Fusion protein pan-sarbecovirus vaccines elicit broadly protective immune responses targeting Clade 1a, 1b, and 3 sarbecoviruses

Fusion protein pan-sarbecovirus vaccines elicit broadly protective immune responses targeting Clade 1a, 1b, and 3 sarbecoviruses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fusion protein pan-sarbecovirus vaccines elicit broadly protective immune responses targeting Clade 1a, 1b, and 3 sarbecoviruses Alyson Kelvin, Matthew Rogers, Zahed Katooni, Eva-Maria Uhlemann, and 24 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8544714/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Broadly protective vaccines are needed to prevent health emergencies caused by emerging and circulating coronaviruses. Previously emerging viruses of the sarbecovirus subgenus (SARS-CoV (Severe Acute Respiratory Syndrome coronavirus) and SARS-CoV-2) have caused significant impacts on human and animal health and are anticipated to spillover again. We developed novel pan-sarbecovirus vaccines using next-generation technology producing plug-and-play fusion proteins. The scaffold of the platform links an N-terminal S1 domain with receptor binding domains (RBDs) from various targeted sarbecoviruses and is easily and quickly biomanufactured to be responsive to health emergencies. The design of our vaccines was guided by epitope prediction and molecular dynamic simulations. In vivo immunization studies showed the antigens induce broadly reactive T and B cell responses against several clades of sarbecoviruses including broadly neutralizing antibodies against pre-emergent sarbecoviruses using WIV16, PangGX, and PangGD pseudotyped viruses. Immunized Syrian hamsters were protected from mortality following heterologous challenge with SARS-CoV-2 Delta (Clade 1b) or SARS-CoV (Tor2) (Clade 1a). Significant reductions in tissue viral titer and lung pathology were observed in immunized animals compared to control groups (Pfizer BA4/5 vaccine and PBS). Taken together, our pan-sarbecovirus vaccines are protective against diverse sarbecoviruses and the platform is a strategy for addressing health emergencies. Biological sciences/Microbiology/Vaccines/Protein vaccines Biological sciences/Microbiology/Virology/SARS virus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Coronaviruses (order Nidovirales; family Coronaviridae ) are a continual threat to human health as seen by the recent COVID-19 (coronavirus disease from 2019) pandemic, circulating seasonal coronaviruses, and the potential for new spillover viruses from animal reservoirs 1 . Coronaviruses have positive sense, single-stranded RNA genomes characterized by one of four genera: alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses 2 . The betacoronavirus genera has been a significant threat to humans as illustrated by the emergence of three coronaviruses that cause severe disease in humans: SARS-CoV (severe acute respiratory coronavirus) (2002), Middle East Respiratory Syndrome coronavirus (MERS-CoV) (2012), and SARS-CoV-2 which causes COVID-19 (2019) and led to an estimate of over 20 million deaths worldwide 2–4 . Of those three coronaviruses, both SARS-CoV and SARS-CoV-2 are members of the sarbecovirus subgenus of betacoronaviruses. Investigation of sarbecoviruses in the animal reservoir suggest pre-emergent sarbecoviruses have the potential for additional spillover events causing future human health emergencies 5–9 . Furthermore, as SARS-CoV-2 continues to circulate in people, the virus has continued to diversify with the emergence of variants of concern (VOCs) that can evade pre-existing immunity from infection and vaccination 10–15 . The immunogenic viral envelope Spike (S) protein is responsible for host cell binding through the human ACE2 (angiotensin-converting enzyme 2) receptor 16–19 . Although the S protein is often the chosen target for vaccines, there is great antigenic diversity among S proteins limiting vaccine effectiveness 1 . Vaccines rapidly developed against the SARS-CoV-2 virus during the COVID-19 pandemic leveraged more modern and/or less common vaccine platforms such as mRNA or viral vector as the main workhorse 20–22 . First generation COVID-19 vaccines targeted single strains eliciting narrow immune responses leading to the need for frequent formulation updates. Considering coronavirus vaccine limitations, new strategies are urgently needed to induce more broadly protective immune responses 5,6 such as pan-sarbecovirus vaccines 23–29 . Much focus has been given to nanoparticles as next-generation broadly protective vaccine platforms 30 . Engineered nanoparticles displaying monovalent or multivalent repetitive arrays of viral surface proteins (such as full exodomains of coronavirus S proteins) or surface protein domains (such as the Receptor Binding Domain (RBD) of S) recapitulate the immunogenic 3D structure of viral surface proteins including trimerization, which is more immunogenic than free trimers 23,24 . Examples of promising nanoparticle platforms include SpyCatcher-SpyTag system, bacteriophage T4 nanoparticles, and ferritin-based nanoparticles 24,26,27 . Protein nanoparticles developed through plug-and-play platforms such as these have been shown to successfully induce broadly protective immune responses toward viral targets such as coronaviruses, influenza viruses, and HIV 23–25,31 . Although these vaccine candidates show promise, there are drawbacks to nanoparticle platforms such as the inability to design the order and amount of each target in the nanoparticle and the need to assemble individually translated proteins into a 3D conformation. In the case of ferritin, 24 ferritin-fusion protein subunits must self-assemble into the quaternary level to create octahedral symmetry 30 . Previously we reported on a flexible protein subunit vaccine platform with plug-and-play capabilities that linked together the S1 domain and RBDs from various SARS-CoV-2 variants to create a single fusion protein antigen during protein translation 32 . The pan-SARS-CoV-2 variant candidate vaccines formulated with SWE (squalene-and-water emulsion) induced cross-reactive immune responses against various SARS-CoV-2 variants. Here we expanded on our previous work to rationally design vaccine candidates using a quantitative approach that guides target virus selection and domain placement with the goal of inducing broadly protective immune responses against several clades of sarbecoviruses. We show that our 4 designed vaccine candidates, targeting Clade 1a, Clade 1b, and Clade 3 of sarbecovirus viruses, elicited cross-reactive humoral and T cell responses and were protective against heterologous challenge with Clade 1a (SARS-CoV Tor2) and Clade 1b (SARS-CoV-2 Delta) viruses reducing viral load, mortality, and lung pathology. RESULTS Design, predictions, physical characterization, and immunogenicity of pan-sarbecovirus vaccine candidates We previously developed a flexible multivalent protein subunit vaccine platform to target several SARS-CoV-2 variants with one antigen-adjuvant formulation 32 . The platform links several domains of the SARS-CoV-2 spike (S) protein together with each domain represented a distinct SARS-CoV-2 variant. In the present study we expanded the breadth of immune responses and protection offered by vaccine candidates by including targets within the subgenus of sarbecoviruses, theoretically covering entire clades within the subgenus. The subgenus of sarbecoviruses include SARS-CoV and SARS-CoV-2, as well as pre-emergent bat viruses such as WIV1, RaTG13, and RsSHC014 that have been suggested to pose a threat to humans 5,6,33 . We performed phylogenetic analysis to understand the relatedness among the subgenus of sarbecoviruses and guide target virus selection. Over 1000 raw sarbecovirus spike sequences from viral strains were acquired from GISAID or other freely available databases such as NextStrain. We removed sequences that were incomplete or duplicated and trimmed sequences to focus on the RBD segment. RBD amino acid sequences were aligned and a phylogenetic tree was constructed using IQtree2 (routed on SARS-CoV-2 ancestral strain spike protein sequence (QHD43416.1; positions 319–541)) 34,35 . Similar to previously reported analyses 5,36 , our analysis identified three clades, Clade 1, Clade 2, and Clade 3, with notable subclades within the sarbecovirus subgenus (Clade 1a and Clade 1b) (Fig. 1 ). Clade 1a (red bubble) encompasses viral sequences related to SARS-CoV including all published SARS-CoV sequences and bat-related sequences WIV1 and RsSHC014. Clade 3 (orange bubble) representing sarbecoviruses collected outside of Asia including BtKY72 and Khosta-2 (from Kenya and Russia, respectively) was most proximal to Clade 1a 7,8,37–39 . Clade 1b (green bubble) illustrates the relatedness of SARS-CoV-2 and variant viruse sequences as well as viruses from bats and other animals including the bat sarbecovirus RaTG13 and pangolin virus Pang17. Finally, the most distant clade, Clade 2 (purple bubble) was characterized by two main subclades representing HKU3 and mostly bat sequences. We designed both trivalent and quadrivalent candidates having 3 and 4 targets, respectively. All candidates leveraged the full ancestral SARS-CoV-2 S1 subunit as their first target in the N-terminal anchoring position. Following the S1 subunit, RBD targets were linked using a 3 GS linker (6 aa total). The trivalent candidate (S1-RBD-RBD) only included targets from Clade 1a and Clade 1b, as there were less positions in the antigen to target more clades or subclades. The quadrivalent candidates (S1-RBD-RBD-RBD) included targets from Clade 1b, Clade 3, and Clade 1a depending on the specific candidate. Two quadrivalent candidates incorporated two sequences from Clade 1a viruses (RsSHC014 and WIV1 or Urbani (SARS-CoV)), while the third candidate included two Clade 1b targets (ancestral SARS-CoV-2 and RaTG13). Specifically, the candidates are SARS-CoV-2-Pang17-WIV (VIDO4500 (4500 onward)); SARS-CoV-2-Khosta2-RsSHC014-WIV1 (VIDO 4501 (4501 onward)); SARS-CoV-2–Khosta2–RsSHC014-Urbani (VIDO4502 (4502 onward)); SARS-CoV-2-RaTG13-BtKY72-WIV1 (VIDO4503 (4503 onward)) (Fig. 2 A). The viral sequences more similar in amino acid identity were placed adjacent to each other in the fusion protein so that the order of the components in the candidate reflected the phylogenetic tree. We hypothesized that this placement would encourage the stimulation of cross-reactive antibodies and a more durable immune response 30,40 . We next predicted the 3D protein structure of the 4 candidates using Alpha fold (Fig. 2 B). The S1 protein of all candidates are shown in purple (left side of each structure) while the RBDs are shown in pink, green, blue, and light purple. Characterization of harvested protein on SDS-PAGE gels followed by Coomassie Blue staining and western blotting with antibodies for SARS-CoV-2 S1 indicated that the expressed proteins were of expected molecular weight (Table 1 ) and had evidence of glycosylation (Fig. 2 C). Further physical characterization analyzed the level of expression and size exclusion chromatography with multi-angle light scattering (SEC-MALS) and Circular Dichroism (CD) was conducted (Table 1 ). The predicted molecular weight (MW) of the candidates ranged from 125-151kDa unglycosylated and 163–217 kDa glycosylated. All the proteins contained above 90% monomers, except candidate 4503 which contained 58.2% of the dimer form requiring 0.08% Tween-80 to the vaccine formulation. Analysis also suggested that the secondary structure content determined by CD spectroscopy was consistent with the predicted structure as shown in Fig. 2 B. Partial denaturation was observed in the temperature range between 39-46 o C, indicating that the proteins were stable under standard purification and storage conditions. Loss of α-helix content and partial disruption of disulfide bridges was observed only at low pH (3.3–3.8). Table 1 Pan-sarbecovirus vaccine antigen characterization Description Expression level Predicted mW (unglycosylated) Experimental MW (glycosylated) SEC-MALs CD composition 4500 WHS1 – Pang17 RBD – WIV1 RBD 16 mg/L 125 kDa 163 kDa Monomer: 100% α- helical: 21% β-sheets: 35% coil: 44% 4501 WHS1 – Khosta2 RBD – RsSHCO14 RBD – WIV1 RBD 8 mg/L 151 kDa 209 kDa Monomer: 90%* α- helical: 20% β-sheets: 34% coil: 46% 4502 WHS1 – Khosta2 RBD – RsSHCO14 RBD – Urbani RBD 15 mg/L 151 kDa 200 kDa Monomer: 100% α- helical: 20% β-sheets: 34% coil: 46% 4503 WHS1 – RaTG13 RBD – BtKY72 RBD – WIV1 RBD 17 mg/L 151 kDa 217 kDa Dimer: 58.2% Aggregate: 8.5% α- helical: 20% β-sheets: 35% coil: 45% We next performed antigenicity predictions of our candidates to ensure epitopes would not be buried upon expression and tertiary structure formation. B-cell epitopes were predicted using DiscoTope-3 focusing on residues with scores equal to or greater than 0.9, which by default is used for Moderate confidence (0.90, recall up to ~ 50%) and ignored those with a negative score. The sums of calibrated scores were 458.6, 508.2, 530, and 531.2 for 4500, 4501, 4502 and 4503, respectively (Fig. 3 ). Results showed concentrated peaks above the threshold within the conformation of all four vaccine structures when epitopes were graphed over the protein by amino acid (Supplementary Fig. 1). To investigate if these epitopes are accessible to bind to antibodies providing confidence in the use of the antigen, we performed molecular docking simulation against the neutralizing antibody CT-P59 (PDB: 7CM4) (Supplementary Fig. 2). RBDs of all vaccine candidates are predicted to properly bind strongly or weakly the heavy and light chains of the tested antibody with acceptable accessibility for immune engagement. Molecular dynamics (MD) simulations (200 ns) for one of the vaccine candidates was completed to analyze the dynamic stability and structural motions over time (Supplementary Fig. 3). Simulations of three control proteins were performed for comparison: a full Spike trimer, a single chain spike, and the 4502 construct without the S1 region as we wanted to know if an N-terminal RBD was sufficient for candidate stability or if the entire S1 domain was required. We found the presence of the S1 domain enhanced dynamic stability of the candidate as the candidate with a full S1 domain had a lower overall Root Mean Square Deviation (RMSD) and a reduced degree of structural fluctuation compared to the control protein with only an N-terminal RBD (Supplementary Fig. 3). Moreover, the stability of candidate 4502 was also greater than the other controls. RMSD and Root Mean Square Fluctuations (RMSF) were calculated to measure whether the proteins held their 3D conformations and to get an insight into their flexibility and stability (Supplementary Fig. 4). RMSF analysis indicated the trimeric spike of SARS-CoV-2 (dotted light blue line, Supplementary Fig. 4) to be the most stable while the isolated Spike Chain A (SARS-CoV-2_Spike_Chain_A) behaved oppositely with large fluctuations throughout both S1 (~ 14–685) and S2 (~ 686–1273) regions. The truncated No S1 form of 4502 (4502_No_S1) had considerable structural motion, with flexibility increasing mainly in the N terminus. Candidate 4502 with the S1 in the N terminus was more stable than the S1 alone (average differences of ~ 0.3–0.7 nm for most regions) which this level of flexibility may allows the formation of important loops that play a role in interacting with antibodies. Immunogenicity of pan-sarbecovirus vaccine candidates in mice We next investigated the immunogenic potential of the candidates by immunizing BALB/c mice with 5 µg of candidate protein formulated with SWE (squalene-in-water emulsion) as adjuvant using a prime-boost regime with a 21-day interval. Responses were compared to control immunizations with a known standard coronavirus vaccine (Pfizer BA4/5) and a negative control (PBS). To determine the breadth of antibodies and potential to be cross-reactive, we developed a panel of sarbecovirus RBD proteins (WIV1, Khosta2, RsSHC014, Pang17, Urbani, and RaTG13) for ELISAs. Plasma samples collected Day 42 post prime immunization were incubated against the panel and analyzed with the results presented as a heat map Log 10 ELISA titer (dark purple = low levels of binding; yellow = high levels) (Fig. 4 A) or as separate histograms (Supplementary Fig. 5). Statistical significance of the Log 10 ELISA titer was determined compared to PBS controls. All vaccine candidates and the control Pfizer vaccine induced high titers of binding antibodies to the SARS-COV-2 S1, as well as binding antibodies to the other RBDs (Fig. 4 A). The Pfizer immunized animals had the lowest titers across the other RBDs, especially toward the Clade 3 antigens, Khosta2 and RsSHC014. All our designed candidates induced antibody titers toward the Clade 3 RBDs and had greater titers against the other Clade 1a and Clade 1b antigens. Splenocytes isolated on Day 42 from each immunization group were stimulated with either RBD from SARS-CoV-2 (Ancestral) or SARS-CoV (Urbani) and IFN-γ and IL-5 were quantified (Fig. 4 B). As expected, low amounts of IFN-γ and IL-5 producing cells were observed from the PBS immunized animals for all stimulations; however, Pfizer immunized animals also had low numbers of positive cells for both Th1 and Th2 responses. Conversely, cells from animals immunized with pan-sarbecovirus vaccine candidates exhibited strong Th1 and Th2 responses toward SARS-CoV-2, above that of the Pfizer groups with the exception of IFN-γ production in cells from 4500, 4502, and 4503 immunized animals following stimulation with SARS-CoV-2 and in cells from 4502 following stimulation with Urbani. Cells from immunized animals had strong IFN-γ response above the PBS control group. Interestingly, IL-5 responses were significantly higher even over 500-fold for the cells from pan-sarbecovirus vaccine candidate immunized animals compared to the Pfizer groups. This data suggests that the pan-sarbecovirus vaccine candidates induce both cross-reactive antibody responses as well as strong and balanced cross-reactive T cell responses. Pan-sarbecovirus vaccine candidates induce cross-protection during SARS-CoV-2 Delta heterologous challenge We next investigated if the pan-sarbecovirus vaccine candidates could provide protection against severe disease and infection following challenge with a virus that is not targeted in the vaccine. Using the Syrian hamster model, animals were immunized and boosted with 25 µg of vaccine candidates, control vaccines (Pfizer BA4/5 (5 µg) or PBS) on Day 0 and 28. On Day 48 post prime, the animals were intranasally challenged with SARS-CoV-2 Delta variant (10 5 TCID 50 ) which was not targeted in the vaccine. To determine protection, we assessed viral load in the respiratory tissues, lung pathology, and induction of viral neutralizing antibodies. Blood samples were collected on Day 48 and blood and lung samples were collected on Days 5 and 10 post challenge for analysis (Fig. 5 A). Due to the large size of the study, three trials were performed with each trial having its own set of PBS and Pfizer vaccinated control groups. Analysis of weight post challenge was graphed for each candidate vaccine group compared to the control vaccines of their trial (Fig. 5 B). The Pfizer vaccine was able to protect against weight loss during the Delta virus challenge period with the animals losing less than 5% of their original weight in all of the three trials. Conversely, the negative control PBS immunized groups lost ~ 10% of original weight in each trial. All pan-sarbecovirus vaccine candidates protected the animals from weight loss with the weight loss experienced in each group being statistically less than those animals immunized with PBS. Of note, the hamsters vaccinated with the candidate 4503 (SARS-CoV-2-RaTG13-BtKY72-WIV1) lost the least amount of weight dropping to ~ 98% of original weight. Microneutralization assays indicated high titers of virus neutralizing antibodies against the Delta variant were present in each group immunized with the pan-sarbecovirus candidate vaccines with levels comparable to the Pfizer immunized groups (Fig. 5 C). Specifically, the candidates 4500 and 4503 elicited higher virus neutralizing titers of antibodies than the Pfizer vaccine, with the 4500 immunized animals having elicited statistically higher levels than the Pfizer immunized animals. The candidate 4503 elicited the highest virus neutralizing titers with titers (1:128 to 1:156). Viral loads were evaluated in nasal turbinates and lung tissue collected on Day 5 post challenge from all groups (Fig. 5 D). Negative control PBS groups had average viral titers between 10 3 and 10 4 TCID 50 . The control Pfizer immunized animals did not have infectious virus within either tissue on Day 5. Furthermore, the vaccine candidates 4500, 4501, and 4503 were all also mostly negative for infectious virus in the nasal turbinates and lungs with only one animal from the 4501 immunized group having a positive viral titer in the respiratory tract samples (10 1 TCID 50 in lungs and 10 3 TCID 50 in nasal turbinates). Independent pathological assessment was conducted on lung tissues from Day 5 and 10 post challenge. Lung disease was rated on a scale of 0–4 in seven categories assessing percentage of parenchyma affected; inflammation, pneumonia, hyperplasia, bronchi and bronchiolar pathology, and hemorrhage. Overall Histopathology scores in the candidate immunized animals were lower than the scores of the PBS control groups (Fig. 5 E). ELISAs against our panel of sarbecovirus RBD antigens (WIV1, Khosta2, RsSHC014, Pang17, Urbani, RaTG13, BtKy72, XBB.1.5 (variant of SARS-CoV-2)) (Fig. 6 A; Supplementary Fig. 6) found the Pfizer vaccine was able to induce cross-binding antibodies toward all antigens