Pan-recombinant vaccines based on the consensus sequence of severe acute respiratory syndrome coronavirus-2 spike protein

Pan-recombinant vaccines based on the consensus sequence of severe acute respiratory syndrome coronavirus-2 spike protein | 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 Pan-recombinant vaccines based on the consensus sequence of severe acute respiratory syndrome coronavirus-2 spike protein Yoo Jin Na, Eun Bee Choi, Kwangwook Kim, Seungyeon Kim, Seo Young Moon, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7407141/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract Although multivalent vaccines are being developed to address the rapid mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), universal vaccines are needed to protect against evolving variants and seasonal epidemics. We designed and evaluated the efficacy of three universal SARS-CoV-2 recombinant vaccine candidates based on a consensus sequence from multiple variants. The candidates were designed with specific substitutions, including S-GSAS/6P and S-R/x2, to enhance immunogenicity by stabilizing the prefusion spike-protein conformation. Using murine models, we assessed humoral and cellular immune responses, protective efficacy, and viral load reduction following immunization and challenge with the ancestral and XBB1.5 strains. The pan-COVID-19 vaccine candidates stimulated the production of cytokines, specifically interferon-γ and interleukin-2, and induced neutralizing antibodies targeting the ancestral, Delta, and Omicron variants. Vaccination reduced viral replication rates in pulmonary tissues of mice infected with ancestral and XBB1.5 variants; however, their protective efficacy varied among formulations. Candidates containing the S2 subunit elicited better immune responses and protective effects than those containing solely the S1 subunit, consistent with published evidence supporting the cross-reactivity of the S2 region. The proposed vaccine candidates represent promising broad-spectrum protective agents against rapidly mutating SARS-CoV-2 variants. Biological sciences/Immunology Biological sciences/Microbiology Spike protein stabilization Coronavirus disease 2019 Pan-COVID-19 vaccine S2 subunit cross-reactivity Cellular immune response Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Coronavirus disease 2019 (COVID-19) persisted as a global pandemic from March 2020 to May 2023. Despite the rapid development of vaccines, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has undergone extensive mutations, producing new variants with diminished sensitivity to early United States Food and Drug Administration-approved vaccines, such as those of Moderna and Pfizer-BioNTech [ 1 ]. Although several trials have focused on the development of multivalent vaccine candidates, the continuous evolution of SARS-CoV-2 variants and seasonal coronaviruses necessitates the development of universal vaccines. Multiple research groups have tested pan-COVID-19 vaccines using nanoparticle (NP) delivery, mosaic antigens, computational antigens, and consensus sequence design [ 2 ]. The decavalent mRNA-LNP vaccine (FLUCOV-10), designed using phylogenetic analysis of sequences from influenza viruses and SARS-CoV-2 variants (ancestral, BQ.1.1, BA.2.75.2, and XBB1.5), elicits robust immune responses and protection against influenza and COVID-19 [ 3 ]. A mosaic NP vaccine comprising receptor-binding domains (RBDs) from Omicron (BA.1, BA.2, BA.5, and BA.2.75), Delta, and D614G, fused with highly conserved T-cell epitope sequences derived from sarbecovirus nucleocapsid or spike (S) proteins, exhibits broad cross-protective activity against various SARS-CoV-2 sublineages and enhanced potency compared with a cocktail of NPs [ 4 ]. Furthermore, a universal SARS-CoV-2 subunit vaccine designed using a highly conserved truncated RBD from Omicron BA.1 or the RBDs of Delta, BA.5, and wild-type (WT) induces neutralizing antibodies against Alpha, Beta, Gamma, Delta, Omicron subvariants (BA.1, BA.2, BA.2.75, BA.4.6, and BA.5), and the ancestral strain and prevents Omicron replication and infection [ 5 ]. Vaccine immunogenicity is commonly enhanced by stabilizing the SARS-CoV-2 surface glycoprotein S in its metastable prefusion conformation and preserving the native positioning of RBDs [ 6 , 7 ]. First-generation engineered spike, S-2P, which includes two proline substitutions at positions 986 and 987 [ 8 – 11 ], has been incorporated into effective vaccines, such as mRNA-1273 and BNT162b2. However, S-2P demonstrates limited yield and thermostability [ 12 – 14 ]. HexaPro, a second-generation spike with four additional proline substitutions (positions 817, 892, 899, and 942), addresses some limitations of S-2P, including improved acquisition rates and thermostability, but exhibits structural instability and transitions to a post-fusion state when exposed to receptor [e.g., angiotensin-converting enzyme 2 (ACE2)] binding, antibody-mediated activation, or protease digestion [ 15 , 16 ]. Another engineered spike, S-R/x2, retains a trimeric prefusion assembly through disulfide bond formation between residues 413 and 987, which stabilizes the closed conformation of the protein [ 17 ]. Despite these recent advances, further investigation is required to develop effective and adaptable universal vaccines against emerging SARS-CoV-2 variants and coronaviruses. In this study, we aimed to design three universal recombinant SARS-CoV-2 vaccine candidates derived from a consensus sequence of variants including Alpha, Beta, Gamma, Delta, and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, and BA.5), with specific spike substitutions. Results Design of pan-COVID-19 recombinant vaccines with the consensus sequence of spike protein Spike variant counts were distributed unevenly among the Alpha (31.6%), Beta (0.5%), Delta (20.2%), Gamma (2.6%), and Omicron (45.1%) variants. The consensus sequence was defined by the most common amino acid residues (cut-off, 0.6) and included four mutations: D614G, N501Y, DELH69V70, and DELY144. Additional mutations associated with the Alpha variant (B.1.1.7; A570D, T716I, S982A, and D1118H) were incorporated (Fig. 1 a). To increase stability, we designed three pan-COVID-19 vaccine candidates with S-GSAS/6P and S-R/x2 modifications (Fig. 1 b). For simplicity, candidates are referred to as G1 (candidate 1), G2 (candidate 2), and G3 (candidate 3). The three candidates were expressed in HEK293 cells and purified using HisTrap FF Crude. The expression of the recombinant proteins was confirmed via SDS-PAGE and western blotting (Fig. 1 c). G2 expression was lower than G1 and G3 expression; thus, the half concentration (5 µg) was a high dose only in G2. Universal vaccine candidates exert humoral immune responses to heterogeneous SARS-CoV-2 variants The timeline for evaluating the immunogenicity of the pan-COVID-19 vaccine candidates is presented in Fig. 2 a. Groups of C57BL/6 mice were intramuscularly vaccinated with 1 µg of pan-COVID-19 vaccines at 3-week intervals. In mouse sera collected 1 week after each booster, SARS-CoV-2 spike-specific total IgG levels increased, with the low dose of G1 exhibiting the highest responses, followed by G2 (Fig. 2 b). The total IgG levels were finally induced at a high dose of G3. Neutralizing antibody responses against WT, Alpha, Beta, Delta, and XBB1.5 strains were assessed using the plaque reduction neutralization test (PRNT) (Fig. 2 c). G1 and G2 elicited stronger neutralizing responses across all strains than G3, which showed no change for the Delta variant, even at a high dose (Supplementary Figure S1 ). NT 50 values were slightly increased by the second booster and high dose of G3 in variant XBB1.5. Pan-COVID-19 vaccine candidates induced robust cellular immune responses The cellular immune response following the second booster vaccination is depicted in Fig. 3 a. Interferon-γ (IFN-γ) secretion was detected in splenocytes