Rational Design of a Modular mRNA Vaccine Platform for Rapid Adaptation to SARS-CoV-2 Variants

Rational Design of a Modular mRNA Vaccine Platform for Rapid Adaptation to SARS-CoV-2 Variants | 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 Rational Design of a Modular mRNA Vaccine Platform for Rapid Adaptation to SARS-CoV-2 Variants Julia Rudert, Julia Volckmar, Andreas Jeron, Maike Bälkner, Pia Grimpe, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7726990/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The ongoing emergence of novel SARS-CoV-2 variants due to viral mutations poses a persistent challenge to the efficacy of existing vaccines. To address this challenge, we engineered and comprehensively tested three optimized mRNA vaccine candidates, evaluating the kinetics, quality, and magnitude of antibody responses as well as antigen-specific T cell immunity during a prime-boost vaccination regimen in mice. Among the tested candidates, TP2A encoding secreted receptor-binding domains (RBDs) derived from SARS-CoV-2 wild type, Delta and Omicron variants demonstrated superior immunogenicity, inducing an early and robust IgG2a-dominated antibody response against distinct SARS-CoV-2 spike protein variants. In addition, TP2A elicited IFN-γ-producing T cells in both spleen and draining lymph nodes and antigen-specific cytotoxic T lymphocytes. Notably, beyond the broad immunity induced by the vaccine, TP2A functions as a modular platform, thus enabling flexible antigen assembly and rapid vaccine adaptation to newly emerging variants or even other viral pathogens. These findings position TP2A as a promising next-generation mRNA vaccine candidate. Biological sciences/Computational biology and bioinformatics Biological sciences/Immunology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Since its emergence in 2019, numerous mRNA vaccines against the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) have been licensed for emergency use and are now available worldwide. These vaccines are mainly constructed based on the spike (S) glycoprotein of the earliest Wuhan-Hu-1 strain, with a decreasing trend of immunoprotection against the newly emerging variants 1 , 2 . Thus, the continuous evolution of SARS-CoV-2 challenges existing vaccines with both immune escape and decreasing protection efficacy 3 . For instance, the mutations in the receptor-binding domain (RBD) of the currently circulating SARS-CoV-2 Omicron variants have dramatically reduced vaccine efficacy 4 – 6 . Thus, there is still a need for the design of improved broad-spectrum, scalable mRNA vaccines. The most crucial part of mRNA vaccines are the antigens they encode. The SARS-CoV-2 S protein, a type I membrane protein, forms a trimer that is anchored to the viral membrane 7 . It undergoes structural rearrangements to promote membrane fusion upon binding to the ACE2 receptor on target cells 8 . The full-length S protein consists of various domains, including the N-terminal domain (NTD), RBD, and C-terminal domains (CTD1 and CTD2) in the S1 fragment, and fusion-related domains in the S2 fragment. The NTD and in particular the RBD regions induce robust neutralizing antibody responses 9 , 10 . Licensed vaccines from Moderna and Pfizer-BioNTech use the S-2P antigen containing two proline substitutions to stabilize the prefusion conformation and thus to maintain the three-dimensional structure of the S protein, resulting in enhanced humoral immunity 11 , 12 . However, targeting the RBD and NTD of the S glycoprotein has been reported to be superior compared to S-2P mRNA vaccines 13 , 14 . Also, it has been shown that the HexaPro S protein structure containing six prolines (S-6P) substituted for amino acids in specific positions exhibits higher stability and enhanced immunogenicity than the S-2P variant at lower doses of COVID-19 mRNA vaccines 15 – 17 . To date, most studies have focused on improving the conformational stability of the S protein (e.g., HexaPro mutations) 16 , optimizing RBD trimer splicing strategies 18 , or inducing broadly acting immunity by tendering broad-spectrum epitopes 19 . However, these strategies still face challenges due to the high mutation rate in certain SARS-CoV-2 strains, the occurrence of hyper-mutated variants, antigenic folding interference, decreased 3D conformational stability, and immune competition 20 , 21 . In order to tackle these challenges, we designed three novel mRNA vaccine candidates focusing on the S protein’s key immunodominant regions RBD and NTD. One of these candidates - the XBB-S6P - was designed following the established HexaPro strategy as internal reference control. In vivo prime-boost vaccination experiments in mice revealed that the mRNA construct TP2A, which encodes RBDs from three SARS-CoV-2 variants designed to be secreted as individual proteins, elicited the most robust and broad humoral and cellular immune response, even outperforming the currently favored HexaPro vaccination strategy. These findings highlight TP2A´s promise as a next-generation broad-spectrum modular mRNA vaccine candidate, which convinces with its modular system that can be quickly adapted for future SARS-CoV-2 variants. Results Rational design of novel SARS-CoV-2 mRNA vaccine candidates To improve S protein-specific mRNA vaccine efficacy we designed three novel SARS-CoV-2 mRNA vaccine candidates containing different Open Reading Frames (ORFs) targeting different antigens, optimized untranslated regions (UTRs) and poly A tails (Fig. 1 A). The TP2A candidate’s ORF encodes RBDs from SARS-CoV-2 wild type (WT), Delta, and the Omicron XBB 1.5 variant connected with a P2A peptide linker for cleavage during translation. A human tissue plasminogen activator signal peptide (tPA-SP) coding sequence was added at the 5’ end of each RBD to facilitate protein secretion. The TPOM´s ORF consists of three subunits: i) the NTD selected through homologous sequence alignment of the WT and Omicron strains, showing the highest sequence similarity and being rich in B cell epitopes; ii) the RBD from the Omicron XBB.1.5 strain; iii) a predicted B cell epitope from the S1/S2 cleavage region and the S2 fusion subunit of S proteins, connected to a pan-DR T helper-Cell epitope (PADRE, AKFVAAWTLKAAA) by a GGC linker. These sequences were connected by a flexible linker consisting of three Gly-Gly-Gly-Gly-Ser repeats (G4S) 3 . Additionally, as for TP2A, a tPA-SP coding sequence was incorporated at the N-terminus to facilitate protein secretion. The XBB-S6P´s ORF encodes Omicron XBB.1.5 S-6P glycoprotein containing the mutations K986P, V987P, F817P, A892P, A899P and A942P, according to the previously described HexaPro strategy 16 , 22 . To enhance the expression of the respective mRNA encoded antigens in target cells, we selected for all three constructs UTRs that were previously reported to be predominantly expressed in antigen presenting cells 23 . Of note, we made further improvements in 3’UTR (sequence is shown in Table S1 ). To each mRNA vaccine candidate, an identical 110-nucleotide poly(A) tail was added. The overall concept for the mRNA design is summarized in Fig. 1 B. Sequences were synthesized by GenScript as DNA template and mRNAs for TP2A, TPOM, and XBB-S6P were synthesized by co-transcription using the CleanCap AG from TriLink Biotechnologies with the N1-Me-Pseudouridine substitution method. mRNA integrity and size were evaluated by agarose gel electrophoresis. A distinct single band corresponding to the expected transcript length was observed for each construct, indicating successful transcription with minimal degradation (Supplementary Fig. 1). To predict the protein conformation of the TP2A, TPOM, and XBB-S6P encoded antigens, we employed the AlphaFold 3 database 24 . P2A is known for its high ribosomal skipping efficiency, enabling the co-translational production of three separate proteins from a single open reading frame. This process typically leaves a short peptide remnant at the C-terminus of the upstream protein and an additional proline residue at the N-terminus of the downstream protein 25 – 27 . SignalP 6.0 analysis revealed that an N-terminal proline residue, resulting from P2A cleavage, does not impair signal peptide function (Supplementary Fig. 2). Therefore, TP2A translation would result in the production of three secreted proteins, two of which (RBD-WT and RBD-Delta) retaining a residual P2A peptide sequence (GSGATNFSLLKQAGDVEENPG) at their C-terminus. Since P2A is located at the bottom of the RBD domain, it would not alter the RBD conformation according to the AlphaFold 3 predicted structure (Fig. 1 C). TPOM encodes an entirely newly designed fusion protein, with i) and iii) subunits predominantly being localized at the lower portion of ii) subunit (Fig. 1 D). The protein encoded by XBB-S6P would exhibit a structure similar to that of the previously reported HexaPro protomer 16 . According to the structure prediction, the S protein protomer would be anchored to the cell membrane and spontaneously assemble into trimeric structures (Fig. 1 E). TP2A and XBB-S6P induce robust humoral and cellular immune responses following prime-boost vaccination in mice To first evaluate the nature and strengths of antibody responses elicited by the newly designed mRNA vaccine constructs, BALB/c mice were immunized intramuscularly (i.m.) three times with LNP-formulated mRNAs encoding either TP2A, TPOM, or XBB-S6P on days 0, 14, and 28. As controls, mice were treated with an unrelated LNP-formulated firefly-luciferase (FLUC) mRNA or vehicle only (PBS). Serum samples were collected one day prior to each vaccination