Reduced Cross-Protective Potential of Omicron Compared to Ancestral SARS-CoV-2 Spike Vaccines Against Potentially Zoonotic Coronaviruses

Reduced Cross-Protective Potential of Omicron Compared to Ancestral SARS-CoV-2 Spike Vaccines Against Potentially Zoonotic Coronaviruses | 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 Reduced Cross-Protective Potential of Omicron Compared to Ancestral SARS-CoV-2 Spike Vaccines Against Potentially Zoonotic Coronaviruses Tyler Renner, Matthew Stuible, Brian Cass, Sylvie Perret, Julie Guimond, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4791122/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2024 Read the published version in npj Viruses → Version 1 posted 11 You are reading this latest preprint version Abstract The COVID-19 pandemic has emphasised the importance of vaccines and preparedness against viral threats crossing species barriers. In response, a worldwide vaccination campaign targeting SARS-CoV-2 was implemented, which provides some cross-protective immunological memory to other coronavirus species with zoonotic potential. Following a vaccination regimen against SARS-CoV-2 spike in a preclinical mouse model, we were able to demonstrate the induction of neutralizing antibodies towards multiple human ACE2 (hACE2)-binding sarbecovirus spikes. Importantly, compared to vaccines based on the SARS-CoV-2 Reference strain, vaccines based on Omicron spike sequences induced drastically less broadly cross-protective neutralizing antibodies against other hACE2-binding sarbecoviruses. This observation remained true whether the vaccination regimens were based on protein subunit or mRNA / LNP vaccines. Overall, while it may be necessary to update vaccine antigens to combat the evolving SARS-CoV-2 virus for enhanced protection from COVID-19, Reference-based vaccines may be a more valuable tool to protect against novel coronavirus zoonoses. Biological sciences/Immunology/Vaccines/Protein vaccines Biological sciences/Immunology/Vaccines Biological sciences/Immunology/Vaccines/Rna vaccines Biological sciences/Immunology Biological sciences/Immunology/Adaptive immunity Biological sciences/Immunology/Adaptive immunity/Humoral immunity Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Antibodies Biological sciences/Microbiology Biological sciences/Microbiology/Virology Biological sciences/Microbiology/Virology/Sars cov 2 Biological sciences/Microbiology/Virology/Sars virus Figures Figure 1 Figure 2 Figure 3 Introduction Historically, a multitude of viruses from different families have exhibited zoonotic potential (e.g., Ebola, influenza, HIV, etc.), however within the last couple of decades, a number of coronaviruses (i.e., SARS-CoV-1, MERS, SARS-CoV-2) have emerged as a particularly significant global threat. While the limited human-to-human spread of SARS-CoV-1 and MERS-CoV restricted their associated outbreaks to epidemic status, the enhanced transmissibility of SARS-CoV-2 played an important role in enabling this virus to expose humanity to a global pandemic, before more recently transitioning to an endemic state 1 , 2 . The magnitude of damage caused by the COVID-19 pandemic cannot be overstated, directly causing an estimated 7 million deaths to date 3 . Widespread vaccination efforts were able to blunt the impact of the pandemic, due in part to the accelerated production timelines and emergency approval of mRNA- and viral vector-based vaccines, saving millions of lives and reducing health care costs 4 , 5 . More recently, several alternative vaccine approaches have been developed, with protein subunit vaccines gaining prominence with approvals of Vidprevtyn Beta® (Sanofi) and Nuvaxovid® (Novavax) by the FDA and/or EMA. The efficacy of these various vaccine platforms is tied to their ability to induce neutralizing antibodies against the spike protein, with the chosen protein sequence used (i.e., Reference vs. variant strain) impacting their ability to neutralize infection by a specific variant. With the ongoing evolution of SARS-CoV-2 and emergence of novel variants of concern, the antigen sequence within commercial vaccine products continues to be updated according to the recommendations of the World Health Organization and regulatory agencies. The use of modernized antigens should improve outcomes against COVID-19 infection from the latest circulating strains. Given the high probability of further zoonosis events from the coronavirus family, our group sought to investigate the sarbecovirus cross-neutralizing potential of antibodies induced by protein subunit or mRNA / LNP vaccines based on SARS-CoV-2 Reference, Beta, Delta, Omicron (BA.1) and Omicron (BA.4/BA.5) spike sequences. Results We focused on sarbecovirus spikes that bind human ACE2 (hACE2), as the ability of coronaviruses to bind this receptor has been linked to the risk of efficient zoonotic transmission to humans 6 . Also, existing surrogate neutralization assays for SARS-CoV-2 spike can be directly applied to other hACE2-binding spike proteins. Therefore, a subset of hACE2-binding spikes, namely BANAL-20-52, Pangolin-GX, Pangolin-GD, SHC014, Bat-WIV1, and Bat-SARSL sarbecoviruses, were selected for analysis. To illustrate the homology between these spikes and their receptor binding domains (RBD), the amino acid sequences were aligned using Clustal Omega 7 and the Percent Identity Matrix was plotted in a Heatmap (Fig. 1 A). There is a clear divergence of SARS-CoV-1 from SARS-CoV-2, with the sarbecovirus spikes chosen for the current study separating into two groups, being more similar to either of these two SARS viruses. The phylogenetic trees (Fig. 1 B) highlight the evolutionary relationship among the tested viral spikes and their receptor binding domains (RBD): based on sequence identity, variants of SARS-CoV-2, such as BA.1, have become more dissimilar from the Reference SARS-CoV-2 spike than some animal-origin viral spikes, such as BANAL-20-52 (97.4% vs 98.7% identity, respectively). These differences are more noticeable in rapidly evolving regions, such as the RBD (93.3% vs 97.3% identity, respectively). We produced the sarbecovirus spikes using our CHO-expression platform and trimeric antigen design developed previously for the SARS-CoV-2 spike (SmT1) 8 ; purity was excellent (> 95%) for all constructs with SDS-PAGE/Coomassie staining showing the presence of a single major species at ~ 170 kDa with low levels of high-molecular-weight bands (Fig. 1 C). We confirmed the ability of these purified spike antigens to bind to HEK293T-hACE2 cells (Fig. 1 D), allowing for the assessment of serum cross-neutralization capacity using a flow cytometry-based surrogate neutralization assay established previously 9 – 11 . Next, using mouse serum generated in previous preclinical studies 10 , 11 , we evaluated the cross-protective ability of antibodies induced by SARS-CoV-2 spike protein subunit vaccination using equivalent antigen doses (3 µg) of Reference, Beta, Delta, a Trivalent (1 µg each of Reference + Beta + Delta) or Omicron (BA.1) in AddaS03-adjuvanted formulations. Notably, while Omicron (BA.1) has over 30 mutations in the spike protein compared to Reference, the Beta and Delta variants differ by fewer than 10 amino acids. The immunogenicity of each of these vaccines against SARS-CoV-2 Reference strain and the aforementioned variants has previously been confirmed 10 , 11 . Using a cell-based spike-hACE2 binding assay, which has been previously shown to correlate strongly with the SARS-CoV-2 neutralization activity as determined with other widely-used assays (e.g., PRNT, pseudolentiviral neutralization and in vivo viral challenge 9 – 11 ), the neutralization activities of these serum samples against the selected spikes were measured (Fig. 2 ). Consistent with our previously published data, neutralization activity for a given spike was highest with serum from animals immunized with the matching antigen: Reference and Omicron (BA.1) SARS-CoV-2 were most neutralized by serum from mice vaccinated with Reference (81.3%) and Omicron (BA.1) spike (56.6%), respectively 10 , 11 . The inverse was also true, whereby Reference or Omicron (BA.1) vaccinated mice had the lowest Omicron (BA.1) (22.6%) or Reference (21.6%) neutralizing titer, respectively. Following this trend, the neutralizability of each spike is linked to their respective relationship to SARS-CoV-2. This is highlighted with BANAL-20-52 and Pangolin-GD, which have a nearly identical neutralization profile to Reference SARS-CoV-2. This correlates strongly with the