from immunized mice stimulated with peptide pools representing SARS-CoV-2 ancestral, Delta, BA.1, and BA.2 strains, as well as SARS-CoV (Fig. 3 b). Although a trend was observed at low doses, significant IFN-γ induction was observed at high doses (Fig. 3 b). Although G3 failed to induce neutralizing antibodies in Delta (Fig. 2 c), IFN-γ secretion was induced. In addition, IFN-γ secretion was significantly induced by G1 and G2 in SARS-CoV. The level of cytokines secreted by splenocytes from immunized and stimulated mice was measured to identify antigen-specific T cell responses. Interleukin 2 (IL-2) was strongly induced by G1 and G2 across most strains, whereas G3 exerted no effect (Fig. 3 c). Tumor necrosis factor-α (TNF-α) levels remained unchanged across groups (Supplementary Figure S2). Pan-COVID-19 vaccine candidates demonstrated significant protective efficacy We immunized and subsequently challenged K18–human angiotensin-converting enzyme 2 (hACE2) mice with 2.6×10 5 plaque-forming units (PFU) of ancestral and 2.9×10 5 PFU of XBB1.5 strains (Fig. 4 a) and assessed the protective effects using a SARS-CoV-2 RNA probe targeting the spike gene (21,631–23,303 bp, number of pairs: 20), which helps detect multiple strains, including ancestral, Alpha, Beta, Gamma, and XBB1.5 (Supplementary Figure S3; Fig. 4 b). The in situ hybridization scores for pulmonary tissues were dramatically decreased by G1 and G2 in the ancestral strain and G1 of the XBB1.5 strain. The scores were similarly decreased by G2 and G3 over time and with increasing doses. Viral loads in lung tissues 3 and 5 d post-infection (dpi) were substantially reduced with G1 and G2 in the ancestral strain, with a moderate reduction observed in the XBB1.5 strain (Fig. 4 c). Discussion SARS-CoV-2 has accumulated mutations that enhance its transmissibility, virulence, and evasion of neutralizing antibodies [ 18 – 20 ]. These adaptations primarily involve changes in the spike protein, which is responsible for target recognition, binding, and cellular entry [ 21 ]. Multiple sequence alignment (MSA) analysis identified mutations, such as DELH69V70, DELY144, N501Y, A570D, and D614G, in the S1 subunit. This region, comprising the NTD and RBD, facilitates recognition and binding to hACE2 receptors [ 22 ]. The D614G mutation in the S1 subunit emerged in mid-2020 and rapidly became dominant [ 23 ]. It was detected in > 95% of SARS-CoV-2 variants sequenced by January 2021, which increased to 99% by March 2022 [ 24 ]. D614G enhances the binding affinity between the S protein and ACE2 by promoting conformational changes that facilitate the RBD-up conformation, achieved by disrupting the D614–K854 salt bridge [ 25 – 27 ]. Consequently, the mutation significantly increases SARS-CoV-2 infectivity [ 28 ]. The A570D mutation, although less infectious than A570 in the pseudovirus revertant system B.1.1.7 + A570 [ 29 ], plays a structural role in the S trimer by increasing the spacing between its individual chains, potentially improving furin cleavage accessibility and modulating RBD conformational transitions. These transitions act as a molecular switch, cycling between open and closed states [ 30 ]. We also identified mutations (including T716I, S982A, and D1118H) in the S2 subunit, which contains a fusion peptide (FP), two heptad repeats (HR1 and 2), transmembrane subdomain, and C-terminal tail [ 31 ]. The S2 subunit facilitates membrane fusion, viral entry, cell-to-cell fusion, syncytia formation, and viral dispersion from infected to neighboring cells [ 32 , 33 ]. The S982A substitution disrupts a hydrogen bond with T574, promoting the RBD-up conformation that facilitates binding with ACE2 [ 34 ]. To compensate for this structural change, the A570D substitution forms a hydrogen bond with N856 that stabilizes to an RBD-down conformation. Similarly, D1118H compensates for the destabilization of RBD formation caused by T716I. These complementary effects among substitutions within the Alpha SARS-CoV-2 variant collectively promote the stabilization of the prefusion S conformation [ 24 ]. We incorporated the four mutations showing ~ 40% concordance in the MSA analysis into the consensus sequence to design the coronavirus vaccine candidates. The S1 subunit and its RBD are major targets for neutralizing antibodies, but these regions experience frequent mutations under immune pressure [ 35 – 37 ]. Conversely, the S2 subunit, containing conserved elements, such as the FP and HRs, remains relatively stable across variants [ 38 ]. Cross-reactive antibodies targeting conserved regions of the S2 subunit, induced by prior seasonal coronavirus infections (such as human coronaviruses OC43, NL63, and 229E), are boosted following SARS-CoV-2 infection or vaccination [ 39 – 43 ]. Several S2-specific monoclonal antibodies have exhibited potent neutralizing activity [ 44 – 49 ]. Consequently, targetable S2 elements have been widely utilized in the design of pan-coronavirus vaccines [50,51]. For example, the RBD-HR chimeric nanoparticle vaccine induces robust cross-reactive T and B cell immune responses against pseudotyped SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, and RATG13, outperforming the RBD monomer vaccine [ 52 ]. The RBD-HR/trimer vaccine not only elicits strong neutralizing antibodies against ancestral, Alpha, Beta, Delta, and Omicron pseudoviruses but also provides protection against Omicron and Delta challenges [ 53 ]. S2-only antigens with six proline substitutions (HexaPro), stabilized in prefusion trimers through the introduction of interprotomer disulfide bonds, induce broadly neutralizing responses against pseudotyped ancestral SARS-CoV-2, Omicron BA.1, SARS-CoV, and SHC014 [ 54 ]. Immunization with these prefusion-stabilized S2 vaccine candidates conferred complete and partial protection against lethal challenges with SARS-CoV-2 and SARS-CoV, respectively. The superior efficacies of G1 and G2, which included the S2 subunit (Fig. 1 b), over G3, which included only S1, in providing protection against SARS-CoV-2 and eliciting both humoral and cellular immune responses (Figs. 2 – 4 ) align with the findings of these prior studies. Studying T cell responses is essential for understanding long-term adaptive immunity [ 55 – 57 ]. IFN-γ, a key cytokine released by T cells, enhances antigen presentation, inhibits viral replication, and stimulates inflammatory gene expression [ 58 ]. IL-2 is critical for the development, proliferation, and maintenance of memory T cells in response to specific antigens [59–61]. Recent studies have reported higher IFN-γ levels in COVID-19 convalescent serum than in serum obtained from the acute disease phase, whereas IFN-γ was not detected in individuals fully vaccinated with SARS-CoV-2 mRNA vaccines [ 62 , 63 ]. Cellular IFN-γ and IL-2 levels are reportedly higher in hybrid immune contexts (vaccination following infection) than with vaccination alone, including mRNA-1273 (Moderna) and BNT162b2 (Pfizer-BioNTech) [ 64 ]. Conversely, both IFN-γ and IL-2 T cell responses were triggered by SARS-CoV-2 vaccination and increased with disease severity in convalescent individuals, with severe cases exhibiting higher responses than vaccinated individuals. Furthermore, no relationship was observed between T cell responses and time since vaccination. IgG responses demonstrate a moderate correlation with IFN-γ and IL-2 levels in cellular and humoral immunity [ 65 ]. Despite variability in previous findings, memory T cell responses are critical for protection against emerging variants and reinfection, especially when humoral immunity wanes or faces newly emerged variants of concern (VOCs) [66,67]. Our vaccine candidates elicited robust IFN-γ and IL-2 responses without inducing the pro-inflammatory cytokine TNF-α (Fig. 3 ). This study has certain limitations. First, owing to the unavailability of XBB1.5 peptide pools, BA.1 and BA.2 peptide pools were used for assessing cellular immune