and two weeks after the final vaccination (Fig. 2 A and B). Quantification of antibody responses by ELISA revealed that in contrast to the two other candidates, already a single immunization with the TP2A vaccine elicited high WT S-specific IgG titers (Fig. 2 C). While considerable WT S-specific IgG titers were obtained following the first and second booster immunization with XBB-S6P, they remained significantly lower compared to that observed after prime-boost vaccination with the TP2A mRNA (Fig. 2 C). Of note, TPOM failed to induce any detectable WT S-specific IgG responses. Interestingly, XBB.1.5 S-specific IgG response followed a similar pattern. Here, both the XBB-S6P and TP2A mRNA candidates induced an equally strong antibody response upon prime-boost vaccination, which was however delayed for the XBB-S6P mRNA vaccine (Fig. 2 D). Again, as for the WT S protein, no XBB.1.5 S-specific IgG response was observed following TPOM vaccination (Fig. 2 D). Determination of antibody subclasses revealed prime-boost immunization with TP2A and XBB-S6P mRNA to generally induce higher levels of IgG2a than IgG1 (Fig. 2 E and 2 F), which was highly significant for TP2A (**** p < 0.0001). This implies that especially the TP2A construct preferentially induces a Th1-dominated immune response, which is known to promote antiviral immunity. To validate the Th1-polarization elicited by the different mRNA candidates, IFN-γ ELISpot assays were performed using leukocytes isolated from spleen, inguinal lymph nodes (iLN) and popliteal lymph nodes (pLN). Re-stimulation was done with WT or XBB.1.5 S protein or a peptide mix containing 53 15-mer peptides with an 11 amino acid overlap covering the RBD of the XBB.1.5 S protein (Fig. 3 ). This peptide pool contained the G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y and Y505 mutations of the lineage B.1.1.529 (XBB.1.5 Omicron). Compared to the respective control groups, the number of IFN-γ producing splenocytes, representing a systemic proxy of vaccination-induced T cells, was significantly increased following XBB-S6P mRNA vaccination and re-stimulation with WT S protein (Fig. 3 B). Similar results were obtained upon re-stimulation with the XBB.1.5 S protein, with the addition that TPOM vaccinated mice also showed significant spot-forming cell (SFC) counts (Fig. 3 B). Re-stimulation with the XBB.1.5 S protein RBD peptide pool induced a far stronger (~ 20-fold) IFN-γ response in splenocytes for all three mRNA candidates tested, with TP2A inducing equally high numbers of IFN-γ producing cells as TPOM (Fig. 3 C). Taken together, prime-boost vaccination with TP2A, TPOM and XBB-S6P mRNA vaccines induces a systemic Th1 cell response in mice. We further quantified the numbers of IFN-γ producing leukocytes in inguinal and popliteal lymph nodes draining the site of mRNA vaccine application. Interestingly, as for the spleen, re-stimulation with WT or XBB.1.5 S protein revealed significant numbers of IFN-γ producers following XBB-S6P and, in contrast to the spleen, as well for TP2A immunization (Fig. 3 D, E). However, apart from XBB.1.5 S protein re-stimulation of popliteal lymph nodes, no IFN-γ response was detectable in TPOM immunized mice. Taken together, the TP2A mRNA candidate induces the strongest and fastest IgG antibody response comprising both Th1-related IgG2a and Th2-related IgG1 antibody subclasses, with a significant bias towards Th1 immunity (Fig. 2 ). Both, TP2A and XBB-S6P efficiently induce IFN-γ producing lymphocytes. Interestingly, while compared to TP2A, the XBB-S6P construct induced a delayed yet efficient humoral immune response, TPOM completely failed to induce spike-specific IgG (Fig. 2 ). This is contrasted by the presence of IFN-γ producing cells observed in splenic leukocytes from TPOM vaccinated mice, demonstrating its capacity to induce cellular immunity (Fig. 3 C). TP2A and TPOM elicit robust cytotoxic CD8⁺ T Cell responses Next, we sought to investigate the ability of each vaccine construct to induce functional antigen-specific CD8⁺ T cell responses in vivo . To this end, we tested immunized BALB/c mice for the presence of cytotoxic CD8 + T cells, recognizing the H-2D d -restricted peptide epitope CGPKKSTNL derived from the RBD of the XBB.1.5 S protein (Supplementary Fig. 3). This peptide was previously described by Muraoka and colleagues 28 . To determine, whether antigen-specific CD8⁺ T cells were induced upon vaccination and were also functionally competent, we conducted an in vivo cytotoxic T lymphocyte (CTL) assay using adoptive transfer of 5-(6)-Carboxyfluorescein succinimidyl ester (CFSE)-labeled target cells prior pulsed with the CGPKKSTNL peptide (Fig. 4 A, B). Mice vaccinated three times with TP2A and TPOM exhibited a marked antigen-specific killing activity, with an average of 58.5% and 57.7% specific target cell lysis, respectively. XBB-S6P immunized mice, however, showed far less pronounced cytotoxicity, which did not exceed background level. Notably, the specific lysis efficiency for TPOM and TP2A increased steadily over the course of three immunizations, whereas this was not observed for the XBB-S6P mRNA candidate. These results demonstrate that prime-boost vaccination with LNP-formulated TP2A and TPOM induces the expansion of antigen-specific CD8⁺ T cells and primes them to exert effective cytolytic activity in vivo . Collectively, our results show that among the three candidates tested the TP2A construct induces both quantitatively and functionally the most potent S protein-specific immune response which comprises the rapid induction of WT and Omicron S protein-specific antibody responses, a Th1-mediated cellular immune response favorable to antiviral immunity, and the effective induction of S protein-specific cytotoxic CD8 + T cells. Unlike conventional RBD fusion constructs, the TP2A design enables multiple distinct RBDs to be independently expressed and presented to the immune system, resulting in a robust and diverse immune activation. Such broad humoral and cellular immune response is considered critical for effective viral clearance and long-term protection against divergent SARS-CoV-2 variants. Materials and methods mRNA vaccine design and synthesis Three different recombinant SARS-CoV-2 mRNA vaccine candidates (TP2A, TPOM, and XBB-S6P) were generated, each containing distinct antigen ORFs, 5’ and 3’ untranslated regions, and a poly(A) tail. The FLUC ORF was used as a negative control (sequence provided in Table S1 ). The SARS-CoV-2 variants included in this study are the wild type (hCoV-19/Croatia/ZG-297-20/2020, GISAID database ID: EPI_ISL_451934), Delta (B.1.617.2 lineage, GenBank: OK091006.1), and Omicron (XBB.1.5, GenBank: OQ164410.1). All sequences were preceded by a T7 promoter (TAATACGACTCACTATA), inserted into the pUC57 vector, and synthesized by GenScript. mRNAs were produced using the HiScribe® T7 High Yield RNA Synthesis Kit (New England BioLabs, E2040S) following the manufacturer’s protocol, with some modifications. In each reaction, uridine was replaced with N1-methylpseudouridine (m1𝚿) (Jena Bioscience, Germany). Additionally, CleanCap ® AG (TriLink Biotech, USA) was added to the 20-µl reaction at a final concentration of 6 mM. After transcription, DNase I was added for 15 minutes, followed by purification using lithium chloride (LiCl). The RNA concentration was then measured using a Nanodrop. Protein structure prediction Protein structure prediction was performed using AlphaFold3 through AlphaFold Server ( doi.org/10.1038/s41586-024-07487-w ). The default parameters were used and predicted structure was visualized and analyzed using ChimeraX 1.9. RNA electrophoresis RNA samples (1 µg) were mixed with 2×RNA Gel Loading Dye (Thermo Scientific, USA), then loaded onto a 1% agarose gel and run at 120 V for 40 minutes in 3-(N-morpholino) propanesulfonic acid (MOPS) buffer. RNA bands were visualized under UV illumination using an Intas GelStick Imager (Intas Science Imaging Instruments GmbH, Göttingen, Germany) to assess RNA integrity. Lipid nanoparticle–mRNA preparation The mRNA nanoparticles were prepared as previously described with some modifications 23 . In brief, SM-102 (MCE, 251104), DSPC (MCE, 261435), cholesterol (Sigma-Aldrich, 57-88-5), and DMG-PEG2000 (Avanti, 880151p-1g-A-025) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. mRNA was prepared in 50 mM citrate buffer (pH 4.0) at a concentration of 0.17 mg/mL. The lipid and mRNA solutions were mixed at an N/P ratio of 6:1 using the NanoAssemblr Ignite microfluidic platform (Precision Nanosystems) with a total flow rate of 12 mL/min and a flow rate ratio of 3:1 (aqueous phase: organic phase). The resulting LNP-formulated mRNA samples were dialyzed against PBS (pH 7.4) for 24 hours using dialysis bags (Viskase, USA) and subsequently stored at 4°C until use. The encapsulation efficiency of mRNA was determined following the protocol provided with the Quant-iT RiboGreen RNA Assay Kit (Invitrogen, USA) and quantified using a microplate reader (FLUOstar Omega, BMG, Germany). Mice Female BALB/cJRj mice were purchased at an age of about 8 weeks from Janvier (France) and housed under specific pathogen-free conditions according to national and institutional guidelines. All animal experiments were performed in accordance with the ARRIVE guidelines and approved by the local government agency (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit; file number 33.19-42502-04-23-00353). Immunization protocol Mice were anesthetized by inhalation of isoflurane and immunized intramuscularly with LNP-formulated mRNA of the three different vaccine candidates: TP2A, TPOM, or XBB-S6P (2 µg/mouse). LNP-formulated FLUC mRNA and PBS were used as controls. Each mouse was immunized with 2 µg of LNP-formulated RNA diluted in a total volume of 30 µl PBS, administered intramuscularly on days 0, 14, and 28 (Fig. 2 B). Anesthesia and euthanasia For immunization and blood collection on days − 1, 13 and 27, mice were briefly anesthetized with isoflurane inhalation using a small animal anesthesia system for mice (XGI-8 Gas Anesthesia System, Caliper). Euthanasia was carried out by intraperitoneal (i.p.) injection of an anesthetic overdose of ketamine (200 mg/kg body weight) and xylazine (20 mg/kg body weight). Death was confirmed by the onset of respiratory arrest, after which terminal cardiac blood collection was performed. This method ensures rapid loss of consciousness and deep anesthesia, thereby minimizing pain and distress prior to death. Sample collection Blood samples were collected from the retro-orbital sinus on days − 1, 13 and 27 and by cardiac puncture on day 42. Sera for the analysis of antigen-specific IgG and IgG subclasses (IgG1 and IgG2a) were obtained by incubation of the samples for 30 minutes at 37°C followed by 30 minutes incubation at 4°C and subsequent centrifugation (10 minutes at 420 x g and 4°C). For the isolation of lymphocytes, organs were pooled for the experimental groups, transferred to PBS and passed through a 100 µm cell strainer (BD Biosciences, USA). Erythrocyte lysis was performed by osmotic shock (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.2)) and cells were resuspended in RPMI medium supplemented with 10% v/v FCS, 1% v/v penicillin/streptomycin. Detection of antigen-specific total serum IgG and subclasses Antigen-specific total IgG as well as IgG1 and IgG2c subclasses were determined in serum samples by enzyme-linked immunosorbent assay (ELISA) as previously described 29 . In brief, 96-well Nunc-Immuno MaxiSorp plates (Nunc, Germany) were coated with 1 µg/ml SARS-CoV-2 (2019-nCoV) WT S protein (S1 + S2 ECD, His Tag) (Sino Biological Europe GmbH, Deutschland) or SARS-CoV-2 XBB.1.5 (Omicron) S protein (S1 + S2 ECD, His Tag) (Sino Biological Europe GmbH, Deutschland) in 0.05 M carbonate buffer (pH 9.6). After overnight incubation, differential washing steps and blocking, serial 2-fold dilutions of sera in 3% BSA/PBS were added. Antibody binding was detected using biotin-conjugated goat α-mouse IgG (Sigma, Germany), rabbit α-mouse IgG1 (Rockland Immunochemicals, USA) or rat α-mouse IgG2a (BioLegend, USA) antibodies (1 hour, 37°C), respectively, and streptavidin-HRPO (BD Biosciences, Germany) (30 minutes, 37°C). Endpoint titers were expressed as the reciprocal value of the last serum dilution, which yielded an absorbance two times above the values of negative controls. ELISPOT assay Enzyme-linked immunosorbent spot (ELISPOT) kits for the detection of murine IFN-γ (BD Biosciences, Germany) were used according to the manufacturer’s instructions. In brief, isolated lymphocytes pooled for the experimental groups were cultured in the presence of 10 µg/ml SARS-CoV-2 (2019-nCoV) S protein (S1 + S2 ECD, His Tag) (Sino Biological Europe GmbH, Germany) or SARS-CoV-2 XBB.1.5 (Omicron) S protein (S1 + S2 ECD, His Tag) (Sino Biological Europe GmbH, Deutschland) or 5 µg/ml PepMix™ SARS-CoV-2 (S-RBD XBB.1.5) (PT Peptide Technologies GmbH, Deutschland) in triplicates (4 x 10 6 or 2 x 10 5 per well) for 18 h (IFN-γ detection). Positive spots were counted with the ImmunoSpot® Analyzer (Cellular Technology Limited, USA) and analyzed using the CTL Switchboard 2.7.2 software 2.7.2 (Cellular Technology Limited, USA). In vivo cytotoxic T cell assay Spleen and lymph node (mandibular, inguinal) cells were isolated from naive donor BALB/cJRj mice. Equal cell numbers were pulsed with 1 µg/ml CGPKKSTNL peptide (Thermo Fisher Scientific, custom peptide synthesis) in serum-free Iscove’s Modified Dulbecco’s Medium (IMDM, Thermo Fisher, USA) for 30 min at 37°C or left untreated. Following staining with either 3 µM CFSE of the peptide-loaded fraction (CFSE high ) or 0.3 µM CFSE of the non-loaded cells (CFSE low ) for 20 min at 37°C by using the CFSE Cell Division Tracker Kit (Biolegend, USA) the CFSE high and CFSE low cell fractions were mixed in a 1:1 cell-to-cell ratio. A total of 2 x 10 7 target cells was injected intravenously into previously vaccinated or PBS control BALB/cJRj mice. After 16 h, the splenocytes of recipient mice were isolated and analyzed by flow cytometry. The percentages of CFSE high and CFSE low in reference to the total CFSE-population (CFSE high + CFSE low ) was determined, respectively. The ratio r [r = % CFSE low / % CFSE high ] and the specific cell lysis L [L = 100 x (1 - (r control / r vaccinated )] was calculated. Data analysis All statistical analyses were conducted using GraphPad Prism V9.0 software. The ANOVA-One way was employed for statistical comparisons between groups. Discussion In this study, three innovative mRNA vaccine constructs were designed and comprehensively characterized in vivo regarding their capacity to induce humoral and cellular immune responses against SARS-CoV-2 S-proteins. Although the TPOM construct integrated multiple common B-cell epitope regions, experimental data showed that it failed to induce detectable humoral immune responses against the SARS-CoV-2 S-protein after immunization. This result suggests that the conformational organization of TPOM may not be conducive to the spatial exposure or conformational maintenance of key RBD epitopes of the S-protein. The RBD structure contains multiple highly conformationally sensitive neutralizing epitopes such as the receptor binding module (RBM), and their correct three-dimensional presentation is crucial for B-cell recognition and the generation of neutralizing antibodies 30 , 31 . If spatial interference or structural folding changes occur between the multi-modal modules of TPOM, it may mask these key epitopes or disrupt their natural conformation, thereby affecting B-cell recognition and the formation of humoral immune responses. An alternative explanation for the observed lack of a humoral response might be that the TPOM encoded protein is not efficiently released from its producing cells. Although the TPOM construct failed to induce significant humoral immunity against RBD (Fig. 2 ), enhanced T-cell responses were observed (Fig. 3 , 4 ). This phenomenon suggests that TPOM is more suitable in its design or structural organization for antigen processing and MHC class I/II antigen presentation within the context of CD4⁺ and/or CD8⁺ T cell activation. The XBB-S6P candidate vaccine was designed and synthesized based on the HexaPro strategy. Consistent with previous reports 32 , this vaccine design can elicit a strong humoral and cellular immune response, especially showing significant immune efficacy against the Omicron variant. However, it failed to induce cytotoxic T cells (Fig. 3 B), which can be considered a major disadvantage when it comes to sterilizing immunity. Compared to the other two candidates, the TP2A mRNA vaccine with the P2A segmentation strategy showed optimal performance in both inducing humoral and cellular immune responses. Our data shows a high TP2A-induced IgG2a/IgG1 ratio, suggesting a Th1-driven humoral immune response (Fig. 2 E, F). In line with this, the ELISpot data also demonstrate the generation of antigen-specific IFN-γ-secreting lymphocytes (Fig. 3 ), further corroborating a TP2A-induced Th1 response, an immune profile, which is in favor of supporting an antiviral mode of action. Although the IFN-γ cell response against the S protein induced by TP2A was somewhat weaker compared to that induced by XBB-S6P vaccination, TP2A very effectively promoted the expansion of antigen-specific cytotoxic CD8⁺ T cells (Fig. 4 B), a functional advantage that clearly prioritizes TP2A over XBB-S6P as the vaccine candidate inducing the broadest spectrum of humoral and cellular immune responses. The efficacy of TP2A is attributed to some innovative features. First, the TP2A construct utilizes the self-shearing mechanism of P2A peptide to generate three independent SARS-CoV-2 RBD proteins during mRNA translation 26 , 33 , 34 . Despite the P2A peptides retained at the C-terminus of the RBD, our computational predictions revealed that these residues disrupt neither the spatial conformation nor the antigenicity of the RBD. In agreement with this notion, TP2A efficiently induced robust immune responses in vivo . Second, we added tPA-SP upstream to each RBD to improve release of antigen to the extracellular space. Several mRNA vaccine approaches including bivalent and trivalent chimeric S constructs 35 – 37 have demonstrated the feasibility of combining S components from multiple coronaviruses within a single mRNA formulation. The RBD trimeric antigenic structure widely used in the current literature 38 – 40 , although enhancing immunogenicity, is structurally prone to misfolding, aggregation 41 , 42 or epitope masking problems 43 , limiting its broad applicability. To our knowledge, no studies have implemented this innovative design of an independently configured, multi-modular antigen construct to date. Our strategy solves the issue of conformational interference and immune competition caused by conventional RBD trimer splicing. It does not only enhance antigenic stability, but can also cover a broader spectrum of mutations, providing a more flexible update path for future vaccines to face continuously mutating viruses. Previous reports clearly indicate that Th1-type immune responses and activation of multifunctional T cells play a critical