bio Reagents Ltd.

S, RBD or nucleocapsid proteins (1µg/mL, Sino Biological) were coated onto 96-well plates for 16 h. Plates were then blocked with SuperBlock (Pierce). Five-fold serial dilutions of hamster serum were then added to the plates (assay diluent -PBS + 0.05% Tween-20 + 5% goat serum) and incubated for 2 hours at 37°C. Bound antibodies were detected with HRPconjugated goat anti-hamster IgG (1:10,000 Abcam AB7146). Following the addition of TMB substrate (SeraCare #5120-0077) and TMB stop solution (SeraCare #5150-0021), the absorbance was measured at OD 450 nm. Titers were determined using a four-parameter logistic curve fit in GraphPad Prism (GraphPad Software, Inc.) and defined as the reciprocal dilution at approximately OD450nm = 1.5 (normalized to a hamster standard on each plate).

Two-fold serial dilutions of heat-inactivated serum at an initial dilution of 1:10 were prepared in serum-free MEM media and incubated with SARS-CoV-2-mNG for 1 hour at 37°C at a final concentration of 100 PFU. Virus-serum mixtures then were absorbed onto Vero-E6 monolayers in black optical 96 well plates for 1 hour at 37°C and replaced with MEM/Methylcellulose/2% FBS overlay. After 2 days of incubation at 37°C in humidified 5% CO2, neon green plaques were visualized and counted. Neutralization titers at an end point of 60% plaque reduction were determined.

Three groups of 6-7 week female golden Syrian hamsters (Envigo) (n = 15 per group) were 

DEGs were identified in scRNA-seq data for MI and VI comparisons to N in CD8 + T cells, NK cells, monocytes and DC subtypes, including pDCs and cDCs, using the non-parametric Wilcoxon rank sum test. The groups of DCα cells were incorporated into cDCs during DEG identification. Differential expression p-values were adjusted for multiple hypothesis testing using the Benjamini-Hochberg procedure (53) . DEGs with FDR < 0.05 and absolute logFC > 0.1 for MI vs. N and VI vs. N comparisons were selected as significant. To study gene function and pathway enrichment among DEGs, golden hamster genes were mapped to homolog genes in the homo sapiens species using Biomart (http://www.ensembl.org/biomart). Significant genes were clustered into functional modules using Louvain community clustering based on the functional similarity between genes in the lung tissue, as predicted by HumanBase DEGs that are specific to either the MI or VI comparisons to N only (Supplemental Figure 10 ).

For pDCs, however, functional clustering and enrichment analysis was only performed on MI vs. N given the insufficient number of DEGs in VI vs. N.

Statistical analysis was performed using GraphPad Prism software, version 6. Two-way ANOVA with Tukey's or Sidak's corrections were respectively performed for multiple comparisons between vaccine groups or between time points. Significance between pre-and post-infection antibody titers was measured by multiple t tests with Holm Sidak's correction for multiple comparisons (Supplemental Figure 4) . ANOVA and post-hoc Tukey's test pairwise comparisons were employed to determine the significance of scRNA-seq-based cell type proportion changes ( Figure 3 ). Correlations were determined by two-sided Spearman s rank tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. 5A . Each gene is represented by a circle and color coded according to module. The size of each circle reflects its connectivity in the network. Edges are not shown, allowing for easy viewing. The module label is shown with the functional processes and pathways identified in each module. Up or down regulation of pathway is indicated by arrow direction.

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We thank members of the UTMB Animal Resource Center for technical assistance with the hamster experiment in ABSL-2 and ABSL-4 and husbandry support. We thank Steve Widen and the UTMB Next Generation Sequencing Core for quantification and pooling of scRNA-seq libraries for submission to the New York Genome Center. We thank the Anatomic Pathology Core facility at UTMB for embedding, sectioning and staining lung tissues for histopathology.

Center for Emerging Viruses and Arboviruses at UTMB. This work was supported by Moderna, Inc.