responses (Fig. 3 ). Second, the serum samples from mice in the same group were pooled for PRNT. Although statistical confirmation is not provided, this should represent the group average as equal specimens were mixed (Fig. 2 c). Alongside improvements in multivalent vaccines for the rapidly evolving SARS-CoV-2, the development of universal vaccinations is essential to continue protecting the public against emerging variations and seasonal coronaviruses. We created three universal recombinant vaccine candidates based on consensus sequencing of different variants. To preserve the prefusion form of the spike protein and improve immunogenicity, these candidates included the substitutions S-GSAS/6P and S-R/x2. The proposed vaccine candidates elicited robust humoral and cellular immune responses, particularly in formulations containing the S2 subunit, which demonstrated superior protective efficacy over S1-based vaccines. Collectively, our findings provide novel insights into the potential of S2-based pan-COVID-19 vaccines for eliciting broad-spectrum protection against emerging SARS-CoV-2 variants. Methods Selection of consensus sequence and design of subunit vaccines We retrieved 894,562 SARS-CoV-2 sequences from the Global Initiative for Sharing All Influenza Data by filtering the conditions “complete,” “high coverage,” and “with patient status.” VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, and BA.5), were aligned with the reference sequence (hCoV-19/Wuhan/WIV04/2019) using the NextAlign tool to identify point mutations and insertion-deletion mutations. MSA analysis was performed with the MAFFT tool (cut-off, 0.6) to generate the consensus sequence. Mutations, including A570D, T716I, S982A, and D1118H, were incorporated into the consensus sequence to design pan-COVID-19 recombinant vaccine candidates. To prevent cleavage at the S1 (receptor-binding)/S2 (membrane fusion) junction and enhance immunogenicity, GSAS (residues 682–685), PRRA deletions (R, residues 681–684), HexaPro (6P), and disulfide 2 (x2) were introduced, generating S-GSAS/6P and S-R/x2. We added the signal peptide (MGWSCIILFLVATATGVHS) at the N-terminus and foldon sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL) at the C-terminus to improve protein secretion and stabilize trimeric structure. Viruses and cells The ancestral (NCCP43326), Alpha (GRY clade, B.1.1.7, NCCP43381), Beta (GH clade, B.1.351, NCCP43382), Gamma (GR clade, P.1, NCCP43388), Delta (GK clade, AY.69, NCCP43409), and Omicron (GRA clade, BA.1, NCCP43408; BA.1.1, NCCP43411; BA.2, NCCP43412; XBB.1.5, NCCP43440) SARS-CoV-2 strains were obtained from the National Culture Collection for Pathogens (Cheongju, Republic of Korea) and titrated via plaque assays. All live virus experiments were conducted in a biosafety level 3 facility. Vero E6 African green monkey kidney cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and L-glutamine at 37°C in a humidified 5% CO 2 incubator. Immunization of mice We separately divided 5–7-week-old female C57BL/6 (SAMTACO Bio Korea; Hwaseong, Republic of Korea) and K18–hACE2 mice (Jackson Laboratory, Farmington, CT, USA) with an average weight of 20–25 g into the following seven groups: alum, G1 (S-GSAS/6P, 1 and 10 µg), G2 (S-R/x2, 1 and 5 µg), and G3 [N-terminal domain (NTD)-RBD, 1 and 10 µg]. Vaccine candidates, formulated with alum as an adjuvant, were intramuscularly administered thrice at 3-week intervals to groups of C57BL/6 mice ( n = 5/group). Serum was collected 1 week after each booster and spleens were extracted 1 week post-final vaccination to evaluate humoral and cellular immune responses. K18–hACE2 mice ( n = 5/group) were immunized twice at 3-week intervals via intramuscular injection of the vaccine candidates formulated with alum. At 7 weeks, mice were challenged intranasally with 2.6×10 5 PFUs of the ancestral strain and 2.9×10 5 PFU of the XBB1.5 strain to evaluate protective immunity. Lung tissues were collected 3 and 5 dpi to measure viral load. All infections and surgical procedures were conducted under anesthesia administered by intraperitoneal injection of a combination of Ketamine (100 mg/kg; Yuhan Corporation, Republic of Korea) and Rompun (10 mg/kg; Elanco, Indianapolis, IN, USA). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Disease Control and Prevention Agency (KDCA) in accordance with the laboratory’s animal ethics guideline (KDCA-IACUC-22-032). This study complied with the ARRIVE guidelines during the experiment. All experiments were performed following the relevant guidelines and regulations. PRNT Vero E6 (2.5×10 5 cells/mL/well) were seeded in 12-well plates and infected the next day with a mixture of serially diluted heat-inactivated mouse serum and viruses (50 PFU/well) for 1 h. Cells were cultured with overlay media (1.5% agarose; Lonza, Rockland, ME, USA) in 2× MEM supplemented with 4% FBS (Gibco) and incubated for 2 d. Plaques were visualized using crystal violet staining and counted to calculate the neutralization titer (NT 50 ) using the Kärber formula. Specimens from mice in the same group were pooled for analysis. Enzyme-linked immunosorbent assay To quantify SARS-CoV-2-specific total IgG, 96-well plates were coated with 50 ng SARS-CoV-2 Spike S1 + S2 ECD–His recombinant protein (Sino Biological, Beijing, China). Following blocking with 1% bovine serum albumin, serially diluted mouse serum samples were added to the antigen-precoated plates and incubated for 1 h at 37°C. Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, 1:5,000) was used as the secondary antibody and absorbance at 450 nm was measured after adding tetramethylbenzidine substrate (Invitrogen, Carlsbad, CA, USA) and stop solution (GenDEPOT). We used 0.02% Tween-20 in phosphate-buffered saline for washing between steps. Cytokine expression (IL-2 and TNF-α) was quantified using a quantitative sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN, USA) at 450 nm. ELISpot To evaluate cellular immune responses, IFN-γ secretion was quantified using a mouse IFN-γ ELISpot kit (R&D Systems). Splenocytes were filtered through 50-µm nylon cell strainers (BD Biosciences, San Jose, CA, USA) and digested with ammonium–chloride–potassium buffer. We plated 10 6 splenocytes on 96-well polyvinylidene difluoride membrane plates pre-coated with a monoclonal antibody specific for mouse IFN-γ and stimulated with 1 µg/mL PepMix SARS-CoV-2 Spike variants (JPT, Berlin, Germany) for 24 h at 37°C. The following day, cells were discarded through washing and the captured IFN-γ secreted by the stimulated cells was bound to biotinylated anti-IFN-γ mouse antibody. Subsequently, the plate was incubated with alkaline phosphatase-conjugated streptavidin and BCIP/NBT substrate was added to visualize colored spot formation. Washing was performed between each process. The spot counts were measured using an S6 Universal M2 analyzer (ImmunoSpot, Cleveland, OH, USA). In situ RNA detection Lung tissues were collected 3 and 5 dpi with SARS-CoV-2 variants (ancestral and XBB1.5). Paraformalin-fixed paraffin-embedded tissues were permeabilized, hybridized, and amplified for in situ hybridization. The embedded tissues were cut into 5-µm sections using a microtome (Leica Biosystems, Nußloch, Germany) and deparaffinized tissues were incubated with hydrogen peroxide for 10 min at 25°C. Following retrieval with Target Retrieval reagent for 15 min at 98°C, tissues were treated with Protease Plus for 30 min at 40°C before probe hybridization. An RNAscope probe targeting the spike gene (Advanced Cell Diagnostics, Newark, CA, USA) was added and allowed to hybridize for 2 h at 40°C. Subsequently, tissues were counterstained with hematoxylin and dehydrated in ethanol and xylene. The extent of viral infection was determined using scores assigned based on labeling extent: 0, no labeling; 1, ≤ 20% positive; 2, 21–50% positive; 3, 51–80% positive; 4, 81–100% positive. Reverse transcription-polymerase chain reaction (RT-PCR) After brief lysis of lung tissues (lysis buffer with 10% proteinase K), the samples were loaded onto a Maxwell RSC cartridge. Viral total RNA was automatically extracted using a Maxwell RSC Viral Total Nucleic Acid Purification Kit in combination with a Maxwell RSC platform (Promega, Madison, WI, USA). Viral loads were quantified by detecting the SARS-CoV-2 E gene using a PowerCheck SARS-CoV-2 RT-PCR Kit (Kogen Biotech, Seoul, Republic of Korea). Real-time RT-PCR (Applied Biosystems, Carlsbad, CA, USA) was performed according to the manufacturer's instructions. Viral load (copies/mL) was calculated using standard curves generated from SARS-CoV-2 transcripts rather than cyclic threshold values. Statistical analysis The mean and standard error of the mean were calculated using Prism 5 (GraphPad Software, San Diego, CA, USA) and compared using an unpaired t -test or one-way ANOVA test followed by Tukey’s correction, according to the design of each experiment (shown in figure legends). Statistical significance was set at P < 0.05 indicated with asterisks. Data availability All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Declarations Funding This work was supported by a grant from the National Institute of Health (2022-NI-047-00), Republic of Korea. Author contributions Yoo Jin Na: Conceptualization, Data curation, Investigation, Writing – original draft, Writing – review & editing. Eun Bee Choi : Data curation, Methodology, Investigation, Writing – original draft, Kwangwook Kim : Methodology, Investigation, Seungyeon Kim : Methodology, Investigation, Seo Young Moon : Methodology, Investigation, Yoo-kyoung Lee : Conceptualization, Funding acquisition. Hyun Ju In : Conceptualization, Data curation, Methodology, Investigation, Funding acquisition. Writing – review & editing. Additional information Competing interests statement: The authors declare no competing interests. References Park, A. Here’s How Effective the Original Vaccines Are Against Omicron. Time https://time.com/6215580/original-vaccines-effectiveness-against-omicron/ (2022). Cankat, S., Demael, M. U. & Swadling, L. 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1","display":"","copyAsset":false,"role":"figure","size":416263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of pan-COVID-19 recombinant vaccine candidates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) Proportions of mutations identified using multiple sequence alignment (MSA). \u003cstrong\u003eb\u003c/strong\u003e) Schematic representation of three recombinant vaccine constructs, including MSA-derived mutations and stability-enhancing features such as S-GSAS/6P and S-R/x2. \u003cstrong\u003ec\u003c/strong\u003e) SDS-PAGE and western blot analyses of recombinant vaccine candidates. M, protein marker; R, reducing condition; NR, non-reducing condition; P, multiple-taq as positive control.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7407141/v1/67079e22493f333b488bee23.png"},{"id":94825708,"identity":"e76be856-ada9-418b-a451-4754d512f171","added_by":"auto","created_at":"2025-10-31 06:50:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":333009,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral immune responses associated with recombinant vaccine candidates in C57BL/6 mice\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) Immunization timeline for assessing binding and neutralizing antibodies. C57BL/6 mice were immunized intramuscularly with the respective candidates or alum. \u003cstrong\u003eb\u003c/strong\u003e) SARS-CoV-2 spike-specific IgG was quantified with diluted serum using enzyme-linked immunosorbent assay (ELISA). The values are presented as the average optical density (OD) at 450 nm. Data are presented as the mean±SEM and were evaluated using one-way ANOVA. \u003cstrong\u003ec\u003c/strong\u003e) Neutralizing antibodies against each variant were measured using a plaque reduction neutralization test with pooled serum samples (\u003cem\u003en\u003c/em\u003e=5/group) collected 1 week after each booster (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7407141/v1/62face9dc9e39d75651a0732.png"},{"id":94786019,"identity":"5a7373c0-fbc8-4aaf-a616-86b6a40397c2","added_by":"auto","created_at":"2025-10-30 16:43:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":366928,"visible":true,"origin":"","legend":"\u003cp\u003eCellular immune responses associated with recombinant vaccine candidates in C57BL/6 mice\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) Immunization timeline for evaluating T-cell responses. \u003cstrong\u003eb\u003c/strong\u003e) Spot counts measured using IFN-γ enzyme-linked immunospot (ELISpot) assay and splenocytes from immunized mice 1 week after the third vaccination (analyzed with an ELISpot plate reader). Representative IFN-γ ELISpot images are shown. \u003cstrong\u003ec\u003c/strong\u003e) IL-2 levels quantified using mouse Quantikine ELISA Kit and supernatants from splenocyte cultures of immunized mice. Data are presented as the mean ± SEM and were evaluated using an unpaired \u003cem\u003et\u003c/em\u003e-test compared with the control group (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7407141/v1/dc8625b790ea6a0d34634c73.png"},{"id":94786020,"identity":"c6133fa7-4051-4701-9e34-082483555ba2","added_by":"auto","created_at":"2025-10-30 16:43:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":580739,"visible":true,"origin":"","legend":"\u003cp\u003eProtective immunity associated with recombinant vaccine candidates in K18-hACE2 transgenic mice\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) K18-hACE2 mice were immunized intramuscularly with the vaccine candidates or alum according to the schedule. Four weeks after the boost, mice were challenged intranasally with 2.6×10\u003csup\u003e5\u003c/sup\u003e PFU of the ancestral strain or 2.9×10\u003csup\u003e5\u003c/sup\u003e PFU of the XBB1.5 strain. \u003cstrong\u003eb\u003c/strong\u003e) Lung tissues were collected, permeabilized, hybridized, and amplified using RNAscope to visualize infection status. \u003cem\u003eIn situ\u003c/em\u003e hybridization scores were based on the percentage of tissue staining at 3 and 5 dpi: 0, no lesion; 1, 2–20%; 2, 21–50%; 3, 51–80%; 4, 81–100%. Representative images (10× magnification) of lung tissue staining. Scale bar: 100 μm. \u003cstrong\u003ec\u003c/strong\u003e) Viral loads in lung tissues 3 and 5 dpi, quantified as RNA copies/mL based on cycle threshold values for the E gene. Data are presented as the mean±SEM and were evaluated using an unpaired \u003cem\u003et\u003c/em\u003e-test compared with the control group (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7407141/v1/5f02f96d6769a57215e92456.png"},{"id":105756061,"identity":"984fa698-6e79-4a02-bef6-6b1075288afc","added_by":"auto","created_at":"2026-03-30 16:34:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2625549,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7407141/v1/8bf57d80-9be5-45d5-8428-5f3df1dfa802.pdf"},{"id":94825163,"identity":"687357b1-d3e7-4e04-bc80-594b65afbdad","added_by":"auto","created_at":"2025-10-31 06:49:55","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1145222,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFinalSubmission0926.docx","url":"https://assets-eu.researchsquare.com/files/rs-7407141/v1/da581bef033ddcc52668a29f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pan-recombinant vaccines based on the consensus sequence of severe acute respiratory syndrome coronavirus-2 spike protein","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoronavirus disease 2019 (COVID-19) persisted as a global pandemic from March 2020 to May 2023. Despite the rapid development of vaccines, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has undergone extensive mutations, producing new variants with diminished sensitivity to early United States Food and Drug Administration-approved vaccines, such as those of Moderna and Pfizer-BioNTech [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although several trials have focused on the development of multivalent vaccine candidates, the continuous evolution of SARS-CoV-2 variants and seasonal coronaviruses necessitates the development of universal vaccines.