role in defense against SARS-CoV-2 infection and cannot be ignored in vaccine-induced long-term protection 44 , 45 . Therefore, we believe that TP2A has the potential to be a next-generation mRNA vaccine candidate and deserves to be further evaluated for its protective effects in subsequent studies. These should include (1) viral neutralization experiments to clarify its functionality for humoral immunity, (2) validation in non-human primate models to assess its protective efficacy and safety, and (3) cross-reactivity analyses against different variants to confirm its broad-spectrum immunity potential. In summary, we designed a novel mRNA vaccine that performed well in inducing both humoral and cellular immunity, providing a valuable experimental basis for the iterative upgrading and multi-variant adaptation. As TP2A features RBDs of multiple widely spread variants, it is designed to have cross-immunization potential and can be expected to be applicable as a “modular platform” to deal with emerging variants (e.g., JN.1 or CH.1.1, etc.) in the future. This modular expression framework may be broadly applicable to other RNA viruses characterized by high genetic variability, such as influenza and dengue. Declarations Author contributions Jo.R., J.V., D.B., and X.D. coordinated the project. X.D., Ju.R., J.V. and A.J. conceived and designed the experiments. X.D., Ju.R., J.V., P.G., D.Z., G.B.B. and M.B., performed the experiments. X.D., Ju.R., J.V., and A.J., acquired, analyzed, and interpreted the data. X.D., A.J., Ju.R., G.B.B. and D.B. wrote the manuscript. Jo.R. and D.B. acquired funding. All authors read, reviewed and approved the final manuscript. Acknowledgements This project was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF, Grant No. 03VP10060, Zell-Trans) to Jo.R., and a grant from the European Union (Project “VIROSTAT”) to D.B. We would like to express our gratitude to Daniela Sebah for her assistance with reagent ordering and procurement. We thank Tatjana Hirsch for expert technical assistance and the VMED team at Helmholtz Centre for Infection Research for advice during the application process for animal testing permit. Funding Declaration This project was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF, Grant No. 03VP10060, Zell-Trans) to Joseph Rosenecker, and a grant from the European Union (Project “VIROSTAT”) to Dunja Bruder. Competing interests The authors declare no conflicts of interest. Data availability The authors declare that the data supporting the findings of this study are available within this article and its supplementary information. Source data are provided with this paper. Ethics approval The experiments were approved by the federal ethical bodies and were carried out in accordance with the guidelines of the Lower Saxony State Office for Consumer Protection and Food Safety (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit; file number 33.19-42502-04-23-00353). Corresponding authors Correspondence to Xiaoyan Ding, Joseph Rosenecker or Dunja Bruder. References Wack, S., Patton, T. & Ferris, L. K. 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Painter, M. M. et al. Rapid induction of antigen-specific CD4 + T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination. Immunity 54 , 2133–2142e3 (2021). Scaglione, A. et al. Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity. Front Immunol 12 , (2021). Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":716949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic structure of the three mRNA vaccine candidates and predicted conformation of their encoded proteins. (A)\u003c/strong\u003e Schematic drawing of the three SARS-CoV-2 mRNA vaccine candidates. Untranslated regions (UTRs) were incorporated upstream and downstream of the antigens of interest. All uridine residues in the mRNAs were replaced by N1-methylpseudouridine. tPA-SP: tissue plasminogen activator signal peptide; P2A: porcine teschovirus-1 2A. \u003cstrong\u003e(B)\u003c/strong\u003e Overview of RNA design and synthesis strategy. \u003cstrong\u003e(C, D and E)\u003c/strong\u003e Predicted structure and conformation of the \u003cstrong\u003e(C)\u003c/strong\u003e TP2A, \u003cstrong\u003e(D)\u003c/strong\u003e TPOM, and \u003cstrong\u003e(E)\u003c/strong\u003e XBB-S6P encoded protein. The black arrow points to relevant domains of the protein structures, including the RBDs, epitopes, and fusion peptides.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7726990/v1/7e9e078e0d772aa5a94a4119.png"},{"id":94597693,"identity":"89cc80d9-1707-4e1a-8c22-6b91c5f2e4c5","added_by":"auto","created_at":"2025-10-28 18:48:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrime-boost vaccination with TP2A and XBB-S6P but not TPOM mRNA induces WT and XBB.1.5 S protein-specific antibody responses.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic presentation of LNP mRNA vaccine formulation. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic presentation depicting immunization schedule and sample collection (image created with Biorender). \u003cstrong\u003e(C and D)\u003c/strong\u003e Total IgG titers against WT S or XBB.1.5 S protein. \u003cstrong\u003e(E and F)\u003c/strong\u003e Subclass analysis of IgG titers against WT S or XBB.1.5 S protein. Mice were immunized with LNP-encapsulated mRNA constructs encoding TP2A, TPOM, or XBB-S6P. Sera were collected on day -1, 13, 27 and 42 and analyzed by ELISA. S-specific total IgG, IgG1 and IgG2a levels were measured using serial dilutions against recombinant WT and XBB.1.5 S proteins. Results represent mean ± SEM (n = 10 mice/group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7726990/v1/9ca7d5393de8c293eebbd2e0.png"},{"id":94597690,"identity":"bcccdaf0-3fcf-4ae8-aaf1-ee036abec237","added_by":"auto","created_at":"2025-10-28 18:48:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrime-boost vaccination with TP2A and XBB-S6P mRNA induces WT and XBB.1.5 S protein-specific IFN-γ response. \u003c/strong\u003eSplenocytes and lymph node cells were isolated from immunized mice at day 14 after final vaccination and stimulated \u003cem\u003ein vitro\u003c/em\u003ewith recombinant WT or XBB.1.5 S proteins or XBB.1.5 RBD peptide mix. IFN-γ secretion was quantified by ELISpot. Data represent spot-forming cells (SFCs) per 10⁶ input cells. Data are expressed as means ± SEM of triplicates of pooled groups from one or two experiments (n = 5 / 10 mice/group). Statistical significance assessed by one-way ANOVA followed by Tukey’s multiple comparisons test (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7726990/v1/1cd11cfa85cc96b8ad0b3977.png"},{"id":94599032,"identity":"e7441a2b-6c6a-45de-98c7-035f292d2d75","added_by":"auto","created_at":"2025-10-28 19:02:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTP2A and TPOM prime-boost vaccination elicits antigen-specific cytotoxic T cells response.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Representative FACS histograms of peptide-loaded CFSE\u003csup\u003ehigh\u003c/sup\u003e and reference CFSE\u003csup\u003elow\u003c/sup\u003e cell populations. Numbers represent percent of CFSE\u003csup\u003ehigh/low\u003c/sup\u003e cells. \u003cstrong\u003e(B)\u003c/strong\u003e Antigen-specific lysis of peptide-loaded CFSE\u003csup\u003ehigh\u003c/sup\u003e target cells. BALB/c mice (n = 8 mice/group) were immunized once, twice or three times with LNP-formulated TP2A, TPOM, or XBB-S6P mRNA. Control mice received PBS. 9 days after the last immunization, CFSE-stained splenocytes pulsed with XBB.1.5 RBD-derived peptide (CGPKKSTNL) (CFSE\u003csup\u003ehigh\u003c/sup\u003e) or left untreated (CFSE\u003csup\u003elow\u003c/sup\u003e) were injected intravenously at a ratio of 1:1 into immunized recipient mice to determine CTL-mediated specific lysis by flow cytometry. Specific lysis of peptide-loaded CFSE\u003csup\u003ehigh\u003c/sup\u003e cells was calculated in reference to the according CFSE\u003csup\u003elow\u003c/sup\u003e population and the PBS control. Data represent mean ± SEM. Statistical significance was assessed one-way ANOVA followed by Tukey’s multiple comparisons test (**p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7726990/v1/c28e510338a0bc0bd2ff3db8.png"},{"id":94600045,"identity":"3b3278ca-34f8-4de8-877e-a9fb36f4bc43","added_by":"auto","created_at":"2025-10-28 19:11:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1951665,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7726990/v1/a68788ce-605c-4a93-84fb-78a6dd8c697e.pdf"},{"id":94597013,"identity":"67f41a2c-e85f-4d83-9f8d-3f2de114d9e6","added_by":"auto","created_at":"2025-10-28 18:45:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":651552,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7726990/v1/92594a0874884bfeb57d7048.pdf"},{"id":94597325,"identity":"ebb53f33-2139-4e2b-b199-52f6077bde74","added_by":"auto","created_at":"2025-10-28 18:46:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12797,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfoFile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7726990/v1/f7a7830789645ff634880a59.