mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents

mRNA technologies have recently proven clinical efficacy against coronavirus disease 2019 and are among the most promising technologies to address the current pandemic. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle-encapsulated mRNA vaccine that encodes full-length, pre-fusion stabilised severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. In contrast to previously published approaches, CVnCoV is exclusively composed of naturally occurring nucleotides. Immunisation with CVnCoV induced strong humoral responses with high titres of virus-neutralising antibodies and robust T-cell responses. CVnCoV vaccination protected hamsters from challenge with wild-type SARS-CoV-2, demonstrated by the absence of viral replication in the lungs. Hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication exhibited no evidence of vaccine-enhanced disease. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.


INTRODUCTION
The global coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for novel technologies that allow rapid development and production of human vaccines against newly emerging infectious pathogens. Following pioneering work using mRNA formulated with protamine to target tumours [1][2][3][4] , CureVac has established that mRNA elicits immune responses against target antigens as a prophylactic vaccine [5][6][7][8][9] . CureVac's proprietary mRNA technology is designed to rapidly identify, produce and test stable and immunogenic mRNA molecules 10 . Following preclinical proof of concept with rabies glycoprotein RABV-G mRNA, formulated with protamine 7,8 , a first-in-human study showed that immune responses are elicited in adult volunteers, although protective titres could only be induced when specialised injection devices were used 9 . Further research has demonstrated that RABV-G mRNA encapsulated in lipid nanoparticles (LNP) overcomes these deficiencies and significantly improves vaccine efficacy in animal models 6 , and in human volunteers 11 . mRNA technology is now the basis for several severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine candidates [12][13][14][15][16] . The main antigenic target of SARS-CoV-2 is the glycosylated spike protein (S) that interacts with human angiotensin-converting enzyme 2 (ACE2). Consistent with the mode of action of SARS-CoV, which first emerged in 2002-2003 17 , ACE2 binding allows cellular entry of the virus [18][19][20] . S is a trimeric glycoprotein complex located on the viral surface and is a critical target for viral neutralising antibodies 21 . Each monomer consists of two domains, S1 and S2 that act separately to mediate viral binding and fusion to the host cell membrane, respectively. The S1 domain interacts with cell-surface receptors through a receptorbinding domain (RBD) and monoclonal antibodies (mAb) against the RBD possess neutralising capacity 22 . Fusion with the membrane through S1 leads to a conformational change in the spike protein, proteolytic cleavage of the S1 and S2 domains and, ultimately, viral uptake and replication 21,23 .
CureVac has applied its mRNA technology to the rapid development of CVnCoV, a SARS-CoV-2 vaccine designed for maximal protein expression and balanced immune activation. CVnCoV is comprised of LNP-formulated, non-chemically modified, sequence engineered mRNA encoding full-length S protein with two proline mutations (S-2P). These mutations stabilise protein conformation as previously reported for Middle East respiratory syndrome coronavirus (MERS-CoV) 24 and SARS-CoV 25 .
Here we describe the immunogenicity and protective efficacy of CVnCoV in preclinical studies in rodents. Protective efficacy was assessed in Syrian hamsters, one of the recognised and accepted models to investigate human-relevant immunogenicity and pathogenesis 26 . Hamsters are susceptible to wild-type SARS-CoV-2 infection, resulting in high levels of virus replication and histopathological changes in viral target organs comparable to mild to moderate human lung disease pathology. Studies shown here enabled the start of CVnCoV clinical development 27 , currently in phase 2b/3 clinical studies.

Protein expression in vitro
CVnCoV encodes for full-length SARS-CoV-2 S protein with intact S1/S2 cleavage site and transmembrane domain, as well as K 986 P and V 987 P mutations 24,25 (S-2P) (Fig. 1A). Non-encapsulated SARS-CoV-2 S-2P mRNA translated in a cell-free in vitro system yielded a 140 kDa protein, representing uncleaved full-length S-2P ( Fig. 1B and Supplementary Fig. 1A). Efficient cleavage of the S-2P protein in cell culture was demonstrated by western blot analysis of mRNA-transfected cells, using an antibody directed against the S2 portion of the protein 20,28 . This analysis showed the generation of two main bands of approx. 90 and 180 kDa, likely reflecting glycosylated forms of unprocessed S protein (S0) and the cleaved S2 subunit (Fig. 1C and Supplementary Fig. 1B). In agreement with an intact signal peptide and transmembrane domain, further flow cytometric analyses proved that a large proportion of mRNA encoded SARS-CoV-2 S localised to the surface of mRNAtransfected cells.