\u003c/p\u003e\u003cp\u003eMultiple research groups have tested pan-COVID-19 vaccines using nanoparticle (NP) delivery, mosaic antigens, computational antigens, and consensus sequence design [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The decavalent mRNA-LNP vaccine (FLUCOV-10), designed using phylogenetic analysis of sequences from influenza viruses and SARS-CoV-2 variants (ancestral, BQ.1.1, BA.2.75.2, and XBB1.5), elicits robust immune responses and protection against influenza and COVID-19 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A mosaic NP vaccine comprising receptor-binding domains (RBDs) from Omicron (BA.1, BA.2, BA.5, and BA.2.75), Delta, and D614G, fused with highly conserved T-cell epitope sequences derived from sarbecovirus nucleocapsid or spike (S) proteins, exhibits broad cross-protective activity against various SARS-CoV-2 sublineages and enhanced potency compared with a cocktail of NPs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, a universal SARS-CoV-2 subunit vaccine designed using a highly conserved truncated RBD from Omicron BA.1 or the RBDs of Delta, BA.5, and wild-type (WT) induces neutralizing antibodies against Alpha, Beta, Gamma, Delta, Omicron subvariants (BA.1, BA.2, BA.2.75, BA.4.6, and BA.5), and the ancestral strain and prevents Omicron replication and infection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVaccine immunogenicity is commonly enhanced by stabilizing the SARS-CoV-2 surface glycoprotein S in its metastable prefusion conformation and preserving the native positioning of RBDs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. First-generation engineered spike, S-2P, which includes two proline substitutions at positions 986 and 987 [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], has been incorporated into effective vaccines, such as mRNA-1273 and BNT162b2. However, S-2P demonstrates limited yield and thermostability [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. HexaPro, a second-generation spike with four additional proline substitutions (positions 817, 892, 899, and 942), addresses some limitations of S-2P, including improved acquisition rates and thermostability, but exhibits structural instability and transitions to a post-fusion state when exposed to receptor [e.g., angiotensin-converting enzyme 2 (ACE2)] binding, antibody-mediated activation, or protease digestion [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another engineered spike, S-R/x2, retains a trimeric prefusion assembly through disulfide bond formation between residues 413 and 987, which stabilizes the closed conformation of the protein [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite these recent advances, further investigation is required to develop effective and adaptable universal vaccines against emerging SARS-CoV-2 variants and coronaviruses. In this study, we aimed to design three universal recombinant SARS-CoV-2 vaccine candidates derived from a consensus sequence of variants including Alpha, Beta, Gamma, Delta, and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, and BA.5), with specific spike substitutions.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDesign of pan-COVID-19 recombinant vaccines with the consensus sequence of spike protein\u003c/h2\u003e\u003cp\u003eSpike variant counts were distributed unevenly among the Alpha (31.6%), Beta (0.5%), Delta (20.2%), Gamma (2.6%), and Omicron (45.1%) variants. The consensus sequence was defined by the most common amino acid residues (cut-off, 0.6) and included four mutations: D614G, N501Y, DELH69V70, and DELY144. Additional mutations associated with the Alpha variant (B.1.1.7; A570D, T716I, S982A, and D1118H) were incorporated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To increase stability, we designed three pan-COVID-19 vaccine candidates with S-GSAS/6P and S-R/x2 modifications (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). For simplicity, candidates are referred to as G1 (candidate 1), G2 (candidate 2), and G3 (candidate 3). The three candidates were expressed in HEK293 cells and purified using HisTrap FF Crude. The expression of the recombinant proteins was confirmed via SDS-PAGE and western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). G2 expression was lower than G1 and G3 expression; thus, the half concentration (5 \u0026micro;g) was a high dose only in G2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eUniversal vaccine candidates exert humoral immune responses to heterogeneous SARS-CoV-2 variants\u003c/h3\u003e\n\u003cp\u003eThe timeline for evaluating the immunogenicity of the pan-COVID-19 vaccine candidates is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. Groups of C57BL/6 mice were intramuscularly vaccinated with 1 \u0026micro;g of pan-COVID-19 vaccines at 3-week intervals. In mouse sera collected 1 week after each booster, SARS-CoV-2 spike-specific total IgG levels increased, with the low dose of G1 exhibiting the highest responses, followed by G2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The total IgG levels were finally induced at a high dose of G3. Neutralizing antibody responses against WT, Alpha, Beta, Delta, and XBB1.5 strains were assessed using the plaque reduction neutralization test (PRNT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). G1 and G2 elicited stronger neutralizing responses across all strains than G3, which showed no change for the Delta variant, even at a high dose (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). NT\u003csub\u003e50\u003c/sub\u003e values were slightly increased by the second booster and high dose of G3 in variant XBB1.5.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePan-COVID-19 vaccine candidates induced robust cellular immune responses\u003c/h3\u003e\n\u003cp\u003eThe cellular immune response following the second booster vaccination is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. Interferon-γ (IFN-γ) secretion was detected in splenocytes from immunized mice stimulated with peptide pools representing SARS-CoV-2 ancestral, Delta, BA.1, and BA.2 strains, as well as SARS-CoV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Although a trend was observed at low doses, significant IFN-γ induction was observed at high doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Although G3 failed to induce neutralizing antibodies in Delta (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), IFN-γ secretion was induced. In addition, IFN-γ secretion was significantly induced by G1 and G2 in SARS-CoV. The level of cytokines secreted by splenocytes from immunized and stimulated mice was measured to identify antigen-specific T cell responses. Interleukin 2 (IL-2) was strongly induced by G1 and G2 across most strains, whereas G3 exerted no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Tumor necrosis factor-α (TNF-α) levels remained unchanged across groups (Supplementary Figure S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePan-COVID-19 vaccine candidates demonstrated significant protective efficacy\u003c/h3\u003e\n\u003cp\u003eWe immunized and subsequently challenged K18\u0026ndash;human angiotensin-converting enzyme 2 (hACE2) mice with 2.6\u0026times;10\u003csup\u003e5\u003c/sup\u003e plaque-forming units (PFU) of ancestral and 2.9\u0026times;10\u003csup\u003e5\u003c/sup\u003e PFU of XBB1.5 strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and assessed the protective effects using a SARS-CoV-2 RNA probe targeting the spike gene (21,631\u0026ndash;23,303 bp, number of pairs: 20), which helps detect multiple strains, including ancestral, Alpha, Beta, Gamma, and XBB1.5 (Supplementary