observed homology within the RBD and not the entire spike protein sequence, as both BANAL-20-52 and Pangolin-GD have ~ 97% identity to Reference SARS-CoV-2 in this region, while Pangolin-GD has lower (91.2%) homology to the entire Reference spike. Interestingly, serum from the Omicron (BA.1) spike-immunized mice showed much lower neutralization against a number of sarbecoviruses than seen following immunization with Reference and sometimes Beta or Delta spike, which often induced an intermediate neutralizing profile. For example, ~ 10% neutralization of binding of BANAL-20-52 spike to hACE2 with following immunization with the Omicron (BA.1) vs. >90% when the immunization antigen is Reference Spike. Significant differences in the neutralization activity between the sera of Reference and Omicron-immunized mice were also seen against the spike protein based on other SARS-CoV-2-Like viruses (Pangolin-GX & Pangolin-GD) as well as SARS-CoV-1 and the SARS-CoV-1-Like virus, Bat WIV. Despite all of these proteins binding the same receptor (hACE2), there was much lower neutralizing activity detected against sarbecovirus spikes that are closely related to SARS-CoV-1. The highest neutralization was observed with Bat-WIV1 spike, with neutralization activity of ~ 30% seen with serum from mice vaccinated with Reference, Beta or Trivalent SARS-CoV-2 spike, and only ~ 10% with Omicron (BA.1), at the dilution tested. To confirm the impact of the vaccine platform on these observations, we next evaluated the neutralizing potential of antibodies invoked by vaccination with mRNA / lipid nanoparticles (mRNA / LNPs) vaccine formulations encoding different SARS-CoV-2 spikes. We generated DNA templates encoding SARS-CoV-2 Reference and Omicron (BA.4/BA.5) spike proteins, with sequences based on the design used by Pfizer-BioNTech 12 . The mRNA was transcribed in vitro and includes N1-methyl-pseudouridine in place of the canonical uridine, as is typical for the clinically approved mRNA / LNP vaccines 12 , 13 . After encapsulation into commercially available lipids, Balb/c mice (n = 10) were vaccinated with 1µg of mRNA / LNPs on Day 0 and Day 21. As before, the serum from these animals was assessed 7 days post-boost for sarbecovirus spike neutralizing potential (Fig. 3 ). As expected, the binding of Reference and Omicron (BA.4/BA.5) spike proteins were most strongly neutralized by serum from mice vaccinated with mRNA / LNPs encoding Reference (61.8%) and Omicron (BA.4/BA.5) spike (71.1%), respectively 10 , 11 , while the bivalent vaccine induced a more intermediate neutralizing profile. Interestingly, despite containing equivalent doses of each spike mRNA, the Bivalent vaccine induced a more potent neutralizing titer towards the BA.4/BA.5 (56.3%) than the Reference (27.9%) spike. The serum from mice vaccinated with Reference, Omicron (BA.4/BA.5) and Bivalent spike all similarly neutralized (35.8% – 47.4%) the hACE2-binding of Omicron (BA.1) spike, which has a similar degree of homology to the Reference and Omicron (BA.4/BA.5) spike proteins. As seen with the protein subunit vaccines in Fig. 2 , the reference-based mRNA/LNP vaccines induced significantly higher neutralization profiles of BANAL-20-52 and Pangolin-GD than vaccines designed to target Omicron (BA4/BA5). Meanwhile, no significant differences were observed in the neutralization profile of the other spike proteins, including those which had shown less pronounced, but still significant, differences with the protein vaccine formulations (i.e., Pangolin-GX, Bat WIV1 and SARS-CoV-1. Altogether, these data highlight the superior ability of SARS-CoV-2-based vaccine formulations based on the Reference sequence to generate neutralizing antibodies to certain animal-derived sarbecoviruses, whether using protein subunit or mRNA / LNP vaccine platforms. Discussion The COVID-19 pandemic, as well as the relatively recent MERS and SARS outbreaks, have demonstrated the devastating impacts of coronaviral zoonosis and the importance of preparedness to mitigate the impact of similar occurrences in the future. We sought to better understand whether SARS-CoV-2-based vaccines could potentially protect against future coronaviral zoonotic events. Previous studies have demonstrated that SARS-CoV-1/2 infection and/or vaccination can induce potent neutralizing antibodies with cross-neutralizing potential to certain sarbecoviruses found currently in various animal species 14 – 20 . Our results confirm that vaccination with formulations based on the ancestral spike of SARS-CoV-2 induces a potent neutralizing response against several hACE2-binding sarbecovirus spikes (e.g., BANAL-20-52, Pangolin-GD, Pangolin-GX, Bat WIV1) that can be superior to those seen with formulations based on variants of concern, in particular Omicron (Figs. 2 & 3 ). To our knowledge, no other studies have gone further to evaluate the potential impact of updating the SARS-CoV-2 spike sequence to match currently circulating variants in the human population on the ability of COVID-19 vaccines to induce protection against potentially zoonotic sarbecoviruses. This can be more complex to accurately measure in human populations due to the heterologous and multifaceted nature of the immune responses generated through previous rounds of infection and/or immunization. In the current study, we used our animal models to compare the cross-protective potential (specifically in terms of induction of sarbecovirus-neutralizing antibodies) of mono- and multi-valent vaccines based on SARS-CoV-2 Reference-strain spike as well as Beta, Delta and Omicron variants. While all formulations generated similarly weak neutralizing responses to certain spike proteins, namely those more closely related to SARS-CoV-1, they had differing abilities to induce cross-reactive neutralizing antibodies to certain viral strains. When immunized with protein subunit antigens based on Reference, Beta, Delta or Omicron (BA.1), the least potent cross-neutralizing responses to BANAL-20-52 or Pangolin-GD were observed in mice receiving Omicron (BA.1)-based vaccines (Fig. 2 ). Similarly poor cross-neutralizing responses were observed to these spike proteins when mice received mRNA/LNPs based on the Omicron (BA.4/BA.5) sequence (Fig. 3 ), confirming that this effect is dependent upon the antigen sequence and not the platform of vaccination. This may be indicative of “humanization” of the SARS-CoV-2 spike in its more recent iterations (i.e., Omicron) as it has evolved through countless replication cycles in hundreds of millions of infected people. Variants such as Omicron have incorporated critical mutations in their ACE2-binding (and potentially other) motifs to better mediate infection in their new host, simultaneously causing them to diverge away from some of the animal-derived sarbecoviruses tested here, as well as the original SARS-CoV-2 virus that is presumed to have been zoonotically transmitted in 2019. We also found that the tested vaccine candidates induced a lower degree of cross-neutralization against the spike protein of SARS-CoV-1 or its close relatives than seen against the SARS-CoV-2-Like sarbecoviruses. This is in contrast to some previous reports that illustrate effective cross-neutralization of SARS-CoV-1-like and SARS-CoV-2-like sarbecoviruses after an initial exposure to SARS-CoV-1 19,21 . This suggests that vaccination with a more closely related sarbecovirus antigen, such as Bat WIV1, may be required to effectively neutralize this subgroup of coronavirus spikes. While the overall level of neutralization was lower in the case of the SARS-CoV-1-like sarbecoviruses, for protein antigens, the Reference-based vaccine formulation still induced superior neutralization compared to the Omicron (BA.1)-based antigen against the spike protein from Bat WIV1 and SARS-CoV-1 (Fig. 2 ). However, for the mRNA-LNP vaccines, no obvious differences were observed between the Reference and Omicron mRNA / LNPs in terms of induction of SARS-CoV-1-related spike neutralization (Fig. 3 ). Whether this is a feature of vaccination platform (protein vs. mRNA), antigen sequence (BA.1 vs. BA4/BA5) and/or mouse strain (C57BL/6 vs. Balb/c) should be confirmed in future studies. SARS-CoV-2 Reference-based vaccines have continuously proved to be effective boosters even in the context of emerging variants 22 , 23 . Nonetheless, it is increasingly evident that updating of SARS-CoV-2 vaccine antigen sequences to resemble dominant, circulating variants is necessary to achieve optimal protection 24 – 27 . Importantly, our