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rational Design of a Modular mRNA Vaccine Platform for Rapid Adaptation to SARS-CoV-2 Variants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince its emergence in 2019, numerous mRNA vaccines against the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) have been licensed for emergency use and are now available worldwide. These vaccines are mainly constructed based on the spike (S) glycoprotein of the earliest Wuhan-Hu-1 strain, with a decreasing trend of immunoprotection against the newly emerging variants \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Thus, the continuous evolution of SARS-CoV-2 challenges existing vaccines with both immune escape and decreasing protection efficacy \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For instance, the mutations in the receptor-binding domain (RBD) of the currently circulating SARS-CoV-2 Omicron variants have dramatically reduced vaccine efficacy \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Thus, there is still a need for the design of improved broad-spectrum, scalable mRNA vaccines.\u003c/p\u003e\u003cp\u003eThe most crucial part of mRNA vaccines are the antigens they encode. The SARS-CoV-2 S protein, a type I membrane protein, forms a trimer that is anchored to the viral membrane \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. It undergoes structural rearrangements to promote membrane fusion upon binding to the ACE2 receptor on target cells \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The full-length S protein consists of various domains, including the N-terminal domain (NTD), RBD, and C-terminal domains (CTD1 and CTD2) in the S1 fragment, and fusion-related domains in the S2 fragment. The NTD and in particular the RBD regions induce robust neutralizing antibody responses \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Licensed vaccines from Moderna and Pfizer-BioNTech use the S-2P antigen containing two proline substitutions to stabilize the prefusion conformation and thus to maintain the three-dimensional structure of the S protein, resulting in enhanced humoral immunity \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, targeting the RBD and NTD of the S glycoprotein has been reported to be superior compared to S-2P mRNA vaccines \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Also, it has been shown that the HexaPro S protein structure containing six prolines (S-6P) substituted for amino acids in specific positions exhibits higher stability and enhanced immunogenicity than the S-2P variant at lower doses of COVID-19 mRNA vaccines \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo date, most studies have focused on improving the conformational stability of the S protein (e.g., HexaPro mutations) \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, optimizing RBD trimer splicing strategies \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, or inducing broadly acting immunity by tendering broad-spectrum epitopes \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, these strategies still face challenges due to the high mutation rate in certain SARS-CoV-2 strains, the occurrence of hyper-mutated variants, antigenic folding interference, decreased 3D conformational stability, and immune competition \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In order to tackle these challenges, we designed three novel mRNA vaccine candidates focusing on the S protein\u0026rsquo;s key immunodominant regions RBD and NTD. One of these candidates - the XBB-S6P - was designed following the established HexaPro strategy as internal reference control. \u003cem\u003eIn vivo\u003c/em\u003e prime-boost vaccination experiments in mice revealed that the mRNA construct TP2A, which encodes RBDs from three SARS-CoV-2 variants designed to be secreted as individual proteins, elicited the most robust and broad humoral and cellular immune response, even outperforming the currently favored HexaPro vaccination strategy. These findings highlight TP2A\u0026acute;s promise as a next-generation broad-spectrum modular mRNA vaccine candidate, which convinces with its modular system that can be quickly adapted for future SARS-CoV-2 variants.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eRational design of novel SARS-CoV-2 mRNA vaccine candidates\u003c/h2\u003e\u003cp\u003eTo improve S protein-specific mRNA vaccine efficacy we designed three novel SARS-CoV-2 mRNA vaccine candidates containing different Open Reading Frames (ORFs) targeting different antigens, optimized untranslated regions (UTRs) and poly A tails (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The TP2A candidate\u0026rsquo;s ORF encodes RBDs from SARS-CoV-2 wild type (WT), Delta, and the Omicron XBB 1.5 variant connected with a P2A peptide linker for cleavage during translation. A human tissue plasminogen activator signal peptide (tPA-SP) coding sequence was added at the 5\u0026rsquo; end of each RBD to facilitate protein secretion. The TPOM\u0026acute;s ORF consists of three subunits: i) the NTD selected through homologous sequence alignment of the WT and Omicron strains, showing the highest sequence similarity and being rich in B cell epitopes; ii) the RBD from the Omicron XBB.1.5 strain; iii) a predicted B cell epitope from the S1/S2 cleavage region and the S2 fusion subunit of S proteins, connected to a pan-DR T helper-Cell epitope (PADRE, AKFVAAWTLKAAA) by a GGC linker. These sequences were connected by a flexible linker consisting of three Gly-Gly-Gly-Gly-Ser repeats (G4S)\u003csub\u003e3\u003c/sub\u003e. Additionally, as for TP2A, a tPA-SP coding sequence was incorporated at the N-terminus to facilitate protein secretion. The XBB-S6P\u0026acute;s ORF encodes Omicron XBB.1.5 S-6P glycoprotein containing the mutations K986P, V987P, F817P, A892P, A899P and A942P, according to the previously described HexaPro strategy \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. To enhance the expression of the respective mRNA encoded antigens in target cells, we selected for all three constructs UTRs that were previously reported to be predominantly expressed in antigen presenting cells \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Of note, we made further improvements in 3\u0026rsquo;UTR (sequence is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To each mRNA vaccine candidate, an identical 110-nucleotide poly(A) tail was added. The overall concept for the mRNA design is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Sequences were synthesized by GenScript as DNA template and mRNAs for TP2A, TPOM, and XBB-S6P were synthesized by co-transcription using the CleanCap AG from TriLink Biotechnologies with the N1-Me-Pseudouridine substitution method. mRNA integrity and size were evaluated by agarose gel electrophoresis. A distinct single band corresponding to the expected transcript length was observed for each construct, indicating successful transcription with minimal degradation (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eTo predict the protein conformation of the TP2A, TPOM, and XBB-S6P encoded antigens, we employed the AlphaFold 3 database \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. P2A is known for its high ribosomal skipping efficiency, enabling the co-translational production of three separate proteins from a single open reading frame. This process typically leaves a short peptide remnant at the C-terminus of the upstream protein and an additional proline residue at the N-terminus of the downstream protein \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. SignalP 6.0 analysis revealed that an N-terminal proline residue, resulting from P2A cleavage, does not impair signal peptide function (Supplementary Fig.\u0026nbsp;2). Therefore, TP2A translation would result in the production of three secreted proteins, two of which (RBD-WT and RBD-Delta) retaining a residual P2A peptide sequence (GSGATNFSLLKQAGDVEENPG) at their C-terminus. Since P2A is located at the bottom of the RBD domain, it would not alter the RBD conformation according to the AlphaFold 3 predicted structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). TPOM encodes an entirely newly designed fusion protein, with i) and iii) subunits predominantly being localized at the lower portion of ii) subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The protein encoded by XBB-S6P would exhibit a structure similar to that of the previously reported HexaPro protomer \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. According to the structure prediction, the S protein protomer would be anchored to the cell membrane and spontaneously assemble into trimeric structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTP2A and XBB-S6P induce robust humoral and cellular immune responses following prime-boost vaccination in mice\u003c/h3\u003e\n\u003cp\u003eTo first evaluate the nature and strengths of antibody responses elicited by the newly designed mRNA vaccine constructs, BALB/c mice were immunized intramuscularly (i.m.) three times with LNP-formulated mRNAs encoding either TP2A, TPOM, or XBB-S6P on days 0, 14, and 28. As controls, mice were treated with an unrelated LNP-formulated firefly-luciferase (FLUC) mRNA or vehicle only (PBS). Serum samples were collected one day prior to each vaccination and two weeks after the final vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B).