Figure S3; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The \u003cem\u003ein situ\u003c/em\u003e hybridization scores for pulmonary tissues were dramatically decreased by G1 and G2 in the ancestral strain and G1 of the XBB1.5 strain. The scores were similarly decreased by G2 and G3 over time and with increasing doses. Viral loads in lung tissues 3 and 5 d post-infection (dpi) were substantially reduced with G1 and G2 in the ancestral strain, with a moderate reduction observed in the XBB1.5 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSARS-CoV-2 has accumulated mutations that enhance its transmissibility, virulence, and evasion of neutralizing antibodies [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e–\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These adaptations primarily involve changes in the spike protein, which is responsible for target recognition, binding, and cellular entry [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Multiple sequence alignment (MSA) analysis identified mutations, such as DELH69V70, DELY144, N501Y, A570D, and D614G, in the S1 subunit. This region, comprising the NTD and RBD, facilitates recognition and binding to hACE2 receptors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The D614G mutation in the S1 subunit emerged in mid-2020 and rapidly became dominant [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It was detected in \u0026gt; 95% of SARS-CoV-2 variants sequenced by January 2021, which increased to 99% by March 2022 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. D614G enhances the binding affinity between the S protein and ACE2 by promoting conformational changes that facilitate the RBD-up conformation, achieved by disrupting the D614–K854 salt bridge [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Consequently, the mutation significantly increases SARS-CoV-2 infectivity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The A570D mutation, although less infectious than A570 in the pseudovirus revertant system B.1.1.7 + A570 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], plays a structural role in the S trimer by increasing the spacing between its individual chains, potentially improving furin cleavage accessibility and modulating RBD conformational transitions. These transitions act as a molecular switch, cycling between open and closed states [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe also identified mutations (including T716I, S982A, and D1118H) in the S2 subunit, which contains a fusion peptide (FP), two heptad repeats (HR1 and 2), transmembrane subdomain, and C-terminal tail [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The S2 subunit facilitates membrane fusion, viral entry, cell-to-cell fusion, syncytia formation, and viral dispersion from infected to neighboring cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The S982A substitution disrupts a hydrogen bond with T574, promoting the RBD-up conformation that facilitates binding with ACE2 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To compensate for this structural change, the A570D substitution forms a hydrogen bond with N856 that stabilizes to an RBD-down conformation. Similarly, D1118H compensates for the destabilization of RBD formation caused by T716I. These complementary effects among substitutions within the Alpha SARS-CoV-2 variant collectively promote the stabilization of the prefusion S conformation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We incorporated the four mutations showing ~ 40% concordance in the MSA analysis into the consensus sequence to design the coronavirus vaccine candidates.\u003c/p\u003e\u003cp\u003eThe S1 subunit and its RBD are major targets for neutralizing antibodies, but these regions experience frequent mutations under immune pressure [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Conversely, the S2 subunit, containing conserved elements, such as the FP and HRs, remains relatively stable across variants [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Cross-reactive antibodies targeting conserved regions of the S2 subunit, induced by prior seasonal coronavirus infections (such as human coronaviruses OC43, NL63, and 229E), are boosted following SARS-CoV-2 infection or vaccination [\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e–\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Several S2-specific monoclonal antibodies have exhibited potent neutralizing activity [\u003cspan additionalcitationids=\"CR45 CR46 CR47 CR48\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e–\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Consequently, targetable S2 elements have been widely utilized in the design of pan-coronavirus vaccines [50,51]. For example, the RBD-HR chimeric nanoparticle vaccine induces robust cross-reactive T and B cell immune responses against pseudotyped SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, and RATG13, outperforming the RBD monomer vaccine [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The RBD-HR/trimer vaccine not only elicits strong neutralizing antibodies against ancestral, Alpha, Beta, Delta, and Omicron pseudoviruses but also provides protection against Omicron and Delta challenges [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. S2-only antigens with six proline substitutions (HexaPro), stabilized in prefusion trimers through the introduction of interprotomer disulfide bonds, induce broadly neutralizing responses against pseudotyped ancestral SARS-CoV-2, Omicron BA.1, SARS-CoV, and SHC014 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Immunization with these prefusion-stabilized S2 vaccine candidates conferred complete and partial protection against lethal challenges with SARS-CoV-2 and SARS-CoV, respectively. The superior efficacies of G1 and G2, which included the S2 subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), over G3, which included only S1, in providing protection against SARS-CoV-2 and eliciting both humoral and cellular immune responses (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e–\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) align with the findings of these prior studies.\u003c/p\u003e\u003cp\u003eStudying T cell responses is essential for understanding long-term adaptive immunity [\u003cspan additionalcitationids=\"CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e55\u003c/span\u003e–\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. IFN-γ, a key cytokine released by T cells, enhances antigen presentation, inhibits viral replication, and stimulates inflammatory gene expression [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. IL-2 is critical for the development, proliferation, and maintenance of memory T cells in response to specific antigens [59–61]. Recent studies have reported higher IFN-γ levels in COVID-19 convalescent serum than in serum obtained from the acute disease phase, whereas IFN-γ was not detected in individuals fully vaccinated with SARS-CoV-2 mRNA vaccines [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Cellular IFN-γ and IL-2 levels are reportedly higher in hybrid immune contexts (vaccination following infection) than with vaccination alone, including mRNA-1273 (Moderna) and BNT162b2 (Pfizer-BioNTech) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Conversely, both IFN-γ and IL-2 T cell responses were triggered by SARS-CoV-2 vaccination and increased with disease severity in convalescent individuals, with severe cases exhibiting higher responses than vaccinated individuals. Furthermore, no relationship was observed between T cell responses and time since vaccination. IgG responses demonstrate a moderate correlation with IFN-γ and IL-2 levels in cellular and humoral immunity [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Despite variability in previous findings, memory T cell responses are critical for protection against emerging