tested, however, overall the Log 10 ELISA titer plasma from the Pfizer immunized animals was lower toward the majority of antigens compared to the binding of antibodies from our candidate immunized animals. The candidate that induced the highest titers of binding antibodies across the sarbecovirus non-SARS-CoV-2 RBD antigens was candidate 4503 which had titers ranging from 4.66 to 5.49 whereas the Pfizer vaccine had titers ranging from 3.24 to 3.82. We also conducted pseudovirus neutralizations using pseudoviruses of WIV16, PangGX, and PangGD against the plasma from immunized animals (Fig. 6 B). WIV16 is a Clade 1a sarbecovirus related to WIV1, while PangGX and PangGD are Clade 1b viruses related to Pang17. Plasma collected from animals immunized with pan-sarbecovirus candidates had statistically higher neutralization against WIV16 compared to PBS control immunized animals; however, animals immunized with Pfizer were not found to have statistically higher titers of neutralizing antibodies. All candidates as well as the Pfizer vaccine elicited statistically higher titers of neutralizing antibodies against PangGX and PangGD than the PBS control immunized animals. These results suggested that pan-sarbecovirus vaccine candidates are potentially protective against heterologous viruses as the vaccine candidates induced significant levels of cross-reactive antibody responses. Pan-sarbecovirus vaccine candidates protect against SARS-CoV Clade 1a sarbecovirus challenge Since SARS-CoV-2 Delta is a Clade 1b sarbecovirus, we felt it was important to next evaluate the pan-sarbecovirus vaccine candidates against a Clade 1a sarbecovirus challenge to further understand the breadth of the protection offered by the vaccines. We conducted a similar challenge study as above except the immunized hamsters were challenged with the Tor2 strain of SARS-CoV (6.24x10 4 TCID 50 ) as a representative of Clade 1a viruses (Fig. 7 a). Two trials were performed to evaluate all 4 candidates with each trial having its independent controls. Infection with SARS-CoV induced greater disease severity in the control PBS immunized hamsters compared to the other groups. The PBS vaccinated animals lost more than 10% of original weight with some animals reaching humane end points and were removed from the study (euthanized) (1 PBS immunized hamster per PBS group reached humane endpoint in the first trial (control groups for 4502 and 4503) and 2 PBS hamsters reached humane endpoint in the second trial (control groups for 4500 and 4501) (Supplementary Fig. 7)). Pfizer vaccinated animals lost less than 5% of original weight whereas all candidate vaccines lost even less than 5% (Fig. 7 B). All pan-sarbecovirus vaccine candidates induced virus neutralizing antibodies toward SARS-CoV with group average titers at 1:64 or above by Day 48. The upper and lower respiratory tracts had significant viral titers in the control PBS immunized groups with average titers of 10 3 TCID 50 or above. Robust SARS-CoV viral titers were also present in the respiratory tracts of the Pfizer immunized animals in each of the trials with 3 out of the 4 animals having nasal turbinate viral titers above 10 3 TCID 50 (Fig. 7 D). For the pan-sarbecovirus vaccine candidates, the results of the TCID 50 assays indicated that most of the respiratory tract samples were below the limit of detection and each group had significantly lower viral loads in both the nasal turbinates and lungs compared to the PBS and Pfizer groups. Lung pathological assessment found Negative control immunized animals had average scores of 3 on Day 5 while the Pfizer group animals had an average score between 2 and 3 depending on the trial (Fig. 7 E). For all pan-sarbecovirus vaccine candidates, no animal scored above 1. The scores for 4500 and 4501 on Day 5 post challenge were statistically lower than both the PBS and Pfizer groups. The trial with 4502 and 4503 had statistically lower scores than the PBS group for the same day. Taken together, these data indicated that protection from viral infection, disease severity, and lung pathology were provided by all designed pan-sarbecovirus vaccine candidates during heterologous challenge with the Clade 1a virus SARS-CoV. DISCUSSION There is a need for anticipatory vaccines to protect against potential emerging coronaviruses. Here we developed four candidates on a flexible plug-and-play fusion protein subunit platform that strategically incorporated specific sarbecovirus targets from Clade 1a, Clade 1b, and Clade 3 to induce broadly protective immune responses. We predicted our antigens to have binding capacity to a known broadly reactive antibody and have accessible B cell epitopes. Experimentally, we found our vaccines protected against SARS-CoV2 Delta and SARS-CoV challenge with concomitant induction of broadly binding and broadly neutralizing antibodies against pseudoviruses from pre-emergent sarbecoviruses (pangolin viruses and WIV16). Additionally, we found our candidates induced robust broadly reactive T cell responses against diverse sarbecovirus RBD antigens, which was not seen in the Pfizer immunized animals. Specifically, in the Pfizer immunized animals, T cells did not have significant levels IFN-γ or IL-5 after stimulation with Clade 1a or 1b antigens (SARS-CoV-2 (Ancestral) or SARS-CoV (Urbani)). This data suggested that our pan-sarbecovirus vaccine candidates may offer pan-virus protection through both broadly reactive humoral and cell mediated responses with the fusion protein platform being a potential tool against other virus families. In our current study we investigated the protection of protein subunit vaccine candidates by intramuscular (IM) delivery. Although the candidates protected from severe disease including against SARS-CoV challenge, small amounts of infectious virus as well as lung pathology was still detected, which could be attributed to the lack of mucosal protection elicited during intramuscular immunizations. Intranasal vaccination is an approach that has been shown to be successful for COVID-19 vaccines and pan-coronavirus vaccine candidates 41 and should be investigated in the future for our vaccine candidates. Moreover, we showed that both broadly reactive T cell and humoral immune responses were induced following immunization with our candidates. Most published studies on broadly reactive vaccine candidates have not investigated T cell responses where one of only the studies to do so was a study involving the dual antigen mRNA candidate vaccine expressing N and S 42 . However, the breadth of these responses was only evaluated against SARS-CoV-2 variants and not against other viruses within the sarbecovirus subclade as we have done here 42 . Although we conducted a fairly thorough investigation of the immune responses, it was not clear what arm of the immune response had more influence on protection. Further studies with adoptive transfer experiments could tease out the mechanisms of protection associated with vaccines developed on our fusion protein platform. Global thought leaders urge the readiness of not only vaccine candidates that are anticipatory, but also for systems supporting vaccine pipelines ensuring required vaccines are available and readily deployable at the first signs of virus emergence 43 . For complete system readiness, anticipatory vaccine candidates must be evaluated in appropriate models during non-pandemic times. Also, having large scale manufacturing processes developed, technology transferred to large industry partners, and industry and deployment pipelines established for equitable global vaccine distribution at fair cost is needed to avoid Public Health Emergencies of International Concern (PHEIC), as well as pandemics 43 . Published studies of broadly protective vaccines and immune responses have shown that vaccine antigens with repetitive arrays of viral surface proteins strongly induce cross- and broad-protection through enhanced B cell activation suggesting these platforms and pipelines being appropriate for pandemic preparedness 30 . Vaccine platforms have included the protein nanoparticle, protein-lipid nanoparticle, live-but-defective SARS-CoV-2 virus with interferon integration, NanoSTING platform, VLPs (Virus Like Particles), chimeric subunit vaccine, and mRNA candidates 24,27–29,41,42,44,45 . These described vaccine candidates have shown various levels of broad-reactivity such as across SARS-CoV-2 variants 27,28,42 or across Clade 1 sarbecoviruses 28,36 . However, the majority of platforms leveraged for establishing the display of repetitive viral surface proteins have been multivalent protein nanoparticles that require the assembly of fusion protein subunits for subsequent nanoparticle assembly which may take longer and be subject to more manufacturing challenges 24,26,27,29,36,46 . Conversely, our protein subunit platform leverages the translation of a single fusion protein as a vaccine antigen that can display repetitive viral surface protein/protein domains while retaining immunogenic 3D properties. Sustainable biomanufacturing solutions are essential for producing enough product for global distribution quickly during an emergency. Our simplified platform producing a single fusion protein potentially supports more sustainable and streamlined biomanufacturing processes. Here we have shown promising results on the broadly reactive nature of multivalent fusion protein subunit pan-sarbecovirus vaccine candidates. Future studies will be important to establish readiness of these candidates for emergency situations. Additional studies investigating both the longevity of protection as well as antigen stability as it relates to shelf life in stockpiles are needed 32 . Moreover, studies in preimmune animal models will also be important for understanding potential immune skewing toward antigens previously seen to support future human clinical trials 43,47–50 . Coronaviruses are a significant threat to public health, animal welfare, and economic stability as new strains emerge and endemic coronaviruses continue to circulate. It is highly likely that a novel coronavirus with pandemic potential will emerge within the next decade as we have seen three severe coronaviruses emerge over the past 25 years. The path to controlling the public health burden of coronaviruses involves developing next generation vaccines that can anticipate the diversification of circulating strains and/or the spillover of strains yet uncharacterized. Together, the work presented here, and future studies will help build a global portfolio of broadly protective coronavirus vaccines and flexible vaccine platforms. Having anticipatory vaccines that offer broad protection readily available are essential for health emergency preparedness. MATERIALS AND METHODS Experimental Design This study investigated the design and development of pan-sarbecovirus vaccine candidates developed as a fusion protein. Structural and immunogenic predictions were made of design vaccine antigens. Immunogenicity was assessed in vivo in Syrian hamsters and in mice. Protection from heterologous virus challenge was assessed in Syrian hamsters immunized with candidate antigens compared to control vaccine (Pfizer) or mock vaccine (PBS). Ethics Statement All work was conducted in accordance with the Canadian Council of Animal Care (CCAC) guidelines, AUP numbers 20200016 and 20240002 by the University Animal Care Committee (UACC) Animal Research Ethics Board at the University of Saskatchewan. Hamster and mouse procedures were performed under 5% isoflurane anesthesia. Phylogenetic analysis Raw sarbecovirus spike gene sequences were collected from GISAID and other open databases such as NextStrain and the NCBI nt database. Initially, 407 sequences were compared, but sequences that were highly similar, overrepresented within certain clades (e.g., multiple strains of human SARS-CoV), or had incomplete receptor-binding domains (RBDs) were removed. After filtering, 105 sequences remained for analysis. The spike gene sequences were translated and aligned using the MUSCLE algorithm implemented in the SEAVIEW phylogenetic package 51 . All sites with the exception of the RBD region (positions 319–541, based on the ancestral SARS-CoV-2 spike protein QHD43416.1) were masked from the analysis. A phylogenetic tree of the RBD nucleotide sequences was constructed with IQ-TREE2 52 , using a amino acid substitution model selected by ModelFinder (Q.yeast) and rates across sites approximated on a gamma distribution of four categories of variable sites and one invariable site category. Branch support was evaluated with 1,000 ultrafast bootstrap replicates. Protein Structure Prediction The 3-dimensional modeling of the candidate vaccine protein antigens were constructed using AlphaFold2 (AlphaFold2 (Colab-Fold) (ChimeraX plugin for AlphaFold2/Colab-Fold with default criteria). The structural predictions were then validated using SWISS-MODEL. Protein structures were visualized in PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) and ChimeraX. Epitope Prediction and Antibody Molecular Docking Prediction The conformation of our vaccine constructs was predicted through using TAMARIND BIO – Boltz-2, which is based on AlphaFold3 reproduction. The conformations were used for B-cell epitope prediction through DiscoTope 3.0, with the threshold being set to 0.9, and HADDOCK2.4, and default criteria for molecular docking. Molecular docking was predicted to the neutralizing antibody CT-P59 (PDB: 7CM4). Molecular Dynamics (MD) Simulations GROMACS version 2020.1 with CHARMM36 all-atom force field was employed to conduct the Molecular dynamics (MD) simulations. All our MD systems with vaccine constructs solvated in a cubic simulation box filled with TIP3P water molecules, with a minimum distance of 1.0 nm between the protein surface and the box boundary. All systems were neutralized by adding counterions Na⁺ and Cl⁻. Energy Minimization and Equilibration were achieved before the production run using the steepest descent algorithm. The NVT (constant Number, Volume, Temperature) for one ns, equilibration for 100 ps and NPT for five ns were performed. Finally, the NPT, NVT equilibrated systems were subjected to a production MD run for 200 ns, using a 2 fs MD integrator. The temperature and pressure at 300 K and 1 bar remained stable through a modified Berendsen thermostat (V-rescale) with time constant τ_t = 0.1 ps and Parrinello-Rahman pressure coupling with the compressibility of 4.5e-5 and τ_p = 2 ps. The LINCS algorithm was used for bond constraints. Lennard-Jones and Coulombic non-bonded interactions were treated with a cutoff of 1.2 nm. Long-range electrostatics were calculated by the Particle Mesh Ewald (PME) method. Periodic boundary conditions were applied in all directions. Vaccine Design, Vaccine Expression and Purification Targeted sequences were identified and linked via 3x GS linker to S1 WT sequence to generate the desired vaccine candidates. The DNA sequence was cloned into the pCDNA3.4 expression vector (Genscript Biotech; Piscataway, New Jersey, USA), transformed into chemically competent OneShot®Top10 E. coli cells (Thermo Fisher Scientific), and amplified and then extracted using Qiagen Endofree Maxi prep kit (Qiagen Cat. 12362). Inserts were verified by restriction digestion and sequencing (Plasmidsaurus, San Francisco, California). ExpiCHO™ cells (6x10 6 cells/ml) were transfected with 0.7 µg/ml of candidate vaccine plasmid using ExpiFectamine™. Vaccine candidate proteins were transiently expressed following the Gibo ExpCHO TM max titer expression protocol as outlined by ThermoFisher Scientific (Mississauga, Ontario, Canada), which includes a temperature shift to 32 o C on day 1 after transfection. Cells were cultured for 3 days for optimal protein expression. Supernatants containing secreted vaccine candidate proteins were collected and centrifuged at 4,000 x g for 30 min at 4 o C. After the centrifugation, 0.05% Tween-80 and 300 mM NaCl were added to the clarified supernatant containing the expressed proteins. A final filtration through a 0.2 µm PES filter was subsequently performed followed by tangential flow filtration with a Vivaflow® 50R 30kDa filter (Sartorius; Oakville, Ontario, Canada), and the supernatant was concentrated 8-10 fold. Vaccine candidates were purified using Repligen NGL Covid-19 Spike protein AR 2.0 resin (Repligen, Walktham, Massachusetts, United States) as previous described 32 . Immediately after elution from the column, the protein was exchanged into a buffer containing 277 mM NaCl, 14 mM NaH 2 PO 4 , 5.7 mM Na 2 HPO 4 , pH 7.4, using Amicon® Ultra centrifugal filter with a 30 kDa MWCO (UFC903024). A Pierce™ BCA assay (ThermoFisher Scientific) was conducted according to manufacturer procedure to verify final protein yield. Protein samples were dialyzed with 1X PBS for 1 day at 4°C. For deglycosylation of samples, 5 µg of purified pan-sarbeco protein was incubated with NEB Biolabs PNGaseF enzyme for 1 h at 37°C following the manufacturer’s protocol. Western blots were conducted to confirm protein size and expression. Coomassie gels and Western blotting To visualize and estimate the size of the expressed proteins, supernatants were collected from the culture media and loaded onto 4-20% Mini-PROTEAN® TGX™ acrylamide gels (Cat: 4561094; BioRad, Mississauga, Ontario, Canada). Precision Plus Protein™ ladder (Cat: 161-0374, BioRad) was loaded at 3 μL/well as a standard. The electrophoresis was performed in a Tris-Glycine-SDS buffer (BioRad) at 200 V for 50 min. Following electrophoresis, total protein in each sample was visualized by BioRad Bio-Safe Coomassie stain G-250 (BioRad) following standard protocol or transferred onto 0.2 μm nitrocellulose membranes (Cytiva, Vancouver, British Columbia, Canada). Membranes were blocked overnight in 3% (w/v) Amersham™ ECL Blocking reagent (Cytiva) followed by incubation with in-house generated rabbit polyclonal primary antibody S1-CoV19 (1:5,000) diluted in 1.5% blocking buffer for 1 hr at room temperature. Blots were washed in PBSA before secondary antibody incubations (donkey anti-rabbit IRDye 800CW (1:10,000), Li-COR®; Lincoln, Nebraska, USA) at 1:10,000 dilution for 1 hr at room temperature on a rocker. Resulting blots were washed in PBSA for 5 min before being scanned on a Li-Cor NIR scanner. Physical Characterization Size exclusion chromatography with multi-angle light scattering (SEC-MALS) Purified proteins were analyzed on a Wyatt Technology multi-angle light scattering instrument (MALS) with refractive index (RI) detector in-line with a Cytiva Superdex 200 Increase 10/300 GL column connected to a Bio-Rad NGS FPLC system. The column was calibrated using bovine serum albumin. Samples contained 1 mg protein. The size-exclusion chromatography was performed in a buffer containing 277 mM NaCl, 14 mM NaH2PO4, 5.7 mM Na2HPO4, pH 7.4, at a flow rate of 0.5 ml/min. Acquired data was analyzed using Wyatt software package ASTRA 7.3.2. Circular Dichroism spectroscopy Prior to analysis, protein samples were dialyzed against buffer containing 155 mM NaCl, 2.97 mM Na2HPO4 and 1.06 mM KH2PO4, pH 7.4 (Gibco). Secondary structure content and thermal stability of the proteins were analyzed by CD spectroscopy using Applied Photophysics CHIRASCAN Plus CD spectrometer at a protein concentration of 0.7 to 1 mg/ml. Measurements were obtained from 200 to 280 nm with a bandwidth setting of 2 nm in a 0.5 mm quartz glass cell. The final CD spectrum of each protein was a mean of three measurements. The resulting CD data was analyzed using the Beta Structure Selection (BeStSel) online tool (https://bestsel.elte.hu/ssfrompdb.php). The data was normalized using the following equation: Θnorm= □((Θ (λ) x ε205)/(10 xA205 x (N-1))), where Θnorm is normalized molar ellipticity, Θ(λ) is raw ellipticity in millidegrees, ε205 protein extinction coefficient at 205nm, A205 absorbance at 205 nm, and N is the number of amino acid residues. Extinction coefficients ε205 for protein constructs were calculated as described previously 53 . For the thermal stability assay, CD spectra were successively recorded while the sample was gradually heated from 5 °C to 95 °C at a rate of 1 °C/min, and then cooled back to 20°C. Changes in the secondary structure were analyzed with CHIRASCAN software Pro-Data viewer to determine the melting temperature of each construct. The experimentally determined secondary structure content was compared to the predicted using PSI-BLAST - based prediction tool (PSIPRED) https://bio.tools/psipred. Protein Identification via mass spectrometry analysis Samples containing 5 µg of the purified protein were subjected to SDS-PAGE on a 4-20% TGX gel (BioRad). The gels were stained with BioRad Bio-Safe Coomassie stain G-250 (BioRad) following the manufacturer’s protocol. Protein bands were excised and analyzed by ESI-LS-MS at the Alberta Proteomics and Mass Spectrometry Facility following in-gel digestion. SARS-CoV and SARS-CoV-2 variant viruses The SARS-CoV-2 pango lineage B isolate /Canada/ON/VIDO-01/2020 was used as a representative for the prototypic B lineage virus (Ancestral virus) (GISAID–EPI_ISL_425177) 54 . SARS-CoV-2 Delta was isolated at VIDO from a patient sample (GenBank PP077059). SARS-CoV Tor2 was obtained from the National Microbiology Laboratory (N. Bastien). All viruses were cultured in vDMEM (viral DMEM (Dulbecco’s Modified Eagle Medium), 2% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1 μg/mL TPCK-trypsin) on Vero-76 cells. All work with infectious SARS-CoV-2 viruses, variants, and SARS-CoV Tor2 was performed in the Vaccine and Infectious Disease Organization’s (VIDO) Containment Level 3 (CL3) facility (InterVac) in Saskatoon, Saskatchewan, Canada. Mouse studies – vaccinations and tissue collections BALB/c mice were