results indicate that these modernized vaccines may be less capable of inducing protective humoral responses towards other sarbecovirus spikes which have the potential to transmit zoonotically. Furthermore, our data in Fig. 3 illustrates that the use of bivalent vaccine strategies may not equally induce neutralizing antibodies to both antigens, as Omicron (BA.4/BA.5) appears to be immunodominant. This immunodominance over the Reference spike has also been observed previously even when limiting the vaccine to the RBD region 28 . As highlighted with COVID-19, the swift timing of vaccine deployment is critical to minimize the global impacts at the onset of any pandemic 4 , 5 . With its cross-neutralizing potential against closely related sarbecoviruses, the ability to quickly deploy a stockpile of Reference SARS-CoV-2 vaccines may prove effective in the event of another outbreak caused by a zoonotic transmission event involving an ACE2-binding coronavirus. The same may be true for other coronaviruses, such as SARS-CoV-1 and MERS-CoV families. Capabilities should be established to quickly develop/manufacture and/or stockpile vaccines based on one (or a few) viruses in their respective families, as they may prove to be effective during an emergency response to a future outbreak. Indeed, with the current immunological status of the overall human population following infection by/vaccination against COVID-19, viruses from other subgroups may now have a higher likelihood of zoonoses than sarbecoviruses closely related to SARS-CoV-2. In a future pandemic, previously developed vaccines could provide some level of protection during the time delay required for viral identification, de novo synthesis of antigen sequences and regulatory approval of novel vaccines. Materials and Methods Antigen Production and Homology CHO codon-optimized sequences encoding spike ectodomains of animal-origin sarbecoviruses (with prefusion-stabilizing 2P mutations) fused to human resistin and purification tags (FLAG-dual-Strep-6His or dual-Strep-6His) at the C-terminus were synthesized at Genscript and cloned into the pTT241® plasmid 29 . These stabilizing prolines are in well-conserved regions for all the selected spikes and were positioned by alignment with the SARS-CoV-2 spike sequence. To mediate trimerization, human resistin was fused to the C-terminus followed by purification tags. Stably transfected pools were generated using CHO 2353 ™ cells and protein productions were performed with expression induced with cumate as described 29 . For the different sarbecovirus spikes, protein expression using stably transfected CHO cell pools generated quite variable production yields ranging from 0.1-2 g/L. In comparison, the corresponding SARS-CoV-2 reference-strain construct yields ~ 0.7 g/L under the same conditions 29 . Clarified CHO culture supernatants harvested at 7 or 10 days post-induction were purified as described by IMAC followed by Strep-affinity chromatography 8 . Purified products were buffer-exchanged using Centripure 100 desalting columns (emp BIOTECH, Berlin, Germany) into DPBS, pH 7.8, or into HEPES (50 mM, pH 7.8) buffer containing 100 mM NaCl. Purified proteins were analyzed by SDS-PAGE using NuPAGE 4–12% Bis-Tris gels (ThermoFisher, Massachusetts, USA) followed by Coomassie Blue staining. The absence of endotoxin contamination was verified using Endosafe cartridge-based Limulus amebocyte lysate tests (Charles River Laboratories, Charleston, SC, USA). Clustal Omega 7 was used to generate the Percent Identity Matrix and Phylograms to depict the relationship of the spike sequences used in this study. To better relate identity to immunological data, the antigen sequences (which are limited to the spike ectodomains) were used for the homology analyses. Preparation of mRNA/LNPs The plasmid DNA templates used to generate the tested mRNAs were designed based on the publicly available Pfizer-BioNTech mRNA/LNP vaccine sequence 12 and synthesized by Genscript (Piscataway, NJ, USA) in a pUC57 backbone. DNA templates were linearized with XbaI and purified by phenol–chloroform extraction. RNA was generated using Megascript T7 transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) and capped using CleanCap Reagent AG (TriLink BioTechnologies, San Diego, CA, USA) to generate RNA with stabilized Cap 1 structure. In place of uridine, m1ψ (TriLink BioTechnologies) was included to generate the mRNAs with modified nucleotide chemistry. Following IVT of RNA, plasmid DNA was digested using Turbo DNase (Invitrogen, Waltham, MA, USA) as per manufacturer’s instructions. RNA was purified by lithium chloride precipitation, washed with 70% ethanol and resuspended in RNase-free water. To assess RNA size and integrity, all mRNA samples were resolved on formaldehyde (2.6%, v/v) denaturing agarose (1%, w/v) gel. The concentrations of mRNA samples were measured using Invitrogen’s RiboGreen assay reagent (Thermo Fisher Scientific), as per manufacturer’s recommendations. IVT mRNA encoding SARS-CoV-2 reference or BA.4/BA.5 spike was encapsulated within Genvoy-ILM ionizable lipid mixture (Precision NanoSystems, Vancouver, BC, Canada) at an N:P ratio of 8. Formation of LNPs was achieved by microfluidic mixing of RNA (aqueous) and ionizable lipids (organic) using the NanoAssemblr Ignite system (Precision NanoSystems) according to manufacturer’s recommendations with a flow rate ratio of 3 (aqueous:organic) and total flow rate of 12 (ml/min). Formulations were then diluted in Mg2+/Ca2+-free PBS, before undergoing buffer exchange and concentration using Amicon Ultra Centrifugal Filters (Millipore Sigma, St. Louis, MO, USA). Encapsulation efficiency of RNA was determined by RiboGreen assay (Thermo Fisher Scientific) as per instructions accompanying the Genvoy-ILM ionizable lipid and found to be > 90%. In addition, particle size and polydispersity index (PDI) of the LNPs were verified using a Zetasizer NanoZS (Malvern Instruments, Malvern, UK) and found to be 122–136nm and 0.10–0.12, respectively. Immunizations and Sample Collection Female C57BL/6 or Balb/c mice (6–8 weeks old) were obtained from Charles River Laboratories (Saint-Constant, QC, Canada) and maintained at the small animal facility of the NRC Canada in accordance with the guidelines of the Canadian Council of Animal Care. Protein subunit vaccinations (Fig. 2 ) were described in previously published studies 10 , 11 . Briefly, C57BL/6 mice (n = 9–10 per group) were immunized by i.m. injection (50 µl) into the left tibialis anterior muscle on Days 0 and 21 with 3 µg SARS-CoV-2 spike SmT1 antigen (reference, Beta, Delta, a 1:1:1 ratio of each reference, Beta and Delta, or Omicron (BA.1)) diluted in PBS (Spectrum, Gardena, CA, USA) and mixed with AddaS03 (Invivogen, San Diego, CA, USA) according to manufacturer’s instructions. Vaccinations of Balb/c mice (n = 10 per group) with mRNA/LNPs (Fig. 3 ) were performed in the same fashion as the protein subunit vaccines except normalized to a 1 µg dose of encapsulated mRNA encoding SARS-CoV-2 spike (reference, Omicron (BA.4/BA.5) or a 1:1 ratio of each). Mice were bled via the submandibular vein on day 28 with recovered serum used for quantification neutralization activity. Samples were simultaneously collected from 10 naïve animals for the assessment of background immune responses. Each of the samples from the individual mice was tested separately in the various readouts. Cell-based Spike-hACE2 Binding Assay Serum was assessed for its ability to neutralize the binding of labeled spike trimers to hACE2-expressing cells. The following reagent was obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK-293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK-293T-hACE2 Cell Line, NR-52511. This assay was performed as previously described, using HEK-293T-hACE2 cells 11 , 30 . Purified spike was biotinylated with EZ-Link™ Sulfo-NHS-LC-LC-Biotin (ThermoFisher, Massachusetts, USA) and purified via molecular weight cut off (MWCO) columns according to manufacturer’s instructions. Mouse serum was diluted 1 in 250, mixed with 250 ng of biotinylated spike and 1 × 10 5 HEK-293T-hACE2 cells. The amount of bound spike was quantified using Streptavidin-R-PE (ThermoFisher, Massachusetts, USA), conjugate by acquiring cells on an LSR Fortessa (Becton Dickinson, New Jersey, USA) and analyzing data on FlowJo (Becton Dickinson). For illustration/analysis purposes, samples with calculated values ≤ 0 were assigned a value of 0. Statistical analysis Data were analyzed using GraphPad Prism version 