Enhanced stability and efficacy of a lyophilized mRNA SARS-CoV-2 vaccine incorporating novel Ionizable lipids after one year storage at 25ºC

Enhanced stability and efficacy of a lyophilized mRNA SARS-CoV-2 vaccine incorporating novel Ionizable lipids after one year storage at 25ºC | 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 Enhanced stability and efficacy of a lyophilized mRNA SARS-CoV-2 vaccine incorporating novel Ionizable lipids after one year storage at 25ºC Elena Mata, Esther Broset, Carlos Matute, Andrei Mihai Stoian, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5780846/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in npj Vaccines → Version 1 posted 16 You are reading this latest preprint version Abstract mRNA vaccines have shown great efficacy against SARS-CoV-2, yet challenges remain in optimizing vaccine components to achieve enhanced immune response and vaccine stability. In this study, we developed CPVax-CoV, a new lyophilized mRNA vaccine that features novel thiolactone-based ionizable lipids and newly designed untranslated regions (UTRs) for enhanced expression. Incorporation of these optimized components into our vaccine candidate CPVax-CoV significantly improved immune responses in mice compared to commercially available mRNA vaccines. Moreover, lyophilized CPVax-CoV has proven to be thermostable, maintaining its biological activity for up to one year at 4°C and 25°C after lyophilization, overcoming the cold-chain limitations of current mRNA vaccines. This vaccine demonstrates protective efficacy against ancestral SARS-CoV-2 and Omicron XBB variant, offering a scalable solution for global distribution and pandemic preparedness. These findings underscore the potential of this platform for future next-generation mRNA vaccine development. Biological sciences/Immunology/Vaccines/Rna vaccines Biological sciences/Immunology/Infectious diseases/Viral infection Biological sciences/Biotechnology/Applied immunology/Vaccines/Rna vaccines Health sciences/Diseases/Infectious diseases/Viral infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION The outbreak of coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has significantly influenced global health, social dynamics, and economic activity. Vaccination remains the most potent strategy for preventing infection and controlling virus spread. Consequently, considerable efforts have been directed towards the rapid development and approval of various vaccines worldwide 1 . Among these, mRNA technology has led the race of anti-SARS-CoV-2 vaccines and emerged as a promising vaccine platform due to their rapid development, manufacturing versatility, safety profile, and ability to elicit broad immune responses. Unlike traditional platforms, mRNA vaccines offer advantages such as rapid design based solely on sequence information, bypassing the need for virus culture or recombinant protein production. They also circumvent issues like insertional mutagenesis and pre-existing immunity that may hinder other vaccine types such as DNA or viral vector vaccines 2,3 . Approved mRNA vaccines, including BNT162b2/Comirnaty (Pfizer/BioNTech) 4 and mRNA-1273/SpikeVax (Moderna) 5 , have validated the potential of mRNA technology, demonstrating significant success in mitigating viral spread, reducing hospitalizations, and lowering mortality rates since their emergency authorization in 2020 6 Additionally, bivalent mRNA vaccines targeting specific SARS-CoV-2 variants have been approved, providing enhanced protection against emerging strains 7 , and ongoing research is focused on the development of combined vaccines that address both COVID-19 and seasonal influenza 8 . Despite their proven efficacy and rapid development, there is still considerable room for improvement in the field of mRNA vaccine components and stability to enhance protective efficacy against emerging variants or new pathogens and to ensure equal distribution and accessibility worldwide 9–13 . Here we present a new mRNA vaccine platform that incorporates novel optimizations on mRNA sequence design and lipid formulation that is suitable for lyophilization and stable for long term storage. Optimization of mRNA sequence, comprising antigen-coding sequence and non-coding regulatory elements directly impacts protein expression and vaccine efficacy 14 . mRNA vaccines typically comprise a codon-optimized nucleotide sequence encoding the antigen of interest 15 along with other stabilizing elements, including two non-coding regulatory sequences flanking the coding sequence (5'UTR and 3'UTR). Untranslated Regions (UTRs) contain multiple regulatory elements and are essential for mRNA stability and translation efficiency. Here we have incorporated novel UTR sequences optimized for efficient protein expression after intramuscular injection 16 into Spike coding mRNA. Along with mRNA sequence optimization, efficient delivery of mRNA into the cytoplasm of target cells is crucial to exert its therapeutic function. Lipid nanoparticles (LNPs) are currently the leading delivery system for mRNA vaccines. LNPs are commonly composed of 4 lipid components: an ionizable lipid, a helper phospholipid, cholesterol and a PEGylated lipid 17 . These lipids encapsulate the antigen-coding mRNA and protect the nucleic acid from degradation. The ionizable lipid is the most critical component of LNPs, as it determines the efficacy of transfection and endosomal escape. They typically feature a tertiary amine that remains deprotonated at physiological pH, improving the biocompatibility of LNPs, but become positively charged at lower pH conditions. Protonation of ionizable lipids at the endosomal acidic pH promote membrane destabilization and delivery of nucleic acid cargo into the cytosol of target cells 18 . Furthermore, ionizable lipid impacts vaccine adjuvanticity, which directly affect the therapeutic effect of mRNA vaccines 19 . Currently there are only two ionizable lipids (SM-102 and ALC-0315) approved for commercial use as part of mRNA vaccines 20,21 . We recently developed a new library of rationally designed ionizable lipids able to induce high protein expression levels in mice inoculated by the intramuscular route 22,23 , the most common route for administration of mRNA vaccines. However, their applicability as part of mRNA vaccines and their ability to induce immunity has not been addressed yet. In this study we have characterized this new family of ionizable lipids as part of mRNA vaccines against SARS-CoV-2, showing a strong potential for their use in mRNA-LNPs based vaccines. A critical limitation in the global distribution of current mRNA vaccines is their dependence on cold-chain logistics to preserve mRNA integrity and maintain the physicochemical properties of LNPs 24,25 . The need for ultra-cold storage conditions poses a major barrier to ensure higher coverage in resource limited-settings which is essential for ensuring equitable protection. 11 . To address this challenge, lyophilization, or freeze-drying, has long been utilized to stabilize pharmaceuticals and vaccines, prolonging their shelf life and simplifying storage and distribution logistics. Applying this technique to mRNA vaccines holds immense potential to overcome the cold chain requirements associated with traditional liquid formulations, thereby enabling broader global access to advanced immunization technologies, since cryogenic transport and storage are needed for the current licensed mRNA vaccines 26 . In this study, we developed a lyophilized formulation of our candidate mRNA vaccine, CPVax-CoV, which maintains LNP physicochemical properties and biological activity showing comparable results to the standard liquid formulation. COVID-19 crisis underscored the critical need for novel vaccine platforms able to provide rapid and effective solutions in response to future outbreaks and emerging pathogens. Here we have applied innovative optimizations on delivery systems, mRNA sequence design and vaccine stability to develop CPVax-CoV, a novel SARS-CoV-2 mRNA-based vaccine that is thermostable for long-term storage in lyophilized format and elicits robust protective immunity compared to commercially available SARS-CoV-2 vaccines. Importantly, lyophilized CPVax-CoV induced protective immunity in mice challenged with SARS-CoV-2 even after one year of storage at 4 and 25°C, while maintaining intact LNP physicochemical properties. This platform has been validated against ancient and Omicron SARS-CoV-2 variants of concern (VOCs), and in a recently developed mouse model using a mouse adapted SARS-CoV-2 virus that resembles human COVID19 27 . Our findings support the efficacy of CPVax-CoV as a robust and flexible mRNA vaccine platform and provide significant impact on pandemic preparedness, highlighting the potential novel ionizable lipids, UTR sequences and lyophilization technology to be transferred to future mRNA vaccines against infectious diseases. RESULTS Novel ionizable lipid-based mRNA vaccines induce robust anti-SARS-CoV-2 immune response We have recently reported the development of an extensive library of novel ionizable lipids proven to be safe and effective for mRNA expression in vivo , showing enhanced results compared to approved lipid based LNPs 22,23 . Here we have selected the most promising novel ionizable lipids in terms of protein expression after intramuscular administration to evaluate their potential to induce immunity for mRNA vaccine applications (Structures shown in Supplementary Fig. 1.). We incorporated these lipids into LNP formulations to construct various mRNA vaccine candidates, all using the Spike-coding mRNA sequence from the commercial Comirnaty vaccine. Both the mRNA and LNPs were prepared in-house via in vitro transcription and microfluidic mixing, respectively. These vaccine candidates were compared to a control LNP formulation, which replicated the Comirnaty vaccine using ALC-0315 as the ionizable lipid. Thus, ALC-0315 was formulated using Comirnaty lipid components at molar lipid ratios of 46.6:9.4:42.7:1.6 (ALC-0315: DSPC: cholesterol: ALC-0159) and the evaluated candidate lipids were formulated at molar ratios of 50:10:38.5:1.5 (ionizable lipid:DOPE:cholesterol:DMG-PEG2000). Physicochemical characterization of the resulting LNPs showed optimal values of diameter and encapsulation efficiency with no significant differences between the various candidates (Table S1 ). To evaluate the immune response elicited by the different vaccine formulations, BALB/c mice were intramuscularly immunized with 1 µg of mRNA/mice following a 21-day prime-boost regimen and specific B and T cell responses were analyzed (Fig. 1 a). Production of specific anti-RBD IgG antibodies were detected in serum from all the vaccinated animals and they were significantly enhanced after boost immunization. Mice vaccinated with LNPs formulated with our novel ionizable lipids showed comparable antibody levels to mice vaccinated with the control vaccine using ALC-0315 as ionizable lipid. Importantly, antibody levels in mice vaccinated with LNPs containing CP-LC-0729 lipid were significantly higher than in those vaccinated with control vaccine after boost (Fig. 1 b). T-cell response was evaluated 3 weeks post-boost in splenocytes after specific stimulation with SARS-CoV-2 Spike peptide mix. IFNγ-producing cells (Fig. 1 d) and cytokine secretion of IFNγ and IL-4 (Fig. 1 e) were measured by ELISPOT and ELISA respectively, showing T cell induction in all vaccinated animals with strong IFNγ and low IL-4 response, indicating a Th1 biased response. Although all groups exhibited elevated T cell responses, IFNγ production was notably higher in mice vaccinated with CP-LC-0729, CP-LC-0867, CP-LC-0743 or ALC-0315 compared to those vaccinated with CP-LC-0431 or CP-LC-0474 based vaccines. Taken together, these results demonstrate that LNPs formulated with our proprietary novel ionizable lipids exhibit robust induction of both B and T cell responses in vivo , providing evidence for their potential use in next generation mRNA vaccines against SARS-CoV-2 and, potentially, other pathogens. CP-LC-0729 lipid was chosen to be further characterized in the next assays as it exhibits superior antibody response after boost compared to the evaluated candidates and the control vaccine. mRNA sequence optimization enhances S-specific B and T-cell responses Despite their significant impact on therapeutic protein expression, current mRNA vaccines candidates often utilize 5′ and 3′UTRs derived from commonly used human genes with minimal optimization. We have previously explored novel UTRs sequences following a semi-rational RNA sequence design approach leading to the discovery of new RNA motifs that enhance mRNA expression in vivo 16 . These optimized UTR sequences, when incorporated into mRNA, significantly improve protein production and outperform UTR designs used in commercially approved mRNA vaccines. Herein, we designed a codon-optimized mRNA sequence based on full length Spike protein of the Wuhan SARS-CoV-2 virus incorporating our proprietary UTR sequences and we tested its efficacy as part of a new mRNA vaccine candidate. First, we evaluated Spike in vitro expression in HEK293T to verify antigen production of our newly developed mRNA Spike sequence based on Wuhan strain (CP-S) by Western Blot and flow cytometry (Figure S1 ). Then, we encapsulated Comirnaty or CP-S mRNA in LNPs formulated with CP-LC-0729 lipid at molar lipid ratios of 40.7:34.9:23.3:1.2 (CP-LC-0729:DOPE:cholesterol:DMG-PEG2000). These specific molar ratios were chosen based on our previous optimization studies of the LNP