variants and reinfection, especially when humoral immunity wanes or faces newly emerged variants of concern (VOCs) [66,67]. Our vaccine candidates elicited robust IFN-γ and IL-2 responses without inducing the pro-inflammatory cytokine TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study has certain limitations. First, owing to the unavailability of XBB1.5 peptide pools, BA.1 and BA.2 peptide pools were used for assessing cellular immune responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Second, the serum samples from mice in the same group were pooled for PRNT. Although statistical confirmation is not provided, this should represent the group average as equal specimens were mixed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eAlongside improvements in multivalent vaccines for the rapidly evolving SARS-CoV-2, the development of universal vaccinations is essential to continue protecting the public against emerging variations and seasonal coronaviruses. We created three universal recombinant vaccine candidates based on consensus sequencing of different variants. To preserve the prefusion form of the spike protein and improve immunogenicity, these candidates included the substitutions S-GSAS/6P and S-R/x2. The proposed vaccine candidates elicited robust humoral and cellular immune responses, particularly in formulations containing the S2 subunit, which demonstrated superior protective efficacy over S1-based vaccines. Collectively, our findings provide novel insights into the potential of S2-based pan-COVID-19 vaccines for eliciting broad-spectrum protection against emerging SARS-CoV-2 variants.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eSelection of consensus sequence and design of subunit vaccines\u003c/h2\u003e\u003cp\u003eWe retrieved 894,562 SARS-CoV-2 sequences from the Global Initiative for Sharing All Influenza Data by filtering the conditions “complete,” “high coverage,” and “with patient status.” VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, and BA.5), were aligned with the reference sequence (hCoV-19/Wuhan/WIV04/2019) using the NextAlign tool to identify point mutations and insertion-deletion mutations. MSA analysis was performed with the MAFFT tool (cut-off, 0.6) to generate the consensus sequence. Mutations, including A570D, T716I, S982A, and D1118H, were incorporated into the consensus sequence to design pan-COVID-19 recombinant vaccine candidates. To prevent cleavage at the S1 (receptor-binding)/S2 (membrane fusion) junction and enhance immunogenicity, GSAS (residues 682–685), PRRA deletions (R, residues 681–684), HexaPro (6P), and disulfide 2 (x2) were introduced, generating S-GSAS/6P and S-R/x2. We added the signal peptide (MGWSCIILFLVATATGVHS) at the N-terminus and foldon sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL) at the C-terminus to improve protein secretion and stabilize trimeric structure.\u003c/p\u003e\n\u003ch3\u003eViruses and cells\u003c/h3\u003e\n\u003cp\u003eThe ancestral (NCCP43326), Alpha (GRY clade, B.1.1.7, NCCP43381), Beta (GH clade, B.1.351, NCCP43382), Gamma (GR clade, P.1, NCCP43388), Delta (GK clade, AY.69, NCCP43409), and Omicron (GRA clade, BA.1, NCCP43408; BA.1.1, NCCP43411; BA.2, NCCP43412; XBB.1.5, NCCP43440) SARS-CoV-2 strains were obtained from the National Culture Collection for Pathogens (Cheongju, Republic of Korea) and titrated via plaque assays. All live virus experiments were conducted in a biosafety level 3 facility. Vero E6 African green monkey kidney cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and L-glutamine at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunization of mice\u003c/h2\u003e\u003cp\u003eWe separately divided 5\u0026ndash;7-week-old female C57BL/6 (SAMTACO Bio Korea; Hwaseong, Republic of Korea) and K18\u0026ndash;hACE2 mice (Jackson Laboratory, Farmington, CT, USA) with an average weight of 20\u0026ndash;25 g into the following seven groups: alum, G1 (S-GSAS/6P, 1 and 10 \u0026micro;g), G2 (S-R/x2, 1 and 5 \u0026micro;g), and G3 [N-terminal domain (NTD)-RBD, 1 and 10 \u0026micro;g]. Vaccine candidates, formulated with alum as an adjuvant, were intramuscularly administered thrice at 3-week intervals to groups of C57BL/6 mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5/group). Serum was collected 1 week after each booster and spleens were extracted 1 week post-final vaccination to evaluate humoral and cellular immune responses.\u003c/p\u003e\u003cp\u003eK18\u0026ndash;hACE2 mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5/group) were immunized twice at 3-week intervals via intramuscular injection of the vaccine candidates formulated with alum. At 7 weeks, mice were challenged intranasally with 2.6\u0026times;10\u003csup\u003e5\u003c/sup\u003e PFUs of the ancestral strain and 2.9\u0026times;10\u003csup\u003e5\u003c/sup\u003e PFU of the XBB1.5 strain to evaluate protective immunity. Lung tissues were collected 3 and 5 dpi to measure viral load. All infections and surgical procedures were conducted under anesthesia administered by intraperitoneal injection of a combination of Ketamine (100 mg/kg; Yuhan Corporation, Republic of Korea) and Rompun (10 mg/kg; Elanco, Indianapolis, IN, USA). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Disease Control and Prevention Agency (KDCA) in accordance with the laboratory\u0026rsquo;s animal ethics guideline (KDCA-IACUC-22-032). This study complied with the ARRIVE guidelines during the experiment. All experiments were performed following the relevant guidelines and regulations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePRNT\u003c/h2\u003e\u003cp\u003eVero E6 (2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL/well) were seeded in 12-well plates and infected the next day with a mixture of serially diluted heat-inactivated mouse serum and viruses (50 PFU/well) for 1 h. Cells were cultured with overlay media (1.5% agarose; Lonza, Rockland, ME, USA) in 2\u0026times; MEM supplemented with 4% FBS (Gibco) and incubated for 2 d. Plaques were visualized using crystal violet staining and counted to calculate the neutralization titer (NT\u003csub\u003e50\u003c/sub\u003e) using the K\u0026auml;rber formula. Specimens from mice in the same group were pooled for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-linked immunosorbent assay\u003c/h2\u003e\u003cp\u003eTo quantify SARS-CoV-2-specific total IgG, 96-well plates were coated with 50 ng SARS-CoV-2 Spike S1\u0026thinsp;+\u0026thinsp;S2 ECD\u0026ndash;His recombinant protein (Sino Biological, Beijing, China). Following blocking with 1% bovine serum albumin, serially diluted mouse serum samples were added to the antigen-precoated plates and incubated for 1 h at 37\u0026deg;C. Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, 1:5,000) was used as the secondary antibody and absorbance at 450 nm was measured after adding tetramethylbenzidine substrate (Invitrogen, Carlsbad, CA, USA) and stop solution (GenDEPOT). We used 0.02% Tween-20 in phosphate-buffered saline for washing between steps. Cytokine expression (IL-2 and TNF-α) was quantified using a quantitative sandwich enzyme immunoassay (R\u0026amp;D Systems, Minneapolis, MN, USA) at 450 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eELISpot\u003c/h2\u003e\u003cp\u003eTo evaluate cellular immune responses, IFN-γ secretion was quantified using a mouse IFN-γ ELISpot kit (R\u0026amp;D Systems). Splenocytes were filtered through 50-\u0026micro;m nylon cell strainers (BD Biosciences, San Jose, CA, USA) and digested with ammonium\u0026ndash;chloride\u0026ndash;potassium buffer. We plated 10\u003csup\u003e6\u003c/sup\u003e splenocytes on 96-well polyvinylidene difluoride membrane plates pre-coated with a monoclonal antibody specific for mouse IFN-γ and stimulated with 1 \u0026micro;g/mL PepMix SARS-CoV-2 Spike variants (JPT, Berlin, Germany) for 24 h at 37\u0026deg;C. The following day, cells were discarded through washing and the captured IFN-γ secreted