10 (GraphPad, Massachusetts, USA). Statistical significance of the difference between groups was calculated by one-way ANOVA followed by Dunnett’s multiple comparisons test. Differences were considered to be not significant with p > 0.05. Significance was indicated in the graphs as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Declarations Institutional review board statement Mice were maintained at the small animal facility of the National Research Council (NRC) Canada in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were approved by our Institutional Review Board (NRC Human Health Therapeutics Animal Care Committee) and covered under animal use protocols 2020.10. All experiments were carried out in accordance with the ARRIVE guidelines. Data availability statement The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s. Acknowledgments The authors would like to acknowledge John Shelvey and Perry Fleming for producing the archaeal biomass. We also thank the technical contribution of many members of the Mammalian Cell Expression Section of the NRC-HHT and all of the staff of the Human Health Therapeutics Animal Resource Group. This work was supported in part by the NRC’s Pandemic Response Challenge Program and by a CQDM partnership with Oragenics Inc., Inspirevax Inc., and the NRC. The following reagents were obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK-293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK-293T-hACE2 Cell Line, NR-52511. Author Contributions BA, TMR, MS, YD and MJM conceived and/or designed the studies. MS, BC, SP, JG, SL-D and YD designed, produced and/or purified the spike proteins used in this study. TMR conducted the homology, phylogeny, vaccinations and neutralization analyses. TMR and BA took the lead in writing the manuscript. All authors contributed to the article and approved the submitted version. Conflict of interest The authors declare no competing non-financial interests but the following competing financial interests: BA and MJM are inventors on an SLA archaeosome-related patent application. YD is an inventor of a patent application related to the SmT1 antigen (publication number 20230174591). 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Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature 602, 657–663 (2022). Yisimayi, A. et al. Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting. Nature 625, 148–156 (2024). Uraki, R. et al. An mRNA vaccine encoding the SARS-CoV-2 receptor-binding domain protects mice from various Omicron variants. Npj Vaccines 9, 1–10 (2024). Joubert, S. et al. A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens. Biotechnol. Bioeng. 120, 1746–1761 (2023). Akache, B. et al. Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant. Sci. Rep. 11, 21849 (2021). Additional Declarations Competing interest reported. The authors declare no competing non-financial interests but the following competing financial interests: BA and MJM are inventors on an SLA archaeosome-related patent application. YD is an inventor of a patent application related to the SmT1 antigen (publication number 20230174591). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2024 Read the published version in npj Viruses → Version 1 posted Editorial decision: Revision requested 22 Aug, 2024 Reviews received at journal 19 Aug, 2024 Reviews received at journal 19 Aug, 2024 Reviews received at journal 13 Aug, 2024 Reviewers agreed at journal 13 Aug, 2024 Reviewers agreed at journal 13 Aug, 2024 Reviewers agreed at journal 11 Aug, 2024 Reviewers invited by journal 11 Aug, 2024 Editor assigned by journal 01 Aug, 2024 Submission checks completed at journal 01 Aug, 2024 First submitted to journal 23 Jul, 2024 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. 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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-4791122","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":343713538,"identity":"bd15f720-e043-432f-a7c9-443c34a943db","order_by":0,"name":"Tyler Renner","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Tyler","middleName":"","lastName":"Renner","suffix":""},{"id":343713539,"identity":"26f2ebd1-3b4c-40d0-bc18-1de6973ef4c2","order_by":1,"name":"Matthew Stuible","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Stuible","suffix":""},{"id":343713540,"identity":"a4855c3b-73f6-4876-b13e-b0dd1f5aed3b","order_by":2,"name":"Brian Cass","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"","lastName":"Cass","suffix":""},{"id":343713541,"identity":"77b21f5b-99da-458d-872c-c1d3a2afb990","order_by":3,"name":"Sylvie Perret","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Sylvie","middleName":"","lastName":"Perret","suffix":""},{"id":343713542,"identity":"262cb660-69a5-4d04-b64b-5ba070cd82e1","order_by":4,"name":"Julie Guimond","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Guimond","suffix":""},{"id":343713543,"identity":"9c595c43-1b61-4e36-965f-4f763bd34dfa","order_by":5,"name":"Simon Lord-Dufour","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Lord-Dufour","suffix":""},{"id":343713544,"identity":"2853bd31-45ff-402a-8a21-80f9362505bb","order_by":6,"name":"Michael J. McCluskie","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"J.","lastName":"McCluskie","suffix":""},{"id":343713545,"identity":"b2b2bde3-dfc9-4c96-be34-f517c2888523","order_by":7,"name":"Yves Durocher","email":"","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Yves","middleName":"","lastName":"Durocher","suffix":""},{"id":343713546,"identity":"7cd4f6d4-2593-4f92-be6c-1d5a170ebb14","order_by":8,"name":"Bassel Akache","email":"data:image/png;base64,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","orcid":"","institution":"National Research Council Canada, Human Health Therapeutics","correspondingAuthor":true,"prefix":"","firstName":"Bassel","middleName":"","lastName":"Akache","suffix":""}],"badges":[],"createdAt":"2024-07-23 20:38:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4791122/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4791122/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s44298-024-00067-9","type":"published","date":"2024-11-21T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63462789,"identity":"4dae1b05-fd08-4f50-a5ab-2515f5f6530a","added_by":"auto","created_at":"2024-08-28 11:46:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157264,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of hACE2-binding sarbecovirus spike proteins. (a) Heatmap relating the % identity of sarbecovirus spikes (left) or solely based on the receptor binding domain (RBD) sequences (right). (b) Phylograms depicting the points of divergence of the same spike sequences (left) or RBD sequences (right). (c) SDS-PAGE with Coomassie blue staining on purified recombinant sarbecovirus spikes. (d) Interaction of purified spike proteins with HEK293T-hACE2 cells as visualized by flow cytometry. Omicron refers to Omicron (BA.1).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4791122/v1/04dfc8de86b12f040d0b9b9a.png"},{"id":63462792,"identity":"60bca998-df74-447e-8af0-d51a83e41e7c","added_by":"auto","created_at":"2024-08-28 11:46:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":255913,"visible":true,"origin":"","legend":"\u003cp\u003eAnimal-derived sarbecovirus spikes are better neutralized by serum from mice vaccinated with protein subunit vaccines specific to SARS-CoV-2 Reference than more divergent variants, such as Omicron (BA.1). Neutralizing activity within serum samples from C57BL/6 mice immunized with SARS-CoV-2 Reference, Beta, Delta, Trivalent (Reference-Beta-Delta), or Omicron (BA.1)-based vaccines was assessed using a surrogate cell-based hACE2 binding assay at a dilution of 1:250. Statistical significance of differences among groups vs. the Reference vaccinated mice are shown: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 and ****p\u0026lt;0.0001 by one-way ANOVA followed by Dunnett’s multiple comparison test.