formulation for CP-LC-0729, which demonstrated enhanced intramuscular mRNA expression using these lipid ratios 23 . In order to evaluate vaccine immunity in vivo , BALB/c mice were immunized in a 21-day prime-boost regimen. Serum antibody responses were analyzed at different time-points and T-cell response was evaluated in spleen 3 weeks post-boost (Fig. 2 a). Anti-RBD total IgG antibody levels in serum were 2.5-fold higher in the animals vaccinated with LNPs formulated with CP-S mRNA compared to the ones encapsulating Comirnaty mRNA or the control vaccine after the first immunization. After the second immunization, these differences increased significantly, showing 5-fold and 4.3-fold higher titers in the CP-S mRNA group (Fig. 2 b). Neutralizing antibodies were analyzed through a pseudovirus-based assay using GFP-expressing lentivirus pseudotyped with SARS-CoV-2 Spike protein from Wuhan strain. Neutralizing titer 50 (NT50) post-boost was comparable in all the experimental groups and slightly higher, although not significantly, in serum from mice vaccinated with CP-LC-0729 encapsulating CP-S mRNA, in accordance with the results of total anti-RBD binding IgG titers (Fig. 2 c). Specific T cell response was analyzed in splenocytes at 3 weeks post-boost. Cytokine secretion after specific stimulation showed enhanced production of IFNy in mice vaccinated with CP-LC-0729 LNPs compared with the cell samples from animals immunized with the control vaccine. Notably, this difference was significantly higher in CP-LC-0729 LNPs encapsulating CP-S mRNA, both in terms of IFNy secreting splenocytes (Fig. 2 e) and total IFNy secretion (Fig. 2 f). T cell populations in spleen were evaluated by flow cytometry, showing higher levels of total CD4 T cells in mice vaccinated with CP-LC-0729 LNPs encapsulating CP-S mRNA. No significant differences were found in total CD8 T cell levels between the evaluated vaccines (Fig. 2 g). To further characterize the immune response triggered by these mRNA vaccine candidates, we evaluated early germinal center (GC) B cell and T helper follicular (Thf) cell responses in the draining lymph nodes. Thf are critical for GC formation, which directly impacts B-cell differentiation and long-lived plasma cell expansion. Early Thf and GC responses have been previously related to protective humoral immunity in mRNA vaccines 28 . BALB/c mice were immunized with a single dose of 5 µg mRNA/mouse. One-week post-prime Thf and GC responses were evaluated in the draining lymph nodes by flow cytometry (Fig. 2 h). As shown in Fig. 2 I, significant expansion of GC B cells and Thf cells populations was observed in the vaccinated animals compared to the non-vaccinated controls. This result correlates with a robust antibody specific response previously observed in the serum analysis (Fig. 2 b). In summary, these data indicate that our optimized mRNA sequence enhances the immune response induced by our novel lipid-based vaccine, underscoring the importance of UTR optimization in the development of more effective mRNA vaccines. In base of these results, we defined our final vaccine candidate, CPVax-CoV, that incorporates novel ionizable lipid CP-LC-0729 and CP-S mRNA sequence. Our results indicate that this novel vaccine candidate induces robust immune B and T cell immune responses and outperforms Comirnaty vaccine. CPVax-CoV biodistribution and preliminary safety evaluation Safety is an essential requisite for any newly developed vaccine. LNPs, especially ionizable lipids, are frequently identified as a safe concern in mRNA vaccines, since they exhibit adjuvant properties and are determinant for the pharmacokinetics and biodistribution profile of mRNA vaccines 29,30 . Consequently, special attention needs to be addressed to the tolerability and safety profile of novel ionizable lipids as part of LNPs-based therapies. We have preliminarily evaluated our ionizable lipid candidate CP-LC-0729 in mice after systemic administration of high dose of FLuc mRNA-LNPs by intravenous injection, showing a good safety profile with no signs of hepatic or systemic toxicity 23 . To further characterize the safety and pharmacokinetics (PK) profile of CPVax-CoV vaccine, we carried out a biodistribution study to track LNPs accumulation and elimination in vivo . LNPs were labeled with indium-111 oxinate and intramuscularly injected in male and female BALB/c mice. Images were acquired using microSPECT/CT at 1, 3 and 6 h and 1, 2, 3, 4 and 7 days post-injection. The visual analysis of the images showed a strong signal at the injection site, but LNPs signal was also found in the sacral ganglion chain, bone marrow (long bone heads/iliac), and kidneys (elimination pathway) (Fig. 3 a). Maximum signal intensity at the injection site decreased over time, but persisted for at least 4 days, reflecting a primarily distribution of LNPs at the injection site within the first 24 hours post-administration (Fig. 3 b). Draining through the retroperitoneal and medial sacral lymph nodes was observed in all animals starting from 1 h post-administration, with signal intensity peaking at 6 h and gradually decreasing thereafter (Fig. 3 c). At 6 h post-administration, the appearance of signal in the bone marrow was visually detected, peaking approximately at 1–2 days post-injection (Figure S5). Ex vivo analysis on day 7 supported the in vivo imaging data, showing clear drainage through the lymph nodes when normalized by organ weight (Fig. 3 d). Migration to draining lymph nodes is compatible with the observed Thf cell expansion, GC formation and immune response elicited by vaccination, the signal observed in kidneys is compatible with urine excretion and no accumulation in liver or other organs was detected. To corroborate vaccine tolerability, hepatic enzyme levels in serum were evaluated 24 h post-vaccination. No increment in AST and ALT levels were observed compared to the serum analysis data pre-vaccination (Fig. 3 E). These data provide preliminary evidence of vaccine safety and support CPVax-CoV for further evaluation as mRNA vaccine. CPVax-CoV induces protection against SARS-CoV-2 challenge To evaluate vaccine-induced protection of CPVax-CoV against SARS-CoV-2, we conducted protection experiments in vivo and validated the results in two different mouse infection models: one using a mouse adapted SARS-CoV-2 strain and the other using a hACE2-K18 transgenic mice. First, a mouse-adapted SARS-CoV-2 strain (MA20) was used to infect wild type BALB/c mice. MA20 was obtained from a clinical isolate of the SARS-CoV-2 Alpha variant (Pango lineage Nomenclature B.1.1.7) after 20 serial passages in one-year-old C57BL/6 mice. MA20 is able of inducing severe lung pathology in mice and recapitulates key pathological features of COVID-19 in humans including higher severity in males, lymphodepletion and increase in inflammatory cytokine response and acture respiratory distress syndrome (ARDS) 27 . BALB/c female mice were immunized in a 21-day prime-boost regimen with 1 µg of mRNA/animal of CPVax-CoV and the control vaccine. Four weeks post-boost mice were intranasally infected with MA20. Survival, viral load and cytokine profile in lungs were determined (Fig. 4 A). All vaccinated animals survived over a 25-day period after virus challenge (endpoint of the experiment) without showing weight loss or clinical signs of disease (Fig. 4 B-C). In contrast, unvaccinated control mice showed severe weight loss and were euthanized on day 4 post-infection according to previously established human endpoint criteria (Fig. 4 B-C). In accordance with survival outcomes, infecting virus were detected by TCID50 assay in lungs from unvaccinated mice but not in lungs from vaccinated mice at day 2 or 4 post-challenge, except for one mouse in the control vaccine group, where low viral load was detected (Fig. 4 D). Inflammatory virus-associated cytokine and chemokine markers were analyzed in lung homogenates using a multiplex assay as a complementary indicator of vaccine-induced protection. These cytokines are typically associated to virus-induced inflammation as well as lung damage and correlate with disease progression 32 . Cytokine and chemokine levels were significantly incremented in the unvaccinated compared to the vaccinated animals, especially at day 2 post-infection. These cytokines were not upregulated in the lungs from the vaccinated groups in any of the evaluated time-points after virus challenge (Fig. 4 E). The results obtained with the virus adapted strain were validated in a transgenic C57BL/6 hACE2-K18 mouse model infected with an Alpha variant SARS-CoV-2 strain (Fig. 5 A). The K18-hACE2 model of SARS-CoV-2 infection has been previously described as closely mimicking many aspects of severe COVID-19 and has been widely used in the evaluation of vaccines and antiviral therapies 33 . In accordance with the results for the mouse-adapted strain, no weight loss or clinical signs of disease were observed in vaccinated animals during the course of the experiment, in contrast with the non-vaccinated controls (Fig. 5 B). Lung viral load was not detected in lungs from any of the vaccinated groups at day 4 post-infection (Fig. 5 C). In addition, no significant increment in lung inflammatory cytokines and chemokines was detected in the vaccinated mice, in contrast with the non-vaccinated controls (Fig. 5 D). Collectively, these data reveal that CPVax-CoV exhibit a strong vaccine-induced protection against viral SARS-CoV-2 challenge in wild type and ACE-2 humanized mouse models. CPVax-CoV platform validation for SARS-CoV-2 Variants of Concern Despite the remarkable efficacy demonstrated by SARS-CoV-2 vaccines, the emergence of novel variants poses a persistent challenge, compromising the level of protection conferred by current immunization strategies. As the virus continues to evolve, these variants exhibit diverse genetic mutations that may impact vaccine effectiveness. This underscores the need for continuous surveillance and adaptation of vaccination strategies to address the evolving challenge posed by these variants. To validate the versatility of CPVax-CoV vaccine platform to be adapted to emerging variants of concern, we developed a vaccine candidate against Omicron XBB1.5 variant using CPVax-CoV technology. We chose XBB lineage since it was one of the most recently emerged and prevalent variants at the time these experiments were conducted 34,35 . XBB variants exhibited superior growth advantages over most omicron mutants and have shown a strong evasive ability against the neutralization of plasma and serum from vaccinated or convalescent individuals 36,37 . We first verified antigen expression in vitro of the new XBB mRNA after HEK293T transfection by Western Blot and Flow Cytometry and we confirmed that Spike XBB mRNA was correctly translated in eukaryotic cells (Fig S1 ). Then, XBB and Wuhan mRNAs were formulated into LNPs with CPVax-CoV lipid composition resulting in CPVax-CoV-XBB vaccine, which exhibited comparable physicochemical properties to that of CPVax-CoV (Table SX). Immunity of these vaccines against ancient strain and XBB variant was evaluated in BALB/c mice following a 21-day prime-boost vaccination (Fig. 6 A). To evaluate variant-specific antibody neutralizing ability, an XBB Spike pseudovirus expressing GFP was constructed. Infection ability of XBB-Pseudovirus was confirmed in Vero E6-ACE2-TMPRSS2 cells (Figure S3). Serum neutralization was determined after boost immunization against both Wuhan and XBB variants and neutralization titers were found to be variant specific. No neutralization was observed in serum from CPVax-CoV-XBB against the ancient Wuhan variant. Conversely, NT50 of Wuhan Spike-based vaccines were not detected against XBB variant (Fig. 6 B). Spleen specific T-cell response was also evaluated at week 6 post prime. Variant selectivity was lower for specific-T cell response than that observed for antibody-response. IFN-y secreting cells were detected in splenocytes from all vaccinated animals after stimulation with specific peptides mixes of Wuhan or XBB Spike variants (Fig. 6 C). A strong cross-reactive T cell response against various SARS-CoV-2 VOCs has been previously reported, highlighting the importance of robust T-cell induction in SARS-CoV-2 immunization strategies 38 . Finally, we evaluated vaccine-induced protection of our XBB based-vaccine candidate. Infection kinetics of XBB strain in hACE2-k18 and BALB/c mouse models was first evaluated to choose the optimal infection model for the vaccine efficacy study. In contrast to ancient strains, no weight loss or clinical signs of disease were detected over the first week of infection. However, infecting viral particles were present in lungs of infected mice in both mouse models, although virus titers were higher in hACE2-K18 mice (Fig S5), which was selected as infection model for XBB strain. These pathological outcomes are in accordance with previous results observed in rodent infection models of SARS-CoV-2 for XBB.1 stain 39,40 . K18 mice were immunized with CPVax-CoV and CPVax-CoV-XBB following a 21-day prime-boost regimen (Fig. 6 D). Serum neutralization titers against XBB-PSV were analyzed before infection. Neutralization against XBB Pseudovirus was not detected for the CPVax-CoV (Fig. 6 E), showing comparable results to those obtained in the BALB/c mouse model (Fig. 6 B). Protective efficacy was evaluated by determination of viral load in lungs. Despite the differences observed between Wuhan and XBB Spike based vaccines in serum neutralizing ability, no infecting viruses were detected in lungs from any vaccinated mice (Fig. 6 