Immunogenicity in mice
To characterise mRNA-induced innate and adaptive immune responses, Balb/c mice were immunised with 2 µg of LNPformulated mRNA encoding SARS-CoV-2 S-2P (CVnCoV). Innate responses were assessed in sera of CVnCoV-vaccinated mice 14 h post injection, the time point when mRNA-induced pro-inflammatory cytokines and chemokines peak 6 . These analyses demonstrated the induction of a balanced immune response upon CVnCoV injection that exhibited no bias towards IFNγ or IL-4, IL-5 and IL-13, indicative of a T h 1 and T h 2 response, respectively. Low levels of pro-inflammatory cytokines IL-6, IFNα were detectable in serum, while TNF and IL-1β remained undetectable ( Fig. 2A).
To determine immunisation schedule impact, two injections of CVnCoV were given 4, 3, 2 or 1 week apart, i.e., on day 0/d28, day 7/d28, day 14/d28 or day 21/d28. Spike ectodomain (S ECD )-binding antibodies, found to correlate with the induction of virusneutralising antibodies in COVID-19 patients 29 , developed rapidly upon a single injection with CVnCoV. Seven days after a single vaccination, robust IgG1 and IgG2a titres were present and increased over time (Fig. 2B). Median endpoint titres on day 7 were 3.1 × 10 4 and 4.7 × 10 5 , for IgG1 and IgG2a, respectively, and increased to 3.9 × 10 6 for both IgG1 and IgG2a on d28. Second injection with CVnCoV induced an increase in S ECD -binding IgG1 and IgG2a antibodies, while a trend towards lower immune responses induced by shorter intervals between first and second vaccination remained detectable until termination of the experiment in week 7 (day 49). As expected, Alum-adjuvanted S ECD protein, employed as a control for T h 2 biased responses, induced high titres of IgG1 but comparatively low levels of IgG2a. Induction of virus-neutralising titres (VNTs) in mouse sera was assessed in a cytopathic effect (CPE)-based assay with wild-type SARS-CoV-2. Detectable levels of VNTs started to develop 4 weeks after a single injection, while a second injection led to significant VNT increase in all groups. Analyses 1 and 3 weeks after the second vaccination showed that responses rose over time (Fig. 2C). By termination on day 49, all sera derived from animals vaccinated on day 0/28 were able to neutralise 50% of infecting virus (100 TCID 50 ) in cell culture at a dilution of 1:5120. Of note, two serum samples of symptomatic convalescent subjects chosen for the presence of high levels of binding S antibodies exhibited titres of 1:320-1:640 in this assay.
Strong induction of IFNγ/TNF double-positive T cells in CVnCoVvaccinated mice (Fig. 2D) was demonstrated by T cell analysis via intracellular cytokine staining (ICS) of splenocytes isolated 3 weeks after the second vaccination (day 49) (gating strategy Supplementary Fig. 2). T cell responses benefited from longer intervals between first and second vaccination. Highest responses were detected in animals vaccinated on day 0/d28 with median values of IFNγ + /TNF + CD4 + and CD8 + T cells of 0.34% and 10.5%, respectively. In contrast, IFNγ + /TNF + CD4 + or CD8 + T cells remained undetectable in the protein control group.
Mice vaccinated with CVnCoV at 0.25, 1 or 4 µg elicited dosedependent humoral responses. IgG1 and IgG2a antibodies induced by CVnCoV interacted with S ECD , the isolated S RBD, the trimeric form of the S protein (S trimer) and with the isolated Nterminal domain (NTD) (Fig. 3A). CVnCoV induced comparable levels of S ECD , RBD and trimeric S reactive antibodies, with the exception of the lowest 0.25 µg dose, which generated lower RBD responses. Of note, antibodies directed against the NTD, an alternative target of VNTs next to the RBD 29 , were detectable for all doses but overall remained lower than levels of S ECD-, RBD-and S trimer-binding antibodies. As observed previously, VNTs started to develop 3 weeks after a single vaccination and robust levels were detectable after a second vaccination with median titres of 1:2560 and 1: 5120 for the 1 and 4 µg dose groups, respectively. Even the lowest dose of 0.25 µg induced detectable levels of VNTs of approximately 1:14 (Fig. 3B). Further experiments demonstrated comparable relative avidity for S ECD -specific IgG1 and IgG2a antibodies (Fig. 3C). CVnCoV-induced antibodies that efficiently competed with a neutralising mAb for binding to native SARS-CoV-2 S expressed on the cellular surface (Fig. 3D).
Immunogenicity and protective efficacy in Syrian hamsters Next, we sought to address the protective efficacy of CVnCoV in Syrian hamsters. Animals vaccinated with two CVnCoV doses of 2 or 10 µg in a 4-week interval developed dose-dependent S ECDbinding IgG antibodies after the first vaccination that increased upon the second (Fig. 4A). Median endpoint titres of animals vaccinated with 10 µg of CVnCoV were 1.6 × 10 5 after one dose   IgG2a endpoint titer 1st vacc 2nd vacc d28 none d28 CVnCoV 2µg protein + adj. Buffer comparison, control animals infected with wild-type SARS-CoV-2 on day 0 developed VNTs of 1:191. A control group that received Alum-adjuvanted S ECD protein developed IgG antibodies but undetectable VNT levels (Fig. 4B). On day 56, 4 weeks after the second vaccination, all animals were challenged with 10 2 TCID 50 /dose of SARS-CoV-2 featuring D614G. While this infection dose is low compared to other studies that employed doses of 2 × 10 3 -2 × 10 6 TCID 50 30-36 , our results show robust levels of viral replication in the upper and lower respiratory tract (Fig. 4) as well as typical weight loss for this model system 30,31,33 (Supplementary Fig. 3) in the buffer control group.
Analysis of animal weight upon challenge on days 56-60 revealed a maximal weight loss of 10% in buffer control animals with a steady decline throughout the observation period, while untreated and mock-infected animals gained 3% of weight. All other groups, including animals previously infected with SARS-CoV-2, exhibited intermediate values of 5-6% on day 60 compared to day 56 with the weight drop stabilising on day 59 ( Supplementary Fig. 3). Buffer control animal throat swabs, taken daily from day 56 to termination on day 60, showed peak replication-competent viral titres of approximately 10 3 TCID 50 /ml 2 days post challenge. These titres were nearly undetectable on day 60. Animals previously infected with SARS-CoV-2 remained negative throughout the experiment. Viral levels were significantly reduced in throat swabs of both CVnCoV-vaccinated groups. Vaccination with 10 µg of CVnCoV resulted in significantly diminished and delayed viral peaks at 10 1.5 TCID 50 /ml 3 days post challenge. At least two out of five animals in this group remained negative throughout the testing period (Fig. 4C). Viral levels in nasal turbinates revealed less pronounced, but detectable dosedependent reduction of viral replication (Fig. 4D). Importantly, animals vaccinated with 10 µg of CVnCoV exhibited no detectable virus in the lungs, proving the ability of CVnCoV to protect animals from viral replication in the lower respiratory tract (Fig. 4E).