by the stimulated cells was bound to biotinylated anti-IFN-γ mouse antibody. Subsequently, the plate was incubated with alkaline phosphatase-conjugated streptavidin and BCIP/NBT substrate was added to visualize colored spot formation. Washing was performed between each process. The spot counts were measured using an S6 Universal M2 analyzer (ImmunoSpot, Cleveland, OH, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn situ\u003c/b\u003e \u003cb\u003eRNA detection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLung tissues were collected 3 and 5 dpi with SARS-CoV-2 variants (ancestral and XBB1.5). Paraformalin-fixed paraffin-embedded tissues were permeabilized, hybridized, and amplified for \u003cem\u003ein situ\u003c/em\u003e hybridization. The embedded tissues were cut into 5-\u0026micro;m sections using a microtome (Leica Biosystems, Nu\u0026szlig;loch, Germany) and deparaffinized tissues were incubated with hydrogen peroxide for 10 min at 25\u0026deg;C. Following retrieval with Target Retrieval reagent for 15 min at 98\u0026deg;C, tissues were treated with Protease Plus for 30 min at 40\u0026deg;C before probe hybridization. An RNAscope probe targeting the spike gene (Advanced Cell Diagnostics, Newark, CA, USA) was added and allowed to hybridize for 2 h at 40\u0026deg;C. Subsequently, tissues were counterstained with hematoxylin and dehydrated in ethanol and xylene. The extent of viral infection was determined using scores assigned based on labeling extent: 0, no labeling; 1, \u0026le;\u0026thinsp;20% positive; 2, 21\u0026ndash;50% positive; 3, 51\u0026ndash;80% positive; 4, 81\u0026ndash;100% positive.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eReverse transcription-polymerase chain reaction (RT-PCR)\u003c/h2\u003e\u003cp\u003eAfter brief lysis of lung tissues (lysis buffer with 10% proteinase K), the samples were loaded onto a Maxwell RSC cartridge. Viral total RNA was automatically extracted using a Maxwell RSC Viral Total Nucleic Acid Purification Kit in combination with a Maxwell RSC platform (Promega, Madison, WI, USA). Viral loads were quantified by detecting the SARS-CoV-2 E gene using a PowerCheck SARS-CoV-2 RT-PCR Kit (Kogen Biotech, Seoul, Republic of Korea). Real-time RT-PCR (Applied Biosystems, Carlsbad, CA, USA) was performed according to the manufacturer's instructions. Viral load (copies/mL) was calculated using standard curves generated from SARS-CoV-2 transcripts rather than cyclic threshold values.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe mean and standard error of the mean were calculated using Prism 5 (GraphPad Software, San Diego, CA, USA) and compared using an unpaired \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA test followed by Tukey\u0026rsquo;s correction, according to the design of each experiment (shown in figure legends). Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated with asterisks.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis\u0026nbsp;work was supported by a grant from the National Institute of Health (2022-NI-047-00), Republic of Korea.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Author contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYoo Jin Na:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eEun Bee Choi\u003c/strong\u003e: Data curation, Methodology, Investigation, Writing \u0026ndash; original draft, \u003cstrong\u003eKwangwook Kim\u003c/strong\u003e: Methodology, Investigation, \u003cstrong\u003eSeungyeon Kim\u003c/strong\u003e: Methodology, Investigation, \u003cstrong\u003eSeo Young Moon\u003c/strong\u003e: Methodology, Investigation, \u003cstrong\u003eYoo-kyoung Lee\u003c/strong\u003e: Conceptualization, Funding acquisition. \u003cstrong\u003eHyun Ju In\u003c/strong\u003e: Conceptualization, Data curation, Methodology, Investigation, Funding acquisition. Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests statement: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePark, A. Here\u0026rsquo;s How Effective the Original Vaccines Are Against Omicron. \u003cem\u003eTime \u003c/em\u003ehttps://time.com/6215580/original-vaccines-effectiveness-against-omicron/ (2022).\u003c/li\u003e\n\u003cli\u003eCankat, S., Demael, M. U. \u0026amp; Swadling, L. In search of a pan-coronavirus vaccine: next-generation vaccine design and immune mechanisms. \u003cem\u003eCell. Mol. 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We designed and evaluated the efficacy of three universal SARS-CoV-2 recombinant vaccine candidates based on a consensus sequence from multiple variants. The candidates were designed with specific substitutions, including S-GSAS/6P and S-R/x2, to enhance immunogenicity by stabilizing the prefusion spike-protein conformation. Using murine models, we assessed humoral and cellular immune responses, protective efficacy, and viral load reduction following immunization and challenge with the ancestral and XBB1.5 strains. The pan-COVID-19 vaccine candidates stimulated the production of cytokines, specifically interferon-γ and interleukin-2, and induced neutralizing antibodies targeting the ancestral, Delta, and Omicron variants. Vaccination reduced viral replication rates in pulmonary tissues of mice infected with ancestral and XBB1.5 variants; however, their protective efficacy varied among formulations. Candidates containing the S2 subunit elicited better immune responses and protective effects than those containing solely the S1 subunit, consistent with published evidence supporting the cross-reactivity of the S2 region. The proposed vaccine candidates represent promising broad-spectrum protective agents against rapidly mutating SARS-CoV-2 variants.\u003c/p\u003e","manuscriptTitle":"Pan-recombinant vaccines based on the consensus sequence of severe acute respiratory syndrome coronavirus-2 spike protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 16:43:42","doi":"10.21203/rs.3.rs-7407141/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-07T06:11:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-02T21:49:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T19:25:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-29T22:54:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161967972077223100789944201795140897549","date":"2025-10-29T15:35:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25780285558222089508034843745302940381","date":"2025-10-24T21:20:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T09:20:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156277168384474772441637901498102797045","date":"2025-10-21T10:02:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30367589843066976289687431869320046797","date":"2025-10-21T03:08:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171005495830978238823024413600614899971","date":"2025-10-20T19:50:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-20T19:46:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T20:08:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-29T17:55:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-26T09:17:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-26T09:09:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ed9a4ca-c6cd-4b63-991f-a30f179c4d46","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57121612,"name":"Biological sciences/Immunology"},{"id":57121613,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-30T16:31:23+00:00","versionOfRecord":{"articleIdentity":"rs-7407141","link":"https://doi.org/10.1038/s41598-026-45295-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-25 16:10:10","publishedOnDateReadable":"March 25th, 2026"},"versionCreatedAt":"2025-10-30 16:43:42","video":"","vorDoi":"10.1038/s41598-026-45295-6","vorDoiUrl":"https://doi.org/10.1038/s41598-026-45295-6","workflowStages":[]},"version":"v1","identity":"rs-7407141","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7407141","identity":"rs-7407141","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}