F). Accordingly, no weight loss was observed in any of the vaccinated groups after challenge (Figure S5). These results demonstrate the potential of the CP vaccine platform to be effectively used with different antigen sequences for the development of SARS-CoV-2 vaccines against VOCs. Notably, the CPVax-CoV wild type vaccine also provided significant protection against XBB variant, highlighting its broad efficacy. Lyophilized CPVax-CoV exhibits long-term thermostability Thermostability and storage requirements are major factors limiting universal access to RNA vaccines. The two currently licensed mRNA vaccines, Comirnaty and Spikevax, require cryogenic transport and storage at − 80°C to − 60°C and − 20°C, respectively 41 . Consequently, developing thermostable mRNA vaccines is crucial to ensure accessibility in medium and low-income settings. To address this challenge, we have developed a lyophilized formulation for CPVax-CoV vaccine that maintains LNPs physicochemical properties and effectively preserves vaccine induced immune response after long-term storage at 4°C and 25°C. To this end, we employed an optimized lyophilization method based on a previously described protocol​ 42 . Furthermore, a long-term stability study was conducted for up to one year, demonstrating that the lyophilization process effectively preserved the characteristics and in vivo functionality of LNPs under both refrigerated and ambient temperature conditions throughout the study period, without the need of freezing. To evaluate the long-term thermostability and biological efficacy of our lyophilized CPVax-CoV formulation, lyophilized CPVax-CoV LNPs from the same production batch were stored at 4 and 25°C for up to one year. Non-lyophilized CPVax-CoV and Comirnaty vaccines stored at -80°C were included as experimental controls as the standard conditions used for the long-term storage of LNP based vaccines 43 . At various time points throughout this time-period, we assessed the physicochemical properties of the LNPs, including particle size, zeta potential, polydispersity index (PDI) and encapsulation efficiency, alongside their in vivo biological activity. BALB/c mice were immunized via a single intramuscular injection at each indicated time point using the same dosage as in the non-lyophilized vaccine formulation (1 µg of mRNA per animal). Serum samples were collected 21 days post-vaccination for specific antibody quantification. After 12 months of storage, a protective efficacy study was also conducted to assess the vaccine's ability to confer protection against SARS-CoV-2 challenge in the mouse model (Fig. 7 a). As shown in Fig. 7 b, the size of the LNPs increased slightly after lyophilization, a result that is consistent with previous reports in the literature 44 . PDI, RNA encapsulation efficiency, and zeta potential showed no significant deviations between the lyophilized and non-lyophilized LNPs. Over the course of the one-year storage period, these parameters remained largely constant under the tested conditions. Lyophilized CPVax-CoV stored at 4°C maintained their initial particle size for up to one year after lyophilization, while LNPs stored at 25°C exhibited a slight increase in size after six months, which then stabilized and remained consistent for up to one year. In line with the physicochemical characterization results, the lyophilized CPVax-CoV vaccine maintained robust in vivo biological activity throughout the entire storage period, irrespective of the storage temperature. IgG anti-RBD levels remained consistently high across all experimental groups at each evaluated time point. Importantly, no significant differences were observed between the controls stored at -80°C and the lyophilized CPVax-CoV stored at 4°C or 25°C. Notably, the lyophilized vaccine stored at 25°C successfully induced specific antibody levels that were comparable to those of the − 80°C stored controls after one year, demonstrating outstanding preservation of immunogenicity (Fig. 7 c). These results underscore the long-term thermostability of our lyophilized CPVax-CoV formulation, highlighting its potential for effective deployment in diverse temperature conditions without compromising efficacy. In addition to assessing the long-term stability of the lyophilized CPVax-CoV, we evaluated the short-term stability of the resuspended lyophilized vaccine, a critical aspect for practical medical applications. To this end, the reconstituted CPVax-CoV was stored at 4°C for up to 96 h, during which we monitored the physicochemical properties of the LNPs and their in vivo biological activity. Our analysis revealed that the LNP size and encapsulation efficiency remained consistent throughout the storage period, showing no significant deviations (Fig. 7 d). Furthermore, the vaccine's ability to induce specific antibody production in mice was unaffected, with no significant differences observed between the freshly resuspended vaccine and the one stored for 96 h (Fig. 7 e). These findings underscore the potential utility of lyophilized CPVax-CoV in clinical settings. Lyophilized CPVax-CoV maintains protective efficacy after one year at 25°C Protection induced by lyophilized CPVax-CoV after one year storage at 4 and 25°C was evaluated in the mouse model. BALB/c mice were vaccinated in a prime boost regimen and challenged with MA20 mouse adapted SARS-CoV-2 strain 27 . In addition to the one year stored lyophilized and control vaccines, freshly prepared CPVax-CoV and Comirnaty vaccines were included in this study as additional controls. Serum neutralization was evaluated at 3 weeks post-boost and viral load, cytokine production and histopathology were evaluated in lungs at day 3 post-infection. Prior to virus challenge, anti-RBD IgG antibodies (Figure S8) and serum neutralization were evaluated (Fig. 8 a), showing high binding and neutralizing antibody titers with no significant differences between lyophilized vaccines stored at 4 and 25 ºC and freshly formulated LNPs. No weight loss or clinical signs of disease progression were observed in any of the vaccinated groups (Fig. 8 b). Accordingly, infecting virus in lungs were only detected in the non-vaccinated animals in the TCID50 assay (Fig. 8 c). These results are consistent with the cytokine profile observed in lung homogenates, since the inflammatory cytokines and chemokines evaluated were significantly higher in lungs from unvaccinated mice and no significant differences in cytokine levels were found between mice vaccinated with controls and 4 and 25°C lyophilized CPVax-CoV (Fig. 8 d). Finally, we validated our lyophilization technology to be applied to different mRNA payloads and developed a lyophilized CPVax-CoV-XBB. Physicochemical parameters of the lyophilized CPVax-CoV-XBB were comparable to those obtained with CPVax-CoV (Table S2). Lyophilized CPVax-CoV-XBB and CPVax-CoV protective efficacy was evaluated in the humanized K18 mouse model after infection with XBB or alpha SARS-CoV-2 strains respectively (Fig. 8 f). In line with the results obtained in the mouse-adapted infection model vaccinated with the Wuhan based Spike sequence, no viral load in lungs were detected in K18 mice immunized with lyophilized CPVax-CoV-XBB or CPVax-CoV at day 3 after infection with their respective viral strains (Fig. 8 f). Collectively, these findings demonstrate that lyophilized CPVax-CoV, stored at both 4°C and 25°C for up to one year, provides robust and consistent protection against SARS-CoV-2 infection in mice. Furthermore, this lyophilization technology proved to effectively maintain the efficacy of our LNP formulations with different mRNA spike sequences, demonstrating its adaptability to other mRNA sequences from SARS-CoV-2 and potentially other pathogens. DISCUSSION The rapid development of mRNA vaccines has proven to be a transformative approach to combating the COVID-19 pandemic 45 . Despite their success, challenges remain in enhancing vaccine stability and optimizing the delivery systems to ensure easily deployment and broader accessibility. This study addresses these challenges by developing an mRNA vaccine platform that incorporates optimized UTR sequences and rationally designed ionizable lipids in a novel LNP formulation that can be successfully lyophilized for long-term storage to overcome the limitations of existing mRNA vaccine formulations. LNPs are the leading delivery system for mRNA vaccines and the ionizable lipids play a critical role in mRNA transfection efficiency and endosomal escape 18 . Although many ionizable lipids have been already described, very few have been approved for clinical use are currently licensed for mRNA vaccines applications 4,5,46 . We have recently described a new library of ionizable lipids developed using a high-throughput screening using a multicomponent thiolactone-based synthesis platform that allowed us to synthesize many lipid candidates in a cost-effective, scalable and time-efficient manner. This screening process enabled us to establish critical structure-function relationships, particularly related to the hydrophobic tails and functional groups that influence endosomal escape and membrane-disruption activity. As a result, we identified several lipid candidates with enhanced in vivo mRNA delivery than gold standard ionizable lipids, as evidenced by superior performance in luciferase protein expression after intramuscular injection. Additionally, the use of our thiolactone-based lipids offers significant advantages due to an optimized synthetic method, featuring a one-pot synthesis with short reaction times (2 hours) and room temperature 23 , in contrast to lipids such as SM-102 or ALC-0315 47,48 . In the present study, we have first evaluated the immunogenic potential of some of our top-performing ionizable lipids in the context of a SARS-CoV-2 vaccination model, hypothesizing that the increased protein expression observed after intramuscular administration would lead to a stronger antigen presentation, thereby enhancing robust immune responses. Our results indicate that all the ionizable lipids tested here efficiently encapsulated the Spike mRNA in a standard LNP formulation and were capable of inducing both humoral and cellular immune responses. Although a strong Th1 polarized immune response was observed across all the experimental groups, lipids CP-LC-0431 and CP-LC-0474 were excluded due to their comparatively lower induction of IFNγ secretion compared to the other lipids and the commercial vaccine formulation. CP-LC-0729 was selected as the lead candidate as it induced one of the strongest immune responses, which correlates with our previous observations for intramuscular protein expression 23 . Furthermore, CP-LC-0729 has been extensively characterized in our previous work and has proven to be safe and well tolerated after systemic injection 23 . Once the ionizable lipid CP-LC-0729 was selected, we incorporated an optimized mRNA sequence encoding the SARS-CoV-2 Spike protein, featuring proprietary UTRs designed to enhance translation efficiency 16 . The incorporation of this optimized sequence significantly improved the immune response associated with the LNP formulated with CP-LC-0729 in terms of B and T cell responses. Notably, the optimization of UTRs can lead to increased protein expression, allowing for a reduction in the required vaccine dose without compromising immunogenicity. For instance, the Comirnaty vaccine uses 30 µg of mRNA per dose, while the Spikevax vaccine requires a higher dose of 100 µg of mRNA per dose. These differences in mRNA dosage likely reflect variations in the efficiency of their respective UTRs and LNP formulations, with Comirnaty’s UTRs potentially offering more efficient translation at a lower mRNA concentration 21 . Our findings reinforce the critical role of UTR optimization, along with the synergy between mRNA design and LNP delivery systems, in producing robust and durable immune responses with potentially lower mRNA doses. Based on these data, we defined our final vaccine candidate, CPVax-CoV, which combines the ionizable lipid CP-LC-0729 with our optimized mRNA sequence. CPVax-CoV demonstrated complete protection against SARS-CoV-2 infection in both mouse-adapted and humanized murine infection models, as it was able to fully protect vaccinated mice from productive SARS-CoV-2 infection and virus associated inflammatory response in the lungs. Additionally, the biodistribution study of CPVax-CoV revealed that the vaccine primarily localizes at the injection site and in the draining lymph nodes after intramuscular injection, which is likely contributing to its strong induced immune response. Although more detailed studies are needed to fully characterize the pharmacokinetic profile of CPVax-CoV, renal excretion was observed and no accumulation of the vaccine was detected in major organs after 7 days, preliminarily indicating a favorable tolerability profile. One of the most significant achievements of this study is the successful lyophilization of CPVax-CoV. While previous studies on lyophilized mRNA vaccines have demonstrated efficacy with freshly prepared lyophilized formulations and stability for shorter time-periods 44,49–51 , this is the first report, to our knowledge, that demonstrates vaccine-induced efficacy in a relevant in vivo infection model after one year of storage at 25°C. We have previously described an optimized lyophilization protocol for LNPs encapsulating luciferase-coding mRNA using SM102 as the ionizable lipid, which successfully preserved LNP physicochemical properties and maintained protein expression in vivo after extended storage