Histopathology upon challenge infection in hamsters
Histopathological analyses demonstrated the occurrence of alveolar damage and moderate to severe inflammation of upper (nasal turbinates) and lower (alveoli, bronchi, trachea) respiratory tract in the buffer control group upon SARS-CoV-2 infection, proving the suitability of this model to assess virus-induced pathology. Consistent with protection from viral replication in the lungs, two vaccinations with 10 µg of CVnCoV significantly reduced histopathological changes in the lungs. Animals in this group featured significantly decreased inflammation of the lower respiratory tract, i.e., tracheitis, bronchitis, bronchiolitis and alveolitis. Importantly, a dose of 2 µg, which led to the induction of binding antibodies with low levels of VNTs, did not induce increased histopathology scores. Group comparisons for differential gene expression in lung homogenates showed no significant change in the induction of IL-4 or IL-5 in the mRNA groups compared to buffer or mock infection groups (data not shown). The presented data give some indication that Alumadjuvanted protein vaccine, which induces a T h 2-shifted immune response with no detectable levels of VNTs but high levels of binding antibodies, causes increased histopathology scores in hamsters (Fig. 5).
In order to exclude that potential pathological changes upon CVnCoV vaccination were induced but remained undetected at day 4 post challenge, a parallel experiment in hamsters terminated on day 7 was conducted. This later time point prevented the determination of viral levels in lungs that were undetectable in all groups (data not shown) but provided overall higher levels of pathological changes in the respiratory tract. The analysis of median gross pathology revealed values of 30% affected lung tissue in buffer control animals. In contrast, all animals in the highest dose group of 8 µg and 3/5 of 2 µg CVnCoVvaccinated animals featured no visually detectable levels of pathological changes of the lungs (Supplementary Fig. 4A). In agreement with analyses on day 4 post challenge, histopathology performed on day 7 post challenge demonstrated significant reduction of inflammation in both of upper and lower respiratory tract samples in both CVnCoV-vaccinated groups with no indication of increased inflammation in either dose group ( Supplementary Fig. 4B).