\u003c/p\u003e\u003cp\u003eQuantification of antibody responses by ELISA revealed that in contrast to the two other candidates, already a single immunization with the TP2A vaccine elicited high WT S-specific IgG titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). While considerable WT S-specific IgG titers were obtained following the first and second booster immunization with XBB-S6P, they remained significantly lower compared to that observed after prime-boost vaccination with the TP2A mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Of note, TPOM failed to induce any detectable WT S-specific IgG responses. Interestingly, XBB.1.5 S-specific IgG response followed a similar pattern. Here, both the XBB-S6P and TP2A mRNA candidates induced an equally strong antibody response upon prime-boost vaccination, which was however delayed for the XBB-S6P mRNA vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Again, as for the WT S protein, no XBB.1.5 S-specific IgG response was observed following TPOM vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eDetermination of antibody subclasses revealed prime-boost immunization with TP2A and XBB-S6P mRNA to generally induce higher levels of IgG2a than IgG1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), which was highly significant for TP2A (**** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This implies that especially the TP2A construct preferentially induces a Th1-dominated immune response, which is known to promote antiviral immunity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate the Th1-polarization elicited by the different mRNA candidates, IFN-γ ELISpot assays were performed using leukocytes isolated from spleen, inguinal lymph nodes (iLN) and popliteal lymph nodes (pLN). Re-stimulation was done with WT or XBB.1.5 S protein or a peptide mix containing 53 15-mer peptides with an 11 amino acid overlap covering the RBD of the XBB.1.5 S protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This peptide pool contained the G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y and Y505 mutations of the lineage B.1.1.529 (XBB.1.5 Omicron). Compared to the respective control groups, the number of IFN-γ producing splenocytes, representing a systemic proxy of vaccination-induced T cells, was significantly increased following XBB-S6P mRNA vaccination and re-stimulation with WT S protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similar results were obtained upon re-stimulation with the XBB.1.5 S protein, with the addition that TPOM vaccinated mice also showed significant spot-forming cell (SFC) counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Re-stimulation with the XBB.1.5 S protein RBD peptide pool induced a far stronger (~\u0026thinsp;20-fold) IFN-γ response in splenocytes for all three mRNA candidates tested, with TP2A inducing equally high numbers of IFN-γ producing cells as TPOM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Taken together, prime-boost vaccination with TP2A, TPOM and XBB-S6P mRNA vaccines induces a systemic Th1 cell response in mice. We further quantified the numbers of IFN-γ producing leukocytes in inguinal and popliteal lymph nodes draining the site of mRNA vaccine application. Interestingly, as for the spleen, re-stimulation with WT or XBB.1.5 S protein revealed significant numbers of IFN-γ producers following XBB-S6P and, in contrast to the spleen, as well for TP2A immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). However, apart from XBB.1.5 S protein re-stimulation of popliteal lymph nodes, no IFN-γ response was detectable in TPOM immunized mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTaken together, the TP2A mRNA candidate induces the strongest and fastest IgG antibody response comprising both Th1-related IgG2a and Th2-related IgG1 antibody subclasses, with a significant bias towards Th1 immunity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Both, TP2A and XBB-S6P efficiently induce IFN-γ producing lymphocytes. Interestingly, while compared to TP2A, the XBB-S6P construct induced a delayed yet efficient humoral immune response, TPOM completely failed to induce spike-specific IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This is contrasted by the presence of IFN-γ producing cells observed in splenic leukocytes from TPOM vaccinated mice, demonstrating its capacity to induce cellular immunity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eTP2A and TPOM elicit robust cytotoxic CD8⁺ T Cell responses\u003c/h3\u003e\n\u003cp\u003eNext, we sought to investigate the ability of each vaccine construct to induce functional antigen-specific CD8⁺ T cell responses \u003cem\u003ein vivo\u003c/em\u003e. To this end, we tested immunized BALB/c mice for the presence of cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells, recognizing the H-2D\u003csup\u003ed\u003c/sup\u003e-restricted peptide epitope CGPKKSTNL derived from the RBD of the XBB.1.5 S protein (Supplementary Fig.\u0026nbsp;3). This peptide was previously described by Muraoka and colleagues \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To determine, whether antigen-specific CD8⁺ T cells were induced upon vaccination and were also functionally competent, we conducted an \u003cem\u003ein vivo\u003c/em\u003e cytotoxic T lymphocyte (CTL) assay using adoptive transfer of 5-(6)-Carboxyfluorescein succinimidyl ester (CFSE)-labeled target cells prior pulsed with the CGPKKSTNL peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Mice vaccinated three times with TP2A and TPOM exhibited a marked antigen-specific killing activity, with an average of 58.5% and 57.7% specific target cell lysis, respectively. XBB-S6P immunized mice, however, showed far less pronounced cytotoxicity, which did not exceed background level. Notably, the specific lysis efficiency for TPOM and TP2A increased steadily over the course of three immunizations, whereas this was not observed for the XBB-S6P mRNA candidate. These results demonstrate that prime-boost vaccination with LNP-formulated TP2A and TPOM induces the expansion of antigen-specific CD8⁺ T cells and primes them to exert effective cytolytic activity \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCollectively, our results show that among the three candidates tested the TP2A construct induces both quantitatively and functionally the most potent S protein-specific immune response which comprises the rapid induction of WT and Omicron S protein-specific antibody responses, a Th1-mediated cellular immune response favorable to antiviral immunity, and the effective induction of S protein-specific cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells. Unlike conventional RBD fusion constructs, the TP2A design enables multiple distinct RBDs to be independently expressed and presented to the immune system, resulting in a robust and diverse immune activation. Such broad humoral and cellular immune response is considered critical for effective viral clearance and long-term protection against divergent SARS-CoV-2 variants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003emRNA vaccine design and synthesis\u003c/h2\u003e\u003cp\u003eThree different recombinant SARS-CoV-2 mRNA vaccine candidates (TP2A, TPOM, and XBB-S6P) were generated, each containing distinct antigen ORFs, 5\u0026rsquo; and 3\u0026rsquo; untranslated regions, and a poly(A) tail. The FLUC ORF was used as a negative control (sequence provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The SARS-CoV-2 variants included in this study are the wild type (hCoV-19/Croatia/ZG-297-20/2020, GISAID database ID: EPI_ISL_451934), Delta (B.1.617.2 lineage, GenBank: OK091006.1), and Omicron (XBB.1.5, GenBank: OQ164410.1). All sequences were preceded by a T7 promoter (TAATACGACTCACTATA), inserted into the pUC57 vector, and synthesized by GenScript. mRNAs were produced using the HiScribe\u0026reg; T7 High Yield RNA Synthesis Kit (New England BioLabs, E2040S) following the manufacturer\u0026rsquo;s protocol, with some modifications. In each reaction, uridine was replaced with N1-methylpseudouridine (m1\u0026#120511;) (Jena Bioscience, Germany). Additionally, CleanCap \u0026reg; AG (TriLink Biotech, USA) was added to the 20-\u0026micro;l reaction at a final concentration of 6 mM. After transcription, DNase I was added for 15 minutes, followed by purification using lithium chloride (LiCl). The RNA concentration was then measured using a Nanodrop.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eProtein structure prediction\u003c/h2\u003e\u003cp\u003eProtein structure prediction was performed using AlphaFold3 through AlphaFold Server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1038/s41586-024-07487-w\u003c/span\u003e\u003cspan address=\"10.1038/s41586-024-07487-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The default parameters were used and predicted structure was visualized and analyzed using ChimeraX 1.9.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA electrophoresis\u003c/h3\u003e\n\u003cp\u003eRNA samples (1 \u0026micro;g) were mixed with 2\u0026times;RNA Gel Loading Dye (Thermo Scientific, USA), then loaded onto a 1% agarose gel and run at 120 V for 40 minutes in 3-(N-morpholino) propanesulfonic acid (MOPS) buffer. RNA bands were visualized under UV illumination using an Intas GelStick Imager (Intas Science Imaging Instruments GmbH, G\u0026ouml;ttingen, Germany) to assess RNA integrity.\u003c/p\u003e\n\u003ch3\u003eLipid nanoparticle–mRNA preparation\u003c/h3\u003e\n\u003cp\u003eThe mRNA nanoparticles were prepared as previously described with some modifications \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In brief, SM-102 (MCE, 251104), DSPC (MCE, 261435), cholesterol (Sigma-Aldrich, 57-88-5), and DMG-PEG2000 (Avanti, 880151p-1g-A-025) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. mRNA was prepared in 50 mM citrate buffer (pH 4.0) at a concentration of 0.17 mg/mL. The lipid and mRNA solutions were mixed at an N/P ratio of 6:1 using the NanoAssemblr Ignite microfluidic platform (Precision Nanosystems) with a total flow rate of 12 mL/min and a flow rate ratio of 3:1 (aqueous phase: organic phase). The resulting LNP-formulated mRNA samples were dialyzed against PBS (pH 7.4) for 24 hours using dialysis bags (Viskase, USA) and subsequently stored at 4\u0026deg;C until use. The encapsulation efficiency of mRNA was determined following the protocol provided with the Quant-iT RiboGreen RNA Assay Kit (Invitrogen, USA) and quantified using a microplate reader (FLUOstar Omega, BMG, Germany).