at 4°C, compared to their liquid counterparts 42 . Applying this optimized protocol to CPVax-CoV, we demonstrated that our recently described ionizable lipids are compatible with lyophilization and the LNPs retain their integrity and their ability to induce immune response after prolonged storage. The demonstration that the lyophilized LNPs remain stable after reconstitution in liquid buffer for at least 96 h at 4°C further enhances the practicality of this platform in clinical settings, allowing for more flexibility in vaccine administration and in diverse medical environments. It is important to note that this assessment was conducted with freshly prepared lyophilized LNPs, and further studies are needed to evaluate the stability of the reconstituted LNPs after long-term storage and at different temperatures including room temperature. Notably, current SARS-CoV-2 mRNA vaccines have a limited recommended timeframe for use once diluted, with usability restricted to up to 6 hours for Comirnaty and 12 hours for Spikevax 52 . The results presented here with mRNA encoding the Spike protein of the XBB variant demonstrate that this platform is highly adaptable to other mRNA sequences. Interestingly, despite the lack of detectable neutralizing antibodies against XBB, CPVax-CoV is able to induce protection in lungs. Further supporting this finding and highlighting the potential for broad protection against other VOCs, it should be noted that MA20 strain carries several mutations in the Spike protein and in other viral proteins such as ORF1a and N, as a consequence of mouse adaptation. Despite these genetic changes, our vaccine formulation demonstrated strong protective efficacy. In this regard, immunity studies demonstrated that the vaccine elicited a strong variant-independent T cell response, which likely contributed to the observed protection in mice of CPVax-CoV against XBB challenge. This finding highlights the role of cellular immunity in protecting against viral variants 38 and suggests that CPVax-CoV could provide cross-protection against emerging variants. However, further studies of immunity and other correlates of protection against XBB and different VOCs are required to confirm these preliminary findings and to fully understand the mechanisms of protection conferred by CPVax-CoV against VOCs. The findings presented in this study also open up valuable opportunities for future research. First, further investigation into long-term immunity is essential to fully understand the durability of both humoral and cellular responses, particularly the persistence of T cell-mediated protection against variants of concern (VOCs). Exploring long-term immune memory, mucosal immunity, and viral dissemination to other organs, as well as conducting studies in additional relevant animal models, will provide a deeper understanding of CPVax-CoV’s full protective potential, PK and safety. Additionally, while the lyophilized formulation of CPVax-CoV has demonstrated stability for one year at 25°C, further research into stability at higher temperatures and under diverse environmental conditions would expand its applicability. This will be especially important for optimizing its widespread use in various climates and global vaccination campaigns, especially in regions where maintaining cold-chain logistics is challenging. In summary, CPVax-CoV represents a significant advancement in mRNA vaccine development by addressing key challenges in stability, delivery and efficacy. The combination of novel ionizable lipids and optimized mRNA sequences, along with successful lyophilization, offers a scalable and accessible solution that can overcome the logistical barriers of cold-chain dependency, making it highly suitable for global deployment in future pandemics. Although further research will be crucial to fully unlock its potential and ensure its adaptability across different contexts and pathogens, our findings provide a solid foundation for further research into the use of this technology to broader applications beyond SARS-CoV-2. MATERIALS AND METHODS DNA template design The DNA template for Comirnaty vaccine was constructed by cloning the publicly available mRNA vaccine sequence 53 into a pUC-based plasmid under the control of a T7 promoter. For CPVax-CoV vaccine the coding sequence of the Spike SARS-CoV-2 protein from Wuhan-H1 strain (GISAID 54 accession number EPI_ISL_402124) was codon-optimized for human expression. This sequence included the K986P and V987P (2P) stabilizing mutations and the substitution of the RRAR furin cleavage site at residues 682–685 with GSAS. For CPVax-CoV -XBB vaccine, the DNA template was generated using the XBB.1.5 strain sequence (GISAID accession number EPI_ISL_15851788). The same 2P and RRAR to GSAS modifications were introduced, and the sequence was similarly codon-optimized for human use. Both CPVax-CoV or CPVax-CoV-XBB, optimized sequences were inserted in-frame immediately downstream the 5´UTR into a pUC based plasmid. This plasmid featured the following elements arranged in a 5′ to 3′ orientation: a T7 RNA polymerase promoter, an optimized 5′ UTR from human APOA2 16 , the 3′ UTR from human beta-globin (GenBank: NM_000518), a polyadenylation tail consisting of 100 adenines, and a BspQI restriction enzyme site. Gene synthesis, cloning, and plasmid preparations were outsourced to Genscript. The purified plasmids were subsequently used for in vitro transcription. In vitro transcription and mRNA purification Each plasmid containing the DNA template sequence of interest was digested with BspQI (ON-124, HONGENE), which cleaved the plasmid immediately after the segment to be transcribed. The linearization reaction was then purified with the Wizard SV Gel and PCR Clean-Up (Promega A7270), in accordance with the manufacturer's instructions. The purified linear DNA was subsequently employed for mRNA production by in vitro transcription using T7 RNA polymerase following manufacturer's instructions. Briefly, transcription reactions were performed at 37°C for 3 h using the following materials: Template linear DNA (50 µg/mL), T7 RNA polymerase (5000 U/mL; HONGENE, ON-004), RNAse inhibitor (1000 U/mL; HONGENE, ON-039), Inorganic Pyrophosphatase (2 U/mL; HONGENE, ON-025), ATP (5 µg /mL; HONGENE, R1331), GTP (5 µg/mL; HONGENE, R2331), CTP (5 µg/mL; HONGENE, R3331), N1-Methylpseudouridine (5 µg/mL, HONGENE, R5-027), CAP AG (4 µg mL, Hongene, ON-134) and RNAse-free double-distilled water. The generated mRNA transcripts were initially treated using DNaseI incubation (Hongene, ON-109) according to manufacturer's instructions. In order to reduce double-stranded RNA (dsRNA) contaminants, the resulting mRNA was purified through a process involved two sequential chromatography steps, starting with anion exchange chromatography (AEX) followed by affinity chromatography 55 . Initially, the mRNA sample was purified using the CIMmultus PrimaS Chromatography Column (BIA Separations). IVT mixture was diluted once in sample loading/equilibrate buffer A (20 mM Tris, 20 mM BTP, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH = 8.0) and loaded onto the column. After unbound IVT components eluted in flow-through, a wash 1 with equilibration buffer was necessary, followed by a high-salt wash with 50 mM Tris, 3.0 M guanidine-HCl, 20 mM EDTA, pH 8.0. And after a wash 3 with buffer A until UV returns to baseline, a step elution was performed with buffer C (20 mM Tris, 20 mM BTP, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH = 11.0). Fractions were neutralized immediately after elution. Following, additional purification was carried out through affinity chromatography using POROS Oligo (dT)25 column (ThermoFisher). Specifically, the buffers employed were as follows: Buffer A which contained 50 mM disodium phosphate, 0.5 M NaCl, 5 mM EDTA, pH = 7.0 and Buffer B which contained 50 mM sodium dihydrogen phosphate, 5 mM EDTA, pH = 7.0. The mRNA samples were initially half-diluted in Buffer A 2x. Following this, the column was equilibrated with 100% Buffer A, loaded with mRNA, washed with Buffer B, and ultimately eluted using double-deionized water. To completely remove Buffer B, mRNA was washed with a 30KDa Amicon® filter and then equilibrated through a one-tenth dilution in citrate buffer 10x with a pH of 6.5. The concentration of mRNA was determined by measuring the optical density at 260 nm, then adjusted to a final concentration of 1 mg/ml, aliquoted and stored at -80°C until needed. For quality assurance, all mRNAs underwent analysis through automated electrophoresis (2100 Bioanalyzer G2938B, Agilent). Subsequently, the mRNA samples were aliquoted and stored at -80°C until needed. Synthesis and characterization of ionizable lipids All intermediate compounds and ionizable lipids were synthesized following protocols based on the Sequential Thiolactone Amine Acrylate Reaction 23 . Briefly, following the synthesis of thiolactone derivatives, the ionizable lipid is obtained via a multicomponent one-pot reaction. Thus, the corresponding thiolactone derivatives (0.15 mmol, 1 equiv.), acrylate (0.15 mmol, 1 equiv.) amine (0.15 mmol, 1 equiv.) were dissolved in 300 µL of tetrahydrofuran (THF) at room temperature. After stirring for two hours, the THF was removed under reduced pressure and the product was purified using a CombiFlash NextGen 300+ (gradient of elution: from 100% dichloromethane to 50% of an 80/20/1 mixture of DCM/MeOH/NH 4 OH (aq)). All synthesized ionizable lipids were characterized by high-performance liquid chromatography coupled with a charged aerosol detector (HPLC-CAD) and mass spectrometry (ThermoFisher ISQ). Noteworthy, in previous publications CP-LC-0743 was also referred to as A4B2C1. Molecular structures are shown in Figure S1 . Theoretical and experimental molecular weights (MW): CP-LC-0729 theoretical [M + H] + = 740.63, experimental [M + H] + = 740.85 CP-LC-0867 theoretical [M + H] + = 684.57, experimental [M + H] + = 684.74 CP-LC-0431 theoretical [M + H] + = 766.65, experimental [M + H] + = 766.85 CP-LC-0474 theoretical [M + H] + = 768.66, experimental [M + H] + = 768.76 CP-LC-0743 theoretical [M + H] + = 766.65, experimental [M + H] + = 766.81 mRNA encapsulation into LNPs LNPs formulations were prepared following a previously described method with modifications 20 . Briefly, the purified mRNAs were initially diluted in 10 mM sodium citrate buffer at pH = 4 reaching a final concentration of 266 µg/ml. Simultaneously, the lipid mixture was prepared in ethanol at the different molar ratios used of 46.6:9.4:42.7:1.6 (Comirnaty), 50:10:38.5:1.5 (CP ionizable lipids formulations in Fig. 1 ) and 40.7:34.9:23.3:1.2 (CPVax-CoV) for ionizable lipids CP-LC-0729, CP-LC-0867, CP-LC-0431, CP-LC-0743, CP-LC-0474 or ALC-0315 (Merck 586224): helper lipids DSPC (Merck 850365P) or DOPE (Merck 850725P): Cholesterol (Sigma C3045) and DMG-PEG2000 (Cayman 33945-1) or ALC-0159 (Cayman 34336). Aqueous mRNA solution and lipid mixture were combined at a molar N/P ratio of 6:1 (Moderna/Pfizer) or ionizable lipid/RNA weight ratio of 10:1 (CPVax-CoV formulation). Microfluidic technique was used for the synthesis of LNPs, thereby NanoAssemblr® Ignite microfluidic device (Precision Nanosystems) was set at a total flow rate (TFR) of 12 ml/min and a aqueous:ethanol flow rate ratio (FRR) of 3:1. The resulting LNPs were dialyzed (Pur-A-Lyzer™ Midi Dialysis Kit) overnight against Tris buffer containing cryoprotectants. Each resulting LNP solution was then collected and adjusted to a final concentration of mRNA of 100 µg/mL, filtered through a 0.22 mm filter and stored at -80°C for further use. The average size, polydispersity index (PDI) and zeta potential of LNPs were determined using a Malvern Zetasizer Advance Lab Blue Label (Malvern Instruments Ltd., UK) with a capillary cell (DTS1070) and diluting the sample (typically 1:100) in KCl 10 mM filtrated solution. The concentration of mRNA in LNPs was measured using Quant-iT™ RiboGreen™ RNA Assay Kit from Thermo Fisher Scientific following the manufacturer’s protocols. Thus, the % of RNA encapsulated was calculated by comparing the total RNA obtained by the lysis of mRNA-LNPs using 0.5% Triton X-100 and the non-encapsulated RNA obtained when the LNPs are not lysed in absence of detergent. Fluorescence was quantified in a Fluostar Omega microplate reader (BMG Labtech). Agarose gel electrophoresis was additionally used to determine the encapsulation of mRNA in LNPs. The quantification of encapsulated mRNA was determined by band densitometry using ImageJ software. Samples were loaded in a 1% agarose gel including SYBR-Safe, and the electrophoresis was run at 120 V for 30 min. Gels were visualized with a UV transilluminator iBright™ CL750 imaging system, using the adequate exposure times to avoid image saturation. LNP lyophilization After adjusting the concentration of mRNA-LNPs to 100 µg/mL, the mRNA-LNPs suspension was aliquoted into glass vials with a volume of 300 µL per vial. Lyophilization was conducted using a Genesis Pilot Freeze Dryer, following a three-stage process: initial freezing, primary drying, and secondary drying. After lyophilization, vials were backfilled with pure nitrogen, capped, and transferred to various temperatures for stability assessments. The lyophilized mRNA-LNPs were stored at either 4 or 25 o C for different times and compared with the non-lyophilized solution stored at -80 o C as control. To reconstitute lyophilized samples, 300 µL of RNase-free water were added to each vial and softly mixed until the solution turns into a homogeneous slightly white clear suspension. Cell lines