Effects of N-glycan modifications on spike expression, virus infectivity, and neutralization sensitivity in ancestral compared to Omicron SARS-CoV-2 variants.

The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI- cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, growth of live (authentic) SARS-CoV-2 in the presence of kifunensine resulted in a greater reduction of titers for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization.

Differences in New Variant of Concern Replication at Physiological Temperatures In Vitro.

Using multiple cell types and isolates of Delta and Omicron variants of SARS-CoV-2, we report differences in virus production, replication, and infectivity in vitro. Ancestral and Delta SARS-CoV-2 variant exhibit reduced virus production and replication at 34°C compared to 37°C while Omicron replication is balanced between temperatures.

Pharmacokinetics and Efficacy of Human Hyperimmune Intravenous Immunoglobulin Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Adult Syrian Hamsters.

BACKGROUND: After the failure of antibody therapies in treating hospitalized patients with coronavirus disease 2019 (COVID-19), we investigated the impact of viral replication on the pharmacokinetics and efficacy of a hyperimmune severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin (CoVIG) product in treating SARS-CoV-2 infection using an adult Syrian hamster model. METHODS: The CoVIG was manufactured from plasma donors who had recovered from COVID-19. The dose used (400 mg/kg) was based on the dose given in clinical trials to hospitalized patients with COVID-19. Hamsters were given a single dose of CoVIG 2 days after challenge with the SARS-CoV-2 virus (isolate NY/PV08410/2020), followed by sampling of blood, nasal, tracheal, and lung tissues at different time points. The blood samples were assayed for anti-SARS-CoV-2 spike binding and used to calculate pharmacokinetic (PK) parameters. Nasal wash, tracheal, and lung tissue samples were assayed for viral replication by polymerase chain reaction (subgenomic messenger RNA). RESULTS: CoVIG-treated hamsters showed a reduction in viral replication in the lower respiratory tract, but minimal reduction in the upper respiratory tract, after challenge with SARS-CoV-2. Challenge resulted in altered PK parameters proportionate to viral replication, resulting in decreased area under the curve, accelerated clearance, and shorter half-life of CoVIG. CONCLUSIONS: These data indicate that in the presence of actively replicating SARS-CoV-2 virus, PK parameters are altered and should trigger an adjustment in CoVIG dosing.

Long-term immunity in convalescent Syrian hamsters provides protection against new-variant SARS-CoV-2 infection of the lower but not upper respiratory tract.

COVID-19 vaccines provide high levels of protection against severe disease and hospitalization due to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection. Vaccination may be less effective in preventing shedding of infectious viruses from otherwise immune patients. In this study, we describe breakthrough infections and shedding of infectious viruses in convalescent hamsters without significant replication in the lower respiratory tract following reinfection by Alpha and Delta variants despite high levels of circulating antibodies in sera. Using convalescent hamsters with long-term immunity (up to 1 year) following infection by ancestral SARS-CoV-2, we can model aspects of recurring COVID-19 in the context of preexisting immunity.

HIF-1α-Dependent Metabolic Reprogramming, Oxidative Stress, and Bioenergetic Dysfunction in SARS-CoV-2-Infected Hamsters.

The mechanistic interplay between SARS-CoV-2 infection, inflammation, and oxygen homeostasis is not well defined. Here, we show that the hypoxia-inducible factor (HIF-1α) transcriptional pathway is activated, perhaps due to a lack of oxygen or an accumulation of mitochondrial reactive oxygen species (ROS) in the lungs of adult Syrian hamsters infected with SARS-CoV-2. Prominent nuclear localization of HIF-1α and increased expression of HIF-1α target proteins, including glucose transporter 1 (Glut1), lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase-1 (PDK1), were observed in areas of lung consolidation filled with infiltrating monocytes/macrophages. Upregulation of these HIF-1α target proteins was accompanied by a rise in glycolysis as measured by extracellular acidification rate (ECAR) in lung homogenates. A concomitant reduction in mitochondrial respiration was also observed as indicated by a partial loss of oxygen consumption rates (OCR) in isolated mitochondrial fractions of SARS-CoV-2-infected hamster lungs. Proteomic analysis further revealed specific deficits in the mitochondrial ATP synthase (Atp5a1) within complex V and in the ATP/ADP translocase (Slc25a4). The activation of HIF-1α in inflammatory macrophages may also drive proinflammatory cytokine production and complement activation and oxidative stress in infected lungs. Together, these findings support a role for HIF-1α as a central mediator of the metabolic reprogramming, inflammation, and bioenergetic dysfunction associated with SARS-CoV-2 infection.