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eFemale BALB/cJRj mice were purchased at an age of about 8 weeks from Janvier (France) and housed under specific pathogen-free conditions according to national and institutional guidelines. All animal experiments were performed in accordance with the ARRIVE guidelines and approved by the local government agency (Nieders\u0026auml;chsisches Landesamt f\u0026uuml;r Verbraucherschutz und Lebensmittelsicherheit; file number 33.19-42502-04-23-00353).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImmunization protocol\u003c/h2\u003e\u003cp\u003eMice were anesthetized by inhalation of isoflurane and immunized intramuscularly with LNP-formulated mRNA of the three different vaccine candidates: TP2A, TPOM, or XBB-S6P (2 \u0026micro;g/mouse). LNP-formulated FLUC mRNA and PBS were used as controls. Each mouse was immunized with 2 \u0026micro;g of LNP-formulated RNA diluted in a total volume of 30 \u0026micro;l PBS, administered intramuscularly on days 0, 14, and 28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAnesthesia and euthanasia\u003c/h2\u003e\u003cp\u003eFor immunization and blood collection on days \u0026minus;\u0026thinsp;1, 13 and 27, mice were briefly anesthetized with isoflurane inhalation using a small animal anesthesia system for mice (XGI-8 Gas Anesthesia System, Caliper). Euthanasia was carried out by intraperitoneal (i.p.) injection of an anesthetic overdose of ketamine (200 mg/kg body weight) and xylazine (20 mg/kg body weight). Death was confirmed by the onset of respiratory arrest, after which terminal cardiac blood collection was performed. This method ensures rapid loss of consciousness and deep anesthesia, thereby minimizing pain and distress prior to death.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSample collection\u003c/h2\u003e\u003cp\u003eBlood samples were collected from the retro-orbital sinus on days \u0026minus;\u0026thinsp;1, 13 and 27 and by cardiac puncture on day 42. Sera for the analysis of antigen-specific IgG and IgG subclasses (IgG1 and IgG2a) were obtained by incubation of the samples for 30 minutes at 37\u0026deg;C followed by 30 minutes incubation at 4\u0026deg;C and subsequent centrifugation (10 minutes at 420 x g and 4\u0026deg;C). For the isolation of lymphocytes, organs were pooled for the experimental groups, transferred to PBS and passed through a 100 \u0026micro;m cell strainer (BD Biosciences, USA). Erythrocyte lysis was performed by osmotic shock (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.2)) and cells were resuspended in RPMI medium supplemented with 10% v/v FCS, 1% v/v penicillin/streptomycin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDetection of antigen-specific total serum IgG and subclasses\u003c/h2\u003e\u003cp\u003eAntigen-specific total IgG as well as IgG1 and IgG2c subclasses were determined in serum samples by enzyme-linked immunosorbent assay (ELISA) as previously described \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In brief, 96-well Nunc-Immuno MaxiSorp plates (Nunc, Germany) were coated with 1 \u0026micro;g/ml SARS-CoV-2 (2019-nCoV) WT S protein (S1\u0026thinsp;+\u0026thinsp;S2 ECD, His Tag) (Sino Biological Europe GmbH, Deutschland) or SARS-CoV-2 XBB.1.5 (Omicron) S protein (S1\u0026thinsp;+\u0026thinsp;S2 ECD, His Tag) (Sino Biological Europe GmbH, Deutschland) in 0.05 M carbonate buffer (pH 9.6). After overnight incubation, differential washing steps and blocking, serial 2-fold dilutions of sera in 3% BSA/PBS were added. Antibody binding was detected using biotin-conjugated goat α-mouse IgG (Sigma, Germany), rabbit α-mouse IgG1 (Rockland Immunochemicals, USA) or rat α-mouse IgG2a (BioLegend, USA) antibodies (1 hour, 37\u0026deg;C), respectively, and streptavidin-HRPO (BD Biosciences, Germany) (30 minutes, 37\u0026deg;C). Endpoint titers were expressed as the reciprocal value of the last serum dilution, which yielded an absorbance two times above the values of negative controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eELISPOT assay\u003c/h2\u003e\u003cp\u003eEnzyme-linked immunosorbent spot (ELISPOT) kits for the detection of murine IFN-γ (BD Biosciences, Germany) were used according to the manufacturer\u0026rsquo;s instructions. In brief, isolated lymphocytes pooled for the experimental groups were cultured in the presence of 10 \u0026micro;g/ml SARS-CoV-2 (2019-nCoV) S protein (S1\u0026thinsp;+\u0026thinsp;S2 ECD, His Tag) (Sino Biological Europe GmbH, Germany) or SARS-CoV-2 XBB.1.5 (Omicron) S protein (S1\u0026thinsp;+\u0026thinsp;S2 ECD, His Tag) (Sino Biological Europe GmbH, Deutschland) or 5 \u0026micro;g/ml PepMix\u0026trade; SARS-CoV-2 (S-RBD XBB.1.5) (PT Peptide Technologies GmbH, Deutschland) in triplicates (4 x 10\u003csup\u003e6\u003c/sup\u003e or 2 x 10\u003csup\u003e5\u003c/sup\u003e per well) for 18 h (IFN-γ detection). Positive spots were counted with the ImmunoSpot\u0026reg; Analyzer (Cellular Technology Limited, USA) and analyzed using the CTL Switchboard 2.7.2 software 2.7.2 (Cellular Technology Limited, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ecytotoxic T cell assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSpleen and lymph node (mandibular, inguinal) cells were isolated from naive donor BALB/cJRj mice. Equal cell numbers were pulsed with 1 \u0026micro;g/ml CGPKKSTNL peptide (Thermo Fisher Scientific, custom peptide synthesis) in serum-free Iscove\u0026rsquo;s Modified Dulbecco\u0026rsquo;s Medium (IMDM, Thermo Fisher, USA) for 30 min at 37\u0026deg;C or left untreated. Following staining with either 3 \u0026micro;M CFSE of the peptide-loaded fraction (CFSE\u003csup\u003ehigh\u003c/sup\u003e) or 0.3 \u0026micro;M CFSE of the non-loaded cells (CFSE\u003csup\u003elow\u003c/sup\u003e) for 20 min at 37\u0026deg;C by using the CFSE Cell Division Tracker Kit (Biolegend, USA) the CFSE\u003csup\u003ehigh\u003c/sup\u003e and CFSE\u003csup\u003elow\u003c/sup\u003e cell fractions were mixed in a 1:1 cell-to-cell ratio. A total of 2 x 10\u003csup\u003e7\u003c/sup\u003e target cells was injected intravenously into previously vaccinated or PBS control BALB/cJRj mice. After 16 h, the splenocytes of recipient mice were isolated and analyzed by flow cytometry. The percentages of CFSE\u003csup\u003ehigh\u003c/sup\u003e and CFSE\u003csup\u003elow\u003c/sup\u003e in reference to the total CFSE-population (CFSE\u003csup\u003ehigh\u003c/sup\u003e + CFSE\u003csup\u003elow\u003c/sup\u003e) was determined, respectively. The ratio r [r = % CFSE\u003csup\u003elow\u003c/sup\u003e / % CFSE\u003csup\u003ehigh\u003c/sup\u003e] and the specific cell lysis L [L\u0026thinsp;=\u0026thinsp;100 x (1 - (r\u003csub\u003econtrol\u003c/sub\u003e / r\u003csub\u003evaccinated\u003c/sub\u003e)] was calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were conducted using GraphPad Prism V9.0 software. The ANOVA-One way was employed for statistical comparisons between groups.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, three innovative mRNA vaccine constructs were designed and comprehensively characterized \u003cem\u003ein vivo\u003c/em\u003e regarding their capacity to induce humoral and cellular immune responses against SARS-CoV-2 S-proteins. Although the TPOM construct integrated multiple common B-cell epitope regions, experimental data showed that it failed to induce detectable humoral immune responses against the SARS-CoV-2 S-protein after immunization. This result suggests that the conformational organization of TPOM may not be conducive to the spatial exposure or conformational maintenance of key RBD epitopes of the S-protein. The RBD structure contains multiple highly conformationally sensitive neutralizing epitopes such as the receptor binding module (RBM), and their correct three-dimensional presentation is crucial for B-cell recognition and the generation of neutralizing antibodies \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. If spatial interference or structural folding changes occur between the multi-modal modules of TPOM, it may mask these key epitopes or disrupt their natural conformation, thereby affecting B-cell recognition and the formation of humoral immune responses. An alternative explanation for the observed lack of a humoral response might be that the TPOM encoded protein is not efficiently released from its producing cells. Although the TPOM construct failed to induce significant humoral immunity against RBD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), enhanced T-cell responses were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This phenomenon suggests that TPOM is more suitable in its design or structural organization for antigen processing and MHC class I/II antigen presentation within the context of CD4⁺ and/or CD8⁺ T cell activation.\u003c/p\u003e\u003cp\u003eThe XBB-S6P candidate vaccine was designed and synthesized based on the HexaPro strategy. Consistent with previous reports \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, this vaccine design can elicit a strong humoral and cellular immune response, especially showing significant immune efficacy against the Omicron variant. However, it failed to induce cytotoxic T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which can be considered a major disadvantage when it comes to sterilizing immunity.