HEK293T cells were obtained from the American Type Culture Collection (ATCC, CRL-3216 and CRL-1586). Vero E6 cells were kindly provided by Júlia Vergara from the Centro de Investigación en Sanidad Animal IRTA-CReSA (Barcelona, Spain). HEK293T-ACE2-TMPRSS2 cells were obtained from the National Institute for Biological Standards and Controls (NIBSC, 101008). Cells were cultured on complete DMEM, which consists of high-glucose Dulbecco’s Modified Eagle’s Medium (Merck, D6429) supplemented with 10% Fetal Bovine Serum (Sigma, F7524), 1% Penicillin-Streptomycin Solution (Gibco, 15140122) and 2 mM Glutamax (Fisher, 35050038). Vero E6 cells were additionally supplemented with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperanzineethanesulfonic acid) (Biowest) and HEK293T-ACE2-TMPRSS2 cultures were additionally supplemented with 1 µg/mL puromycin dihydrochloride (Sigma, P8833). Animals All animal experiments were conducted in agreement with European and national directives for protection of experimental animals, and experimental procedures were approved by the Ethics Committee for Animal Experiments of University of Zaragoza (PI59/21 and PI36/22) or University of Navarra (Protocol ref. CEEA026/20). In addition, animal experiments using the SARS-CoV-2 strains were approved by the Biosafety Committee from University of Zaragoza (Refs: 124/20, 156/21 and 184/22) and GMO Interministerial Council (A/ES/20/99). For the immunity studies, BALB/cAnNRj mice were purchased from Janvier Labs. For the viral challenge studies, BALB/cOlaHsd and C57BL/6J K18-hACE2 transgenic mice were purchased from Envigo and Charles River (K18-hACE2 JAX Mice Strain), respectively. Male and female mice aged 8–10 weeks and weighing 18–28 g were used for all the experiments. All mice underwent an acclimation period lasting 3–7 days to adapt to the experimental conditions upon arrival at the research facilities, and were housed and maintained in specific pathogen–free conditions in the facilities of Centro de Investigaciones Biomédicas de Aragón (Zaragoza, Spain; reference ES 50 297 0012 011) for immunity studies and Centro de Investigación de Encefalopatías y Enfermedades Transmisibles Emergentes (Zaragoza, Spain; reference ES 50 297 0012 009) for challenge studies. Housing conditions were controlled, maintaining a room temperature of 20–24°C, humidity levels ranging from 50–70%, and a light intensity of 60 lux, with a light-dark cycle lasting 12 h. During the studies, all animals were monitored by animal resources center or laboratory staff daily. For the procedures requiring anesthesia (SPECT-CT imaging and SARS-CoV-2 challenge) all animals were anesthetized via inhalation using isoflurane (IsoVet) mixed with oxygen at a flow rate of 1 L/min in a rodent-specific anesthesia station. Anesthesia was induced with 5% isoflurane and maintained at 2%. Animals were monitored continuously during anesthesia and observed post-procedure until fully recovered. No anesthesia was used for intramuscular injections, blood collection and weighing. Mice were humanely euthanized with CO 2 asphyxiation followed by cervical dislocation. Virus strains The SARS-CoV-2, hCoV-19/Sweden/20-53846/2020, (Lineage B.1.1.7; Alpha variant) was provided by The Public Health Agency of Sweden. The mouse adapted strain SARS-CoV-2 MA20 was obtained by serial passaging of SARS-CoV-2, hCoV-19/Sweden/20-53846/2020, (Lineage B.1.1.7; Alpha variant) in mice as described in 27 . The SARS-CoV-2 hCoV19/USA/CA-Stanford-109_S21/2022 (Lineage XBB; Omicron Variant) (GISAID: EPI_ISL_15509864) was isolated from a human on October 10, 2022, in California and was obtained from BEI resources (NR-58925). All procedures involving infectious viruses, including in vivo experiments, were performed under biosafety level 3 (BSL-3) conditions. mRNA in vitro transfection HEK293T cells were seeded in 6-well cell culture plates at a density of 4 x 10 5 cells/well and incubated overnight. The SARS-CoV-2 Spike-coding mRNAs were transfected using Lipofectamine MessengerMax Transfection Reagent (Invitrogen, 15397974) according to manufacturer's protocol. Briefly, a mixture of each mRNA (2.5 µg/well) and Lipofectamine MessengerMAX (5 µL/well) were pre-incubated in OptiMEM media. The mRNA-lipofectamine mixture was added to the corresponding wells in duplicate, resulting in a final mRNA concentration of 2.5 µg/well. The cells were incubated for 24 h at 37°C and 5% CO 2 . For Western Blot, cells were lysed after incubation with 0,1% TritonX and centrifuged at 13000 x g for 10 min. The cell lysates were run under reducing conditions by SDS-PAGE (SurePAGE, 4–12% Genscript, M00653) and transferred to a nitrocellulose PVDF membrane (Bio-Rad). The membranes were blocked with 3% BSA in TBST buffer (Tris-buffered saline + 0.1% Tween 20) and incubated with a rabbit polyclonal SARS-CoV-2 Spike antibody (SinoBiological, 40591-T62) overnight at 4°C. Membrane was washed and incubated with a goat anti-rabbit Ig, Human ads-HRP (Southern Biotech, 4010-05) as a secondary antibody. Colorimetric detection of the samples was performed with Pierce ECL Western Blotting Substrate (Thermo, 32106) and images were acquired using iBright CL750 Imaging system (Invitrogen). For flow cytometry analysis, cells were trypsinized after incubation. Collected cells were incubated with human FcR blocking reagent (Miltenyi, 130-059-901) for 15 min at 4°C. Cells were washed and incubated with fixation buffer (eBioscience, 88-8824-00) for 30 min at 4°C followed by 3 washing steps with permeabilization buffer (eBioscience, 88-8824-00) and incubation with SARS-CoV-2 Spike S1 Subunit Antibody (R&D systems, MAB105403) diluted in permeabilization buffer for 1 h at room temperature. Subsequently, cells were washed and incubated with FITC Goat Anti-Mouse IgG/IgM (BD, 555988) for 1 h at room temperature. After final washing steps, labeled cells were resuspended in PBS buffer and acquisition was performed using Gallios flow cytometer (Beckman Coulter). In vivo immunization and blood collection Mice were immunized with LNPs prepared as described above, receiving an intramuscular injection of 1 µg of the specified mRNA-LNP into the right thigh muscle, diluted in Tris buffer containing 15% sucrose in a final volume of 30 µL, and administered using a 30G insulin syringe. An equal booster dose was administered on day 21 following the initial immunization. Blood samples were collected from the submandibular vein on days 0, 21, and 42 post-prime. Blood was allowed to clot and then centrifuged at 6500 g and 4°C for 10 min. The sera were collected and stored at -80°C for antibody analysis. Determination of antibody levels in serum The serum samples from immunized mice were analyzed for RBD-specific IgG antibodies titers using an indirect enzyme-linked immunosorbent assay (ELISA). 96-well Nunc MaxiSorp microplates (Thermo Scientific) were coated with 50 ng/well of recombinant RBD (Certest Biotec) diluted in carbonate/bicarbonate buffer pH 9.6 and incubated overnight. The next day, plates were washed with phosphate buffer saline – Tween 20 0.05% (PBST) followed by blocking with 3% BSA (Seqens) in PBST for 1 h. After washing, serially diluted mouse sera were added to the plate and incubated for 1.5 h at 37°C, followed by washing steps and addition of IgG goat anti-mouse horseradish peroxidase (HRP) antibody (Southern Biotech) at a dilution of 1:10.000. The plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Abcam) and 0.2M H 2 SO 4 (aq) to stop the reaction. Finally, the absorbance was measured at 450 and 630 nm using a FLUOstar Omega microplate reader (BMG Labtech). The reciprocal endpoint titer was defined as the highest dilution at which the optical density (OD 450–630 nm) of the sample reached a predetermined cutoff of 0.1 or greater. Organ single cell suspensions Organs were harvested and placed in RPMI 1640 medium (Fisher, 10379144). Spleens and lymph nodes (LNs) were homogenized using a syringe plunger and filtered through a 70 µm cell strainer. The cell strainer was washed with 10 ml of RPMI and cells were centrifuged at 450 g for 5 min. Red blood cells (RBCs) in spleens were lysed using 1 ml of eBioscience RBC Lysis Buffer (Thermofisher, 00-4300-54) for 1 min. After lysing, the cells were washed with 10 ml of RPMI and centrifuged at 450 g for 5 min. Cells from tissues were resuspended in 1 ml of RPMI 1640 medium supplemented with 10% heat inactivated Fetal Bovine Serum (Sigma F7524), 1% Glutamax (Fisher, 35050038), 1% Penicillin-Streptomycin (Gibco, 15140122) and 0.00035% β-mercaptoethanol (Merck, M3148). The resuspended cells were immediately used for counting, culture, or staining. ELISPOT assay Enzyme-linked immunospot (ELISPOT) assay was employed to quantify the frequency of cytokine-secreting splenocytes. ELISpot assays were performed using mouse IFN-γ ELISpot kits (CTL Immunospot) according to the manufacturer’s instructions. 96-well immunospot plates were coated with 60 µL of murine IFN-γ capture solution and incubated at 4°C overnight. Next day, plates were washed with PBS and 4×10 5 splenocytes/well were stimulated with 50 µg/mL of a peptide pool covering the immunodominant sequence domains of the S protein (Miltenyi, 130-126-700). Phorbol-12-myristate-13-acetate (PMA) 50 ng/ml (Merck, 524400) and ionomycin 500 ng/ml (Merck, 407953) were used as positive control while splenocytes without stimulation were utilized as negative control. Samples were incubated for 20 h at 37°C and 5% CO 2 . The following day, plates were washed and 10 µL of anti-murine IFN-γ detection solution was added. After washing, a tertiary solution containing streptavidin-HRP was used followed by the addition of a chromogen substrate for enzyme activity detection. Finally, plates were scanned using an ImmunoSpotS6 Ultra-V analyzer (CTL EUROPE GMBH) and spot numbers were assessed using ImmunoSpot 7.0.34.0 Professional Analyzer DC software. ELISA cytokine assay 1x10 6 splenocytes/well were seeded in 96-well plates and stimulated with 50 µg/mL of a peptide pool covering the immunodominant sequence domains of the S protein (Miltenyi, 130-126-700). Phorbol-12-myristate-13-acetate (PMA) 50 ng/ml (Merck, 524400) and ionomycin 500 ng/ml (Merck, 407953) were used as positive control while splenocytes without stimulation were utilized as negative control. Samples were incubated for 20 h at 37°C and 5% CO 2 . Supernatants were collected after centrifugation at 1500 g for 5 min, and samples were stored at -80°C until analysis. Cytokine quantification was conducted using Mabtech ELISA Flex (HRP) kits following manufacturer’s instructions. Briefly, Nunc MaxiSorp 96-well plates (Thermo Fisher) were coated with capture antibody and incubated at 4°C overnight. Next day, plates were washed with PBST and blocked with 0.1% BSA in PBST buffer for 1 h at room temperature. After washing, samples and standards dilutions were added and incubated for 2 h at room temperature, followed by the addition of detection biotinylated antibody and incubation for 1 h. Streptavidin-HRP was added and incubated for 1 h at RT. Finally, the plates were washed and TMB substrate (Abcam) was added. Reaction was stopped with 0.2M H 2 SO 4 and the absorbance was measured at 450 and 630 nm using a FLUOstar Omega microplate reader (BMG Labtech). Luminex assay Lung samples were acquired from a weighed portion of murine lung, previously homogenized in 1 ml of DMEM using a GentleMACS Dissociator (Miltenyi), and then clarified by collecting the supernatant after centrifugation at 450 rpm for 5 min. Before analysis, samples were inactivated by the addition of 0.5% Triton X100 and incubated for 30 min at 4°C. Protease inhibitor cocktail (Complete, Roche) was added to prevent protein degradation during incubation. Samples were stored at -80°C until Luminex assay was conducted. Cytokine/Chemokine array analysis was carried out using Luminex Mouse Discovery Assay 11-plex kit (R&D Systems). Samples were centrifuged at 1000 × g for 10 min and then diluted 1:2 in calibrator diluent and the assay was conducted following manufacturer’s instructions. Measurements were carried out using Luminex LABSCAN 100 and quantified by comparison to a standard curve. Data collection and analysis were performed utilizing LUMINEX 100 IS software. Flow cytometry analysis of Thf and GC B cells Lymph node single cell suspensions were incubated with mouse FcR blocking reagent (Miltenyi, 130-092-575) for 15 min at 4°C. Cells were washed and incubated with anti-mouse antibodies for surface staining for 15 min at 4 ºC. Cells were washed and fixed with 4% paraformaldehyde for 30 minutes at room temperature and resuspended in PBS buffer. Acquisition was performed using Gallios flow cytometer (Beckman Coulter). Thf cells were defined as CD45R − CD4 + , CD44 high , CXCR5 + , PD-1 + and the following antibodies were used for surface staining: CD45R (B220)-APC-Vio770 (Miltenyi, 130-110-849), CD4-VioBright FITC (Miltenyi, 130-118-692), CD44-PerCP-Vio700 (Miltenyi, 130-128-625), CD185 (CXCR5)-APC (Miltenyi, 130-119-129) and CD279 (PD1)-PE (Miltenyi, 130-111-953). GC B cells were defined as CD19 + GL7 + CD95 + and the following antibodies were used for surface staining: CD19-APC (Miltenyi, 130-112-036), CD95 (FAS)-PE (Miltenyi, 130-112-036) and GL7-Alexa Fluor 488 eBioscience (Thermofisher, 53-5902-82). Pseudotyped SARS‑CoV‑2 neutralization assay For the neutralization assay, mouse sera were serially diluted in complete DMEM and added to black 96-well microplates, alongside the previously established volume of pseudovirus. The mixture was then incubated at 37°C and 5% CO 2 for 1 h to allow neutralization. After