purchased from Charles River Laboratories (Wilmington, Massachusetts USA). Mice were anesthetized with 2% isoflurane for in life procedures. Mice were immunized by intramuscular injection with pan-sarbecovirus vaccine candidates (5 µg per dose) adjuvanted with Sepivac SWE™ (Vaccine Formulation Institute (VFI), Ouates, Switzerland) on Days 0 and 21. For the control groups, mice were immunized by intramuscular injection with either Pfizer-BioNTech (Mainz, Germany) Comirnaty BA4/5 (5 µg per dose), or with PBS in 50 µl volumes on Day 0 and 21. Candidates were adjuvanted with SWE (Vaccine Formulation Institute (VFI), Ouates, Switzerland). Eight mice were immunized per group. Blood was collected in BD Vacutainer ® EDTA coated blood collection tubes for plasma separation and spleens were removed for single cell isolation on Day 42 post immunizations and subsequent ELISpot. Syrian hamster studies – vaccinations, infections, and tissue collection Male Syrian hamsters aged 8-weeks were purchased from Charles River Laboratories. Ten hamsters per group were anesthetized with 5% isoflurane for in life procedures. Hamsters were vaccinated by intramuscular injection with pan-sarbecovirus vaccine candidates (25 µg per dose), with Pfizer-BioNTech Comirnaty (5 µg per dose), or with PBS in 50 µl volumes on Day 0 and 28. Candidates were adjuvanted with Sepivac SWE™ (VFI). All virus inoculations were performed intranasally with 100mL of virus at a concentration of either 1x10 5 TCID 50 for SARS-CoV-2 Delta or 6.24x10 4 TCID 50 for SARS-CoV per animal on Day 48 post immunization. Five hamsters were randomly selected and euthanized for necropsies on Day 5 post challenge and the remaining animals were necropsied on Day 10 post challenge. To calculate weight loss and survival, hamsters were monitored until end day post challenge, as we and others have done previously 55,56 . Blood and respiratory tissues (nasal turbinates and lungs) were collected from the animals removed from the study on Day 5 post challenge. At necropsy, nasal turbinates and lungs were collected for virological, immunological, pathological analysis. The left lung lobe was collected for histopathology while the right lobes were used for viral load. Weight was measured daily and then calculated as a percentage of original values from Day 0. Blood was collected in BD Vacutainer ® EDTA coated blood collection tubes for plasma separation. Immune Assays – ELISAs and ELISPOTS ELISAs Plasma IgG antibodies from immunized BALB/c mice or Syrian hamsters were quantified for binding capabilities against the S1 subunit of the S protein from SARS-CoV-2 (Sino Biological 40591-V08H) or against a panel of sarbecovirus RBDs (WIV1, Khosta2, RsSHC014, Pang17, Urbani, RaTG13, BtKy72, XBB.1.5) (Cat: SC9305PF, GenScript) using ELISAs, according to standard protocol. Ninety-six-well ELISA plates (Cat: 3655TS, ThermoFisher Scientific) were coated with 1 μg/mL antigen in coating buffer and incubated at 4°C overnight. Plates were washed four times with 1x TBS with 0.05% Tween-20 (Cat: P1379, MilliporeSigma; Oakville, Ontario, Canada) (TBST) then blocked for 1 hr in 5% (w/v) skim milk powder in TBST. Plasma samples were diluted 1:4 then serially diluted 1:100 in 1% skim milk in TBST then incubated at room temperature for 1 hr, then washed, and then alkaline phosphatase labelled anti-mouse IgG (Cat: 5220-0355 (KPL), MilliporeSigma) secondary was added 1:10,000 or goat anti-Hamster IgG HRP at 1:7000 dilution (ThermoFisher Scientific) to 1% skim milk in TBST (1 hr). After washing, OPD substrate (Cat: 34074, ThermoFisher Scientific) and stop solution was added. Plates were read at 405 and 490 nm on an 800TS reader (BioTek; Winooski, Vermont, USA). Delta values were blanked to wells not containing plasma. Cutoffs were determined based on mean absorbance plus 3 standard deviation values of negative controls. ELISpots To evaluate T cell responses following stimulation with RBD from SARS-CoV-2 (Ancestral) or SARS-CoV (Urbani), ELISpot assays were performed with splenocytes harvested from vaccinated mice. MultiScreen® 96-well PVDF plates (Cat: MSIPS4510, MilliporeSigma) were prepared and coated 2 μg/ml anti-IL-5 (Cat:554395, BD Biosciences) or anti-IFN-γ (Cat: 551216, BD Biosciences) the day prior to the assay. Splenocyte single cell suspensions were prepared from harvested spleens in Gey’s solution after washing. Two ml aliquots of suspended cells at a concentration of 10 7 cells per ml were prepared in culture media (AIM-V (Cat: 12055083, ThermoFisher Scientific), non-essential amino acids (Cat: 11140050, ThermoFisher Scientific) at 1:100, sodium pyruvate (Cat: 11360070, ThermoFisher Scientific) at 1:100, HEPES buffer (Cat: 15630080, ThermoFisher Scientific) at 1:100, and 50 mM β-mercaptoethanol (Cat: 21985023, ThermoFisher Scientific) at 1:1000). Plates were blocked with 1% BSA (Cat: A4737, MilliporeSigma) and cells were added at 10 6 cells/well 1:1 with 4 μg/mL antigen in triplicate. Concanavalin A (Cat: 00-4978-93, ThermoFisher) was used as a positive control and was plated at 2 μg/mL. Splenocytes were incubated at 37°C for 18 hours overnight with antigen. PBST was added to lyse cells and plates were washed 3 times with PBST (0.05% Tween20 (Cat: P1379, MilliporeSigma) in PBS) then twice with dH 2 O before addition of secondary antibody (IL-5 (Cat: 554397, BD Bioscience) and IFN-γ (Cat: 554410, BD Bioscience)). After secondary antibody incubation, plates were washed and streptavidin conjugated alkaline phosphatase (Cat: 016-050-084, Cedarlane Labs; Burlington, Ontario, Canada) was diluted 1:1000 in 1% BSA in PBS was added to plates and incubated for 1.5 hr. Plates were washed and BCIP/NBT substrate (Cat: B5655, MilliporeSigma). After washing, plates were scanned using an ELISpot Reader (Autoimmun Diagnostika GmbH; Straßberg, Germany) and spots were counted using ELISpot v7.0 software (Autoimmun Diagnostika GmbH) and evaluated by eye. Viral titers Tissues collected at necropsy were homogenized in serum free DMEM using a TissueLyserII (Qiagen; Toronto, Ontario, Canada). To determine TCID 50 , a 1:10 dilution series of sample was prepared in the appropriate viral growth media in 96-well plates. Samples were assessed in triplicate. Cells were incubated for 1h at 37 o C. Sample dilutions were removed and replaced with fresh viral media. Cytopathic effect (CPE) was monitored daily for 5 days. On Day 5 post inoculation of cells, CPE was recorded indicating the presence of infectious virus. These data were used to calculate TCID 50 /mL using the Spearman-Kärber method . Microneutralization assay Sera isolated from blood samples were heat-inactivated at 56 o C for 30 min and then serially diluted 1:2 in a low serum viral media, in round bottom 96-well plates. Virus was diluted to 25 TCID 50 per well in viral media and used at a 1:1 ratio to serum, where 60 mL of serum was placed in each well and 60 mL of diluted virus was added. The serum-virus mixture was incubated at 37 o C for 1 h and then added to cultured virus-designated cells in 96 well plates. Plates were incubated at 37 o C and were monitored for CPE over five days. Endpoint neutralization titer was based on inhibition of CPE observed on Day 5 after cell infection. The endpoint titer is reciprocal of the highest dilution of serum that is able to suppress CPE. Pseudoviral neutralization assays Pseudoparticle Production – Pangolin Assay Pseudotyped viral particles were produced by transfecting HEK 293T/17 cells (ATCC CRL-11268) with plasmid DNA using polyethylenimine (PEIpro; Polyplus) similarly as done previously 60 . Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Cytiva HyClone) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin. At 70–80% confluency, plasmid–PEI complexes were applied, and after 7–10h, medium was replaced with supplemented DMEM. Supernatants were harvested 48h post-transfection, clarified by centrifugation and filtration (0.45 µm), aliquoted, and stored at –80°C. When required, particles were concentrated using Lenti-X Concentrator (Takara Bio) according to the manufacturer’s protocol. Pseudoparticle Infectivity and Neutralization Assay – Pangolin Assay Neutralization activity was assessed using 293T-hsACE2 cells (Integral Molecular). Serum samples were heat-inactivated (56°C, 30min) and serially diluted in non-supplemented DMEM before incubation with pseudoparticles for 1h at 37°C. The mixtures were then added to 96-well plates containing 20,000 cells/well. Following a 72h incubation at 37°C in 5% CO₂, infectivity was quantified using pseudoparticles encoding luciferase. Following incubation, luciferase activity was determined with the Bright-Glo Luciferase Assay System (Promega) on a Beckman Coulter DTX 880 multimode detector. Results were expressed as relative luminescence units (RLUs). Pseudoviral Infectivity and Neutralization Assay – WIV16 Pseudovirus assays and production of WIV16 pseudovirus for WIV16 were completed similarly as previously described 60 . All sera were diluted from 1:40 and then serial dilution of 1:2 was performed. Cell-only and pseudopartical-only controls were included on each plate. WIV16 pseudoparticles were diluted to a concentration of 1x10 8 RLU per plate (5 ml) as determined via titration, and 50 µl was added to all wells except the cell only controls. Plates were incubated for 1 hour at 37°C, 5% CO 2 . ACE2/TMPRSS2 expressing 293T cells were added to all wells at a concentration of 3x10 5 /ml in a volume of 50 µl per well. Plates were then incubated at 37°C, 5% CO 2 for 48 hrs before being read using the GloMax® Navigator (ProMega) using the Promega GloMax® Luminescence Quick-Read protocol. Plates were analyzed using PRISM as described 61 . All negative % neutralization values were plotted as 0 and all neutralization above 100% was plotted at max titre. Lung Histopathology Lung tissues collected for histopathology were kept in formalin in the CL3 laboratory. After 7 days tissues were transferred to fresh formalin and removed to be paraffin-embedded, sectioned, slide-mounted, and hematoxylin and eosin H&E stained at Prairie Diagnostic Services (PDS) (Saskatoon, Saskatchewan). Slides were analyzed in-house as well by an independent blinded board-certified pathologist. The pathologist gave the lungs a score ranging from 0 to 4 in the following categories: Inflammation; Pneumonia; Hemorrhaging; and Overall Histopathology. Only the Overall Histopathology score is reported here where the Overall gives a summary score taking all factors into account. 0, absent (no lesion); 1, slight or questionable; 2, clearly present, but not conspicuously so; 3, marked; 4, severe. Statistical Analysis Prism GraphPad (San Diego, California, USA) was used to analyze the data. A mixed effect model with repeated measures, multiple comparisons done by Tukeys multiple comparison test was used to evaluate weight data. ELISA and neutralizing antibody data was Log transformed and evaluated using One-way ANOVA with multiple comparisons. Two-way ANOVA with repeated measures and Tukeys multiple comparisons test was used for evaluating viral load quantifications. A Kruskal Wallis test was used with Dunn’s multiple comparisons for comparing histopathology. A p value of ≤ 0.05 was considered statistically significant with p <0.05:*; p <0.01:**; p <0.001:***; p <0.0001:****. Survival was calculated according to the experimental design considering predetermined necropsy days for randomized hamster removals. Therefore, survival was calculated out of 8 hamsters until Day 5 and out of 4 hamsters for end day on Day 10. Declarations ACKNOWLEDGMENTS The authors would like to acknowledge the team at CEPI (Coalition for Epidemic Preparedness and Innovation) for their support through the project. Additionally, the authors would like to thank Dr. Nigel Temperton and his team for support with the WIV16 pseudovirus neutralization assays, as well as Dr. Nathalie Bastien from the National Microbiology Laboratory at the Public Health Agency of Canada for supplying the SARS-CoV Tor2 strain of virus. Published as VIDO manuscript series no. 1147. Funding has been provided by the Coalition for Epidemic Preparedness Innovations (CEPI) seed funding (T.R.); Canadian Institutes for Heath Research (CIHR) Project Grant (PJT-190107) (A.A.K.); Canadian Institutes of Health Research (CIHR) and Coalition for Epidemic Preparedness Innovations (CEPI) Leadership Award in Vaccine Research (A.A.K.); Canadian Institutes of Health Research (CIHR) grants OV5-170349, VRI-173022 and VS1-175531 (D.F.). VIDO receives operational funding from Innovation Saskatchewan (Government of Saskatchewan) and the Ministry of Agriculture. VIDO is also supported by the Canada Foundation for Innovation through the Major Science Initiatives (Government of Canada). AUTHOR CONTRIBUTIONS T.R. and A.A.K. conceptualized and designed the experiments. M.B.R., Z.K., E.-M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., and A.A.K. established the methodology. M.B.R., Z.K., E.-M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., and A.A.K. performed the experiments and/or analyzed the data. M.B.R., Z.K., E.-M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., A.A.K. constructed visualizations of the data for figures. A.A.K. and T.R. supervised the study. A.A.K., T.R., D.F., and V.G. contributed materials/funding. A.A.K. wrote the original draft. A.A.K., T.R., M.B.R., Z.K., E. M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., A.V.K., V.G., Q.L., A.B., and D.F. reviewed and edited the manuscript. DATA AVAILABILITY All data are available in the main text or the supplementary materials COMPETING INTERESTS Authors declare that they have no competing interests References Masters, P. S. The molecular biology of coronaviruses. Adv Virus Res 66 , 193–292 (2006). https://doi.org/10.1016/S0065-3527(06)66005-3 Wertheim, J. O., Chu, D. K., Peiris, J. S., Kosakovsky Pond, S. L. & Poon, L. L. A case for the ancient origin of coronaviruses. J Virol 87 , 7039–7045 (2013). https://doi.org/10.1128/JVI.03273-12 Zhuang Pinghui, J. W. Wuhan pneumonia: how the search for the source of the mystery illness unfolded , < https://www.scmp.com/news/china/society/article/3046233/wuhan-pneumonia-how-search-source-mystery-illness-unfolded%3E (2020). Cucinotta, D. & Vanelli, M. WHO Declares COVID-19 a Pandemic. Acta Biomed 91 , 157–160 (2020). https://doi.org/10.23750/abm.v91i1.9397 Menachery, V. D. et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21 , 1508–1513 (2015). https://doi.org/10.1038/nm.3985 Menachery, V. D. et al. SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci U S A 113 , 3048–3053 (2016). https://doi.org/10.1073/pnas.1517719113 Su, C. et al. Structural characteristics of BtKY72 RBD bound to bat ACE2 reveal multiple key residues affecting ACE2 usage of sarbecoviruses. mBio 15 , e0140424 (2024). https://doi.org/10.1128/mbio.01404-24 Alkhovsky, S. et al. SARS-like Coronaviruses in Horseshoe Bats (Rhinolophus spp.) in Russia, 2020. Viruses 14 (2022). https://doi.org/10.3390/v14010113 Pena-Hernandez, M. A. et al. SARS-CoV-2-related bat viruses evade human intrinsic immunity but lack efficient transmission capacity. Nat Microbiol 9 , 2038–2050 (2024). https://doi.org/10.1038/s41564-024-01765-z Holmes, E. C. et al. The origins of SARS-CoV-2: A critical review. Cell 184 , 4848–4856 (2021). https://doi.org/10.1016/j.cell.2021.08.017 Khan, K. et al. Omicron BA.4/BA.5 escape neutralizing immunity elicited by BA.1 infection. Nat Commun 13 , 4686 (2022). https://doi.org/10.1038/s41467-022-32396-9 Lyke, K. E. et al. Rapid decline in vaccine-boosted neutralizing antibodies against SARS-CoV-2 Omicron variant. Cell Rep Med 3 , 100679 (2022). https://doi.org/10.1016/j.xcrm.2022.100679 Reynolds, C. J. et al. Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure. Science 377 , eabq1841 (2022). https://doi.org/10.1126/science.abq1841 van der Straten, K. et al. Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron. Immunity (2022). https://doi.org/10.1016/j.immuni.2022.07.018 Mykytyn, A. Z. et al. Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct. Sci Immunol , eabq4450 (2022). https://doi.org/10.1126/sciimmunol.abq4450 Duan, L. et al. The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens. Front Immunol 11 , 576622 (2020). https://doi.org/10.3389/fimmu.2020.576622 Mahajan, S. et al. Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals. Sci Rep 11 , 13164 (2021). https://doi.org/10.1038/s41598-021-92521-4 Levy, Y. et al. Immunodominant Linear B-Cell Epitopes of SARS-CoV-2 Spike, Identified by Sera from K18-hACE2 Mice Infected with the WT or Variant Viruses. Vaccines (Basel) 10 (2022). https://doi.org/10.3390/vaccines10020251 Hoffmann, M. et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181 , 271–280 e278 (2020). https://doi.org/10.1016/j.cell.2020.02.052 Folegatti, P. M. et al. Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet 396 , 467–478 (2020). https://doi.org/10.1016/S0140-6736(20)31604-4 Oliver, S. E. et al. The Advisory Committee on Immunization Practices' Interim Recommendation for Use of Pfizer-BioNTech COVID-19 Vaccine - United States, December 2020. MMWR Morb Mortal Wkly Rep 69 , 1922–1924 (2020). https://doi.org/10.15585/mmwr.mm6950e2 Oliver, S. E. et al. The Advisory Committee on Immunization Practices' Interim Recommendation for Use of Moderna COVID-19 Vaccine - United States, December 2020. MMWR Morb Mortal Wkly Rep 69 , 1653–1656 (2021). https://doi.org/10.15585/mmwr.mm695152e1 Wang, W., Huang, B., Zhu, Y., Tan, W. & Zhu, M. Ferritin nanoparticle-based SARS-CoV-2 RBD vaccine induces a persistent antibody response and long-term memory in mice. Cell Mol Immunol 18 , 749–751 (2021). https://doi.org/10.1038/s41423-021-00643-6 Joyce, M. G. et al. SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity. Cell Rep 37 , 110143 (2021). https://doi.org/10.1016/j.celrep.2021.110143 Joyce, M. G. et al. A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates. Sci Transl Med 14 , eabi5735 (2022). https://doi.org/10.1126/scitranslmed.abi5735 Cohen, A. A. et al. Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice. Science 371 , 735–741 (2021). https://doi.org/10.1126/science.abf6840 Zhu, J. et al. A universal bacteriophage T4 nanoparticle platform to design multiplex SARS-CoV-2 vaccine candidates by CRISPR engineering. Sci Adv 7 , eabh1547 (2021). https://doi.org/10.1126/sciadv.abh1547 Yuen, C. K. et al. An interferon-integrated mucosal vaccine provides pan-sarbecovirus protection in small animal models. Nat Commun 14 , 6762 (2023). https://doi.org/10.1038/s41467-023-42349-5 Hutchinson, G. B. et al. Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera. Nat Commun 14 , 6195 (2023). https://doi.org/10.1038/s41467-023-41661-4 Lopez-Sagaseta, J., Malito, E., Rappuoli, R. & Bottomley, M. J. Self-assembling protein nanoparticles in the design of vaccines. Comput Struct Biotechnol J 14 , 58–68 (2016). https://doi.org/10.1016/j.csbj.2015.11.001 Wahome, N. et al. Conformation-specific display of 4E10 and 2F5 epitopes on self-assembling protein nanoparticles as a potential HIV vaccine. Chem Biol Drug Des 80 , 349–357 (2012). https://doi.org/10.1111/j.1747-0285.2012.01423.x Garg, R. et al. Efficacy of a stable broadly protective subunit vaccine platform against SARS-CoV-2 variants of concern. Vaccine 42 , 125980 (2024). https://doi.org/10.1016/j.vaccine.2024.05.028 Zech, F. et al. Spike residue 403 affects binding of coronavirus spikes to human ACE2. Nat Commun 12 , 6855 (2021). https://doi.org/10.1038/s41467-021-27180-0 Minh, B. Q., Nguyen, M. A. & von Haeseler, A. Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol 30 , 1188–1195 (2013). https://doi.org/10.1093/molbev/mst024 Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14 , 587–589 (2017). https://doi.org/10.1038/nmeth.4285 Halfmann, P. J. et al. Broad protection against clade 1 sarbecoviruses after a single immunization with cocktail spike-protein-nanoparticle vaccine. Nat Commun 15 , 1284 (2024). https://doi.org/10.1038/s41467-024-45495-6 Tao, Y. & Tong, S. Complete Genome Sequence of a Severe Acute Respiratory Syndrome-Related Coronavirus from Kenyan Bats. Microbiol Resour Announc 8 (2019). https://doi.org/10.1128/MRA.00548-19 Wang, J. et al. SARS-related coronavirus S-protein structures reveal synergistic RBM interactions underpinning high-affinity human ACE2 binding. Sci Adv 11 , eadr8772 (2025). https://doi.org/10.1126/sciadv.adr8772 Roelle, S. M., Shukla, N., Pham, A. T., Bruchez, A. M. & Matreyek, K. A. Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains. PLoS Biol 20 , e3001738 (2022). https://doi.org/10.1371/journal.pbio.3001738 Cohen, A. A. et al. Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models. Science 377 , eabq0839 (2022). https://doi.org/10.1126/science.abq0839 Leekha, A. et al. Multi-antigen intranasal vaccine protects against challenge with sarbecoviruses and prevents transmission in hamsters. Nat Commun 15 , 6193 (2024). https://doi.org/10.1038/s41467-024-50133-2 Hajnik, R. L. et al. Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Sci Transl Med 14 , eabq1945 (2022). https://doi.org/10.1126/scitranslmed.abq1945 Gouglas, D., Christodoulou, M., Plotkin, S. A. & Hatchett, R. CEPI: Driving Progress Toward Epidemic Preparedness and Response. Epidemiol Rev 41 , 28–33 (2019). https://doi.org/10.1093/epirev/mxz012 Wang, G. et al. Universal subunit vaccine protects against multiple SARS-CoV-2 variants and SARS-CoV. NPJ Vaccines 9 , 133 (2024). https://doi.org/10.1038/s41541-024-00922-z Martinez, D. R. et al. Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice. Science (2021). https://doi.org/10.1126/science.abi4506 Ueda, G. et al. Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens. Elife 9 (2020). https://doi.org/10.7554/eLife.57659 Francis, M. E. et al. Historical H1N1 influenza virus imprinting increases vaccine protection by influencing the activity and sustained production of antibodies elicited at vaccination in ferrets. Vaccines 7 (2019). Francis, T. J. On the Doctrine of Original Antigenic Sin. Proceedings of the American Philosophical Society 104 , 572–578 (1960). Francis, M. E. et al. Previous infection with seasonal coronaviruses does not protect male Syrian hamsters from challenge with SARS-CoV-2. Nat Commun 14 , 5990 (2023). https://doi.org/10.1038/s41467-023-41761-1 Cohen, A. A. et al. Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals. Cell 187 , 5554–5571 e5519 (2024). https://doi.org/10.1016/j.cell.2024.07.052 Gouy, M., Tannier, E., Comte, N. & Parsons, D. P. Seaview Version 5: A Multiplatform Software for Multiple Sequence Alignment, Molecular Phylogenetic Analyses, and Tree Reconciliation. Methods Mol Biol 2231 , 241–260 (2021). https://doi.org/10.1007/978-1-0716-1036-7_15 Minh, B. Q. et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol 37 , 1530–1534 (2020). https://doi.org/10.1093/molbev/msaa015 Anthis, N. J. & Clore, G. M. Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci 22 , 851–858 (2013). https://doi.org/10.1002/pro.2253 Marchand-Senecal, X. et al. Diagnosis and Management of First Case of COVID-19 in Canada: Lessons Applied From SARS-CoV-1. Clin Infect Dis 71 , 2207–2210 (2020). https://doi.org/10.1093/cid/ciaa227 Francis, M. E. et al. SARS-CoV-2 infection in the Syrian hamster model causes inflammation as well as type I interferon dysregulation in both respiratory and non-respiratory tissues including the heart and kidney. PLoS Pathog 17 , e1009705 (2021). https://doi.org/10.1371/journal.ppat.1009705 Imai, M. et al. Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development. Proc Natl Acad Sci U S A 117 , 16587–16595 (2020). https://doi.org/10.1073/pnas.2009799117 Additional Declarations There is NO Competing Interest. Supplementary Files Racineetal2026NatComssupplementarymaterials.pdf Supplementary Figures DataaccessionforreviewersNCOMMS26001693.docx Data accession - For reviewers ReportingsummaryNCOMMS26001693.pdf Reporting summary Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8544714","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581124201,"identity":"43add975-ef79-43be-a08b-1b62776a1f28","order_by":0,"name":"Alyson Kelvin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIie3PsUrDQBzH8X85SBz+IesFIfcKdwQCYsBXOXBwFqEoOKQErqNrA+I7FKE4Bv7QLlVwdBN8gZZMgoN3FalDTzoK3ndIjh/5cAQgFPqLxV8v6Q4dXG4Xf+ybMEeW22U/AgOzB0kbLFbrByhShorO70ikTfzcXxkQab2bcMIya5dQZg1KamekJoQX2aMBNek811jCEgOVpEhTMiNtF52NDGjwEEFY9I6cbMgtabucvjsiPEQSykNLSslYR0lNWlI839wiPURRNMxawwtOluD8TE0Jo6P6iauph+SL5n61NpW6GY+aHq+PRb4Yv73Uw0rkvt+HA2kf/OeCEgYR93zuil93LR+/iFAoFPpvfQKUClW4Jh80hAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Calgary","correspondingAuthor":true,"prefix":"","firstName":"Alyson","middleName":"","lastName":"Kelvin","suffix":""},{"id":581124202,"identity":"a46aed2d-bc59-4d66-976b-894943242cd2","order_by":1,"name":"Matthew Rogers","email":"","orcid":"","institution":"University of Pittsburgh School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Rogers","suffix":""},{"id":581124203,"identity":"34b6fe5b-2b5e-4ca2-a84b-6fd3ed4241f4","order_by":2,"name":"Zahed Katooni","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Zahed","middleName":"","lastName":"Katooni","suffix":""},{"id":581124204,"identity":"27e146f5-0e85-4941-a1a4-1fdbc8d639d2","order_by":3,"name":"Eva-Maria Uhlemann","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Eva-Maria","middleName":"","lastName":"Uhlemann","suffix":""},{"id":581124205,"identity":"12d79948-547a-4d29-93b7-2c6d633e6169","order_by":4,"name":"Rachelle Buchanan","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization (VIDO)","correspondingAuthor":false,"prefix":"","firstName":"Rachelle","middleName":"","lastName":"Buchanan","suffix":""},{"id":581124206,"identity":"06beb7f3-5fdf-481b-9ee7-546e970d0ab2","order_by":5,"name":"Erin Scruten","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Erin","middleName":"","lastName":"Scruten","suffix":""},{"id":581124207,"identity":"9bd47445-3eb2-4a79-8a87-af64970a55b2","order_by":6,"name":"Anthony Yourkowski","email":"","orcid":"https://orcid.org/0009-0009-7994-6168","institution":"Vaccine and Infectious Disease Organization (VIDO)","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"","lastName":"Yourkowski","suffix":""},{"id":581124208,"identity":"e522f293-f94a-4e45-b30d-8ecd042478f0","order_by":7,"name":"Jocelyne Lew","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Jocelyne","middleName":"","lastName":"Lew","suffix":""},{"id":581124209,"identity":"92c22bfe-5918-4391-a99e-1ce6830ef845","order_by":8,"name":"Akarin Asavajaru","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Akarin","middleName":"","lastName":"Asavajaru","suffix":""},{"id":581124210,"identity":"29e51683-0506-4fa4-b685-7aa4963a4584","order_by":9,"name":"Jill Van Kessel","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Jill","middleName":"Van","lastName":"Kessel","suffix":""},{"id":581124211,"identity":"d1e88e90-adae-4fe0-bab3-5d2ed04b847d","order_by":10,"name":"Paul Pennington","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Pennington","suffix":""},{"id":581124212,"identity":"73cca091-1b74-4fe4-8fd1-e338f1a04366","order_by":11,"name":"Andrea Kroeker","email":"","orcid":"","institution":"VIDO","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Kroeker","suffix":""},{"id":581124213,"identity":"1a1b724a-573a-4efd-92a9-80c11449872e","order_by":12,"name":"Cynthia Swan","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization (VIDO)","correspondingAuthor":false,"prefix":"","firstName":"Cynthia","middleName":"","lastName":"Swan","suffix":""},{"id":581124214,"identity":"c65ff2f7-eb99-486b-90ef-b1f5684db8d1","order_by":13,"name":"Rinku Patel","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Rinku","middleName":"","lastName":"Patel","suffix":""},{"id":581124215,"identity":"803957d3-da7f-4a86-944d-7ebd9222fa0e","order_by":14,"name":"Brittany Thivierge","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization (VIDO)","correspondingAuthor":false,"prefix":"","firstName":"Brittany","middleName":"","lastName":"Thivierge","suffix":""},{"id":581124216,"identity":"72dc3cfe-40f8-4009-b527-e35bd91f2fe3","order_by":15,"name":"Willian Deck","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Willian","middleName":"","lastName":"Deck","suffix":""},{"id":581124217,"identity":"f4cd0d49-bdbf-40fe-8682-dec62b91f644","order_by":16,"name":"Ravendra Garg","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Ravendra","middleName":"","lastName":"Garg","suffix":""},{"id":581124218,"identity":"28c26572-a374-4c75-a41a-809422863661","order_by":17,"name":"Glenn Hamonic","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Glenn","middleName":"","lastName":"Hamonic","suffix":""},{"id":581124219,"identity":"0d3e4711-a49c-4f8a-956b-67297d4e8d35","order_by":18,"name":"Wolfgang Koster","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Wolfgang","middleName":"","lastName":"Koster","suffix":""},{"id":581124220,"identity":"0e8cbab9-bc8e-49fd-b6f2-4de8f6fd5f20","order_by":19,"name":"Tracy Prysliak","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Tracy","middleName":"","lastName":"Prysliak","suffix":""},{"id":581124221,"identity":"dc1118e0-9faa-4600-8238-0ce171f34e76","order_by":20,"name":"Anne-Catherine Fluckiger","email":"","orcid":"","institution":"Bionaria Consulting","correspondingAuthor":false,"prefix":"","firstName":"Anne-Catherine","middleName":"","lastName":"Fluckiger","suffix":""},{"id":581124222,"identity":"bbaaf587-8b70-443a-98f7-ff621b3009aa","order_by":21,"name":"Jasminka Bozic","email":"","orcid":"https://orcid.org/0009-0002-0566-8416","institution":"VBI Vaccines","correspondingAuthor":false,"prefix":"","firstName":"Jasminka","middleName":"","lastName":"Bozic","suffix":""},{"id":581124223,"identity":"e0da8980-b30f-4afa-9291-709ed149ac68","order_by":22,"name":"Andrew Van Kessel","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"Van","lastName":"Kessel","suffix":""},{"id":581124224,"identity":"3d7be12a-921c-4146-b566-e3f8277e49c6","order_by":23,"name":"Volker Gerdts","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Volker","middleName":"","lastName":"Gerdts","suffix":""},{"id":581124225,"identity":"fb93e6bd-05d0-4020-9636-4dc7183d7455","order_by":24,"name":"Qiang Liu","email":"","orcid":"https://orcid.org/0000-0003-1508-7172","institution":"Vaccine and Infectious Disease Organization (VIDO), University ofSaskatchewan; School of Public Health, University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Liu","suffix":""},{"id":581124226,"identity":"7ca5a648-5e5d-47a3-b2e0-c31a6d491fbe","order_by":25,"name":"Arinjay Banerjee","email":"","orcid":"https://orcid.org/0000-0002-2821-8357","institution":"Vaccine and Infectious Disease Organization, University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Arinjay","middleName":"","lastName":"Banerjee","suffix":""},{"id":581124227,"identity":"c4b06acf-e7cf-4269-bc9c-c6adeeae9943","order_by":26,"name":"Darryl Falzarano","email":"","orcid":"https://orcid.org/0000-0002-8805-8068","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Darryl","middleName":"","lastName":"Falzarano","suffix":""},{"id":581124228,"identity":"d0805567-0f16-4140-afa7-726d746b413c","order_by":27,"name":"Trina Racine","email":"","orcid":"","institution":"Vaccine and Infectious Disease Organization","correspondingAuthor":false,"prefix":"","firstName":"Trina","middleName":"","lastName":"Racine","suffix":""}],"badges":[],"createdAt":"2026-01-07 19:20:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8544714/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8544714/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101398540,"identity":"5844c470-18f2-4317-bb8f-2d18bc3be91d","added_by":"auto","created_at":"2026-01-29 09:42:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":339161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of sarbecovirus RBD sequences and design of multivalent protein subunit vaccine candidates. \u003c/strong\u003eAligned RBD peptide sarbecovirus sequences were phylogenetically analyzed for relatedness and visualized as a tree using IQtree2. The three classical clades of sarbecoviruses, Clade 1, Clade 2, and Clade 3 were identified. The subclades of Clade 1, Clade 1a and Clade 1b, were also determined. Clade 1a viruses are highlighted in the red bubble, Clade 1b viruses in the green bubble, Clade 2 viruses in the purple bubble, and Clade 3 viruses in the orange bubble. Viruses of interest in each clade are marked by their common names. 1000 ultrafast bootstraps were performed.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/141dd1e786153398de243dbe.jpg"},{"id":101398405,"identity":"3270ff4c-14a9-4e60-9725-2848313278cb","added_by":"auto","created_at":"2026-01-29 09:41:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":487244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein prediction and physical characterization of multivalent protein subunit pan-sarbecovirus vaccine candidates. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of trivalent and quadrivalent pan-sarbecovirus vaccine candidates illustrating the S1, RBD, and GS linkers. The trivalent candidate is referred to as 4500 and the three quadrivalent candidates are referred to as 4501, 4502, and 4503. Each S1 component of the candidates is the Ancestral (Wuhan) S1 sequences. (\u003cstrong\u003eB\u003c/strong\u003e) Protein conformation predictions of the 4500, 4501, 4502, and 4503 candidates. 3D protein conformation predictions were performed using AlphaFold. (\u003cstrong\u003eC\u003c/strong\u003e) Candidate sequences were cloned into pCDNA3.4 and transfected in ExpiCHO cells for protein expression and purification. Proteins were characterized by Coomassie protein staining gel (left) and western blot probing for anti-SARS-CoV-2 S1 (right). Both glycosylated and deglycosylated protein samples were analyzed and shown on the Coomassie and western blot.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/c3c36534e13eab55a0335109.jpg"},{"id":101332986,"identity":"eb382ca0-4eb4-4e01-9a16-9948bc01e6fc","added_by":"auto","created_at":"2026-01-28 15:05:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":693742,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of the B-cell epitopes shown as sphere representations across four vaccine candidates. Predicted B-cell epitopes (shown in black spheres) are mapped on the surface of four vaccine candidates: 4500 (A), 4501 (B), 4502 (C), and 4503 (D). Epitopes are highlighted as black spheres on the candidate ribbon diagrams. Each candidate is comprised of an S1 domain from SARS CoV-2 Ancestral virus and RBD domains from various coronaviruses, including WIV1, Pang17, RsSHC014, BtKY72, RatG13, Khosta2, and Urbani viruses. Epitopes are mainly located on exposed regions: cyan, yellow, and blue represent the first to third RBDs for 4500 and the same colors in addition to orange for the fourth RBDs, respectively, for 4501 to 4503. This visualization highlights epitope distribution shifts dependent on the sequence composition of each construct.\u003c/p\u003e","description":"","filename":"PanSarbFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/006a72df3ffb772c6ad10fd7.jpg"},{"id":101332991,"identity":"df4b788d-e362-4071-89e2-29028842050f","added_by":"auto","created_at":"2026-01-28 15:05:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":352960,"visible":true,"origin":"","legend":"\u003cp\u003eMultivalent protein subunit pan-sarbecovirus vaccine candidates induce broadly-reactive antibodies and T cell responses in mice. BALB/c mice (n=8/grp) were vaccinated on Day 0 and 21 with 5 µg of multivalent protein subunit pan-sarbecovirus vaccine candidates (4500, 4501, 4502, and 4503) formulated with SWE or control vaccine (Pfizer BA4/5, 5 µg) or negative control (PBS). On Day 42 all animals were euthanized and blood and spleens were collected. (A) ELISA assays were performed with isolated plasma to evaluate binding antibodies. Plates were coated with RBD protein from a panel of known sarbecoviruses (Ancestral SARS-CoV-2, WIV1, Khosta2, RsSHC014, Pang17, Urbani, and RatG13). Plasma from collected blood was incubated against coated plates to quantify binding antibodies. Results are depicted as a heat map of Log10 ELISA titer for each group (dark purple = low levels of binding; yellow = high levels). Group results are compared to antibody levels from the control PBS group. (B) ELISPOTs were used to evaluate cellular responses in splenocytes isolated on end day. IFN-g and IL-5 ELISPOTs were carried out by stimulating splenocytes with select protein domains or vaccine candidates. Specifically, for each candidate immunizations, splenocytes were stimulated with Wuhan S1, Urbani RBD or the vaccine candidate itself (ex. 4500 stimulated with 4500 protein). Statistical analysis was conducted by Log transforming the raw and analyzing differences among groups using a Kruskal Wallis (One-way ANOVA) test with Dunn’s multiple comparisons. A p value of ≤ 0.05 was considered statistically significant with p\u0026lt;0.05:*; p\u0026lt;0.01:**; p\u0026lt;0.001:***; p\u0026lt;0.0001:****. P values in A refer to comparisons to PBS.\u003c/p\u003e","description":"","filename":"PanSarbFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/8d97fc430c13ec11e1dce02a.jpg"},{"id":101332989,"identity":"6b71c1f7-85aa-4d65-8866-595ab385c63b","added_by":"auto","created_at":"2026-01-28 15:05:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":408974,"visible":true,"origin":"","legend":"\u003cp\u003ePan-sarbecovirus vaccine immunization in Syrian hamsters protects against heterologous virus challenge with a Clade 1b sarbecovirus. (A) Schematic of heterologous challenge studies in immunized Syrian hamsters. Syrian hamsters (n=8/group) were immunized with 25 µg of multivalent protein subunit pan-sarbecovirus vaccine candidates (4500, 4501, 4502, or 4503) formulated with SWE on Day 0 and 28 post immunization. On Day 48, animals were intranasally challenged with 105 TCID50 of SARS-CoV-2 Delta variant. Blood was collected on Day 48 while blood and respiratory tissues were collected on Day 5 (n=4) and 10 (n=4) post challenge for quantifying virus neutralizing antibodies and viral load in the nasal turbinates and lungs. (B) Body weight was measured daily for 10 days following challenge and percent of original weight was calculated and the group average was graphed for candidates (4500, 4501, 4502, and 4503) compared to control groups. (C) Neutralizing antibodies (Day 48 post immunization) and (D) respiratory tissue viral titres (Day 5 post challenge) were evaluated by microneutralization assays and TCID50 assay, respectively, for each candidate. (E) At necropsy lungs were collected and fixed in formalin. Sectioned H\u0026amp;E stained slides were assessed and scored by a pathologist for Overall Histopathological scores. Statistical analysis was conducted using a mixed effect model with repeated measures, multiple comparisons done by Tukeys multiple comparison test (weight data); Kruskal Wallis (One-way ANOVA) with Dunn’s multiple comparisons (neutralization data and histopathology data); or Two-way ANOVA with repeated measures and Tukeys multiple comparisons test (viral load data). * indicates a p-value between 0.05 and 0.005, ** indicates a p-value between 0.005 and 0.0005, *** indicates a p-value between 0.0005 and 0.0001, and **** indicates a p-value \u0026lt; 0.0001. Data are presented as mean values with error bars indicating ±SD as appropriate.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/7b377a3d8cd8a14a27029a3c.jpg"},{"id":101751342,"identity":"58e3b879-7010-4bb1-a11f-8f18677e29a1","added_by":"auto","created_at":"2026-02-03 10:19:28","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":287949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultivalent protein subunit pan-sarbecovirus vaccine candidates induce broadly-reactive antibodies in Syrian hamsters. \u003c/strong\u003ePlasma isolated from Syrian hamsters post immunization in challenge studies were evaluated for broad-binding capabilities using ELISAs. Plasma was collected on Day 48 post immunization and boost with 25 µg of multivalent protein subunit pan-sarbecovirus vaccine candidates (4500, 4501, 4502, and 4503) formulated with SWE, control vaccine (Pfizer Bivalent BA4/5, 5 ug) or negative control (PBS). (\u003cstrong\u003eA\u003c/strong\u003e) Antibodies were evaluated for binding abilities toward a panel of sarbecovirus spike protein RBD (Ancestral SARS-CoV-2, WIV1, Khosta2, RsSHC014, Pang17, Urbani, and RatG13) using ELISAs. Results are depicted as a heat map of Log10 ELISA titer for each immunization group with dark purple indicating low levels of binding compared to yellow indicating high levels. Group results are compared to antibody levels from the control PBS group for statistical evaluation. (\u003cstrong\u003eB\u003c/strong\u003e) Pseudoneutralization assays were performed with pseudoviruses of WIV1, PangGX, PangGD after incubation with plasma from immunized hamsters. Data was Log transformed and evaluated using One-way ANOVA with multiple comparisons. A p value of ≤ 0.05 was considered statistically significant with p\u0026lt;0.05:*; p\u0026lt;0.01:**; p\u0026lt;0.001:***; p\u0026lt;0.0001:****. P values in A refer to comparisons to PBS.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/80882e22e34fd1cc01dd8b41.jpg"},{"id":101332982,"identity":"6a005575-48bf-4fce-a473-28e127d69610","added_by":"auto","created_at":"2026-01-28 15:05:22","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":395813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultivalent protein subunit pan-sarbecovirus vaccine candidates protect against Clade 1a sarbecovirus challenge.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Study plan for heterologous challenge with SARS-CoV in Syrian hamsters immunized with\u003cstrong\u003e \u003c/strong\u003emultivalent protein subunit pan-sarbecovirus vaccine candidates (4500, 4501, 4502, or 4503) or\u003cstrong\u003e \u003c/strong\u003econtrols. Animals were prime and boosted on Day 0 and 28, respectively, and then intranasally\u003cstrong\u003e \u003c/strong\u003echallenged on Day 48 with SARS-CoV Tor2 strain (6.24x104 TCID50). Blood was collected on Day 48\u003cstrong\u003e \u003c/strong\u003ewhile blood and nasal turbinates and lungs were collected on Day 5 and 10 post challenge for quantifying\u003cstrong\u003e \u003c/strong\u003eantibodies and viral titre. (\u003cstrong\u003eB\u003c/strong\u003e) Weight (percent original weight), (\u003cstrong\u003eC\u003c/strong\u003e) neutralizing antibodies\u003cstrong\u003e \u003c/strong\u003e(microneutralization assays), (\u003cstrong\u003eD\u003c/strong\u003e) lung viral titre (TCID50 assay), and (\u003cstrong\u003eE\u003c/strong\u003e) Overall lung pathology were\u003cstrong\u003e \u003c/strong\u003eevaluated for each candidate group. For histopathological assessments, collected lungs from each group\u003cstrong\u003e \u003c/strong\u003epost inoculation were formalin perfused, sectioned, mounted, and H\u0026amp;E stained followed by\u003cstrong\u003e \u003c/strong\u003evisualization and analysis by a board-certified pathologist. Overall pathology scores were given a score\u003cstrong\u003e \u003c/strong\u003eranging from 0 to 4: 0, absent (no lesion); 1, slight or questionable; 2, clearly present, but not\u003cstrong\u003e \u003c/strong\u003econspicuously so; 3, marked; 4, severe. Statistical analyses were conducted as previously described\u003cstrong\u003e \u003c/strong\u003ewith mixed effect model and Tukeys multiple comparison test (weight data); Kruskal Wallis with\u003cstrong\u003e \u003c/strong\u003eDunn’s multiple comparisons (neutralization data and histopathology data); or Two-way ANOVA with\u003cstrong\u003e \u003c/strong\u003erepeated measures and Tukeys multiple comparisons test (viral titre data). * indicates a p-value\u003cstrong\u003e \u003c/strong\u003ebetween 0.05 and 0.005, ** indicates a p-value between 0.005 and 0.0005, *** indicates a p-value\u003cstrong\u003e \u003c/strong\u003ebetween 0.0005 and 0.0001, and **** indicates a p-value \u0026lt; 0.0001. Data are presented as mean values\u003cstrong\u003e \u003c/strong\u003ewith error bars indicating ±SD as appropriate.