\u003c/p\u003e\u003cp\u003eCompared to the other two candidates, the TP2A mRNA vaccine with the P2A segmentation strategy showed optimal performance in both inducing humoral and cellular immune responses. Our data shows a high TP2A-induced IgG2a/IgG1 ratio, suggesting a Th1-driven humoral immune response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). In line with this, the ELISpot data also demonstrate the generation of antigen-specific IFN-γ-secreting lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), further corroborating a TP2A-induced Th1 response, an immune profile, which is in favor of supporting an antiviral mode of action. Although the IFN-γ cell response against the S protein induced by TP2A was somewhat weaker compared to that induced by XBB-S6P vaccination, TP2A very effectively promoted the expansion of antigen-specific cytotoxic CD8⁺ T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), a functional advantage that clearly prioritizes TP2A over XBB-S6P as the vaccine candidate inducing the broadest spectrum of humoral and cellular immune responses.\u003c/p\u003e\u003cp\u003eThe efficacy of TP2A is attributed to some innovative features. First, the TP2A construct utilizes the self-shearing mechanism of P2A peptide to generate three independent SARS-CoV-2 RBD proteins during mRNA translation \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Despite the P2A peptides retained at the C-terminus of the RBD, our computational predictions revealed that these residues disrupt neither the spatial conformation nor the antigenicity of the RBD. In agreement with this notion, TP2A efficiently induced robust immune responses \u003cem\u003ein vivo\u003c/em\u003e. Second, we added tPA-SP upstream to each RBD to improve release of antigen to the extracellular space. Several mRNA vaccine approaches including bivalent and trivalent chimeric S constructs \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e have demonstrated the feasibility of combining S components from multiple coronaviruses within a single mRNA formulation. The RBD trimeric antigenic structure widely used in the current literature \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, although enhancing immunogenicity, is structurally prone to misfolding, aggregation \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e or epitope masking problems \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, limiting its broad applicability. To our knowledge, no studies have implemented this innovative design of an independently configured, multi-modular antigen construct to date. Our strategy solves the issue of conformational interference and immune competition caused by conventional RBD trimer splicing. It does not only enhance antigenic stability, but can also cover a broader spectrum of mutations, providing a more flexible update path for future vaccines to face continuously mutating viruses.\u003c/p\u003e\u003cp\u003ePrevious reports clearly indicate that Th1-type immune responses and activation of multifunctional T cells play a critical role in defense against SARS-CoV-2 infection and cannot be ignored in vaccine-induced long-term protection \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Therefore, we believe that TP2A has the potential to be a next-generation mRNA vaccine candidate and deserves to be further evaluated for its protective effects in subsequent studies. These should include (1) viral neutralization experiments to clarify its functionality for humoral immunity, (2) validation in non-human primate models to assess its protective efficacy and safety, and (3) cross-reactivity analyses against different variants to confirm its broad-spectrum immunity potential.\u003c/p\u003e\u003cp\u003eIn summary, we designed a novel mRNA vaccine that performed well in inducing both humoral and cellular immunity, providing a valuable experimental basis for the iterative upgrading and multi-variant adaptation. As TP2A features RBDs of multiple widely spread variants, it is designed to have cross-immunization potential and can be expected to be applicable as a \u0026ldquo;modular platform\u0026rdquo; to deal with emerging variants (e.g., JN.1 or CH.1.1, etc.) in the future. This modular expression framework may be broadly applicable to other RNA viruses characterized by high genetic variability, such as influenza and dengue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJo.R., J.V., D.B., and X.D. coordinated the project. X.D., Ju.R., J.V. and A.J. conceived and designed the experiments. X.D., Ju.R., J.V., P.G., D.Z., G.B.B. and M.B., performed the experiments. X.D., Ju.R., J.V., and A.J., acquired, analyzed, and interpreted the data. X.D., A.J., Ju.R., G.B.B. and D.B. wrote the manuscript. Jo.R. and D.B. acquired funding. All authors read, reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by a grant from the Bundesministerium f\u0026uuml;r Bildung und Forschung (BMBF, Grant No. 03VP10060, Zell-Trans) to Jo.R., and a grant from the European Union (Project \u0026ldquo;VIROSTAT\u0026rdquo;) to D.B. We would like to express our gratitude to Daniela Sebah for her assistance with reagent ordering and procurement. We thank Tatjana Hirsch for expert technical assistance and the VMED team at Helmholtz Centre for Infection Research for advice during the application process for animal testing permit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by a grant from the Bundesministerium f\u0026uuml;r Bildung und Forschung (BMBF, Grant No. 03VP10060, Zell-Trans) to Joseph Rosenecker, and a grant from the European Union (Project \u0026ldquo;VIROSTAT\u0026rdquo;) to Dunja Bruder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within this article and its supplementary information. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were approved by the federal ethical bodies and were carried out in accordance with the guidelines of the Lower Saxony State Office for Consumer Protection and Food Safety (Nieders\u0026auml;chsisches Landesamt f\u0026uuml;r Verbraucherschutz und Lebensmittelsicherheit; file number 33.19-42502-04-23-00353).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Xiaoyan Ding, Joseph Rosenecker or Dunja Bruder.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWack, S., Patton, T. \u0026amp; Ferris, L. K. COVID-19 vaccine safety and efficacy in patients with immune-mediated inflammatory disease: Review of available evidence. \u003cem\u003eJ. Am. Acad. 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Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, (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":"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},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7726990/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7726990/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ongoing emergence of novel SARS-CoV-2 variants due to viral mutations poses a persistent challenge to the efficacy of existing vaccines. To address this challenge, we engineered and comprehensively tested three optimized mRNA vaccine candidates, evaluating the kinetics, quality, and magnitude of antibody responses as well as antigen-specific T cell immunity during a prime-boost vaccination regimen in mice. Among the tested candidates, TP2A encoding secreted receptor-binding domains (RBDs) derived from SARS-CoV-2 wild type, Delta and Omicron variants demonstrated superior immunogenicity, inducing an early and robust IgG2a-dominated antibody response against distinct SARS-CoV-2 spike protein variants. In addition, TP2A elicited IFN-γ-producing T cells in both spleen and draining lymph nodes and antigen-specific cytotoxic T lymphocytes. Notably, beyond the broad immunity induced by the vaccine, TP2A functions as a modular platform, thus enabling flexible antigen assembly and rapid vaccine adaptation to newly emerging variants or even other viral pathogens. These findings position TP2A as a promising next-generation mRNA vaccine candidate.\u003c/p\u003e","manuscriptTitle":"Rational Design of a Modular mRNA Vaccine Platform for Rapid Adaptation to SARS-CoV-2 Variants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-28 18:00:01","doi":"10.21203/rs.3.rs-7726990/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T14:18:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T13:45:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T06:02:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272245892558652904148771658379548419883","date":"2025-10-19T22:17:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99557435353171379070083751541393424818","date":"2025-10-14T18:50:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50617203010421845496591264127437888512","date":"2025-10-14T10:40:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T00:48:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T00:43:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-13T10:27:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-10T16:41:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-10T16:38:06+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":"ea1223e1-c3ca-4031-8bc9-188c7d2ed580","owner":[],"postedDate":"October 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56960317,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":56960318,"name":"Biological sciences/Immunology"},{"id":56960319,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-08T05:10:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-28 18:00:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7726990","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7726990","identity":"rs-7726990","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}