incubation, 2 x 10 4 HEK293T-ACE2-TMPRSS2 cells were added to each well followed by 48 h of further incubation. The cells were then fixed as previously described, and the fluorescent spots were counted using a C.T.L. S6 Ultra-V analyzer with the FluoroSpot-X suite selected. The NT50 titer was defined as the reciprocal of the highest dilution at which a 50% or higher reduction in the number of spots compared to the unnaturalized condition was achieved. In vivo biodistribution In vivo biodistribution assays were performed using Indium-111 radiolabeled LNPs. Lipid nanoparticle suspensions were incubated with 55 MBq Indium-111 oxine for 15 min at 37°C. Labelled LNPs were afterwards purified using centrifugal concentrators (Vivaspin 500, 10.000 MWCO PES). In vivo biodistribution assays were performed on BALB/c mice (n = 3 male, n = 3 female) on the U-SPECT6/E-class (MILabs) single photon emission computed tomography (SPECT) system. Radiolabeled LNPs were administrated via intramuscular injection as described above. At 1, 3, 6, 24, 48, 72, 96 and 168 h post-administration animals were anesthetized using isoflurane (2% in 100% O 2 ) and SPECT images were acquired. Computer tomography (CT) scan was performed immediately after adjusting voltage and current to 55 kV and 0,33 mA respectively. After image acquisition these were reconstructed using indium-111 photopeaks with a windo with of 20% and a calibration factor to obtain the exact activity information (MBq/mL). Images were analyzed using PMOD v3.2 software (PMOD Technologies, Switzerland). Signal values were corrected using the specific indium-111 radioactive decay correction factor and then transformed to standardized uptake value (SUV) units using the formula: SUV = [tisular activity concentration (MBq /cm 3 )/dose (MBq)] × bodyweight (g). Quantitative analysis was performed on organs/areas where signal could be correctly detected, setting a volume of interest (VOI) and quantifying during time, calculating the average signal in the VOI (Suvmean). Due to the difficulty in quantifying bone marrow signal, a visual analysis was performed to identify the timing of signal appearance in various bones. At 168 h, the animals were euthanized, and the following organs/tissues were dissected: lungs, spleen, liver, kidneys, bone, muscle at the injection site, contralateral muscle, presacral lymph nodes, inguinal lymph nodes, and brain. Tissue activity was measured using a gamma counter (Hidex) to determine counts per minute (cpm) for each tissue. To quantitatively assess the amount of radioactivity in each sample, a ratio was calculated using the brain (background) as the reference tissue, and the values were normalized to tissue weight (g). Serum biochemical analysis Serum samples were collected as specified above. Biochemical analysis of AST and ALT levels was performed using Cobas c-311 automatic analyzer (Roche Diagnostics). SARS-CoV-2 challenge Mice were anaesthetized with isoflurane (5% for induction and 2.5% for maintenance) and infected intranasally with the correspondent amount of virus to achieve the indicated doses, which was diluted in PBS in a total volume of 40 µL. After virus challenge, mice were periodically weighed and assessed using a clinical scoring system evaluating various parameters including mouse appearance, level of consciousness, activity, response to stimuli, eye appearance and frequency and quality of respiration 56 . Humane endpoints were established on a weight loss threshold of 25% or greater, alongside clinical scoring criteria. Viral load determination by TCID50 assay Lungs were harvested, weighed and homogenized in 1 ml of DMEM using a GentleMACS Dissociator (Miltenyi). Homogenates were centrifuged at 450 g for 5 min and the supernatant was taken for titration. Virus titration was determined by TCID50 in Vero E6 cells. Vero E6 cells were seeded in 96 well plates at a density of 10 4 cells/well and cultured overnight at 37°C and 5% CO 2 . Lung homogenates serial 1-log dilutions were prepared in complete DMEM with only 2% FBS and added to the cultured cells. 72 h after cell infection plates were evaluated for cell death and TCID50 was calculated using Ramakrishan newly proposed method formula 57 and normalized to weight (g) of lung and amount of buffer (ml) used for lung homogenization. Statistical analysis In experimental studies, GraphPad Prism 10 software was used for representation and statistical analyses. We used ordinary one-way or two-way ANOVA with Tukey’s multiple comparison post-test to compare experimental groups. Statistical significances are denoted in the Figures by asterisks, as follows: *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001 or ****P-value 0.05. Outliers were identified and removed using Grubbs' test, applying a significance threshold of p < 0.05. Data availability The data supporting the conclusions of this research are accessible within the article and its supplementary files. The source data is included in this paper. Declarations Competing Interests EM, EB, CM, AS, SA, TA, AL, BA, DDM, JH, JGW, VL, DC, JM and EP are employees at the Certest Pharma Department, Certest Biotec S.L. EB, JH, DDM, JM and JGW are inventors on patents related to this publication FUNDING This study was supported by “Ministerio de Ciencia e Innovación” (Spain) through project STABVAC4COV (Desarrollo de vacunas termoestables basadas en mRNA frente a la variante delta del SARS-CoV-2). Work in JP lab is funded by PID2020- 113963RBI00 from AEI (Agencia Estatal de Investigación), Aragon Government (B29-20R), Postdoctoral Juan de la Cierva Contract (MA); Health National Institute Carlos III (COV20-00308 and CIBERINFEC, CB21/13/ 00087). Author Contribution Conceptualization: EM, EB, EP, JM; mRNA sequence design: EB; mRNA synthesis: VL, DC; Lipid molecular design, synthesis and characterization: JH, JGW; LNP formulation and characterization: TA, DDM; Biological and immune assays: CM, AS, SA, AL, BA, CG, AC; Biodistribution study: IP, GQ; Establishment of COVID-19 Infection models and in vivo vaccine-induced protection experiments: JP, IU, NP, MA, CM, AS, SA, BM, JB; Supervision: EM, EP, JM; Writing original draft and figure preparation: EM, CM, AS, AL; Manuscript review: all authors. Acknowledgement We would like to extend our sincere gratitude to all the members of Certest Biotec, especially the Certest Pharma group, whose valuable insights and expertise significantly contributed to the success of this research. Authors would like to acknowledge the use of “Servicios Científico Técnicos” at CIBA (IACS-University of Zaragoza), especially the collaboration of the animal facilities and flow cytometry services, as well as the use of “Servicio de análisis Bioquímicos” at CIMA (University of Navarra). Data Availability The data supporting the conclusions of this research are accessible within the article and its supplementary files. References Tregoning, J. S., Flight, K. E., Higham, S. L., Wang, Z. & Pierce, B. F. Progress of the COVID-19 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape. Nat. Rev. Immunol. 21 , 626–636 (2021). Verbeke, R., Lentacker, I., De Smedt, S. C. & Dewitte, H. The dawn of mRNA vaccines: The COVID-19 case. J. Control. release Off. J. Control. Release Soc. 333 , 511–520 (2021). Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17 , 261–279 (2018). Polack, F. P. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 383 , 2603–2615 (2020). Baden, L. R. et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N. Engl. J. Med. 384 , 403–416 (2021). Pawlowski, C. et al. FDA-authorized mRNA COVID-19 vaccines are effective per real-world evidence synthesized across a multi-state health system. Med (New York, N.Y.) 2 , 979–992.e8 (2021). Scheaffer, S. M. et al. Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant. bioRxiv : the preprint server for biology at https://doi.org/10.1101/2022.09.12.507614 (2022). Ye, Q. et al. Rational development of a combined mRNA vaccine against COVID-19 and influenza. NPJ vaccines 7 , 84 (2022). Cheng, L., Chan, W. K., Zhu, L., Chao, M. H. & Wang, Y. Confronting Inequalities and Bridging the Divide: A Retrospective Study Assessment of Country-Level COVID-19 Vaccine Equality with a Cox Regression Model. Vaccines 12 , (2024). Acharya, K. P., Ghimire, T. R. & Subramanya, S. H. Access to and equitable distribution of COVID-19 vaccine in low-income countries. npj Vaccines 6 , 54 (2021). Duroseau, B., Kipshidze, N. & Limaye, R. J. The impact of delayed access to COVID-19 vaccines in low- and lower-middle-income countries. Front. Public Heal. 10 , (2023). Chi, W.-Y. et al. COVID-19 vaccine update: vaccine effectiveness, SARS-CoV-2 variants, boosters, adverse effects, and immune correlates of protection. J. Biomed. Sci. 29 , 82 (2022). Echaide, M. et al. mRNA Vaccines against SARS-CoV-2: Advantages and Caveats. Int. J. Mol. Sci. 24 , (2023). Kim, S. C. et al. Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency. Mol. Cell. Toxicol. 18 , 1–8 (2022). Luna-Cerralbo, D. et al. A statistical-physics approach for codon usage optimisation. Comput. Struct. Biotechnol. J. 23 , 3050–3064 (2024). Broset Blasco, E. & Martínez Oliván, J. E. Artificial polynucleotides for expressing proteins, WO2024110381A1. (2022). Ramachandran, S., Satapathy, S. R. & Dutta, T. Delivery Strategies for mRNA Vaccines. Pharmaceut. Med. 36 , 11–20 (2022). Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6 , 1078–1094 (2021). Hald Albertsen, C. et al. The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Deliv. Rev. 188 , 114416 (2022). Hassett, K. J. et al. Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines. Mol. Ther. Nucleic Acids 15 , 1–11 (2019). Zhang, L. et al. Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability. NPJ vaccines 8 , 156 (2023). Gimenez-Warren, J. Ionizable lipids and lipid nanoparticles containing thereof, WO2024110381A1. (2022). Pena, A. et al. Multicomponent Thiolactone-Based Ionizable Lipid Screening Platform for an Efficient and Tunable mRNA Delivery to the Lungs. bioRxiv 2024.10.09.617396 (2024) doi:10.1101/2024.10.09.617396. Crommelin, D. J. A., Anchordoquy, T. J., Volkin, D. B., Jiskoot, W. & Mastrobattista, E. Addressing the Cold Reality of mRNA Vaccine Stability. J. Pharm. Sci. 110 , 997–1001 (2021). Schoenmaker, L. et al. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 601 , 120586 (2021). Oude Blenke, E. et al. The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case. J. Pharm. Sci. 112 , 386–403 (2023). Albert, M.-C. et al. Identification of FasL as a crucial host factor driving COVID-19 pathology and lethality. Cell Death Differ. (2024) doi:10.1038/s41418-024-01278-6. Lederer, K. et al. SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation. Immunity 53 , 1281–1295.e5 (2020). Verbeke, R., Hogan, M. J., Loré, K. & Pardi, N. Innate immune mechanisms of mRNA vaccines. Immunity 55 , 1993–2005 (2022). Sharma, P., Hoorn, D., Aitha, A., Breier, D. & Peer, D. The immunostimulatory nature of mRNA lipid nanoparticles. Adv. Drug Deliv. Rev. 205 , 115175 (2024). Chang, H.-D. & Radbruch, A. Maintenance of quiescent immune memory in the bone marrow. Eur. J. Immunol. 51 , 1592–1601 (2021). Dubuc, I. et al. Cytokines and Lipid Mediators of Inflammation in Lungs of SARS-CoV-2 Infected Mice. Front. Immunol. 13 , 893792 (2022). Winkler, E. S. et al. SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. Nat. Immunol. 21 , 1327–1335 (2020). Tamura, T. et al. Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants. Nat. Commun. 14 , 2800 (2023). Tamura, T. et al. Virological characteristics of the SARS-CoV-2 Omicron XBB.1.5 variant. Nat. Commun. 15 , 1176 (2024). Yue, C. et al. ACE2 binding and antibody evasion in enhanced transmissibility of XBB.1.5. The Lancet. Infectious diseases vol. 23 278–280 at https://doi.org/10.1016/S1473-3099(23)00010-5 (2023). Lewnard, J. A. et al. Increased vaccine sensitivity of an emerging SARS-CoV-2 variant. Nat. Commun. 14 , 3854 (2023). Azevedo, P. O. et al. Differential requirement of neutralizing antibodies and T cells on protective immunity to SARS-CoV-2 variants of concern. npj Vaccines 8 , 15 (2023). Powers, J. M. et al. Divergent pathogenetic outcomes in BALB/c mice following Omicron subvariant infection. Virus Res. 341 , 199319 (2024). Halfmann, P. J. et al. Characterization of Omicron BA.4.6, XBB, and BQ.1.1 subvariants in hamsters. Commun. Biol. 7 , 331 (2024). Uddin, M. N. & Roni, M. A. Challenges of Storage and Stability of mRNA-Based COVID-19 Vaccines. Vaccines 9 , (2021). Alejo, T. et al. Comprehensive Optimization of a Freeze-Drying Process Achieving Enhanced Long-Term Stability and In Vivo Performance of Lyophilized mRNA-LNPs. Int. J. Mol. Sci. 25 , (2024). Kafetzis, K. N. et al. The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery. Adv. Healthc. Mater. 12 , e2203022 (2023). Muramatsu, H. et al. Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine. Mol. Ther. 30 , 1941–1951 (2022). Hogan, M. J. & Pardi, N. mRNA Vaccines in the COVID-19 Pandemic and Beyond. Annu. Rev. Med. 73 , 17–39 (2022). Akinc, A. et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 14 , 1084–1087 (2019). Sabnis, S. et al. A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates. Mol. Ther. 26 , 1509–1519 (2018). Ansell, S. M.. US 10, 166, 298 B2. (2019). Li, M. et al. Lyophilization process optimization and molecular dynamics simulation of mRNA-LNPs for SARS-CoV-2 vaccine. NPJ vaccines 8 , 153 (2023). Ai, L. et al. Lyophilized mRNA-lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2. Cell Discov. 9 , 9 (2023). Suzuki, Y. et al. Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates. Mol. Ther. Nucleic Acids 30 , 226–240 (2022). Hermosilla, J. et al. Analysing the In-Use Stability of mRNA-LNP COVID-19 Vaccines Comirnaty™ (Pfizer) and Spikevax™ (Moderna): A Comparative Study of the Particulate. Vaccines 11 , (2023). WHO. Messenger RNA encoding the full-length SARS‐CoV‐2 spike glycoprotein . https://berthub.eu/articles/11889.doc (2021). GISAID. https://gisaid.org/wiv04/. Martínez, J., Lampaya, V., Larraga, A., Magallón, H. & Casabona, D. Purification of linearized template plasmid DNA decreases double-stranded RNA formation during IVT reaction. Front. Mol. Biosci. 10 , (2023). Shrum, B. et al. A robust scoring system to evaluate sepsis severity in an animal model. BMC Res. Notes 7 , 233 (2014). Ramakrishnan, M. A. Determination of 50% endpoint titer using a simple formula. World J. Virol. 5 , 85–86 (2016). Additional Declarations Competing interest reported. EM, EB, CM, AS, SA, TA, AL, BA, DDM, JH, JGW, VL, DC, JM and EP are employees at the Certest Pharma Department, Certest Biotec S.L. EB, JH, DDM, JM and JGW are inventors on patents related to this publication Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in npj Vaccines → Version 1 posted Editorial decision: Revision requested 14 Apr, 2025 Reviews received at journal 31 Mar, 2025 Reviews received at journal 26 Mar, 2025 Reviews received at journal 26 Mar, 2025 Reviews received at journal 25 Mar, 2025 Reviews received at journal 22 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 17 Mar, 2025 Reviewers agreed at journal 17 Mar, 2025 Reviewers agreed at journal 16 Mar, 2025 Reviewers agreed at journal 16 Mar, 2025 Reviewers agreed at journal 16 Mar, 2025 Reviewers invited by journal 16 Mar, 2025 Editor assigned by journal 11 Mar, 2025 Submission checks completed at journal 08 Jan, 2025 First submitted to journal 07 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5780846","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399553037,"identity":"c1939067-b935-4bcc-83b0-1b5e11a1f12b","order_by":0,"name":"Elena Mata","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYNCCChLVMzYwnAFSbEB8gGgtjG2kaOEXO/z8wc95dnLm85sPf/7AYGNPUIvk7DTDxt5tycYyx9jSJA4wpCU2ENJicDvBsIF324HEGWw8ZkCHHU4gaIv97fSPjX/nHKifwcb/+cMBhv+EHWYgnWPYzNtwIEGCjYcB6LADjAQdJnE7p3C2zLFkwxlsaWYSZwySCfuFf3b6ho9vauzkJZgPP/5QUWFH2GHo7iRVwygYBaNgFIwCrAAAvEo7N+oJAc4AAAAASUVORK5CYII=","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":true,"prefix":"","firstName":"Elena","middleName":"","lastName":"Mata","suffix":""},{"id":399553039,"identity":"2ab22b6e-06de-46bc-bdf2-80e16e302b8c","order_by":1,"name":"Esther Broset","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Esther","middleName":"","lastName":"Broset","suffix":""},{"id":399553041,"identity":"3d26ec0a-9326-4663-9429-3ceed381307e","order_by":2,"name":"Carlos Matute","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Matute","suffix":""},{"id":399553044,"identity":"c18f05a5-3982-4dce-9e09-748797e6f4c8","order_by":3,"name":"Andrei Mihai Stoian","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Andrei","middleName":"Mihai","lastName":"Stoian","suffix":""},{"id":399553053,"identity":"be853a8e-d2cb-4524-a1f3-7c9317d1775e","order_by":4,"name":"Susana Adame","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"","lastName":"Adame","suffix":""},{"id":399553055,"identity":"60b793db-a578-44bb-854c-78a7c5b33c84","order_by":5,"name":"Teresa Alejo","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Teresa","middleName":"","lastName":"Alejo","suffix":""},{"id":399553056,"identity":"2df9fe53-ee63-4150-8a5b-57017b2fbbde","order_by":6,"name":"Alexandre López","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"López","suffix":""},{"id":399553057,"identity":"9aa548d0-8f1d-4c05-a10f-9ca3f7375a6c","order_by":7,"name":"Beatriz Andrés","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"","lastName":"Andrés","suffix":""},{"id":399553058,"identity":"79f5d1e3-a31d-49ce-bb4b-d6c968212bfd","order_by":8,"name":"Juan Heredero","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Heredero","suffix":""},{"id":399553059,"identity":"bf7ab0eb-2820-45b1-8413-28f1c7d4d5dc","order_by":9,"name":"Diego de Miguel","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"de Miguel","suffix":""},{"id":399553060,"identity":"f1b8a9fd-573b-42ec-ad7e-b16e6dbc7ab9","order_by":10,"name":"Javier Giménez-Warren","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Giménez-Warren","suffix":""},{"id":399553061,"identity":"0b3ceaa8-4106-41b1-9b94-6d23132e182c","order_by":11,"name":"Verónica Lampaya","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Verónica","middleName":"","lastName":"Lampaya","suffix":""},{"id":399553062,"identity":"147d949b-3e82-4f06-ab66-41c4dda3b3c1","order_by":12,"name":"Diego Casabona","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Casabona","suffix":""},{"id":399553063,"identity":"de601ba7-17a6-46d4-a9c2-bcd1854b0759","order_by":13,"name":"Alba Calvo","email":"","orcid":"","institution":"Department of Microbiology and Parasitology, University of Navarra; IdiSNA, Navarra Institute for Health Research","correspondingAuthor":false,"prefix":"","firstName":"Alba","middleName":"","lastName":"Calvo","suffix":""},{"id":399553064,"identity":"5984ccc6-c85a-433c-b759-a50fae9754f9","order_by":14,"name":"Gema Quincoces","email":"","orcid":"","institution":"Radiopharmacy and Translational Molecular Imaging Unit, University Clinic of Navarra. IdiSNA, Navarra Institute for Health Research","correspondingAuthor":false,"prefix":"","firstName":"Gema","middleName":"","lastName":"Quincoces","suffix":""},{"id":399553065,"identity":"102142cc-d957-459c-81eb-5fbeb525a19f","order_by":15,"name":"Iván Peñuelas","email":"","orcid":"","institution":"Radiopharmacy and Translational Molecular Imaging Unit, University Clinic of Navarra. IdiSNA, Navarra Institute for Health Research","correspondingAuthor":false,"prefix":"","firstName":"Iván","middleName":"","lastName":"Peñuelas","suffix":""},{"id":399553066,"identity":"a7900252-dd7e-45d7-89ea-75c20b34be97","order_by":16,"name":"Carlos Gamazo","email":"","orcid":"","institution":"Department of Microbiology and Parasitology, University of Navarra; IdiSNA, Navarra Institute for Health Research","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Gamazo","suffix":""},{"id":399553067,"identity":"2c41a7c4-5653-4b6b-8686-e9b00f666ef6","order_by":17,"name":"Iratxe Uranga-Murillo","email":"","orcid":"","institution":"CIBA, Instituto de Investigacion Sanitaria Aragon (IISA)","correspondingAuthor":false,"prefix":"","firstName":"Iratxe","middleName":"","lastName":"Uranga-Murillo","suffix":""},{"id":399553068,"identity":"4343a959-a018-48c1-916e-90d146b32754","order_by":18,"name":"Natacha Peña","email":"","orcid":"","institution":"Dpto. Microbiologia, Radiologia, Pediatria y Salud Publica, Fac. Medicina, Universidad de Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"Natacha","middleName":"","lastName":"Peña","suffix":""},{"id":399553069,"identity":"7da0f7a2-4031-4515-aa2a-0bffc6c67713","order_by":19,"name":"Maykel Arias","email":"","orcid":"","institution":"CIBA, Instituto de Investigacion Sanitaria Aragon (IISA)","correspondingAuthor":false,"prefix":"","firstName":"Maykel","middleName":"","lastName":"Arias","suffix":""},{"id":399553070,"identity":"718b89d5-ab90-49a1-9b07-6f597f4a8677","order_by":20,"name":"Julian Pardo","email":"","orcid":"","institution":"CIBA, Instituto de Investigacion Sanitaria Aragon (IISA)","correspondingAuthor":false,"prefix":"","firstName":"Julian","middleName":"","lastName":"Pardo","suffix":""},{"id":399553071,"identity":"b9ee377b-812a-4503-b409-16c9f1c272c6","order_by":21,"name":"Bernardino Moreno","email":"","orcid":"","institution":"Centro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, IA2, Universidad de Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"Bernardino","middleName":"","lastName":"Moreno","suffix":""},{"id":399553072,"identity":"847f86f1-801a-4d2e-9991-b83b8c5461d8","order_by":22,"name":"Juan Badiola","email":"","orcid":"","institution":"Centro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, IA2, Universidad de Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Badiola","suffix":""},{"id":399553073,"identity":"72907c24-517c-4b78-a4f3-445807d17f44","order_by":23,"name":"Juan Martínez","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Martínez","suffix":""},{"id":399553074,"identity":"4152e32c-b3dc-4478-aec2-880007508780","order_by":24,"name":"Esther Pérez","email":"","orcid":"","institution":"Certest Pharma, Certest Biotec S. L","correspondingAuthor":false,"prefix":"","firstName":"Esther","middleName":"","lastName":"Pérez","suffix":""}],"badges":[],"createdAt":"2025-01-07 11:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5780846/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5780846/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41541-025-01201-1","type":"published","date":"2025-07-01T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73513189,"identity":"2428a1f3-53e7-4d63-b9b1-c93d5453cfc4","added_by":"auto","created_at":"2025-01-10 17:12:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":192382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunity induced by SARS-CoV-2 mRNA vaccine candidates formulated with novel ionizable lipids\u003c/strong\u003e. (a) Immunization scheme and sample collection schedule. BALB/c mice (n=5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. Blood samples were obtained at week 3 (prior to boost) and 6 and specific antibody levels were determined in serum. T-cell response was evaluated in splenocytes 3 weeks post-boost. (b) Reciprocal endpoint titers of antigen specific IgG antibodies in serum samples determined by ELISA using RBD recombinant protein from Wuhan variant. (c) IFNγ-secreting splenocytes quantification by ELISPOT after O.N. stimulation with SARS-CoV-2 Spike peptide pool (d-e) IFNγ and IL-4 quantification in supernatants of splenocytes after overnight stimulation with SARS-CoV-2 Spike peptide pool determined by ELISA. Graphs are represented as mean ±SD. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt; 0.001; ****p\u0026lt;0.0001 as determined by two-way OVA (B) and one way ANOVA (C, D) with Tukey post-test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/d74b88ca1c336db360ae3a3f.png"},{"id":73512411,"identity":"5ca2a079-34f3-4a22-8cb5-e17fccab0d5e","added_by":"auto","created_at":"2025-01-10 17:04:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":252563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of mRNA sequence optimization in vaccine-elicited immune response. \u003c/strong\u003e(a) Immunization scheme and sample collection schedule. Mice (n=5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. Blood samples were obtained at week 3 (prior to boost) and 6, and specific antibody levels were determined in serum. T-cell response was evaluated in splenocytes 3 weeks post-boost. (b) Reciprocal endpoint titers of antigen specific IgG antibodies in serum samples determined by ELISA using RBD recombinant protein from Wuhan variant. (c) Neutralization titers in serum samples determined by neutralization assay using Spike-pseudotyped lentivirus and infection in HEK293T-ACE2-TMPRSS2 cells. NT50 titers refers to the dilution of a serum sample at which 50% of the pseudovirus infection is inhibited. (d) IFNγ-secreting splenocytes quantification by ELISPOT after O.N. stimulation with SARS-CoV-2 Spike peptide pool. (e) IFNγ quantification in supernatants of splenocytes after overnight stimulation with SARS-CoV-2 Spike peptide pool determined by ELISA. (f) T-lymphocyte CD4 and CD8 cell frequencies in spleen analyzed by flow cytometry. (g) Thf and GC B cells analysis. Mice (n=5) were immunized intramuscularly with 5 µg of mRNA/animal. Inguinal lymph nodes were extracted on day 7. (h) Thf (CD45R\u003csup\u003e-\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e, CD44hi, CXCR5\u003csup\u003e+\u003c/sup\u003e, PD-1\u003csup\u003e+\u003c/sup\u003e) and GC B cell (CD19\u003csup\u003e+\u003c/sup\u003e, CD95\u003csup\u003e+\u003c/sup\u003e, GL7\u003csup\u003e+\u003c/sup\u003e) frequencies were determined by flow cytometry analysis. Graphs are represented as mean ±SD. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt; 0.001; ****p\u0026lt;0.0001 as determined by two-way ANOVA (a) and one way ANOVA (c-i) with Tukey post-test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/a298edd5677be97bfbdb98fc.png"},{"id":73513691,"identity":"7d971707-c413-4803-b80f-ae108f6ed7f6","added_by":"auto","created_at":"2025-01-10 17:20:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":213897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCPVax-CoV vaccine candidate biodistribution and preliminary safety evaluation. \u003c/strong\u003eBALB/c mice (male n=3, female n=3) were immunized intramuscularly with 5 µg of mRNA/animal encapsulated in Indium-111 labeled LNPs. (a) \u003cem\u003eIn vivo\u003c/em\u003e SPECT/CT representative images of coronal and axial sections acquired at different time points post-immunization for up to one week. The lower image shows the signal progression in the presacral lymph node (red arrow) with an adjusted threshold. (b) Signal quantification at the injection site at different points post-vaccination using standardized uptake value (SUV) units. (c) Signal quantification in the presacral lymph nodeat different points post-vaccination. (d) \u003cem\u003eEx vivo \u003c/em\u003esignal quantification at day 7 post-immunization, represented as injected dose per gram normalized to brain signal. (e) Serum biochemical analysis of hepatic enzymes 24 h post-immunization. Graphs are represented as mean ± SD. ns, non-significant as determined by two-way ANOVA with Tukey post-test (e).