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/5b5d2c33b0c07a40e258a491.jpg"},{"id":101754953,"identity":"88371eb5-8f90-4bf2-b707-0f6ac0d018aa","added_by":"auto","created_at":"2026-02-03 10:48:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4356698,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/e0fa071b-82e0-4932-ac9d-d680d7cb98dc.pdf"},{"id":101332987,"identity":"dc0ebd0e-8fa5-4b5d-ae59-bc0339f4823b","added_by":"auto","created_at":"2026-01-28 15:05:23","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2025527,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"Racineetal2026NatComssupplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/b086f24e0fea29f15883a597.pdf"},{"id":101332981,"identity":"45a5adbc-ac81-44fb-9bbb-d458020037f8","added_by":"auto","created_at":"2026-01-28 15:05:22","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14233,"visible":true,"origin":"","legend":"Data accession - For reviewers","description":"","filename":"DataaccessionforreviewersNCOMMS26001693.docx","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/4f38156f4a21e038c6e9ec2e.docx"},{"id":101398357,"identity":"6dea09b8-0bd7-4018-be26-34015041acd4","added_by":"auto","created_at":"2026-01-29 09:41:06","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2783589,"visible":true,"origin":"","legend":"Reporting summary","description":"","filename":"ReportingsummaryNCOMMS26001693.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8544714/v1/585d44a38a3bd0b3dab81dfb.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fusion protein pan-sarbecovirus vaccines elicit broadly protective immune responses targeting Clade 1a, 1b, and 3 sarbecoviruses","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCoronaviruses (order \u003cem\u003eNidovirales;\u003c/em\u003e family \u003cem\u003eCoronaviridae\u003c/em\u003e) are a continual threat to human health as seen by the recent COVID-19 (coronavirus disease from 2019) pandemic, circulating seasonal coronaviruses, and the potential for new spillover viruses from animal reservoirs \u003csup\u003e1\u003c/sup\u003e. Coronaviruses have positive sense, single-stranded RNA genomes characterized by one of four genera: alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses \u003csup\u003e2\u003c/sup\u003e. The betacoronavirus genera has been a significant threat to humans as illustrated by the emergence of three coronaviruses that cause severe disease in humans: SARS-CoV (severe acute respiratory coronavirus) (2002), Middle East Respiratory Syndrome coronavirus (MERS-CoV) (2012), and SARS-CoV-2 which causes COVID-19 (2019) and led to an estimate of over 20\u0026nbsp;million deaths worldwide \u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. Of those three coronaviruses, both SARS-CoV and SARS-CoV-2 are members of the sarbecovirus subgenus of betacoronaviruses. Investigation of sarbecoviruses in the animal reservoir suggest pre-emergent sarbecoviruses have the potential for additional spillover events causing future human health emergencies \u003csup\u003e5\u0026ndash;9\u003c/sup\u003e. Furthermore, as SARS-CoV-2 continues to circulate in people, the virus has continued to diversify with the emergence of variants of concern (VOCs) that can evade pre-existing immunity from infection and vaccination \u003csup\u003e10\u0026ndash;15\u003c/sup\u003e. The immunogenic viral envelope Spike (S) protein is responsible for host cell binding through the human ACE2 (angiotensin-converting enzyme 2) receptor \u003csup\u003e16\u0026ndash;19\u003c/sup\u003e. Although the S protein is often the chosen target for vaccines, there is great antigenic diversity among S proteins limiting vaccine effectiveness \u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVaccines rapidly developed against the SARS-CoV-2 virus during the COVID-19 pandemic leveraged more modern and/or less common vaccine platforms such as mRNA or viral vector as the main workhorse \u003csup\u003e20\u0026ndash;22\u003c/sup\u003e. First generation COVID-19 vaccines targeted single strains eliciting narrow immune responses leading to the need for frequent formulation updates. Considering coronavirus vaccine limitations, new strategies are urgently needed to induce more broadly protective immune responses \u003csup\u003e5,6\u003c/sup\u003e such as pan-sarbecovirus vaccines \u003csup\u003e23\u0026ndash;29\u003c/sup\u003e. Much focus has been given to nanoparticles as next-generation broadly protective vaccine platforms \u003csup\u003e30\u003c/sup\u003e. Engineered nanoparticles displaying monovalent or multivalent repetitive arrays of viral surface proteins (such as full exodomains of coronavirus S proteins) or surface protein domains (such as the Receptor Binding Domain (RBD) of S) recapitulate the immunogenic 3D structure of viral surface proteins including trimerization, which is more immunogenic than free trimers \u003csup\u003e23,24\u003c/sup\u003e. Examples of promising nanoparticle platforms include SpyCatcher-SpyTag system, bacteriophage T4 nanoparticles, and ferritin-based nanoparticles \u003csup\u003e24,26,27\u003c/sup\u003e. Protein nanoparticles developed through plug-and-play platforms such as these have been shown to successfully induce broadly protective immune responses toward viral targets such as coronaviruses, influenza viruses, and HIV \u003csup\u003e23\u0026ndash;25,31\u003c/sup\u003e. Although these vaccine candidates show promise, there are drawbacks to nanoparticle platforms such as the inability to design the order and amount of each target in the nanoparticle and the need to assemble individually translated proteins into a 3D conformation. In the case of ferritin, 24 ferritin-fusion protein subunits must self-assemble into the quaternary level to create octahedral symmetry \u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePreviously we reported on a flexible protein subunit vaccine platform with plug-and-play capabilities that linked together the S1 domain and RBDs from various SARS-CoV-2 variants to create a single fusion protein antigen during protein translation \u003csup\u003e32\u003c/sup\u003e. The pan-SARS-CoV-2 variant candidate vaccines formulated with SWE (squalene-and-water emulsion) induced cross-reactive immune responses against various SARS-CoV-2 variants. Here we expanded on our previous work to rationally design vaccine candidates using a quantitative approach that guides target virus selection and domain placement with the goal of inducing broadly protective immune responses against several clades of sarbecoviruses. We show that our 4 designed vaccine candidates, targeting Clade 1a, Clade 1b, and Clade 3 of sarbecovirus viruses, elicited cross-reactive humoral and T cell responses and were protective against heterologous challenge with Clade 1a (SARS-CoV Tor2) and Clade 1b (SARS-CoV-2 Delta) viruses reducing viral load, mortality, and lung pathology.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDesign, predictions, physical characterization, and immunogenicity of pan-sarbecovirus vaccine candidates\u003c/h2\u003e \u003cp\u003eWe previously developed a flexible multivalent protein subunit vaccine platform to target several SARS-CoV-2 variants with one antigen-adjuvant formulation \u003csup\u003e32\u003c/sup\u003e. The platform links several domains of the SARS-CoV-2 spike (S) protein together with each domain represented a distinct SARS-CoV-2 variant. In the present study we expanded the breadth of immune responses and protection offered by vaccine candidates by including targets within the subgenus of sarbecoviruses, theoretically covering entire clades within the subgenus. The subgenus of sarbecoviruses include SARS-CoV and SARS-CoV-2, as well as pre-emergent bat viruses such as WIV1, RaTG13, and RsSHC014 that have been suggested to pose a threat to humans \u003csup\u003e5,6,33\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe performed phylogenetic analysis to understand the relatedness among the subgenus of sarbecoviruses and guide target virus selection. Over 1000 raw sarbecovirus spike sequences from viral strains were acquired from GISAID or other freely available databases such as NextStrain. We removed sequences that were incomplete or duplicated and trimmed sequences to focus on the RBD segment. RBD amino acid sequences were aligned and a phylogenetic tree was constructed using IQtree2 (routed on SARS-CoV-2 ancestral strain spike protein sequence (QHD43416.1; positions 319\u0026ndash;541)) \u003csup\u003e34,35\u003c/sup\u003e. Similar to previously reported analyses \u003csup\u003e5,36\u003c/sup\u003e, our analysis identified three clades, Clade 1, Clade 2, and Clade 3, with notable subclades within the sarbecovirus subgenus (Clade 1a and Clade 1b) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Clade 1a (red bubble) encompasses viral sequences related to SARS-CoV including all published SARS-CoV sequences and bat-related sequences WIV1 and RsSHC014. Clade 3 (orange bubble) representing sarbecoviruses collected outside of Asia including BtKY72 and Khosta-2 (from Kenya and Russia, respectively) was most proximal to Clade 1a \u003csup\u003e7,8,37\u0026ndash;39\u003c/sup\u003e. Clade 1b (green bubble) illustrates the relatedness of SARS-CoV-2 and variant viruse sequences as well as viruses from bats and other animals including the bat sarbecovirus RaTG13 and pangolin virus Pang17. Finally, the most distant clade, Clade 2 (purple bubble) was characterized by two main subclades representing HKU3 and mostly bat sequences.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe designed both trivalent and quadrivalent candidates having 3 and 4 targets, respectively. All candidates leveraged the full ancestral SARS-CoV-2 S1 subunit as their first target in the N-terminal anchoring position. Following the S1 subunit, RBD targets were linked using a 3 GS linker (6 aa total). The trivalent candidate (S1-RBD-RBD) only included targets from Clade 1a and Clade 1b, as there were less positions in the antigen to target more clades or subclades. The quadrivalent candidates (S1-RBD-RBD-RBD) included targets from Clade 1b, Clade 3, and Clade 1a depending on the specific candidate. Two quadrivalent candidates incorporated two sequences from Clade 1a viruses (RsSHC014 and WIV1 or Urbani (SARS-CoV)), while the third candidate included two Clade 1b targets (ancestral SARS-CoV-2 and RaTG13). Specifically, the candidates are SARS-CoV-2-Pang17-WIV (VIDO4500 (4500 onward)); SARS-CoV-2-Khosta2-RsSHC014-WIV1 (VIDO 4501 (4501 onward)); SARS-CoV-2\u0026ndash;Khosta2\u0026ndash;RsSHC014-Urbani (VIDO4502 (4502 onward)); SARS-CoV-2-RaTG13-BtKY72-WIV1 (VIDO4503 (4503 onward)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The viral sequences more similar in amino acid identity were placed adjacent to each other in the fusion protein so that the order of the components in the candidate reflected the phylogenetic tree. We hypothesized that this placement would encourage the stimulation of cross-reactive antibodies and a more durable immune response \u003csup\u003e30,40\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next predicted the 3D protein structure of the 4 candidates using Alpha fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The S1 protein of all candidates are shown in purple (left side of each structure) while the RBDs are shown in pink, green, blue, and light purple. Characterization of harvested protein on SDS-PAGE gels followed by Coomassie Blue staining and western blotting with antibodies for SARS-CoV-2 S1 indicated that the expressed proteins were of expected molecular weight (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and had evidence of glycosylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Further physical characterization analyzed the level of expression and size exclusion chromatography with multi-angle light scattering (SEC-MALS) and Circular Dichroism (CD) was conducted (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The predicted molecular weight (MW) of the candidates ranged from 125-151kDa unglycosylated and 163\u0026ndash;217 kDa glycosylated. All the proteins contained above 90% monomers, except candidate 4503 which contained 58.2% of the dimer form requiring 0.08% Tween-80 to the vaccine formulation. Analysis also suggested that the secondary structure content determined by CD spectroscopy was consistent with the predicted structure as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Partial denaturation was observed in the temperature range between 39-46\u003csup\u003eo\u003c/sup\u003eC, indicating that the proteins were stable under standard purification and storage conditions. Loss of α-helix content and partial disruption of disulfide bridges was observed only at low pH (3.3\u0026ndash;3.8).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePan-sarbecovirus vaccine antigen characterization\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExpression\u003c/p\u003e \u003cp\u003elevel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePredicted\u003c/p\u003e \u003cp\u003emW\u003c/p\u003e \u003cp\u003e(unglycosylated)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExperimental\u003c/p\u003e \u003cp\u003eMW\u003c/p\u003e \u003cp\u003e(glycosylated)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSEC-MALs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCD\u003c/p\u003e \u003cp\u003ecomposition\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWHS1 \u0026ndash; Pang17 RBD \u0026ndash; WIV1 RBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e163 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMonomer: 100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eα- helical: 21%\u003c/p\u003e \u003cp\u003eβ-sheets: 35%\u003c/p\u003e \u003cp\u003ecoil: 44%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWHS1 \u0026ndash; Khosta2 RBD \u0026ndash; RsSHCO14 RBD \u0026ndash;\u003c/p\u003e \u003cp\u003eWIV1 RBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e209 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMonomer: 90%*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eα- helical: 20%\u003c/p\u003e \u003cp\u003eβ-sheets: 34%\u003c/p\u003e \u003cp\u003ecoil: 46%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWHS1 \u0026ndash; Khosta2 RBD \u0026ndash; RsSHCO14 RBD \u0026ndash;\u003c/p\u003e \u003cp\u003eUrbani RBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e200 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMonomer: 100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eα- helical: 20%\u003c/p\u003e \u003cp\u003eβ-sheets: 34%\u003c/p\u003e \u003cp\u003ecoil: 46%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWHS1 \u0026ndash; RaTG13 RBD \u0026ndash; BtKY72 RBD \u0026ndash;\u003c/p\u003e \u003cp\u003eWIV1 RBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e217 kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDimer: 58.2%\u003c/p\u003e \u003cp\u003eAggregate: 8.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eα- helical: 20%\u003c/p\u003e \u003cp\u003eβ-sheets: 35%\u003c/p\u003e \u003cp\u003ecoil: 45%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe next performed antigenicity predictions of our candidates to ensure epitopes would not be buried upon expression and tertiary structure formation. B-cell epitopes were predicted using DiscoTope-3 focusing on residues with scores equal to or greater than 0.9, which by default is used for Moderate confidence (0.90, recall up to ~\u0026thinsp;50%) and ignored those with a negative score. The sums of calibrated scores were 458.6, 508.2, 530, and 531.2 for 4500, 4501, 4502 and 4503, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Results showed concentrated peaks above the threshold within the conformation of all four vaccine structures when epitopes were graphed over the protein by amino acid (Supplementary Fig.\u0026nbsp;1). To investigate if these epitopes are accessible to bind to antibodies providing confidence in the use of the antigen, we performed molecular docking simulation against the neutralizing antibody CT-P59 (PDB: 7CM4) (Supplementary Fig.\u0026nbsp;2). RBDs of all vaccine candidates are predicted to properly bind strongly or weakly the heavy and light chains of the tested antibody with acceptable accessibility for immune engagement. Molecular dynamics (MD) simulations (200 ns) for one of the vaccine candidates was completed to analyze the dynamic stability and structural motions over time (Supplementary Fig.\u0026nbsp;3). Simulations of three control proteins were performed for comparison: a full Spike trimer, a single chain spike, and the 4502 construct without the S1 region as we wanted to know if an N-terminal RBD was sufficient for candidate stability or if the entire S1 domain was required. We found the presence of the S1 domain enhanced dynamic stability of the candidate as the candidate with a full S1 domain had a lower overall Root Mean Square Deviation (RMSD) and a reduced degree of structural fluctuation compared to the control protein with only an N-terminal RBD (Supplementary Fig.\u0026nbsp;3). Moreover, the stability of candidate 4502 was also greater than the other controls. RMSD and Root Mean Square Fluctuations (RMSF) were calculated to measure whether the proteins held their 3D conformations and to get an insight into their flexibility and stability (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRMSF analysis indicated the trimeric spike of SARS-CoV-2 (dotted light blue line, Supplementary Fig.\u0026nbsp;4) to be the most stable while the isolated Spike Chain A (SARS-CoV-2_Spike_Chain_A) behaved oppositely with large fluctuations throughout both S1 (~\u0026thinsp;14\u0026ndash;685) and S2 (~\u0026thinsp;686\u0026ndash;1273) regions. The truncated No S1 form of 4502 (4502_No_S1) had considerable structural motion, with flexibility increasing mainly in the N terminus. Candidate 4502 with the S1 in the N terminus was more stable than the S1 alone (average differences of ~\u0026thinsp;0.3\u0026ndash;0.7 nm for most regions) which this level of flexibility may allows the formation of important loops that play a role in interacting with antibodies.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunogenicity of pan-sarbecovirus vaccine candidates in mice\u003c/h3\u003e\n\u003cp\u003eWe next investigated the immunogenic potential of the candidates by immunizing BALB/c mice with 5 \u0026micro;g of candidate protein formulated with SWE (squalene-in-water emulsion) as adjuvant using a prime-boost regime with a 21-day interval. Responses were compared to control immunizations with a known standard coronavirus vaccine (Pfizer BA4/5) and a negative control (PBS). To determine the breadth of antibodies and potential to be cross-reactive, we developed a panel of sarbecovirus RBD proteins (WIV1, Khosta2, RsSHC014, Pang17, Urbani, and RaTG13) for ELISAs. Plasma samples collected Day 42 post prime immunization were incubated against the panel and analyzed with the results presented as a heat map Log\u003csub\u003e10\u003c/sub\u003e ELISA titer (dark purple\u0026thinsp;=\u0026thinsp;low levels of binding; yellow\u0026thinsp;=\u0026thinsp;high levels) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) or as separate histograms (Supplementary Fig.