Structure-Function Analysis of Resistance to Bamlanivimab by SARS-CoV-2 Variants Kappa, Delta, and Lambda.

The newly emerging Kappa, Delta, and Lambda SARS-CoV-2 variants are worrisome, characterized with the double mutations E484Q/L452R, T478K/L452R, and F490S/L452Q, respectively, in their receptor binding domains (RBDs) of the spike proteins. As revealed in crystal structures, most of these residues (e.g., 452 and 484 in RBDs) are not in direct contact with interfacial residues in the angiotensin-converting enzyme 2 (ACE2). This suggests that albeit there are some possibly nonlocal effects, these mutations might not significantly affect RBD's binding with ACE2, which is an important step for viral entry into host cells. Thus, without knowing the molecular mechanism, these successful mutations (from the point of view of SARS-CoV-2) may be hypothesized to evade human antibodies. Using all-atom molecular dynamics (MD) simulation, here, we show that the E484Q/L452R mutations significantly reduce the binding affinity between the RBD of the Kappa variant and the antibody LY-CoV555 (also named as Bamlanivimab), which was efficacious for neutralizing the wild-type SARS-CoV-2. To verify simulation results, we further carried out experiments with both pseudovirions- and live virus-based neutralization assays and demonstrated that LY-CoV555 completely lost neutralizing activity against the L452R/E484Q mutant. Similarly, we show that mutations in the Delta and Lambda variants can also destabilize the RBD's binding with LY-CoV555. With the revealed molecular mechanism on how these variants evade LY-CoV555, we expect that more specific therapeutic antibodies can be accordingly designed and/or a precise mixing of antibodies can be achieved as a cocktail treatment for patients infected with these variants.

Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference.

The protein synthesis machineries of two distinct phyla of the Animal kingdom, insects of Arthropoda and mammals of Chordata, have different preferences for how to best encode proteins. Nevertheless, arboviruses (arthropod-borne viruses) are capable of infecting both mammals and insects just like arboviruses that use insect vectors to infect plants. These organisms have evolved carefully balanced genomes that can efficiently use the translational machineries of different phyla, even if the phyla belong to different kingdoms. Using dengue virus as an example, we have undone the genome encoding balance and specifically shifted the encoding preference away from mammals. These mammalian-attenuated viruses grow to high titers in insect cells but low titers in mammalian cells, have dramatically increased LD50s in newborn mice, and induce high levels of protective antibodies. Recoded arboviruses with a bias toward phylum-specific expression could form the basis of a new generation of live attenuated vaccine candidates.

Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection.

UNLABELLED: Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. IMPORTANCE: VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.

Vaccine-associated respiratory pathology correlates with viral clearance and protective immunity after immunization with self-amplifying RNA expressing the spike (S) protein of SARS-CoV-2 in mouse models.

In this study, we evaluated the immunogenicity and protective immunity of in vitro transcribed Venezuelan equine encephalitis virus (VEEV TC-83 strain) self-amplifying RNA (saRNA) encoding the SARS-CoV-2 spike (S) protein in wild type (S-WT) and stabilized pre-fusion conformations (S-PP). Immunization with S-WT and S-PP saRNA induced specific neutralizing antibody responses in both K18-Tg hACE2 (K18) and BALB/c mice, as assessed using SARS-CoV-2 pseudotyped viruses. Protective immunity was assessed in challenge experiments. Two immunizations with S-WT and S-PP induced protective immunity, evidenced by lower mortality, lower weight loss and more than one log10 lower subgenomic virus RNA titers in the upper and lower respiratory tracts in both K18 and BALB/c mice. Histopathologic examination of lungs post-challenge showed that immunization with S-WT and S-PP resulted in a higher degree of immune cell infiltration and inflammatory changes, compared with control mice, characterized by high levels of T- and B-cell infiltration. No substantial differences were found in the presence and localization of eosinophils, macrophages, neutrophils, and natural killer cells. CD4 and CD8 T-cell depletion post immunization resulted in reduced lung inflammation post challenge but also prolonged virus clearance. These data indicate that immunization with saRNA encoding the SARS-CoV-2 S protein induces immune responses that are protective following challenge, that virus clearance is associated with pulmonary changes caused by T-cell and B-cell infiltration in the lungs, but that this T and B-cell infiltration plays an important role in viral clearance.

Antigenic cartography using hamster sera identifies SARS-CoV-2 JN.1 evasion seen in human XBB.1.5 booster sera.

Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with five to six-fold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a five-fold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.

Comparative Assessment of the Binding and Neutralisation Activity of Bispecific Antibodies Against SARS-CoV-2 Variants.

Background: Neutralising antibodies against SARS-CoV-2 are a vital component in the fight against COVID-19 pandemic, having the potential of both therapeutic and prophylactic applications. Bispecific antibodies (BsAbs) against SARS-CoV-2 are particularly promising, given their ability to bind simultaneously to two distinct sites of the receptor-binding domain (RBD) of the viral spike protein. Such antibodies are complex molecules associated with multi-faceted mechanisms of action that require appropriate bioassays to ensure product quality and manufacturing consistency. Methods: We developed procedures for biolayer interferometry (BLI) and a cell-based virus neutralisation assay, the focus reduction neutralisation test (FRNT). Using both assays, we tested a panel of five BsAbs against different spike variants (Ancestral, Delta and Omicron) to evaluate the use of these analytical methods in assessing binding and neutralisation activities of anti-SARS-CoV-2 therapeutics. Results: We found comparable trends between BLI-derived binding affinity and FRNT-based virus neutralisation activity. Antibodies that displayed high binding affinity against a variant were often followed by potent neutralisation at lower concentrations, whereas those with low binding affinity also demonstrated reduced neutralisation activity. Conclusion: The results support the utility of BLI and FRNT assays in measuring variant-specific binding and virus neutralisation activity of anti-SARS-CoV-2 antibodies.

Intranasal or airborne transmission-mediated delivery of an attenuated SARS-CoV-2 protects Syrian hamsters against new variants.

Detection of secretory antibodies in the airway is highly desirable when evaluating mucosal protection by vaccines against a respiratory virus, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that intranasal delivery of an attenuated SARS-CoV-2 (Nsp1-K164A/H165A) induces both mucosal and systemic IgA and IgG in male Syrian hamsters. Interestingly, either direct intranasal immunization or airborne transmission-mediated delivery of Nsp1-K164A/H165A in Syrian hamsters offers protection against heterologous challenge with variants of concern (VOCs) including Delta, Omicron BA.1, BA.2.12.1 and BA.5. Vaccinated animals show significant reduction in both tissue viral loads and lung inflammation. Similarly attenuated viruses bearing BA.1 and BA.5 spike boost variant-specific neutralizing antibodies in male mice that were first vaccinated with modified vaccinia virus Ankara vectors (MVA) expressing full-length WA1/2020 Spike protein. Together, these results demonstrate that our attenuated virus may be a promising nasal vaccine candidate for boosting mucosal immunity against future SARS-CoV-2 VOCs.

Identification of salivary gland escape barriers to western equine encephalitis virus in the natural vector, Culex tarsalis.

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Intranasal delivery of a rationally attenuated SARS-CoV-2 is immunogenic and protective in Syrian hamsters.

Few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are in pre-clinical or clinical development. We seek to attenuate SARS-CoV-2 (isolate WA1/2020) by removing the polybasic insert within the spike protein and the open reading frames (ORFs) 6-8, and by introducing mutations that abolish non-structural protein 1 (Nsp1)-mediated toxicity. The derived virus (WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A) replicates to 100- to 1000-fold-lower titers than the ancestral virus and induces little lung pathology in both K18-human ACE2 (hACE2) transgenic mice and Syrian hamsters. Immunofluorescence and transcriptomic analyses of infected hamsters confirm that three-pronged genetic modifications attenuate the proinflammatory pathways more than the removal of the polybasic cleavage site alone. Finally, intranasal administration of just 100 PFU of the WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A elicits robust antibody responses in Syrian hamsters and protects against SARS-CoV-2-induced weight loss and pneumonia. As a proof-of-concept study, we demonstrate that live but sufficiently attenuated SARS-CoV-2 vaccines may be attainable by rational design.

The G614 pandemic SARS-CoV-2 variant is not more pathogenic than the original D614 form in adult Syrian hamsters.

Dynamic tracking of variant frequencies among viruses circulating in the global pandemic has revealed the emergence and dominance of a D614G mutation in the SARS-CoV-2 spike protein. To address whether pandemic SARS-CoV-2 G614 variant has evolved to become more pathogenic, we infected adult hamsters (>10 months old) with two natural SARS-CoV-2 variants carrying either D614 or G614 spike protein to mimic infection of the adult/elderly human population. Hamsters infected by the two variants exhibited comparable viral loads and pathology in lung tissues as well as similar amounts of virus shed in nasal washes. Altogether, our study does not find that naturally circulating D614 and G614 SARS-CoV-2 variants differ significantly in pathogenicity in hamsters.