DISCUSSION
Here we show that CVnCoV mRNA is translated into a protein that was cleaved, post-translationally modified and presented on the cell surface, mimicking viral protein presentation during infection. In line with previous results 6 , CVnCoV induced innate immune responses, including systemic IL-6 and IFNα in mice, indicative of a pro-inflammatory environment for the induction of cellular and humoral immune responses. Incorporation of two prolines that prevent structural rearrangement to the post-fusion state of coronavirus S proteins 24,25 in the CVnCoV design was aimed to increase S-induced immune responses as observed in the context of MERS 24 . Indeed, vaccination with CVnCoV induced robust humoral responses featuring high levels of VNTs in mice and hamsters that were dose and vaccination schedule dependent. Antibody responses in mice developed rapidly and to comparatively high levels 37 upon injection and were reactive with both S domains targeted by human VNTs, i.e., the RBD domain as the primary target of neutralising antibodies 38,39 , as well as the NTD 40 . CPE-based detection of virus-neutralising antibody responses revealed that CVnCoV induced high levels of VNTs upon two injections against both viruses featuring D and G on position 614 in S for mice and hamsters, respectively. In hamsters, VNTs were comparable to titres induced upon natural infection with wildtype SARS-CoV-2. As previously shown for mRNA vaccines against SARS-CoV-2 13,37 , CVnCoV was able to induce both S-specific CD4 + and CD8 + T-cell responses in mice that were comparatively high at low vaccine doses 37 . T cells are known to contribute to protection in respiratory infections, with virus-specific CD8 + T cells representing effectors of viral clearance from lung tissue 41 . In a clinical study, influenza-specific CD8 + T cells protected against symptomatic influenza infection in the absence of protective antibodies 42 . Furthermore, memory CD8 + T cells provided substantial protection in a lethal SARS-CoV-1 mouse model 43 . In the context of SARS-CoV-2, recent data demonstrated that CD8+ T cells contribute to viral control in a rhesus macaque model 44 .
Data presented here demonstrate that, in mice, CVnCoV vaccination is able to induce high levels of antigen-specific CD8 + IFNγ/ TNF double-positive T cells, likely to contribute to vaccine efficacy upon viral challenge. Consistent with robust immune responses, CVnCoV protected hamsters against SARS-CoV-2 viral challenge featuring the D614G mutation in S, proving CVnCoV's ability to protect against a prevalent form of the virus. These experiments showed significant reduction in replicating virus levels in the upper respiratory tract and the absence of detectable live virus in the lungs of animals upon two vaccinations with 10 µg of CVnCoV. This result is in line with previous observations in other respiratory viruses as well in SARS-CoV-2 challenge models in hamsters 30 and non-human primates 13,45 , where robust immune responses induced better protection in the lower than in the upper respiratory tract.
Importantly, data presented here support a favourable safety profile of CVnCoV. A potential risk of vaccine development is the induction of vaccine-enhanced disease or pathology, either induced by antibodies (antibody-dependent enhanced disease, ADE) or vaccine-associated enhanced respiratory disease (VAERD) (reviewed in [46][47][48]. ADE, which leads to exacerbated disease by enhancement of viral entry, has been described for a feline coronavirus 49 as well as for other viruses such as respiratory syncytial virus and measles virus. ADE has largely been associated with the presence of non-neutralising antibodies (reviewed in 48 ). VAERD that occurred in the context of whole-inactivated vaccines 50,51 has been linked to inflammation induced by T h 2biased immune responses and high ratios on non-neutralising to neutralising antibodies due to presentation of conformationally suboptimal antigens (reviewed in 47 ). Vaccine-enhanced disease has been described in non-clinical models for SARS and MERS-CoV (reviewed in 52,53 ). There is no evidence of vaccine-enhanced disease in animal models of SARS-CoV-2 12,45,54,55 , nor clinical evidence that this might be a concern in SARS-CoV-2 infections. However, such hypothetical effects need to be carefully excluded during vaccine development. Several lines of evidence suggest that the risk of CVnCoV to induce vaccine-enhanced disease is low. First, immunogenicity data generated in two animal models demonstrated robust levels of VNTs compared to binding antibodies upon vaccination with CVnCoV. Second, the vaccine elicited a balanced T h 1/T h 2 profile, indicated by the induction of equivalent levels of IgG1 and IgG2a antibodies that exhibited comparable relative avidity to SARS-CoV-2 S as well as a cytokine profile in mice that gave no indication of a T h 2 bias, i.e., induction of IL-4, IL-5 and IL-13. In addition, challenge infection in hamsters did not reveal evidence of disease enhancement, neither by enhanced viral levels in the respiratory tract, nor inflammation of lung tissue, which was assessed at both early (day 4) and late (day 7) time points post challenge. This observation remained true in a group of animals vaccinated with 2 µg, a suboptimal dose of CVnCoV that induced high levels of binding antibodies in the absence of significant VNTs. Here, viral challenge led to breakthrough viral replication in the lungs. However, neither viral levels nor tissue inflammation on day 4 or day 7 was enhanced giving strong support for a favourable safety profile of CVnCoV.
We conclude that CVnCoV represents a promising vaccine candidate against SARS-CoV-2 in terms of potency and safety. The presented data supported the start of CVnCoV clinical trials, which is currently in phase 2b/3 testing.