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4791122/v1/e6f2c9b010f93e0c8d1b6454.png"},{"id":63462788,"identity":"4dd52b5f-5261-4537-bbc1-8b846d936c20","added_by":"auto","created_at":"2024-08-28 11:46:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210065,"visible":true,"origin":"","legend":"\u003cp\u003eAnimal-derived sarbecovirus spikes are better neutralized by serum from mice vaccinated with mRNA / LNP vaccines encoding SARS-CoV-2 Reference than the divergent variant, Omicron (BA.4/BA.5). Neutralizing activity within serum samples from Balb/c mice immunized with SARS-CoV-2 Reference, Omicron (BA.4/BA.5) or Bivalent mRNA / LNP vaccines was assessed using a surrogate cell-based hACE2 binding assay at a dilution of 1:250. Statistical significance of differences among groups vs. the Reference vaccinated mice are shown: ****p\u0026lt;0.0001 by one-way ANOVA followed by Dunnett’s multiple comparison test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4791122/v1/2d152614609fe64072e2d9ee.png"},{"id":69641725,"identity":"1e62ac1d-97a4-4715-88ab-ece45bb70a26","added_by":"auto","created_at":"2024-11-22 14:14:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":921429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791122/v1/102dd53f-d116-4d81-ac28-583df681f093.pdf"}],"financialInterests":"Competing interest reported. The authors declare no competing non-financial interests but the following competing financial interests: BA and MJM are inventors on an SLA archaeosome-related patent application. YD is an inventor of a patent application related to the SmT1 antigen (publication number 20230174591).\n\nThe remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.","formattedTitle":"Reduced Cross-Protective Potential of Omicron Compared to Ancestral SARS-CoV-2 Spike Vaccines Against Potentially Zoonotic Coronaviruses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHistorically, a multitude of viruses from different families have exhibited zoonotic potential (e.g., Ebola, influenza, HIV, etc.), however within the last couple of decades, a number of coronaviruses (i.e., SARS-CoV-1, MERS, SARS-CoV-2) have emerged as a particularly significant global threat. While the limited human-to-human spread of SARS-CoV-1 and MERS-CoV restricted their associated outbreaks to epidemic status, the enhanced transmissibility of SARS-CoV-2 played an important role in enabling this virus to expose humanity to a global pandemic, before more recently transitioning to an endemic state\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The magnitude of damage caused by the COVID-19 pandemic cannot be overstated, directly causing an estimated 7\u0026nbsp;million deaths to date\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Widespread vaccination efforts were able to blunt the impact of the pandemic, due in part to the accelerated production timelines and emergency approval of mRNA- and viral vector-based vaccines, saving millions of lives and reducing health care costs\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. More recently, several alternative vaccine approaches have been developed, with protein subunit vaccines gaining prominence with approvals of Vidprevtyn Beta\u0026reg; (Sanofi) and Nuvaxovid\u0026reg; (Novavax) by the FDA and/or EMA. The efficacy of these various vaccine platforms is tied to their ability to induce neutralizing antibodies against the spike protein, with the chosen protein sequence used (i.e., Reference vs. variant strain) impacting their ability to neutralize infection by a specific variant.\u003c/p\u003e \u003cp\u003eWith the ongoing evolution of SARS-CoV-2 and emergence of novel variants of concern, the antigen sequence within commercial vaccine products continues to be updated according to the recommendations of the World Health Organization and regulatory agencies. The use of modernized antigens should improve outcomes against COVID-19 infection from the latest circulating strains. Given the high probability of further zoonosis events from the coronavirus family, our group sought to investigate the sarbecovirus cross-neutralizing potential of antibodies induced by protein subunit or mRNA / LNP vaccines based on SARS-CoV-2 Reference, Beta, Delta, Omicron (BA.1) and Omicron (BA.4/BA.5) spike sequences.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe focused on sarbecovirus spikes that bind human ACE2 (hACE2), as the ability of coronaviruses to bind this receptor has been linked to the risk of efficient zoonotic transmission to humans\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Also, existing surrogate neutralization assays for SARS-CoV-2 spike can be directly applied to other hACE2-binding spike proteins. Therefore, a subset of hACE2-binding spikes, namely BANAL-20-52, Pangolin-GX, Pangolin-GD, SHC014, Bat-WIV1, and Bat-SARSL sarbecoviruses, were selected for analysis.\u003c/p\u003e \u003cp\u003eTo illustrate the homology between these spikes and their receptor binding domains (RBD), the amino acid sequences were aligned using Clustal Omega\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and the Percent Identity Matrix was plotted in a Heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). There is a clear divergence of SARS-CoV-1 from SARS-CoV-2, with the sarbecovirus spikes chosen for the current study separating into two groups, being more similar to either of these two SARS viruses. The phylogenetic trees (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) highlight the evolutionary relationship among the tested viral spikes and their receptor binding domains (RBD): based on sequence identity, variants of SARS-CoV-2, such as BA.1, have become more dissimilar from the Reference SARS-CoV-2 spike than some animal-origin viral spikes, such as BANAL-20-52 (97.4% vs 98.7% identity, respectively). These differences are more noticeable in rapidly evolving regions, such as the RBD (93.3% vs 97.3% identity, respectively).\u003c/p\u003e \u003cp\u003eWe produced the sarbecovirus spikes using our CHO-expression platform and trimeric antigen design developed previously for the SARS-CoV-2 spike (SmT1)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e; purity was excellent (\u0026gt;\u0026thinsp;95%) for all constructs with SDS-PAGE/Coomassie staining showing the presence of a single major species at ~\u0026thinsp;170 kDa with low levels of high-molecular-weight bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We confirmed the ability of these purified spike antigens to bind to HEK293T-hACE2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), allowing for the assessment of serum cross-neutralization capacity using a flow cytometry-based surrogate neutralization assay established previously\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, using mouse serum generated in previous preclinical studies\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, we evaluated the cross-protective ability of antibodies induced by SARS-CoV-2 spike protein subunit vaccination using equivalent antigen doses (3 \u0026micro;g) of Reference, Beta, Delta, a Trivalent (1 \u0026micro;g each of Reference\u0026thinsp;+\u0026thinsp;Beta\u0026thinsp;+\u0026thinsp;Delta) or Omicron (BA.1) in AddaS03-adjuvanted formulations. Notably, while Omicron (BA.1) has over 30 mutations in the spike protein compared to Reference, the Beta and Delta variants differ by fewer than 10 amino acids. The immunogenicity of each of these vaccines against SARS-CoV-2 Reference strain and the aforementioned variants has previously been confirmed\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Using a cell-based spike-hACE2 binding assay, which has been previously shown to correlate strongly with the SARS-CoV-2 neutralization activity as determined with other widely-used assays (e.g., PRNT, pseudolentiviral neutralization and in vivo viral challenge\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e), the neutralization activities of these serum samples against the selected spikes were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consistent with our previously published data, neutralization activity for a given spike was highest with serum from animals immunized with the matching antigen: Reference and Omicron (BA.1) SARS-CoV-2 were most neutralized by serum from mice vaccinated with Reference (81.3%) and Omicron (BA.1) spike (56.6%), respectively\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The inverse was also true, whereby Reference or Omicron (BA.1) vaccinated mice had the lowest Omicron (BA.1) (22.6%) or Reference (21.6%) neutralizing titer, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing this trend, the neutralizability of each spike is linked to their respective relationship to SARS-CoV-2. This is highlighted with BANAL-20-52 and Pangolin-GD, which have a nearly identical neutralization profile to Reference SARS-CoV-2. This correlates strongly with the observed homology within the RBD and not the entire spike protein sequence, as both BANAL-20-52 and Pangolin-GD have ~\u0026thinsp;97% identity to Reference SARS-CoV-2 in