Epitope diversity of SARS-CoV-2 hyperimmune intravenous human immunoglobulins and neutralization of variants of concern.

Hyperimmune immunoglobulin (hCoV-2IG) generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in clinical trials. Here we explored the antibody epitope repertoire, and virus neutralizing capacity of six hCoV-2IG batches as well as nine CP against SARS-CoV-2 and emerging variants of concern (VOCs). Epitope-mapping by gene-fragment phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike that was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of VOCs were reduced to different extent by hCoV-2IG lots but were higher than CP. Significant reduction of hCoV-2IG binding was observed to RBD-E484K followed by RBD-N501Y (but not RBD-K417N). This study suggests that post-exposure treatment with hCoV-2IG could be preferable to CP.

SARS-CoV-2 B.1.1.7 infection of Syrian hamster does not cause more severe disease and is protected by naturally acquired immunity.

Epidemiological studies have revealed the emergence of multiple SARS-CoV-2 variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality (1). To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and re-infection studies in naïve and convalescent Syrian hamsters (>10 months old). Hamsters infected by either a B.1.1.7 variant or a B.1 (G614) variant exhibited comparable viral loads and pathology. Convalescent hamsters that were previously infected by the original D614 variant were protected from disease following B.1.1.7 challenge with no observable clinical signs or lung pathology. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in hamsters and that natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant.

SARS-CoV-2 B.1.1.7 Infection of Syrian Hamster Does Not Cause More Severe Disease, and Naturally Acquired Immunity Confers Protection.

Epidemiological studies have revealed the emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality. To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and reinfection studies in naive and convalescent Syrian hamsters (>10 months old). Nasal wash samples from hamsters infected by a B.1.1.7 variant exhibited slightly higher viral RNA levels but lower infectious titers than those from B.1 (G614) variant-infected hamsters, and the two variants induced comparable lung pathologies in hamsters. Despite a sporadic and transient low-level infection in the nasal cavity, convalescent hamsters that had recovered from a previous USA-WA1 isolate (D614) infection displayed no observable clinical signs or lung pathology following B.1.1.7 rechallenge. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in Syrian hamsters and that a heterologous natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant. IMPORTANCE The rapid emergence of several variants of concern of SARS-CoV-2 calls for evaluations of viral fitness and pathogenicity in animal models in order to understand the mechanism of enhanced transmission and the possible increases in morbidity and mortality rates. Here, we demonstrated that immunity naturally acquired through a prior infection with the first-wave variant does confer nearly complete protection against the B.1.1.7 variant in Syrian hamsters upon reexposure. Strikingly, although the B.1.1.7 variant appears to replicate to a higher level in the nose than the ancestral B.1 variant, it does not induce more severe lung pathology in hamsters.

MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge.

Numerous vaccine candidates against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are under development. The majority of vaccine candidates to date are designed to induce immune responses against the viral spike (S) protein, although different forms of S antigen have been incorporated. To evaluate the yield and immunogenicity of different forms of S, we constructed modified vaccinia virus Ankara (MVA) vectors expressing full-length S (MVA-S), the RBD, and soluble S ectodomain and tested their immunogenicity in dose-ranging studies in mice. All three MVA vectors induced spike-specific immunoglobulin G after one subcutaneous immunization and serum titers were boosted following a second immunization. The MVA-S and MVA-ssM elicited the strongest neutralizing antibody responses. In assessing protective efficacy, MVA-S-immunized adult Syrian hamsters were challenged with SARS-CoV-2 (USA/WA1/2020). MVA-S-vaccinated hamsters exhibited less severe manifestations of atypical pneumocyte hyperplasia, hemorrhage, vasculitis, and especially consolidation, compared to control animals. They also displayed significant reductions in gross pathology scores and weight loss, and a moderate reduction in virus shedding was observed post challenge in nasal washes. There was evidence of reduced viral replication by in situ hybridization, although the reduction in viral RNA levels in lungs and nasal turbinates did not reach significance. Taken together, the data indicate that immunization with two doses of an MVA vector expressing SARS-CoV-2 S provides protection against a stringent SARS-CoV-2 challenge of adult Syrian hamsters, reaffirm the utility of this animal model for evaluating candidate SARS-CoV-2 vaccines, and demonstrate the value of an MVA platform in facilitating vaccine development against SARS-CoV-2.