METHODS mRNA vaccines
The mRNA vaccine is based on the RNActive ® platform (claimed and described in, e.g., WO2002098443 and WO2012019780) and is comprised of a 5′ cap structure, a GC-enriched open reading frame, 3′ UTR, polyA tail and does not include chemically modified nucleosides. LNP-encapsulation of mRNA was performed by Acuitas Therapeutics (Vancouver, Canada) or Polymun Scientific Immunbiologische Forschung GmbH (Klosterneuburg, Austria). The LNPs used in this study are particles of ionisable amino lipid, phospholipid, cholesterol and a PEGylated lipid. The mRNA encoded protein is based on the spike glycoprotein of SARS-CoV-2 NCBI Reference Sequence NC_045512.2, GenBank accession number YP_009724390.1 and encodes for full-length S featuring K 986 P and V 987 P mutations.

In vitro protein expression
In vitro translation was performed using Rabbit Reticulocyte Lysate System, Nuclease Treated (Promega, Cat. L4960) according to the manufacturer's instructions. In brief, 2 µg of mRNA encoding for SARS-CoV-2 S or control mRNA encoding for luciferase was denatured for 3 min at 65°C and incubated with Rabbit Reticulate Lysate, Amino Acid Mixtures, Transcend™ Biotin-Lysyl-tRNA (Promega, Cat.L5061), RNasin ® Ribonuclease Inhibitors (Promega, Cat. N2511) and Nuclease-Free water in a total volume of 50 µl at 30°C for 90 min. In vitro translated proteins in 1× Lämmli buffer were proteins were separated on 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (BioRad) and transferred to an Immobilon-FL PVDF membrane (Merck,