\u0026nbsp;5). Statistical significance of the Log\u003csub\u003e10\u003c/sub\u003e ELISA titer was determined compared to PBS controls. All vaccine candidates and the control Pfizer vaccine induced high titers of binding antibodies to the SARS-COV-2 S1, as well as binding antibodies to the other RBDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The Pfizer immunized animals had the lowest titers across the other RBDs, especially toward the Clade 3 antigens, Khosta2 and RsSHC014. All our designed candidates induced antibody titers toward the Clade 3 RBDs and had greater titers against the other Clade 1a and Clade 1b antigens. Splenocytes isolated on Day 42 from each immunization group were stimulated with either RBD from SARS-CoV-2 (Ancestral) or SARS-CoV (Urbani) and IFN-γ and IL-5 were quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). As expected, low amounts of IFN-γ and IL-5 producing cells were observed from the PBS immunized animals for all stimulations; however, Pfizer immunized animals also had low numbers of positive cells for both Th1 and Th2 responses. Conversely, cells from animals immunized with pan-sarbecovirus vaccine candidates exhibited strong Th1 and Th2 responses toward SARS-CoV-2, above that of the Pfizer groups with the exception of IFN-γ production in cells from 4500, 4502, and 4503 immunized animals following stimulation with SARS-CoV-2 and in cells from 4502 following stimulation with Urbani. Cells from immunized animals had strong IFN-γ response above the PBS control group. Interestingly, IL-5 responses were significantly higher even over 500-fold for the cells from pan-sarbecovirus vaccine candidate immunized animals compared to the Pfizer groups. This data suggests that the pan-sarbecovirus vaccine candidates induce both cross-reactive antibody responses as well as strong and balanced cross-reactive T cell responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePan-sarbecovirus vaccine candidates induce cross-protection during SARS-CoV-2 Delta heterologous challenge\u003c/h3\u003e\n\u003cp\u003eWe next investigated if the pan-sarbecovirus vaccine candidates could provide protection against severe disease and infection following challenge with a virus that is not targeted in the vaccine. Using the Syrian hamster model, animals were immunized and boosted with 25 \u0026micro;g of vaccine candidates, control vaccines (Pfizer BA4/5 (5 \u0026micro;g) or PBS) on Day 0 and 28. On Day 48 post prime, the animals were intranasally challenged with SARS-CoV-2 Delta variant (10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e) which was not targeted in the vaccine. To determine protection, we assessed viral load in the respiratory tissues, lung pathology, and induction of viral neutralizing antibodies. Blood samples were collected on Day 48 and blood and lung samples were collected on Days 5 and 10 post challenge for analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Due to the large size of the study, three trials were performed with each trial having its own set of PBS and Pfizer vaccinated control groups. Analysis of weight post challenge was graphed for each candidate vaccine group compared to the control vaccines of their trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The Pfizer vaccine was able to protect against weight loss during the Delta virus challenge period with the animals losing less than 5% of their original weight in all of the three trials. Conversely, the negative control PBS immunized groups lost\u0026thinsp;~\u0026thinsp;10% of original weight in each trial. All pan-sarbecovirus vaccine candidates protected the animals from weight loss with the weight loss experienced in each group being statistically less than those animals immunized with PBS. Of note, the hamsters vaccinated with the candidate 4503 (SARS-CoV-2-RaTG13-BtKY72-WIV1) lost the least amount of weight dropping to ~\u0026thinsp;98% of original weight. Microneutralization assays indicated high titers of virus neutralizing antibodies against the Delta variant were present in each group immunized with the pan-sarbecovirus candidate vaccines with levels comparable to the Pfizer immunized groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Specifically, the candidates 4500 and 4503 elicited higher virus neutralizing titers of antibodies than the Pfizer vaccine, with the 4500 immunized animals having elicited statistically higher levels than the Pfizer immunized animals. The candidate 4503 elicited the highest virus neutralizing titers with titers (1:128 to 1:156). Viral loads were evaluated in nasal turbinates and lung tissue collected on Day 5 post challenge from all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Negative control PBS groups had average viral titers between 10\u003csup\u003e3\u003c/sup\u003e and 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e. The control Pfizer immunized animals did not have infectious virus within either tissue on Day 5. Furthermore, the vaccine candidates 4500, 4501, and 4503 were all also mostly negative for infectious virus in the nasal turbinates and lungs with only one animal from the 4501 immunized group having a positive viral titer in the respiratory tract samples (10\u003csup\u003e1\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e in lungs and 10\u003csup\u003e3\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e in nasal turbinates). Independent pathological assessment was conducted on lung tissues from Day 5 and 10 post challenge. Lung disease was rated on a scale of 0\u0026ndash;4 in seven categories assessing percentage of parenchyma affected; inflammation, pneumonia, hyperplasia, bronchi and bronchiolar pathology, and hemorrhage. Overall Histopathology scores in the candidate immunized animals were lower than the scores of the PBS control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eELISAs against our panel of sarbecovirus RBD antigens (WIV1, Khosta2, RsSHC014, Pang17, Urbani, RaTG13, BtKy72, XBB.1.5 (variant of SARS-CoV-2)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; Supplementary Fig.\u0026nbsp;6) found the Pfizer vaccine was able to induce cross-binding antibodies toward all antigens tested, however, overall the Log\u003csub\u003e10\u003c/sub\u003e ELISA titer plasma from the Pfizer immunized animals was lower toward the majority of antigens compared to the binding of antibodies from our candidate immunized animals. The candidate that induced the highest titers of binding antibodies across the sarbecovirus non-SARS-CoV-2 RBD antigens was candidate 4503 which had titers ranging from 4.66 to 5.49 whereas the Pfizer vaccine had titers ranging from 3.24 to 3.82. We also conducted pseudovirus neutralizations using pseudoviruses of WIV16, PangGX, and PangGD against the plasma from immunized animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). WIV16 is a Clade 1a sarbecovirus related to WIV1, while PangGX and PangGD are Clade 1b viruses related to Pang17. Plasma collected from animals immunized with pan-sarbecovirus candidates had statistically higher neutralization against WIV16 compared to PBS control immunized animals; however, animals immunized with Pfizer were not found to have statistically higher titers of neutralizing antibodies. All candidates as well as the Pfizer vaccine elicited statistically higher titers of neutralizing antibodies against PangGX and PangGD than the PBS control immunized animals. These results suggested that pan-sarbecovirus vaccine candidates are potentially protective against heterologous viruses as the vaccine candidates induced significant levels of cross-reactive antibody responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePan-sarbecovirus vaccine candidates protect against SARS-CoV Clade 1a sarbecovirus challenge\u003c/h3\u003e\n\u003cp\u003eSince SARS-CoV-2 Delta is a Clade 1b sarbecovirus, we felt it was important to next evaluate the pan-sarbecovirus vaccine candidates against a Clade 1a sarbecovirus challenge to further understand the breadth of the protection offered by the vaccines. We conducted a similar challenge study as above except the immunized hamsters were challenged with the Tor2 strain of SARS-CoV (6.24x10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e) as a representative of Clade 1a viruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Two trials were performed to evaluate all 4 candidates with each trial having its independent controls. Infection with SARS-CoV induced greater disease severity in the control PBS immunized hamsters compared to the other groups. The PBS vaccinated animals lost more than 10% of original weight with some animals reaching humane end points and were removed from the study (euthanized) (1 PBS immunized hamster per PBS group reached humane endpoint in the first trial (control groups for 4502 and 4503) and 2 PBS hamsters reached humane endpoint in the second trial (control groups for 4500 and 4501) (Supplementary Fig.\u0026nbsp;7)). Pfizer vaccinated animals lost less than 5% of original weight whereas all candidate vaccines lost even less than 5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). All pan-sarbecovirus vaccine candidates induced virus neutralizing antibodies toward SARS-CoV with group average titers at 1:64 or above by Day 48. The upper and lower respiratory tracts had significant viral titers in the control PBS immunized groups with average titers of 10\u003csup\u003e3\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e or above. Robust SARS-CoV viral titers were also present in the respiratory tracts of the Pfizer immunized animals in each of the trials with 3 out of the 4 animals having nasal turbinate viral titers above 10\u003csup\u003e3\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). For the pan-sarbecovirus vaccine candidates, the results of the TCID\u003csub\u003e50\u003c/sub\u003e assays indicated that most of the respiratory tract samples were below the limit of detection and each group had significantly lower viral loads in both the nasal turbinates and lungs compared to the PBS and Pfizer groups. Lung pathological assessment found Negative control immunized animals had average scores of 3 on Day 5 while the Pfizer group animals had an average score between 2 and 3 depending on the trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). For all pan-sarbecovirus vaccine candidates, no animal scored above 1. The scores for 4500 and 4501 on Day 5 post challenge were statistically lower than both the PBS and Pfizer groups. The trial with 4502 and 4503 had statistically lower scores than the PBS group for the same day. Taken together, these data indicated that protection from viral infection, disease severity, and lung pathology were provided by all designed pan-sarbecovirus vaccine candidates during heterologous challenge with the Clade 1a virus SARS-CoV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThere is a need for anticipatory vaccines to protect against potential emerging coronaviruses. Here we developed four candidates on a flexible plug-and-play fusion protein subunit platform that strategically incorporated specific sarbecovirus targets from Clade 1a, Clade 1b, and Clade 3 to induce broadly protective immune responses. We predicted our antigens to have binding capacity to a known broadly reactive antibody and have accessible B cell epitopes. Experimentally, we found our vaccines protected against SARS-CoV2 Delta and SARS-CoV challenge with concomitant induction of broadly binding and broadly neutralizing antibodies against pseudoviruses from pre-emergent sarbecoviruses (pangolin viruses and WIV16). Additionally, we found our candidates induced robust broadly reactive T cell responses against diverse sarbecovirus RBD antigens, which was not seen in the Pfizer immunized animals. Specifically, in the Pfizer immunized animals, T cells did not have significant levels IFN-γ or IL-5 after stimulation with Clade 1a or 1b antigens (SARS-CoV-2 (Ancestral) or SARS-CoV (Urbani)). This data suggested that our pan-sarbecovirus vaccine candidates may offer pan-virus protection through both broadly reactive humoral and cell mediated responses with the fusion protein platform being a potential tool against other virus families.\u003c/p\u003e \u003cp\u003eIn our current study we investigated the protection of protein subunit vaccine candidates by intramuscular (IM) delivery. Although the candidates protected from severe disease including against SARS-CoV challenge, small amounts of infectious virus as well as lung pathology was still detected, which could be attributed to the lack of mucosal protection elicited during intramuscular immunizations. Intranasal vaccination is an approach that has been shown to be successful for COVID-19 vaccines and pan-coronavirus vaccine candidates \u003csup\u003e41\u003c/sup\u003e and should be investigated in the future for our vaccine candidates. Moreover, we showed that both broadly reactive T cell and humoral immune responses were induced following immunization with our candidates. Most published studies on broadly reactive vaccine candidates have not investigated T cell responses where one of only the studies to do so was a study involving the dual antigen mRNA candidate vaccine expressing N and S \u003csup\u003e42\u003c/sup\u003e. However, the breadth of these responses was only evaluated against SARS-CoV-2 variants and not against other viruses within the sarbecovirus subclade as we have done here \u003csup\u003e42\u003c/sup\u003e. Although we conducted a fairly thorough investigation of the immune responses, it was not clear what arm of the immune response had more influence on protection. Further studies with adoptive transfer experiments could tease out the mechanisms of protection associated with vaccines developed on our fusion protein platform.\u003c/p\u003e \u003cp\u003eGlobal thought leaders urge the readiness of not only vaccine candidates that are anticipatory, but also for systems supporting vaccine pipelines ensuring required vaccines are available and readily deployable at the first signs of virus emergence \u003csup\u003e43\u003c/sup\u003e. For complete system readiness, anticipatory vaccine candidates must be evaluated in appropriate models during non-pandemic times. Also, having large scale manufacturing processes developed, technology transferred to large industry partners, and industry and deployment pipelines established for equitable global vaccine distribution at fair cost is needed to avoid Public Health Emergencies of International Concern (PHEIC), as well as pandemics \u003csup\u003e43\u003c/sup\u003e. Published studies of broadly protective vaccines and immune responses have shown that vaccine antigens with repetitive arrays of viral surface proteins strongly induce cross- and broad-protection through enhanced B cell activation suggesting these platforms and pipelines being appropriate for pandemic preparedness \u003csup\u003e30\u003c/sup\u003e. Vaccine platforms have included the protein nanoparticle, protein-lipid nanoparticle, live-but-defective SARS-CoV-2 virus with interferon integration, NanoSTING platform, VLPs (Virus Like Particles), chimeric subunit vaccine, and mRNA candidates \u003csup\u003e24,27\u0026ndash;29,41,42,44,45\u003c/sup\u003e. These described vaccine candidates have shown various levels of broad-reactivity such as across SARS-CoV-2 variants \u003csup\u003e27,28,42\u003c/sup\u003e or across Clade 1 sarbecoviruses \u003csup\u003e28,36\u003c/sup\u003e. However, the majority of platforms leveraged for establishing the display of repetitive viral surface proteins have been multivalent protein nanoparticles that require the assembly of fusion protein subunits for subsequent nanoparticle assembly which may take longer and be subject to more manufacturing challenges \u003csup\u003e24,26,27,29,36,46\u003c/sup\u003e. Conversely, our protein subunit platform leverages the translation of a single fusion protein as a vaccine antigen that can display repetitive viral surface protein/protein domains while retaining immunogenic 3D properties. Sustainable biomanufacturing solutions are essential for producing enough product for global distribution quickly during an emergency. Our simplified platform producing a single fusion protein potentially supports more sustainable and streamlined biomanufacturing processes.\u003c/p\u003e \u003cp\u003eHere we have shown promising results on the broadly reactive nature of multivalent fusion protein subunit pan-sarbecovirus vaccine candidates. Future studies will be important to establish readiness of these candidates for emergency situations. Additional studies investigating both the longevity of protection as well as antigen stability as it relates to shelf life in stockpiles are needed \u003csup\u003e32\u003c/sup\u003e. Moreover, studies in preimmune animal models will also be important for understanding potential immune skewing toward antigens previously seen to support future human clinical trials \u003csup\u003e43,47\u0026ndash;50\u003c/sup\u003e. Coronaviruses are a significant threat to public health, animal welfare, and economic stability as new strains emerge and endemic coronaviruses continue to circulate. It is highly likely that a novel coronavirus with pandemic potential will emerge within the next decade as we have seen three severe coronaviruses emerge over the past 25 years. The path to controlling the public health burden of coronaviruses involves developing next generation vaccines that can anticipate the diversification of circulating strains and/or the spillover of strains yet uncharacterized. Together, the work presented here, and future studies will help build a global portfolio of broadly protective coronavirus vaccines and flexible vaccine platforms. Having anticipatory vaccines that offer broad protection readily available are essential for health emergency preparedness.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study investigated the design and development of pan-sarbecovirus vaccine candidates developed as a fusion protein. Structural and immunogenic predictions were made of design vaccine antigens. Immunogenicity was assessed in vivo in Syrian hamsters and in mice. Protection from heterologous virus challenge was assessed in Syrian hamsters immunized with candidate antigens compared to control vaccine (Pfizer) or mock vaccine (PBS). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll work was conducted in accordance with the Canadian Council of Animal Care (CCAC) guidelines, AUP numbers 20200016 and 20240002 by the University Animal Care Committee (UACC) Animal Research Ethics Board at the University of Saskatchewan. Hamster and mouse procedures were performed under 5% isoflurane anesthesia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sarbecovirus spike gene sequences were collected from GISAID and other open databases such as NextStrain and the NCBI nt database. Initially, 407 sequences were compared, but sequences that were highly similar, overrepresented within certain clades (e.g., multiple strains of human SARS-CoV), or had incomplete receptor-binding domains (RBDs) were removed. After filtering, 105 sequences remained for analysis. The spike gene sequences were translated and aligned using the MUSCLE algorithm implemented in the SEAVIEW phylogenetic package\u003csup\u003e51\u003c/sup\u003e. All sites with the exception of the RBD region (positions 319\u0026ndash;541, based on the ancestral SARS-CoV-2 spike protein QHD43416.1) were masked from the analysis. A phylogenetic tree of the RBD nucleotide sequences was constructed with IQ-TREE2\u003csup\u003e52\u003c/sup\u003e, using a amino acid substitution model selected by ModelFinder (Q.yeast) and rates across sites approximated on a gamma distribution of four categories of variable sites and one invariable site category. Branch support was evaluated with 1,000 ultrafast bootstrap replicates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein Structure Prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3-dimensional modeling of the candidate vaccine protein antigens were constructed using AlphaFold2 (AlphaFold2 (Colab-Fold) (ChimeraX plugin for AlphaFold2/Colab-Fold with default criteria). The structural predictions were then validated using SWISS-MODEL. Protein structures were visualized in PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schr\u0026ouml;dinger, LLC) and ChimeraX. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpitope Prediction and Antibody Molecular Docking Prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conformation of our vaccine constructs was predicted through using TAMARIND BIO \u0026ndash; Boltz-2, which is based on AlphaFold3 reproduction. The conformations were used for B-cell epitope prediction through DiscoTope 3.0, with the threshold being set to 0.9, and HADDOCK2.4, and default criteria for molecular docking. Molecular docking was predicted to the neutralizing antibody CT-P59 (PDB: 7CM4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Dynamics (MD) Simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGROMACS version 2020.1 with CHARMM36 all-atom force field was employed to conduct the Molecular dynamics (MD) simulations. All our MD systems with vaccine constructs solvated in a cubic simulation box filled with TIP3P water molecules, with a minimum distance of 1.0 nm between the protein surface and the box boundary. All systems were neutralized by adding counterions Na⁺ and Cl⁻. Energy Minimization and Equilibration were achieved before the production run using the steepest descent algorithm. The NVT (constant Number, Volume, Temperature) for one ns, equilibration for 100 ps and NPT for five ns were performed. Finally, the NPT, NVT equilibrated systems were subjected to a production MD run for 200 ns, using a 2 fs MD integrator. The temperature and pressure at 300 K and 1 bar remained stable through a modified Berendsen thermostat (V-rescale) with time constant \u0026tau;_t = 0.1 ps and Parrinello-Rahman pressure coupling with the compressibility of 4.5e-5 and \u0026tau;_p = 2 ps. \u0026nbsp;The LINCS algorithm was used for bond constraints. Lennard-Jones and Coulombic non-bonded interactions were treated with a cutoff of 1.2 nm. Long-range electrostatics were calculated by the Particle Mesh Ewald (PME) method. Periodic boundary conditions were applied in all directions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVaccine Design, Vaccine Expression and Purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTargeted sequences were identified and linked via 3x GS linker to S1 WT sequence to generate the desired vaccine candidates. The DNA sequence was cloned into the pCDNA3.4 expression vector (Genscript Biotech; Piscataway, New Jersey, USA), transformed into chemically competent OneShot\u0026reg;Top10 \u003cem\u003eE. coli\u003c/em\u003e cells (Thermo Fisher Scientific), and amplified and then extracted using Qiagen Endofree Maxi prep kit (Qiagen Cat. 12362). Inserts were verified by restriction digestion and sequencing (Plasmidsaurus, San Francisco, California). ExpiCHO\u0026trade; cells (6x10\u003csup\u003e6\u003c/sup\u003e cells/ml) were transfected with 0.7 \u0026micro;g/ml of candidate vaccine plasmid using ExpiFectamine\u0026trade;. Vaccine candidate proteins were transiently expressed following the Gibo ExpCHO\u003csup\u003eTM\u003c/sup\u003e max titer expression protocol as outlined by ThermoFisher Scientific (Mississauga, Ontario, Canada), which includes a temperature shift to 32\u003csup\u003eo\u003c/sup\u003eC on day 1 after transfection. Cells were cultured for 3 days for optimal protein expression. Supernatants containing secreted vaccine candidate proteins were collected and centrifuged at 4,000 x g for 30 min at 4\u003csup\u003eo\u003c/sup\u003eC. After the centrifugation, 0.05% Tween-80 and 300 mM NaCl were added to the clarified supernatant containing the expressed proteins. A final filtration through a 0.2 \u0026micro;m PES filter was subsequently performed followed by tangential flow filtration with a Vivaflow\u0026reg; 50R 30kDa filter (Sartorius; Oakville, Ontario, Canada), and the supernatant was concentrated 8-10 fold. Vaccine candidates were purified using Repligen NGL Covid-19 Spike protein AR 2.0 resin (Repligen, Walktham, Massachusetts, United States) as previous described\u003csup\u003e32\u003c/sup\u003e. Immediately after elution from the column, the protein was exchanged into a buffer containing 277 mM NaCl, 14 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5.7 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, pH 7.4, using Amicon\u0026reg; Ultra centrifugal filter with a 30 kDa MWCO (UFC903024). A Pierce\u0026trade; BCA assay (ThermoFisher Scientific) was conducted according to manufacturer procedure to verify final protein yield. Protein samples were dialyzed with 1X PBS for 1 day at 4\u0026deg;C. For deglycosylation of samples, 5 \u0026micro;g of purified pan-sarbeco protein was incubated with NEB Biolabs PNGaseF enzyme for 1 h at 37\u0026deg;C following the manufacturer\u0026rsquo;s protocol. Western blots were conducted to confirm protein size and expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCoomassie gels and Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo visualize and estimate the size of the expressed proteins, supernatants were collected from the culture media and loaded onto 4-20% Mini-PROTEAN\u0026reg; TGX\u0026trade; acrylamide gels (Cat: 4561094; BioRad, Mississauga, Ontario, Canada). Precision Plus Protein\u0026trade; ladder (Cat: 161-0374, BioRad) was loaded at 3 \u0026mu;L/well as a standard. The electrophoresis was performed in a Tris-Glycine-SDS buffer (BioRad) at 200 V for 50 min. Following electrophoresis, total protein in each sample was visualized by BioRad Bio-Safe Coomassie stain G-250 (BioRad) following standard protocol or transferred onto 0.2 \u0026mu;m nitrocellulose membranes (Cytiva, Vancouver, British Columbia, Canada). Membranes were blocked overnight in 3% (w/v) Amersham\u0026trade; ECL Blocking reagent (Cytiva) followed by incubation with in-house generated rabbit polyclonal primary antibody S1-CoV19 (1:5,000) diluted in 1.5% blocking buffer for 1 hr at room temperature. Blots were washed in PBSA before secondary antibody incubations (donkey anti-rabbit IRDye 800CW (1:10,000), Li-COR\u0026reg;; Lincoln, Nebraska, USA) at 1:10,000 dilution for 1 hr at room temperature on a rocker. Resulting blots were washed in PBSA for 5 min before being scanned on a Li-Cor NIR scanner. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysical Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSize exclusion chromatography with multi-angle light scattering (SEC-MALS)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePurified proteins were analyzed on a Wyatt Technology multi-angle light scattering instrument (MALS) with refractive index (RI) detector in-line with a Cytiva Superdex 200 Increase 10/300 GL column connected to a Bio-Rad NGS FPLC system. The column was calibrated using bovine serum albumin. Samples contained 1 mg protein. The size-exclusion chromatography was performed in a buffer containing 277 mM NaCl, 14 mM NaH2PO4, 5.7 mM Na2HPO4, pH 7.4, at a flow rate of 0.5 ml/min. Acquired data was analyzed using Wyatt software package ASTRA 7.3.2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCircular Dichroism spectroscopy\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePrior to analysis, protein samples were dialyzed against buffer containing 155 mM NaCl, 2.97 mM Na2HPO4 and 1.06 mM KH2PO4, pH 7.4 (Gibco). Secondary structure content and thermal stability of the proteins were analyzed by CD spectroscopy using Applied Photophysics CHIRASCAN Plus CD spectrometer at a protein concentration of 0.7 to 1 mg/ml. Measurements were obtained from 200 to 280 nm with a bandwidth setting of 2 nm in a 0.5 mm quartz glass cell. The final CD spectrum of each protein was a mean of three measurements. The resulting CD data was analyzed using the Beta Structure Selection (BeStSel) online tool (https://bestsel.elte.hu/ssfrompdb.php). The data was normalized using the following equation: \u0026nbsp; \u0026Theta;norm= □((\u0026Theta; (\u0026lambda;) \u0026nbsp;x \u0026epsilon;205)/(10 xA205 x (N-1))), where \u0026Theta;norm is normalized molar ellipticity, \u0026Theta;(\u0026lambda;) is raw ellipticity in millidegrees, \u0026epsilon;205 protein extinction coefficient at 205nm, A205 absorbance at 205 nm, and N is the number of amino acid residues. Extinction coefficients \u0026epsilon;205 for protein constructs were calculated as described previously\u003csup\u003e53\u003c/sup\u003e. \u0026nbsp;For the thermal stability assay, CD spectra were successively recorded while the sample was gradually heated from 5 \u0026deg;C to 95 \u0026deg;C at a rate of 1 \u0026deg;C/min, and then cooled back to 20\u0026deg;C. \u0026nbsp;Changes in the secondary structure were analyzed with CHIRASCAN software Pro-Data viewer to determine the melting temperature of each construct.\u003c/p\u003e\n\u003cp\u003eThe experimentally determined secondary structure content was compared to the predicted using PSI-BLAST - based prediction tool (PSIPRED) \u0026nbsp;https://bio.tools/psipred.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eProtein Identification via mass spectrometry analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSamples containing 5 \u0026micro;g of the purified protein were subjected to SDS-PAGE on a 4-20% TGX gel (BioRad). The gels were stained with BioRad Bio-Safe Coomassie stain G-250 (BioRad) following the manufacturer\u0026rsquo;s protocol. \u0026nbsp;Protein bands were excised and analyzed by ESI-LS-MS at the Alberta Proteomics and Mass Spectrometry Facility following in-gel digestion. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eSARS-CoV and SARS-CoV-2 variant viruses\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe SARS-CoV-2 pango lineage B isolate /Canada/ON/VIDO-01/2020 was used as a representative for the prototypic B lineage virus (Ancestral virus) (GISAID\u0026ndash;EPI_ISL_425177)\u003csup\u003e54\u003c/sup\u003e. SARS-CoV-2 Delta was isolated at VIDO from a patient sample (GenBank PP077059). SARS-CoV Tor2 was obtained from the National Microbiology Laboratory (N. Bastien). All viruses were cultured in vDMEM (viral DMEM (Dulbecco\u0026rsquo;s Modified Eagle Medium), 2% fetal bovine serum, 100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin, and 1 \u0026mu;g/mL TPCK-trypsin) on Vero-76 cells. All work with infectious SARS-CoV-2 viruses, variants, and SARS-CoV Tor2 was performed in the Vaccine and Infectious Disease Organization\u0026rsquo;s (VIDO) Containment Level 3 (CL3) facility (InterVac) in Saskatoon, Saskatchewan, Canada.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMouse studies \u0026ndash; vaccinations and tissue collections\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eBALB/c mice were purchased from Charles River Laboratories (Wilmington, Massachusetts USA). Mice were anesthetized with 2% isoflurane for in life procedures. Mice were immunized by intramuscular injection with pan-sarbecovirus vaccine candidates (5 \u0026micro;g per dose) adjuvanted with Sepivac SWE\u0026trade; (Vaccine Formulation Institute (VFI), Ouates, Switzerland) on Days 0 and 21. For the control groups, mice were immunized by intramuscular injection with either Pfizer-BioNTech (Mainz, Germany) Comirnaty BA4/5 (5 \u0026micro;g per dose), or with PBS in 50 \u0026micro;l volumes on Day 0 and 21. Candidates were adjuvanted with SWE (Vaccine Formulation Institute (VFI), Ouates, Switzerland). Eight mice were immunized per group. Blood was collected in BD Vacutainer\u003csup\u003e\u0026reg;\u003c/sup\u003e EDTA coated blood collection tubes for plasma separation and spleens were removed for single cell isolation on Day 42 post immunizations and subsequent ELISpot. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eSyrian hamster studies \u0026ndash; vaccinations, infections, and tissue collection\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eMale Syrian hamsters aged 8-weeks were purchased from Charles River Laboratories.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTen hamsters per group were anesthetized with 5% isoflurane for in life procedures. Hamsters were vaccinated by intramuscular injection with pan-sarbecovirus vaccine candidates (25 \u0026micro;g per dose), with Pfizer-BioNTech Comirnaty (5 \u0026micro;g per dose), or with PBS in 50 \u0026micro;l volumes on Day 0 and 28. Candidates were adjuvanted with Sepivac SWE\u0026trade; (VFI).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll virus inoculations were performed intranasally with 100mL of virus at a concentration of either 1x10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e for SARS-CoV-2 Delta or 6.24x10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e for SARS-CoV per animal on Day 48 post immunization. Five hamsters were randomly selected and euthanized for necropsies on Day 5 post challenge and the remaining animals were necropsied on Day 10 post challenge. To calculate weight loss and survival, hamsters were monitored until end day post challenge, as we and others have done previously \u003csup\u003e55,56\u003c/sup\u003e. Blood and respiratory tissues (nasal turbinates and lungs) were collected from the animals removed from the study on Day 5 post challenge. At necropsy, nasal turbinates and lungs were collected for virological, immunological, pathological analysis. The left lung lobe was collected for histopathology while the right lobes were used for viral load. Weight was measured daily and then calculated as a percentage of original values from Day 0. Blood was collected in BD Vacutainer\u003csup\u003e\u0026reg;\u003c/sup\u003e EDTA coated blood collection tubes for plasma separation. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eImmune Assays \u0026ndash; ELISAs and ELISPOTS\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eELISAs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePlasma IgG antibodies from immunized BALB/c mice or Syrian hamsters were quantified for binding capabilities against the S1 subunit of the S protein from SARS-CoV-2 (Sino Biological 40591-V08H) or against a panel of sarbecovirus RBDs (WIV1, Khosta2, RsSHC014, Pang17, Urbani, RaTG13, BtKy72, XBB.1.5) (Cat: SC9305PF, GenScript) using ELISAs, according to standard protocol. Ninety-six-well ELISA plates (Cat: 3655TS, ThermoFisher Scientific) were coated with 1 \u0026mu;g/mL antigen in coating buffer and incubated at 4\u0026deg;C overnight. Plates were washed four times with 1x TBS with 0.05% Tween-20 (Cat: P1379, MilliporeSigma; Oakville, Ontario, Canada) (TBST) then blocked for 1 hr in 5% (w/v) skim milk powder in TBST. \u0026nbsp;Plasma samples were diluted 1:4 then serially diluted 1:100 in 1% skim milk in TBST then incubated at room temperature for 1 hr, then washed, and then alkaline phosphatase labelled anti-mouse IgG (Cat: 5220-0355 (KPL), MilliporeSigma) secondary was added 1:10,000 or goat anti-Hamster IgG HRP at 1:7000 dilution (ThermoFisher Scientific) to 1% skim milk in TBST (1 hr). After washing, OPD substrate (Cat: 34074, ThermoFisher Scientific) and stop solution was added. Plates were read at 405 and 490 nm on an 800TS reader (BioTek; Winooski, Vermont, USA). Delta values were blanked to wells not containing plasma. Cutoffs were determined based on mean absorbance plus 3 standard deviation values of negative controls.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eELISpots\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate T cell responses following stimulation with RBD from SARS-CoV-2 (Ancestral) or SARS-CoV (Urbani), ELISpot assays were performed with splenocytes harvested from vaccinated mice. MultiScreen\u0026reg; 96-well PVDF plates (Cat: MSIPS4510, MilliporeSigma) were prepared and coated 2 \u0026mu;g/ml anti-IL-5 (Cat:554395, BD Biosciences) or anti-IFN-\u0026gamma; (Cat: 551216, BD Biosciences) the day prior to the assay. Splenocyte single cell suspensions were prepared from harvested spleens in Gey\u0026rsquo;s solution after washing. Two ml aliquots of suspended cells at a concentration of 10\u003csup\u003e7\u003c/sup\u003e cells per ml were prepared in culture media (AIM-V (Cat: 12055083, ThermoFisher Scientific), non-essential amino acids (Cat: 11140050, ThermoFisher Scientific) at 1:100, \u0026nbsp;sodium pyruvate (Cat: 11360070, ThermoFisher Scientific) at 1:100, \u0026nbsp;HEPES buffer (Cat: 15630080, ThermoFisher Scientific) at 1:100, and \u0026nbsp;50 mM \u0026beta;-mercaptoethanol (Cat: 21985023, ThermoFisher Scientific) at 1:1000). Plates were blocked with 1% BSA (Cat: A4737, MilliporeSigma) and cells were added at 10\u003csup\u003e6\u003c/sup\u003e cells/well 1:1 with 4 \u0026mu;g/mL antigen in triplicate. Concanavalin A (Cat: 00-4978-93, ThermoFisher) was used as a positive control and was plated at 2 \u0026mu;g/mL. Splenocytes were incubated at 37\u0026deg;C for 18 hours overnight with antigen. PBST was added to lyse cells and plates were washed 3 times with PBST (0.05% Tween20 (Cat: P1379, MilliporeSigma) in PBS) then twice with dH\u003csub\u003e2\u003c/sub\u003eO before addition of secondary antibody (IL-5 (Cat: 554397, BD Bioscience) and IFN-\u0026gamma; (Cat: 554410, BD Bioscience)). After secondary antibody incubation, plates were washed and streptavidin conjugated alkaline phosphatase (Cat: 016-050-084, Cedarlane Labs; Burlington, Ontario, Canada) was diluted 1:1000 in 1% BSA in PBS was added to plates and incubated for 1.5 hr. Plates were washed and BCIP/NBT substrate (Cat: B5655, MilliporeSigma). After washing, plates were scanned using an ELISpot Reader (Autoimmun Diagnostika GmbH; Stra\u0026szlig;berg, Germany) and spots were counted using ELISpot v7.0 software (Autoimmun Diagnostika GmbH) and evaluated by eye. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eViral titers\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTissues collected at necropsy were homogenized in serum free DMEM using a TissueLyserII (Qiagen; Toronto, Ontario, Canada). To determine TCID\u003csub\u003e50\u003c/sub\u003e, a 1:10 dilution series of sample was prepared in the appropriate viral growth media in 96-well plates. Samples were assessed in triplicate. Cells were incubated for 1h at 37\u003csup\u003eo\u003c/sup\u003eC. Sample dilutions were removed and replaced with fresh viral media. Cytopathic effect (CPE) was monitored daily for 5 days. On Day 5 post inoculation of cells, CPE was recorded indicating the presence of infectious virus. These data were used to calculate TCID\u003csub\u003e50\u003c/sub\u003e/mL using the Spearman-K\u0026auml;rber method .\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMicroneutralization assay\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eSera isolated from blood samples were heat-inactivated at 56\u003csup\u003eo\u003c/sup\u003eC for 30 min and then serially diluted 1:2 in a low serum viral media, in round bottom 96-well plates. Virus was diluted to 25 TCID\u003csub\u003e50\u003c/sub\u003e per well\u003csub\u003e\u0026nbsp;\u003c/sub\u003ein viral media and used at a 1:1 ratio to serum, where 60\u0026nbsp;mL of serum was placed in each well and 60\u0026nbsp;mL of diluted virus was added. The serum-virus mixture was incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 1 h and then added to cultured virus-designated cells in 96 well plates. Plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC and were monitored for CPE over five days. Endpoint neutralization titer was based on inhibition of CPE observed on Day 5 after cell infection. The endpoint titer is reciprocal of the highest dilution of serum that is able to suppress CPE. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePseudoviral neutralization assays\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePseudoparticle Production \u0026ndash; Pangolin Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePseudotyped viral particles were produced by transfecting HEK 293T/17 cells (ATCC CRL-11268) with plasmid DNA using polyethylenimine (PEIpro; Polyplus) similarly as done previously \u003csup\u003e60\u003c/sup\u003e. Cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Cytiva HyClone) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin\u0026ndash;streptomycin. At 70\u0026ndash;80% confluency, plasmid\u0026ndash;PEI complexes were applied, and after 7\u0026ndash;10h, medium was replaced with supplemented DMEM. Supernatants were harvested 48h post-transfection, clarified by centrifugation and filtration (0.45 \u0026micro;m), aliquoted, and stored at \u0026ndash;80\u0026deg;C. When required, particles were concentrated using Lenti-X Concentrator (Takara Bio) according to the manufacturer\u0026rsquo;s protocol.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePseudoparticle Infectivity and Neutralization Assay \u0026ndash; Pangolin Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNeutralization activity was assessed using 293T-hsACE2 cells (Integral Molecular). Serum samples were heat-inactivated (56\u0026deg;C, 30min) and serially diluted in non-supplemented DMEM before incubation with pseudoparticles for 1h at 37\u0026deg;C. The mixtures were then added to 96-well plates containing 20,000 cells/well. Following a 72h incubation at 37\u0026deg;C in 5% CO₂, infectivity was quantified using pseudoparticles encoding luciferase. Following incubation, luciferase activity was determined with the Bright-Glo Luciferase Assay System (Promega) on a Beckman Coulter DTX 880 multimode detector. Results were expressed as relative luminescence units (RLUs).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePseudoviral Infectivity and Neutralization Assay \u0026ndash; WIV16\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePseudovirus assays and production of WIV16 pseudovirus for WIV16 were completed similarly as previously described \u003csup\u003e60\u003c/sup\u003e. All sera were diluted from 1:40 and then serial dilution of 1:2 was performed. Cell-only and pseudopartical-only controls were included on each plate. WIV16 pseudoparticles were diluted to a concentration of 1x10\u003csup\u003e8\u003c/sup\u003e RLU per plate (5 ml) as determined via titration, and 50 \u0026micro;l was added to all wells except the cell only controls. Plates were incubated for 1 hour at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. ACE2/TMPRSS2 expressing 293T cells were added to all wells at a concentration of 3x10\u003csup\u003e5\u003c/sup\u003e/ml in a volume of 50 \u0026micro;l per well. Plates were then incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 48 hrs before being read using the GloMax\u0026reg; Navigator (ProMega) using the Promega GloMax\u0026reg; Luminescence Quick-Read protocol. Plates were analyzed using PRISM as described\u003csup\u003e61\u003c/sup\u003e. All negative % neutralization values were plotted as 0 and all neutralization above 100% was plotted at max titre. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eLung Histopathology\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eLung tissues collected for histopathology were kept in formalin in the CL3 laboratory. After 7 days tissues were transferred to fresh formalin and removed to be paraffin-embedded, sectioned, slide-mounted, and hematoxylin and eosin H\u0026amp;E stained at Prairie Diagnostic Services (PDS) (Saskatoon, Saskatchewan). Slides were analyzed in-house as well by an independent blinded board-certified pathologist. The pathologist gave the lungs a score ranging from 0 to 4 in the following categories: Inflammation; Pneumonia; Hemorrhaging; and Overall Histopathology. Only the Overall Histopathology score is reported here where the Overall gives a summary score taking all factors into account. 0, absent (no lesion); 1, slight or questionable; 2, clearly present, but not conspicuously so; 3, marked; 4, severe. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStatistical Analysis\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003ePrism GraphPad (San Diego, California, USA) was used to analyze the data. A mixed effect model with repeated measures, multiple comparisons done by Tukeys multiple comparison test was used to evaluate weight data. ELISA and neutralizing antibody data was Log transformed and evaluated using One-way ANOVA with multiple comparisons. Two-way ANOVA with repeated measures and Tukeys multiple comparisons test was used for evaluating viral load quantifications. A Kruskal Wallis test was used with Dunn\u0026rsquo;s multiple comparisons for comparing histopathology. A \u003cem\u003ep\u003c/em\u003e value of \u0026le; 0.05 was considered statistically significant with\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05:*;\u003cbr\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01:**; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001:***; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001:****. Survival was calculated according to the experimental design considering predetermined necropsy days for randomized hamster removals. Therefore, survival was calculated out of 8 hamsters until Day 5 and out of 4 hamsters for end day on Day 10.