this region, while Pangolin-GD has lower (91.2%) homology to the entire Reference spike. Interestingly, serum from the Omicron (BA.1) spike-immunized mice showed much lower neutralization against a number of sarbecoviruses than seen following immunization with Reference and sometimes Beta or Delta spike, which often induced an intermediate neutralizing profile. For example, ~\u0026thinsp;10% neutralization of binding of BANAL-20-52 spike to hACE2 with following immunization with the Omicron (BA.1) vs. \u0026gt;90% when the immunization antigen is Reference Spike. Significant differences in the neutralization activity between the sera of Reference and Omicron-immunized mice were also seen against the spike protein based on other SARS-CoV-2-Like viruses (Pangolin-GX \u0026amp; Pangolin-GD) as well as SARS-CoV-1 and the SARS-CoV-1-Like virus, Bat WIV. Despite all of these proteins binding the same receptor (hACE2), there was much lower neutralizing activity detected against sarbecovirus spikes that are closely related to SARS-CoV-1. The highest neutralization was observed with Bat-WIV1 spike, with neutralization activity of ~\u0026thinsp;30% seen with serum from mice vaccinated with Reference, Beta or Trivalent SARS-CoV-2 spike, and only\u0026thinsp;~\u0026thinsp;10% with Omicron (BA.1), at the dilution tested.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the impact of the vaccine platform on these observations, we next evaluated the neutralizing potential of antibodies invoked by vaccination with mRNA / lipid nanoparticles (mRNA / LNPs) vaccine formulations encoding different SARS-CoV-2 spikes. We generated DNA templates encoding SARS-CoV-2 Reference and Omicron (BA.4/BA.5) spike proteins, with sequences based on the design used by Pfizer-BioNTech\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The mRNA was transcribed \u003cem\u003ein vitro\u003c/em\u003e and includes N1-methyl-pseudouridine in place of the canonical uridine, as is typical for the clinically approved mRNA / LNP vaccines\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. After encapsulation into commercially available lipids, Balb/c mice (n\u0026thinsp;=\u0026thinsp;10) were vaccinated with 1\u0026micro;g of mRNA / LNPs on Day 0 and Day 21. As before, the serum from these animals was assessed 7 days post-boost for sarbecovirus spike neutralizing potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As expected, the binding of Reference and Omicron (BA.4/BA.5) spike proteins were most strongly neutralized by serum from mice vaccinated with mRNA / LNPs encoding Reference (61.8%) and Omicron (BA.4/BA.5) spike (71.1%), respectively\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, while the bivalent vaccine induced a more intermediate neutralizing profile. Interestingly, despite containing equivalent doses of each spike mRNA, the Bivalent vaccine induced a more potent neutralizing titer towards the BA.4/BA.5 (56.3%) than the Reference (27.9%) spike. The serum from mice vaccinated with Reference, Omicron (BA.4/BA.5) and Bivalent spike all similarly neutralized (35.8% \u0026ndash; 47.4%) the hACE2-binding of Omicron (BA.1) spike, which has a similar degree of homology to the Reference and Omicron (BA.4/BA.5) spike proteins. As seen with the protein subunit vaccines in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the reference-based mRNA/LNP vaccines induced significantly higher neutralization profiles of BANAL-20-52 and Pangolin-GD than vaccines designed to target Omicron (BA4/BA5). Meanwhile, no significant differences were observed in the neutralization profile of the other spike proteins, including those which had shown less pronounced, but still significant, differences with the protein vaccine formulations (i.e., Pangolin-GX, Bat WIV1 and SARS-CoV-1. Altogether, these data highlight the superior ability of SARS-CoV-2-based vaccine formulations based on the Reference sequence to generate neutralizing antibodies to certain animal-derived sarbecoviruses, whether using protein subunit or mRNA / LNP vaccine platforms.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe COVID-19 pandemic, as well as the relatively recent MERS and SARS outbreaks, have demonstrated the devastating impacts of coronaviral zoonosis and the importance of preparedness to mitigate the impact of similar occurrences in the future. We sought to better understand whether SARS-CoV-2-based vaccines could potentially protect against future coronaviral zoonotic events. Previous studies have demonstrated that SARS-CoV-1/2 infection and/or vaccination can induce potent neutralizing antibodies with cross-neutralizing potential to certain sarbecoviruses found currently in various animal species\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Our results confirm that vaccination with formulations based on the ancestral spike of SARS-CoV-2 induces a potent neutralizing response against several hACE2-binding sarbecovirus spikes (e.g., BANAL-20-52, Pangolin-GD, Pangolin-GX, Bat WIV1) that can be superior to those seen with formulations based on variants of concern, in particular Omicron (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo our knowledge, no other studies have gone further to evaluate the potential impact of updating the SARS-CoV-2 spike sequence to match currently circulating variants in the human population on the ability of COVID-19 vaccines to induce protection against potentially zoonotic sarbecoviruses. This can be more complex to accurately measure in human populations due to the heterologous and multifaceted nature of the immune responses generated through previous rounds of infection and/or immunization. In the current study, we used our animal models to compare the cross-protective potential (specifically in terms of induction of sarbecovirus-neutralizing antibodies) of mono- and multi-valent vaccines based on SARS-CoV-2 Reference-strain spike as well as Beta, Delta and Omicron variants. While all formulations generated similarly weak neutralizing responses to certain spike proteins, namely those more closely related to SARS-CoV-1, they had differing abilities to induce cross-reactive neutralizing antibodies to certain viral strains. When immunized with protein subunit antigens based on Reference, Beta, Delta or Omicron (BA.1), the least potent cross-neutralizing responses to BANAL-20-52 or Pangolin-GD were observed in mice receiving Omicron (BA.1)-based vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly poor cross-neutralizing responses were observed to these spike proteins when mice received mRNA/LNPs based on the Omicron (BA.4/BA.5) sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), confirming that this effect is dependent upon the antigen sequence and not the platform of vaccination. This may be indicative of \u0026ldquo;humanization\u0026rdquo; of the SARS-CoV-2 spike in its more recent iterations (i.e., Omicron) as it has evolved through countless replication cycles in hundreds of millions of infected people. Variants such as Omicron have incorporated critical mutations in their ACE2-binding (and potentially other) motifs to better mediate infection in their new host, simultaneously causing them to diverge away from some of the animal-derived sarbecoviruses tested here, as well as the original SARS-CoV-2 virus that is presumed to have been zoonotically transmitted in 2019.\u003c/p\u003e \u003cp\u003eWe also found that the tested vaccine candidates induced a lower degree of cross-neutralization against the spike protein of SARS-CoV-1 or its close relatives than seen against the SARS-CoV-2-Like sarbecoviruses. This is in contrast to some previous reports that illustrate effective cross-neutralization of SARS-CoV-1-like and SARS-CoV-2-like sarbecoviruses after an initial exposure to SARS-CoV-1\u003csup\u003e19,21\u003c/sup\u003e. This suggests that vaccination with a more closely related sarbecovirus antigen, such as Bat WIV1, may be required to effectively neutralize this subgroup of coronavirus spikes. While the overall level of neutralization was lower in the case of the SARS-CoV-1-like sarbecoviruses, for protein antigens, the Reference-based vaccine formulation still induced superior neutralization compared to the Omicron (BA.1)-based antigen against the spike protein from Bat WIV1 and SARS-CoV-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, for the mRNA-LNP vaccines, no obvious differences were observed between the Reference and Omicron mRNA / LNPs in terms of induction of SARS-CoV-1-related spike neutralization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Whether this is a feature of vaccination platform (protein vs. mRNA), antigen sequence (BA.1 vs. BA4/BA5) and/or mouse strain (C57BL/6 vs. Balb/c) should be confirmed in future studies.