Challenge infections
Animals were challenged by infection IN with 10 2 TCID 50 /dose of SARS-CoV-2 (BetaCoV/Munich/BavPat1/2020) in a total dose volume of 0.1 ml on day 56, 4 weeks following the second immunisation. Animals were followed for 4 or 7 days post challenge and euthanised on day 60 or day 63 of the experiment as indicated.
To assess binding competition of sera from vaccinated animals with neutralising mAb, HeLa cells were transfected with mRNA encoding for SARS-CoV-2 S protein as described above. Twenty-four hours post transfection, approximately 160,000 cells/well were transferred into a 96well V-bottom plate and incubated with 100 µl of 1:10 diluted mouse serum from mice vaccinated with CVnCoV, alum-adjuvanted protein or buffer, at 4°C overnight. As a positive control, an additional sample of transfected cells remained untreated with serum. The cells were then incubated with 200 ng/well of R-PE-labelled rabbit anti-SARS-CoV-2 spike neutralising mAb (Sino Biological, Cat: 40592-R001) at 4°C for 2 h. As a negative control, a sample of transfected cells treated with serum from CVnCoV-vaccinated animals was incubated with buffer without labelled antibody. Antibody labelling with R-PE using the Zenon TM labelling kit for rabbit IgG (Invitrogen, Cat: Z25355) was performed according to the manufacturer's instructions. Specific binding of R-PE-labelled mAb to S protein-expressing HeLa cells was assessed via FACS analysis as described above.
For the analysis of VNTs of mouse sera, serial dilutions of heatinactivated sera (56°C for 30 min) tested in duplicates with a starting dilution of 1:10 followed by 1:2 serial dilutions were incubated with 100 TCID 50 of wild-type SARS-CoV-2 (strain 2019-nCov/Italy-INMI1) for 1 h at 37°C. Every plate contained a dedicated row (eight wells) for cell control that contains only cells and medium, and a dedicated row of virus control that contains only cells and virus. Infectious virus was quantified upon incubation of 100 µl of virus-serum mixture with a confluent layer of Vero E6 cells (ATCC, Cat.1586) followed by incubation for 3 days at 37°C and microscopical scoring for CPE formation. A back titration was performed for each run in order to verify the correct range of TCID 50 of the working virus solution. VN titres were calculated according to the method described by Reed and Muench. If no neutralisation was observed (MNt < 10), an arbitrary value of 5 was reported. Analyses were carried out at VisMederi srl (Siena, Italy).
VN antibody titres of hamster serum samples were analysed upon heat inactivation of samples for 30 min at 56°C. Triplicate, serial two-fold dilutions were incubated with 10 2 TCID 50 /well SARS-CoV-2 virus for 1 h at 37°C leading to a sample starting dilution of 1:10. The virus-serum mixtures were transferred to 96-well plates with Vero E6 cell culture monolayers and incubated for 5 days at 37°C. Plates were then scored using the vitality marker WST8 and (100% endpoint) VN titres were calculated according to the method described by Reed and Muench. Analyses were done at Viroclinics Xplore (Schaijk, The Netherlands).

T cell analysis
The induction of antigen-specific T cells was determined using ICS. For ICS, splenocytes from vaccinated and control mice were isolated and single-cell suspensions were prepared in supplemented medium. Then, 2 × 10 6 splenocytes (200 µl) per well were stimulated for 5-6 h at 37°C using an SARS-CoV-2 peptide library (JPT, PM-WCPV-S2) at 0.5 µg/ml. After 1 h, Golgi Plug (BD Biosciences, Cat: 555029) was added in a dilution of 1:200 (50 µl) to the splenocytes to inhibit the secretion of intracellular cytokines. After stimulation, splenocytes were centrifuged, resuspended in supplemented medium and stored at 4°C overnight. Following this, splenocytes were washed twice in PBS and stained with AquaDye (Invitrogen, Cat: L34957) solution at 4°C for 30 min. After an additional washing step in FACS buffer (PBS with 0.5% BSA), cells were surface stained for Thy1.2, CD4 and CD8 and incubated with FcƴR-block for 30 min at 4°C in FACS buffer. After surface staining, splenocytes were washed in FACS buffer and fixed using Cytofix/Cytoperm (BD Biosciences) according to the manufacturer's instructions. After fixation, splenocytes were washed in perm buffer and stained for IFN-ƴ and TNF for 30 min at 4°C. After staining, the cells were washed with perm buffer, resuspended in FACS buffer supplemented with 2 mM EDTA and 0.01% Natriumacid and stored at 4°C until analysis.
Splenocytes were analysed on a Canto II flow cytometer (BD Biosciences). Flow cytometry data were analysed using FlowJo software (Tree Star, Inc, Ashland, USA.).

Cytokine analysis
Blood samples were taken via retro-orbital bleeding 14 h after administration of CVnCoV or buffer. Serum cytokines (IFN-γ, IL-1β, TNF, IL-6, IL-4, IL-5 and IL-13) were assessed using cytometric bead array using the BD FACS CANTO II. Serum was diluted 1:4 and BD Bioscience mouse cytokine flex sets were used according to the manufacturer's protocol to determine serum cytokine levels.

Viral load in the respiratory tract
Detectable levels of replication-competent virus in throat swabs, lung and nasal turbinate tissues post challenge were analysed. Quadruplicate, tenfold serial dilutions were transferred to 96-well plates with Vero E6 cell culture monolayers and incubated for 1 h at 37°C. Cell monolayers were washed prior to incubation for 5 days at 37°C. Plates were then scored using the vitality marker WST8 and viral titres (Log10 TCID 50 /ml or /g) were calculated using the method of Spearman-Karber. Analyses were done at Viroclinics Xplore (Schaijk, The Netherlands).

Gross lung pathology
At the time of necropsy, gross pathology was performed on each animal and all abnormalities were described. All lung lobes were inspected and the percentage of affected lung tissue evaluated via visual inspection and scoring determined.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.