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the team at CEPI (Coalition for Epidemic Preparedness and Innovation) for their support through the project. Additionally, the authors would like to thank Dr. Nigel Temperton and his team for support with the WIV16 pseudovirus neutralization assays, as well as Dr. Nathalie Bastien from the National Microbiology Laboratory at the Public Health Agency of Canada for supplying the SARS-CoV Tor2 strain of virus. Published as VIDO manuscript series no. 1147. Funding has been provided by the Coalition for Epidemic Preparedness Innovations (CEPI) seed funding (T.R.); Canadian Institutes for Heath Research (CIHR) Project Grant (PJT-190107) (A.A.K.); Canadian Institutes of Health Research (CIHR) and Coalition for Epidemic Preparedness Innovations (CEPI) Leadership Award in Vaccine Research (A.A.K.); Canadian Institutes of Health Research (CIHR) grants OV5-170349, VRI-173022 and VS1-175531 (D.F.). VIDO receives operational funding from Innovation Saskatchewan (Government of Saskatchewan) and the Ministry of Agriculture. VIDO is also supported by the Canada Foundation for Innovation through the Major Science Initiatives (Government of Canada). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eT.R. and A.A.K. conceptualized and designed the experiments. M.B.R., Z.K., E.-M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., and A.A.K. established the methodology. M.B.R., Z.K., E.-M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., and A.A.K. performed the experiments and/or analyzed the data. M.B.R., Z.K., E.-M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., A.A.K. constructed visualizations of the data for figures. A.A.K. and T.R. supervised the study. A.A.K., T.R., D.F., and V.G. contributed materials/funding. A.A.K. wrote the original draft. A.A.K., T.R., M.B.R., Z.K., E. M.U., R.B., E.S., A.Y., J.L., A.A., J.V.K., P.P., A.K., C.L.S., R.P., B.M.T., W.D., R.G., G.H., W.K., T.P., A.-C.F., J.B., A.V.K., V.G., Q.L., A.B., and D.F. reviewed and edited the manuscript. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMasters, P. S. The molecular biology of coronaviruses. \u003cem\u003eAdv Virus Res\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 193\u0026ndash;292 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0065-3527(06)66005-3\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eWertheim, J. O., Chu, D. K., Peiris, J. S., Kosakovsky Pond, S. L. \u0026amp; Poon, L. L. A case for the ancient origin of coronaviruses. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 7039\u0026ndash;7045 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/JVI.03273-12\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eZhuang Pinghui, J. W. \u003cem\u003eWuhan pneumonia: how the search for the source of the mystery illness unfolded\u003c/em\u003e, \u0026lt;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.scmp.com/news/china/society/article/3046233/wuhan-pneumonia-how-search-source-mystery-illness-unfolded%3E\u003c/span\u003e\u003c/span\u003e (2020).\u003c/li\u003e\n \u003cli\u003eCucinotta, D. \u0026amp; Vanelli, M. WHO Declares COVID-19 a Pandemic. \u003cem\u003eActa Biomed\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e, 157\u0026ndash;160 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.23750/abm.v91i1.9397\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMenachery, V. D. \u003cem\u003eet al.\u003c/em\u003e A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1508\u0026ndash;1513 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nm.3985\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMenachery, V. D. \u003cem\u003eet al.\u003c/em\u003e SARS-like WIV1-CoV poised for human emergence. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 3048\u0026ndash;3053 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1517719113\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eSu, C. \u003cem\u003eet al.\u003c/em\u003e Structural characteristics of BtKY72 RBD bound to bat ACE2 reveal multiple key residues affecting ACE2 usage of sarbecoviruses. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, e0140424 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mbio.01404-24\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eAlkhovsky, S. \u003cem\u003eet al.\u003c/em\u003e SARS-like Coronaviruses in Horseshoe Bats (Rhinolophus spp.) in Russia, 2020. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/v14010113\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003ePena-Hernandez, M. A. \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2-related bat viruses evade human intrinsic immunity but lack efficient transmission capacity. \u003cem\u003eNat Microbiol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 2038\u0026ndash;2050 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41564-024-01765-z\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eHolmes, E. C. \u003cem\u003eet al.\u003c/em\u003e The origins of SARS-CoV-2: A critical review. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 4848\u0026ndash;4856 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2021.08.017\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eKhan, K. \u003cem\u003eet al.\u003c/em\u003e Omicron BA.4/BA.5 escape neutralizing immunity elicited by BA.1 infection. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 4686 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-022-32396-9\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eLyke, K. E. \u003cem\u003eet al.\u003c/em\u003e Rapid decline in vaccine-boosted neutralizing antibodies against SARS-CoV-2 Omicron variant. \u003cem\u003eCell Rep Med\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 100679 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.xcrm.2022.100679\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eReynolds, C. J. \u003cem\u003eet al.\u003c/em\u003e Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e377\u003c/strong\u003e, eabq1841 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.abq1841\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003evan der Straten, K. \u003cem\u003eet al.\u003c/em\u003e Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron. \u003cem\u003eImmunity\u003c/em\u003e (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.immuni.2022.07.018\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMykytyn, A. Z. \u003cem\u003eet al.\u003c/em\u003e Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct. \u003cem\u003eSci Immunol\u003c/em\u003e, eabq4450 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/sciimmunol.abq4450\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eDuan, L. \u003cem\u003eet al.\u003c/em\u003e The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 576622 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2020.576622\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMahajan, S. \u003cem\u003eet al.\u003c/em\u003e Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 13164 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-92521-4\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eLevy, Y. \u003cem\u003eet al.\u003c/em\u003e Immunodominant Linear B-Cell Epitopes of SARS-CoV-2 Spike, Identified by Sera from K18-hACE2 Mice Infected with the WT or Variant Viruses. \u003cem\u003eVaccines (Basel)\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/vaccines10020251\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eHoffmann, M. \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e181\u003c/strong\u003e, 271\u0026ndash;280 e278 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2020.02.052\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eFolegatti, P. M. \u003cem\u003eet al.\u003c/em\u003e Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e396\u003c/strong\u003e, 467\u0026ndash;478 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(20)31604-4\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eOliver, S. E. \u003cem\u003eet al.\u003c/em\u003e The Advisory Committee on Immunization Practices\u0026apos; Interim Recommendation for Use of Pfizer-BioNTech COVID-19 Vaccine - United States, December 2020. \u003cem\u003eMMWR Morb Mortal Wkly Rep\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 1922\u0026ndash;1924 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15585/mmwr.mm6950e2\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eOliver, S. E. \u003cem\u003eet al.\u003c/em\u003e The Advisory Committee on Immunization Practices\u0026apos; Interim Recommendation for Use of Moderna COVID-19 Vaccine - United States, December 2020. \u003cem\u003eMMWR Morb Mortal Wkly Rep\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 1653\u0026ndash;1656 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15585/mmwr.mm695152e1\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eWang, W., Huang, B., Zhu, Y., Tan, W. \u0026amp; Zhu, M. Ferritin nanoparticle-based SARS-CoV-2 RBD vaccine induces a persistent antibody response and long-term memory in mice. \u003cem\u003eCell Mol Immunol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 749\u0026ndash;751 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41423-021-00643-6\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eJoyce, M. G. \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 110143 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2021.110143\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eJoyce, M. G. \u003cem\u003eet al.\u003c/em\u003e A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates. \u003cem\u003eSci Transl Med\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, eabi5735 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/scitranslmed.abi5735\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eCohen, A. A. \u003cem\u003eet al.\u003c/em\u003e Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e371\u003c/strong\u003e, 735\u0026ndash;741 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.abf6840\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eZhu, J. \u003cem\u003eet al.\u003c/em\u003e A universal bacteriophage T4 nanoparticle platform to design multiplex SARS-CoV-2 vaccine candidates by CRISPR engineering. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, eabh1547 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/sciadv.abh1547\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eYuen, C. K. \u003cem\u003eet al.\u003c/em\u003e An interferon-integrated mucosal vaccine provides pan-sarbecovirus protection in small animal models. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 6762 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-023-42349-5\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eHutchinson, G. B. \u003cem\u003eet al.\u003c/em\u003e Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 6195 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-023-41661-4\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eLopez-Sagaseta, J., Malito, E., Rappuoli, R. \u0026amp; Bottomley, M. J. Self-assembling protein nanoparticles in the design of vaccines. \u003cem\u003eComput Struct Biotechnol J\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 58\u0026ndash;68 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.csbj.2015.11.001\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eWahome, N. \u003cem\u003eet al.\u003c/em\u003e Conformation-specific display of 4E10 and 2F5 epitopes on self-assembling protein nanoparticles as a potential HIV vaccine. \u003cem\u003eChem Biol Drug Des\u003c/em\u003e \u003cstrong\u003e80\u003c/strong\u003e, 349\u0026ndash;357 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1747-0285.2012.01423.x\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eGarg, R. \u003cem\u003eet al.\u003c/em\u003e Efficacy of a stable broadly protective subunit vaccine platform against SARS-CoV-2 variants of concern. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 125980 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vaccine.2024.05.028\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eZech, F. \u003cem\u003eet al.\u003c/em\u003e Spike residue 403 affects binding of coronavirus spikes to human ACE2. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 6855 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-021-27180-0\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMinh, B. Q., Nguyen, M. A. \u0026amp; von Haeseler, A. Ultrafast approximation for phylogenetic bootstrap. \u003cem\u003eMol Biol Evol\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1188\u0026ndash;1195 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/mst024\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eKalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. \u0026amp; Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 587\u0026ndash;589 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.4285\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eHalfmann, P. J. \u003cem\u003eet al.\u003c/em\u003e Broad protection against clade 1 sarbecoviruses after a single immunization with cocktail spike-protein-nanoparticle vaccine. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1284 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-45495-6\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eTao, Y. \u0026amp; Tong, S. Complete Genome Sequence of a Severe Acute Respiratory Syndrome-Related Coronavirus from Kenyan Bats. \u003cem\u003eMicrobiol Resour Announc\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MRA.00548-19\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eWang, J. \u003cem\u003eet al.\u003c/em\u003e SARS-related coronavirus S-protein structures reveal synergistic RBM interactions underpinning high-affinity human ACE2 binding. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, eadr8772 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/sciadv.adr8772\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eRoelle, S. M., Shukla, N., Pham, A. T., Bruchez, A. M. \u0026amp; Matreyek, K. A. Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains. \u003cem\u003ePLoS Biol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, e3001738 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pbio.3001738\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eCohen, A. A. \u003cem\u003eet al.\u003c/em\u003e Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e377\u003c/strong\u003e, eabq0839 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.abq0839\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eLeekha, A. \u003cem\u003eet al.\u003c/em\u003e Multi-antigen intranasal vaccine protects against challenge with sarbecoviruses and prevents transmission in hamsters. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 6193 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-50133-2\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eHajnik, R. L. \u003cem\u003eet al.\u003c/em\u003e Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. \u003cem\u003eSci Transl Med\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, eabq1945 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/scitranslmed.abq1945\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eGouglas, D., Christodoulou, M., Plotkin, S. A. \u0026amp; Hatchett, R. CEPI: Driving Progress Toward Epidemic Preparedness and Response. \u003cem\u003eEpidemiol Rev\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 28\u0026ndash;33 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/epirev/mxz012\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eWang, G. \u003cem\u003eet al.\u003c/em\u003e Universal subunit vaccine protects against multiple SARS-CoV-2 variants and SARS-CoV. \u003cem\u003eNPJ Vaccines\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 133 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41541-024-00922-z\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMartinez, D. R. \u003cem\u003eet al.\u003c/em\u003e Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice. \u003cem\u003eScience\u003c/em\u003e (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.abi4506\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eUeda, G. \u003cem\u003eet al.\u003c/em\u003e Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/eLife.57659\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eFrancis, M. E. \u003cem\u003eet al.\u003c/em\u003e Historical H1N1 influenza virus imprinting increases vaccine protection by influencing the activity and sustained production of antibodies elicited at vaccination in ferrets. \u003cem\u003eVaccines\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e (2019).\u003c/li\u003e\n \u003cli\u003eFrancis, T. J. On the Doctrine of Original Antigenic Sin. \u003cem\u003eProceedings of the American Philosophical Society\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 572\u0026ndash;578 (1960).\u003c/li\u003e\n \u003cli\u003eFrancis, M. E. \u003cem\u003eet al.\u003c/em\u003e Previous infection with seasonal coronaviruses does not protect male Syrian hamsters from challenge with SARS-CoV-2. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 5990 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-023-41761-1\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eCohen, A. A. \u003cem\u003eet al.\u003c/em\u003e Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e187\u003c/strong\u003e, 5554\u0026ndash;5571 e5519 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2024.07.052\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eGouy, M., Tannier, E., Comte, N. \u0026amp; Parsons, D. P. Seaview Version 5: A Multiplatform Software for Multiple Sequence Alignment, Molecular Phylogenetic Analyses, and Tree Reconciliation. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e2231\u003c/strong\u003e, 241\u0026ndash;260 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-0716-1036-7_15\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMinh, B. Q. \u003cem\u003eet al.\u003c/em\u003e IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. \u003cem\u003eMol Biol Evol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 1530\u0026ndash;1534 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msaa015\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eAnthis, N. J. \u0026amp; Clore, G. M. Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. \u003cem\u003eProtein Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 851\u0026ndash;858 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pro.2253\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMarchand-Senecal, X. \u003cem\u003eet al.\u003c/em\u003e Diagnosis and Management of First Case of COVID-19 in Canada: Lessons Applied From SARS-CoV-1. \u003cem\u003eClin Infect Dis\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 2207\u0026ndash;2210 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/cid/ciaa227\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eFrancis, M. E. \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 infection in the Syrian hamster model causes inflammation as well as type I interferon dysregulation in both respiratory and non-respiratory tissues including the heart and kidney. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e1009705 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.ppat.1009705\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eImai, M. \u003cem\u003eet al.\u003c/em\u003e Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 16587\u0026ndash;16595 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.2009799117\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8544714/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8544714/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBroadly protective vaccines are needed to prevent health emergencies caused by emerging and circulating coronaviruses. Previously emerging viruses of the sarbecovirus subgenus (SARS-CoV (Severe Acute Respiratory Syndrome coronavirus) and SARS-CoV-2) have caused significant impacts on human and animal health and are anticipated to spillover again. We developed novel pan-sarbecovirus vaccines using next-generation technology producing plug-and-play fusion proteins. The scaffold of the platform links an N-terminal S1 domain with receptor binding domains (RBDs) from various targeted sarbecoviruses and is easily and quickly biomanufactured to be responsive to health emergencies. The design of our vaccines was guided by epitope prediction and molecular dynamic simulations. \u003cem\u003eIn vivo\u003c/em\u003e immunization studies showed the antigens induce broadly reactive T and B cell responses against several clades of sarbecoviruses including broadly neutralizing antibodies against pre-emergent sarbecoviruses using WIV16, PangGX, and PangGD pseudotyped viruses. Immunized Syrian hamsters were protected from mortality following heterologous challenge with SARS-CoV-2 Delta (Clade 1b) or SARS-CoV (Tor2) (Clade 1a). Significant reductions in tissue viral titer and lung pathology were observed in immunized animals compared to control groups (Pfizer BA4/5 vaccine and PBS). Taken together, our pan-sarbecovirus vaccines are protective against diverse sarbecoviruses and the platform is a strategy for addressing health emergencies.\u003c/p\u003e","manuscriptTitle":"Fusion protein pan-sarbecovirus vaccines elicit broadly protective immune responses targeting Clade 1a, 1b, and 3 sarbecoviruses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 15:05:13","doi":"10.21203/rs.3.rs-8544714/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c664a59f-7014-49eb-a95e-68cc2ea9903b","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61819929,"name":"Biological sciences/Microbiology/Vaccines/Protein vaccines"},{"id":61819930,"name":"Biological sciences/Microbiology/Virology/SARS virus"}],"tags":[],"updatedAt":"2026-03-20T19:30:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 15:05:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8544714","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8544714","identity":"rs-8544714","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}