\u003c/p\u003e \u003cp\u003eSARS-CoV-2 Reference-based vaccines have continuously proved to be effective boosters even in the context of emerging variants\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Nonetheless, it is increasingly evident that updating of SARS-CoV-2 vaccine antigen sequences to resemble dominant, circulating variants is necessary to achieve optimal protection\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Importantly, our results indicate that these modernized vaccines may be less capable of inducing protective humoral responses towards other sarbecovirus spikes which have the potential to transmit zoonotically. Furthermore, our data in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates that the use of bivalent vaccine strategies may not equally induce neutralizing antibodies to both antigens, as Omicron (BA.4/BA.5) appears to be immunodominant. This immunodominance over the Reference spike has also been observed previously even when limiting the vaccine to the RBD region\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs highlighted with COVID-19, the swift timing of vaccine deployment is critical to minimize the global impacts at the onset of any pandemic\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. With its cross-neutralizing potential against closely related sarbecoviruses, the ability to quickly deploy a stockpile of Reference SARS-CoV-2 vaccines may prove effective in the event of another outbreak caused by a zoonotic transmission event involving an ACE2-binding coronavirus. The same may be true for other coronaviruses, such as SARS-CoV-1 and MERS-CoV families. Capabilities should be established to quickly develop/manufacture and/or stockpile vaccines based on one (or a few) viruses in their respective families, as they may prove to be effective during an emergency response to a future outbreak. Indeed, with the current immunological status of the overall human population following infection by/vaccination against COVID-19, viruses from other subgroups may now have a higher likelihood of zoonoses than sarbecoviruses closely related to SARS-CoV-2. In a future pandemic, previously developed vaccines could provide some level of protection during the time delay required for viral identification, de novo synthesis of antigen sequences and regulatory approval of novel vaccines.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAntigen Production and Homology\u003c/h2\u003e \u003cp\u003eCHO codon-optimized sequences encoding spike ectodomains of animal-origin sarbecoviruses (with prefusion-stabilizing 2P mutations) fused to human resistin and purification tags (FLAG-dual-Strep-6His or dual-Strep-6His) at the C-terminus were synthesized at Genscript and cloned into the pTT241\u0026reg; plasmid \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These stabilizing prolines are in well-conserved regions for all the selected spikes and were positioned by alignment with the SARS-CoV-2 spike sequence. To mediate trimerization, human resistin was fused to the C-terminus followed by purification tags. Stably transfected pools were generated using CHO\u003csup\u003e2353\u003c/sup\u003e\u0026trade; cells and protein productions were performed with expression induced with cumate as described \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. For the different sarbecovirus spikes, protein expression using stably transfected CHO cell pools generated quite variable production yields ranging from 0.1-2 g/L. In comparison, the corresponding SARS-CoV-2 reference-strain construct yields\u0026thinsp;~\u0026thinsp;0.7 g/L under the same conditions \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Clarified CHO culture supernatants harvested at 7 or 10 days post-induction were purified as described by IMAC followed by Strep-affinity chromatography \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Purified products were buffer-exchanged using Centripure 100 desalting columns (emp BIOTECH, Berlin, Germany) into DPBS, pH 7.8, or into HEPES (50 mM, pH 7.8) buffer containing 100 mM NaCl. Purified proteins were analyzed by SDS-PAGE using NuPAGE 4\u0026ndash;12% Bis-Tris gels (ThermoFisher, Massachusetts, USA) followed by Coomassie Blue staining. The absence of endotoxin contamination was verified using Endosafe cartridge-based Limulus amebocyte lysate tests (Charles River Laboratories, Charleston, SC, USA). Clustal Omega\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e was used to generate the Percent Identity Matrix and Phylograms to depict the relationship of the spike sequences used in this study. To better relate identity to immunological data, the antigen sequences (which are limited to the spike ectodomains) were used for the homology analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of mRNA/LNPs\u003c/h2\u003e \u003cp\u003eThe plasmid DNA templates used to generate the tested mRNAs were designed based on the publicly available Pfizer-BioNTech mRNA/LNP vaccine sequence\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and synthesized by Genscript (Piscataway, NJ, USA) in a pUC57 backbone. DNA templates were linearized with XbaI and purified by phenol\u0026ndash;chloroform extraction. RNA was generated using Megascript T7 transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) and capped using CleanCap Reagent AG (TriLink BioTechnologies, San Diego, CA, USA) to generate RNA with stabilized Cap 1 structure. In place of uridine, m1ψ (TriLink BioTechnologies) was included to generate the mRNAs with modified nucleotide chemistry. Following IVT of RNA, plasmid DNA was digested using Turbo DNase (Invitrogen, Waltham, MA, USA) as per manufacturer\u0026rsquo;s instructions. RNA was purified by lithium chloride precipitation, washed with 70% ethanol and resuspended in RNase-free water. To assess RNA size and integrity, all mRNA samples were resolved on formaldehyde (2.6%, v/v) denaturing agarose (1%, w/v) gel. The concentrations of mRNA samples were measured using Invitrogen\u0026rsquo;s RiboGreen assay reagent (Thermo Fisher Scientific), as per manufacturer\u0026rsquo;s recommendations.\u003c/p\u003e \u003cp\u003eIVT mRNA encoding SARS-CoV-2 reference or BA.4/BA.5 spike was encapsulated within Genvoy-ILM ionizable lipid mixture (Precision NanoSystems, Vancouver, BC, Canada) at an N:P ratio of 8. Formation of LNPs was achieved by microfluidic mixing of RNA (aqueous) and ionizable lipids (organic) using the NanoAssemblr Ignite system (Precision NanoSystems) according to manufacturer\u0026rsquo;s recommendations with a flow rate ratio of 3 (aqueous:organic) and total flow rate of 12 (ml/min). Formulations were then diluted in Mg2+/Ca2+-free PBS, before undergoing buffer exchange and concentration using Amicon Ultra Centrifugal Filters (Millipore Sigma, St. Louis, MO, USA). Encapsulation efficiency of RNA was determined by RiboGreen assay (Thermo Fisher Scientific) as per instructions accompanying the Genvoy-ILM ionizable lipid and found to be \u0026gt;\u0026thinsp;90%. In addition, particle size and polydispersity index (PDI) of the LNPs were verified using a Zetasizer NanoZS (Malvern Instruments, Malvern, UK) and found to be 122\u0026ndash;136nm and 0.10\u0026ndash;0.12, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunizations and Sample Collection\u003c/h2\u003e \u003cp\u003e Female C57BL/6 or Balb/c mice (6\u0026ndash;8 weeks old) were obtained from Charles River Laboratories (Saint-Constant, QC, Canada) and maintained at the small animal facility of the NRC Canada in accordance with the guidelines of the Canadian Council of Animal Care. Protein subunit vaccinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were described in previously published studies\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Briefly, C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;9\u0026ndash;10 per group) were immunized by i.m. injection (50 \u0026micro;l) into the left tibialis anterior muscle on Days 0 and 21 with 3 \u0026micro;g SARS-CoV-2 spike SmT1 antigen (reference, Beta, Delta, a 1:1:1 ratio of each reference, Beta and Delta, or Omicron (BA.1)) diluted in PBS (Spectrum, Gardena, CA, USA) and mixed with AddaS03 (Invivogen, San Diego, CA, USA) according to manufacturer\u0026rsquo;s instructions. Vaccinations of Balb/c mice (n\u0026thinsp;=\u0026thinsp;10 per group) with mRNA/LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were performed in the same fashion as the protein subunit vaccines except normalized to a 1 \u0026micro;g dose of encapsulated mRNA encoding SARS-CoV-2 spike (reference, Omicron (BA.4/BA.5) or a 1:1 ratio of each). Mice were bled via the submandibular vein on day 28 with recovered serum used for quantification neutralization activity. Samples were simultaneously collected from 10 na\u0026iuml;ve animals for the assessment of background immune responses. Each of the samples from the individual mice was tested separately in the various readouts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell-based Spike-hACE2 Binding Assay\u003c/h2\u003e \u003cp\u003eSerum was assessed for its ability to neutralize the binding of labeled spike trimers to hACE2-expressing cells. The following reagent was obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK-293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK-293T-hACE2 Cell Line, NR-52511. This assay was performed as previously described, using HEK-293T-hACE2 cells \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Purified spike was biotinylated with EZ-Link\u0026trade; Sulfo-NHS-LC-LC-Biotin (ThermoFisher, Massachusetts, USA) and purified via molecular weight cut off (MWCO) columns according to manufacturer\u0026rsquo;s instructions. Mouse serum was diluted 1 in 250, mixed with 250 ng of biotinylated spike and 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e HEK-293T-hACE2 cells. The amount of bound spike was quantified using Streptavidin-R-PE (ThermoFisher, Massachusetts, USA), conjugate by acquiring cells on an LSR Fortessa (Becton Dickinson, New Jersey, USA) and analyzing data on FlowJo (Becton Dickinson). For illustration/analysis purposes, samples with calculated values\u0026thinsp;\u0026le;\u0026thinsp;0 were assigned a value of 0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using GraphPad Prism version 10 (GraphPad, Massachusetts, USA). Statistical significance of the difference between groups was calculated by one-way ANOVA followed by Dunnett\u0026rsquo;s multiple comparisons test. Differences were considered to be not significant with p\u0026thinsp;\u0026gt;\u0026thinsp;0.05. Significance was indicated in the graphs as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eInstitutional review board statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were maintained at the small animal facility of the National Research Council (NRC) Canada in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were approved by our Institutional Review Board (NRC Human Health Therapeutics Animal Care Committee) and covered under animal use protocols 2020.10. All experiments were carried out in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge John Shelvey and Perry Fleming for producing the archaeal biomass. We also thank the technical contribution of many members of the Mammalian Cell Expression Section of the NRC-HHT and all of the staff of the Human Health Therapeutics Animal Resource Group. This work was supported in part by the NRC\u0026rsquo;s Pandemic Response Challenge Program and by a CQDM partnership with Oragenics Inc., Inspirevax Inc., and the NRC. The following reagents were obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK-293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK-293T-hACE2 Cell Line, NR-52511.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBA, TMR, MS, YD and MJM conceived and/or designed the studies. MS, BC, SP, JG, SL-D and YD designed, produced and/or purified the spike proteins used in this study. TMR conducted the homology, phylogeny, vaccinations and neutralization analyses. TMR and BA took the lead in writing the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing non-financial interests but the following competing financial interests: BA and MJM are inventors on an SLA archaeosome-related patent application. YD is an inventor of a patent application related to the SmT1 antigen (publication number 20230174591).\u003c/p\u003e\n\u003cp\u003eThe remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBiancolella, M. \u003cem\u003eet al.\u003c/em\u003e COVID-19 2022 update: transition of the pandemic to the endemic phase. Hum. Genomics 16, 19 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNesteruk, I. Endemic characteristics of SARS-CoV-2 infection. Sci. 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Rep. 11, 21849 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4791122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4791122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The COVID-19 pandemic has emphasised the importance of vaccines and preparedness against viral threats crossing species barriers. In response, a worldwide vaccination campaign targeting SARS-CoV-2 was implemented, which provides some cross-protective immunological memory to other coronavirus species with zoonotic potential. Following a vaccination regimen against SARS-CoV-2 spike in a preclinical mouse model, we were able to demonstrate the induction of neutralizing antibodies towards multiple human ACE2 (hACE2)-binding sarbecovirus spikes. Importantly, compared to vaccines based on the SARS-CoV-2 Reference strain, vaccines based on Omicron spike sequences induced drastically less broadly cross-protective neutralizing antibodies against other hACE2-binding sarbecoviruses. This observation remained true whether the vaccination regimens were based on protein subunit or mRNA / LNP vaccines. Overall, while it may be necessary to update vaccine antigens to combat the evolving SARS-CoV-2 virus for enhanced protection from COVID-19, Reference-based vaccines may be a more valuable tool to protect against novel coronavirus zoonoses.","manuscriptTitle":"Reduced Cross-Protective Potential of Omicron Compared to Ancestral SARS-CoV-2 Spike Vaccines Against Potentially Zoonotic Coronaviruses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 11:46:26","doi":"10.21203/rs.3.rs-4791122/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-22T13:02:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-20T03:59:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-19T16:44:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-14T01:12:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99557435353171379070083751541393424818","date":"2024-08-13T20:46:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45356301634663406718127265812928934478","date":"2024-08-13T12:28:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324715749203744196625815418238638414622","date":"2024-08-12T01:59:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-11T19:00:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-01T14:44:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-01T08:20:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Viruses","date":"2024-07-23T20:37:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"12f87830-2224-41d8-9350-65b7d8224452","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36422078,"name":"Biological sciences/Immunology/Vaccines/Protein vaccines"},{"id":36422081,"name":"Biological sciences/Immunology/Vaccines"},{"id":36422082,"name":"Biological sciences/Immunology/Vaccines/Rna vaccines"},{"id":36422083,"name":"Biological sciences/Immunology"},{"id":36422084,"name":"Biological sciences/Immunology/Adaptive immunity"},{"id":36422085,"name":"Biological sciences/Immunology/Adaptive immunity/Humoral immunity"},{"id":36422086,"name":"Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Antibodies"},{"id":36422087,"name":"Biological sciences/Microbiology"},{"id":36422088,"name":"Biological sciences/Microbiology/Virology"},{"id":36422089,"name":"Biological sciences/Microbiology/Virology/Sars cov 2"},{"id":36422090,"name":"Biological sciences/Microbiology/Virology/Sars virus"}],"tags":[],"updatedAt":"2024-11-22T14:13:58+00:00","versionOfRecord":{"articleIdentity":"rs-4791122","link":"https://doi.org/10.1038/s44298-024-00067-9","journal":{"identity":"npj-viruses","isVorOnly":false,"title":"npj Viruses"},"publishedOn":"2024-11-21 00:00:00","publishedOnDateReadable":"November 21st, 2024"},"versionCreatedAt":"2024-08-28 11:46:26","video":"","vorDoi":"10.1038/s44298-024-00067-9","vorDoiUrl":"https://doi.org/10.1038/s44298-024-00067-9","workflowStages":[]},"version":"v1","identity":"rs-4791122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4791122","identity":"rs-4791122","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}