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/86d9e4fd9013a583e0f1fce2.png"},{"id":73512417,"identity":"859e5a78-90d0-44fa-a03c-a8e190d0d223","added_by":"auto","created_at":"2025-01-10 17:04:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVaccine-induced protection against mouse-adapted SARS-CoV-2 infection. \u003c/strong\u003e(a) Immunization scheme and sample collection schedule of mouse-adapted infection model. BALB/c mice were immunized intramuscularly at day 0 (prime) and 21 (boost) with 1 µg of mRNA/animal. At week 7 mice were challenged with 1x10\u003csup\u003e4\u003c/sup\u003e TCID50 of the mouse adapted strain MA20. At day 2 and 4 post-challenge, lung viral load and cytokine profile were evaluated (n=5). Clinical signs and survival were monitored during 25 days after challenge in an independent experimental group of each condition (n=10). (b) Body weight monitored over 25 days after challenge calculated as percentage of the initial weight (pre-challenge). (c) Survival rate over 25 days after challenge. (d) Lung viral load determined by virus titration assay in VERO E6 cells at day 2 and 4 post-infection. (e) Cytokine and chemokine profile in lung homogenates collected at day 2 and 4 post-challenge and analyzed by Luminex assay. \u0026nbsp;Graphs are represented as mean ±SD. ns, non-significant; **p\u0026lt;0.01; ****p\u0026lt;0.0001 as determined by two-way ANOVA with Tukey post-test.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/656c33b1d8f7439ee6dce920.png"},{"id":73513190,"identity":"02143a7c-729e-4a58-b30d-685d39a6e43b","added_by":"auto","created_at":"2025-01-10 17:12:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":131050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVaccine-induced protection against SARS-CoV-2 infection in humanized mouse model. \u003c/strong\u003e(a) Immunization scheme and challenge schedule of K18 infection model. C57BL/6-hACE2 K18 mice (n=5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. At week 7, mice were challenged with 1x10\u003csup\u003e5\u003c/sup\u003e TCID50 of SARS-CoV-2 alpha strain. At day 3 post-challenge, lung viral load was evaluated. (b) Body weight monitoring after 4 days of infection calculated as percentage of the initial weight (pre-challenge). (c) Lung viral load determined by virus titration assay in VERO E6 cells at day 3 post-infection. (d) Cytokine and chemokine profile in lung homogenates collected at day 3 post-challenge and analyzed by Luminex assay. Graphs are represented as mean ±SD. ns, non-significant; ***p\u0026lt; 0.001; ****p\u0026lt;0.0001 as determined by two-way ANOVA (b) and one-way ANOVA (c) with Tukey post-test.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/df5339424589c8762e82acaf.png"},{"id":73512418,"identity":"0de55fce-c3a5-43c0-a436-7a3d37b27b37","added_by":"auto","created_at":"2025-01-10 17:04:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":166477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of the CPVax-CoV platform's ability to trigger immune responses against VOCs. \u003c/strong\u003e(a) Timeline of CPVax-CoV-XBB immunity evaluation. BALB/c mice (n=5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. Blood samples were obtained at week 6 and serum neutralizing ability was determined. T-cell response was evaluated in splenocytes 3 weeks post-boost. (b) NT50 neutralization titers in serum of vaccinated mice determined at 3 weeks post-boost by Spike pseudovirus-neutralization assay using Wuhan and XBB Spike variants. \u0026nbsp;(c) IFNγ-secreting splenocytes quantification by ELISPOT after O.N. stimulation with SARS-CoV-2 Spike peptide pools of Wuhan and XBB variants. (d) Immunization scheme and challenge schedule of K18 infection model with XBB strain. C57BL/6-hACE2 K18 mice (n=5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. At week 7, mice were challenged with 5x10\u003csup\u003e3\u003c/sup\u003e TCID50 of SARS-CoV-2 XBB1.5 strain. At day 3 post-challenge, lung viral load was evaluated. (e) NT50 neutralization titers in serum of mice vaccinated determined at 3 weeks post-boost by Spike pseudovirus-neutralization assay using Wuhan and XBB Spike variants.\u0026nbsp; (f) Lung viral load determined by virus titration assay in VERO E6 cells at day 3 post-infection. Graphs are represented as mean ±SD. ns, non-significant; *p\u0026lt;0.05; **p\u0026lt;0.01; as determined by two-way ANOVA (c), unpaired t-test (e) and one-way ANOVA (f) with Tukey post-test.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/dfe195a7051a6cb28376cb30.png"},{"id":73512430,"identity":"365bbeb6-76d5-4066-98a4-52c8b5b00928","added_by":"auto","created_at":"2025-01-10 17:04:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":245854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term stability assessment of lyophilized vaccine formulation. \u003c/strong\u003e(a) Timeline of the stability study. Lyophilized CPVax-CoV was stored at 4 and 25 ºC and standard liquid vaccines were stored at -80 ºC as controls. Aliquots of each condition were reconstitutedat the indicated time-points to characterize their physicochemical properties and to evaluate their \u003cem\u003ein vivo \u003c/em\u003eactivity. \u0026nbsp;At 0, 3, 6 and 9 months, production of specific antibodies in serum was analyzed by ELISA after a single immunization of BALB/c mice with 1 µg of mRNA/animal. At 12 months, an efficacy study was conducted. After prime-boost immunization, animals were infected with mouse adapted SARS-CoV-2 and viral load, cytokines and histopathology were evaluated. (b) Determination of diameter, polydispersity index (PDI), Z potential and encapsulation efficacy of liquid and lyophilized LNPs at the indicated time-points of storage. (c) Reciprocal endpoint titers of anti-RBD IgG antibodies in serum samples collected at day 21 post-immunization determined by ELISA after the indicated time-points of storage. (d) Determination of diameter, PDI, Z potential and encapsulation efficacy at different time-points after resuspension and storage at 4 °C of CPVax-CoV. (e) Reciprocal endpoint titers of anti-RBD IgG antibodies in serum samples collected at day 21 post-immunization determined by ELISA at different time-points after resuspension and storage at 4 °C of CPVax-CoV. Graphs are represented as mean ±SD. ns, non-significant; as determined by one-way ANOVA with Tukey post-test.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/ab1d3da13039f6dcfe472982.png"},{"id":73512422,"identity":"94eb869b-e07b-4ebb-b16b-117aed1a0160","added_by":"auto","created_at":"2025-01-10 17:04:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":223804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective efficacy of lyophilized CPVax-CoV. \u003c/strong\u003eA-E Protection of one-year stored lyophilized CPVax-CoV at 4 and 25 °C in BALB/c mice infected with a viral dose of 1x10\u003csup\u003e4\u003c/sup\u003e TCID50 of MA20 strain. (a) Immunization scheme and challenge schedule of MA20 infection model. (b) NT50 neutralization titers in serum of vaccinated mice determined at 3 weeks post-boost by Spike pseudovirus-neutralization assay. (c) Body weight monitoring during 3 days after infection calculated as percentage of the initial weight (pre-challenge). (d) Lung viral load determined by virus titration assay in VERO E6 cells at day 3 post-infection. (e) Cytokine and chemokine profile in lung homogenates collected at day 3 post-challenge and analyzed by Luminex assay. \u0026nbsp;(f) Protective efficacy of lyophilized CPVax-CoV and CPVax-CoV-XBB in C57BL/6 humanized ACE2 model against 1x10\u003csup\u003e5\u003c/sup\u003e or 5x10\u003csup\u003e3\u003c/sup\u003e TCID50 of alpha or XBB infection respectively. Lung viral load determined by virus titration assay in VERO E6 cells on day 3 post-infection. Graphs are represented as mean ±SD. ns, non-significant; **p\u0026lt;0.01; ****p\u0026lt;0.0001 as determined by one-way ANOVA (b, d, f) and two-way ANOVA (c) with Tukey post-test.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/583fcc60db5cd3422eca5684.png"},{"id":86179123,"identity":"fa6e2eb3-401f-4867-a1b5-b59d09cb9264","added_by":"auto","created_at":"2025-07-07 16:16:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3192137,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/e7600fe6-258d-4b57-88b0-fc4b0bab9e23.pdf"},{"id":73512415,"identity":"5dd99ef9-e373-458f-b752-c46f50a30be9","added_by":"auto","created_at":"2025-01-10 17:04:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1360021,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5780846/v1/c2912f769c6ab8ef06264768.docx"}],"financialInterests":"Competing interest reported. EM, EB, CM, AS, SA, TA, AL, BA, DDM, JH, JGW, VL, DC, JM and EP are employees at the Certest Pharma Department, Certest Biotec S.L. EB, JH, DDM, JM and JGW are inventors on patents related to this publication","formattedTitle":"Enhanced stability and efficacy of a lyophilized mRNA SARS-CoV-2 vaccine incorporating novel Ionizable lipids after one year storage at 25ºC","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe outbreak of coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has significantly influenced global health, social dynamics, and economic activity. Vaccination remains the most potent strategy for preventing infection and controlling virus spread. Consequently, considerable efforts have been directed towards the rapid development and approval of various vaccines worldwide\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong these, mRNA technology has led the race of anti-SARS-CoV-2 vaccines and emerged as a promising vaccine platform due to their rapid development, manufacturing versatility, safety profile, and ability to elicit broad immune responses. Unlike traditional platforms, mRNA vaccines offer advantages such as rapid design based solely on sequence information, bypassing the need for virus culture or recombinant protein production. They also circumvent issues like insertional mutagenesis and pre-existing immunity that may hinder other vaccine types such as DNA or viral vector vaccines\u003csup\u003e2,3\u003c/sup\u003e. Approved mRNA vaccines, including BNT162b2/Comirnaty (Pfizer/BioNTech)\u003csup\u003e4\u003c/sup\u003e and mRNA-1273/SpikeVax (Moderna)\u003csup\u003e5\u003c/sup\u003e, have validated the potential of mRNA technology, demonstrating significant success in mitigating viral spread, reducing hospitalizations, and lowering mortality rates since their emergency authorization in 2020\u003csup\u003e6\u003c/sup\u003e Additionally, bivalent mRNA vaccines targeting specific SARS-CoV-2 variants have been approved, providing enhanced protection against emerging strains\u003csup\u003e7\u003c/sup\u003e, and ongoing research is focused on the development of combined vaccines that address both COVID-19 and seasonal influenza\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite their proven efficacy and rapid development, there is still considerable room for improvement in the field of mRNA vaccine components and stability to enhance protective efficacy against emerging variants or new pathogens and to ensure equal distribution and accessibility worldwide\u003csup\u003e9\u0026ndash;13\u003c/sup\u003e. Here we present a new mRNA vaccine platform that incorporates novel optimizations on mRNA sequence design and lipid formulation that is suitable for lyophilization and stable for long term storage.\u003c/p\u003e \u003cp\u003eOptimization of mRNA sequence, comprising antigen-coding sequence and non-coding regulatory elements directly impacts protein expression and vaccine efficacy\u003csup\u003e14\u003c/sup\u003e. mRNA vaccines typically comprise a codon-optimized nucleotide sequence encoding the antigen of interest\u003csup\u003e15\u003c/sup\u003e along with other stabilizing elements, including two non-coding regulatory sequences flanking the coding sequence (5'UTR and 3'UTR). Untranslated Regions (UTRs) contain multiple regulatory elements and are essential for mRNA stability and translation efficiency. Here we have incorporated novel UTR sequences optimized for efficient protein expression after intramuscular injection \u003csup\u003e16\u003c/sup\u003e into Spike coding mRNA.\u003c/p\u003e \u003cp\u003eAlong with mRNA sequence optimization, efficient delivery of mRNA into the cytoplasm of target cells is crucial to exert its therapeutic function. Lipid nanoparticles (LNPs) are currently the leading delivery system for mRNA vaccines. LNPs are commonly composed of 4 lipid components: an ionizable lipid, a helper phospholipid, cholesterol and a PEGylated lipid \u003csup\u003e17\u003c/sup\u003e. These lipids encapsulate the antigen-coding mRNA and protect the nucleic acid from degradation. The ionizable lipid is the most critical component of LNPs, as it determines the efficacy of transfection and endosomal escape. They typically feature a tertiary amine that remains deprotonated at physiological pH, improving the biocompatibility of LNPs, but become positively charged at lower pH conditions. Protonation of ionizable lipids at the endosomal acidic pH promote membrane destabilization and delivery of nucleic acid cargo into the cytosol of target cells\u003csup\u003e18\u003c/sup\u003e. Furthermore, ionizable lipid impacts vaccine adjuvanticity, which directly affect the therapeutic effect of mRNA vaccines\u003csup\u003e19\u003c/sup\u003e. Currently there are only two ionizable lipids (SM-102 and ALC-0315) approved for commercial use as part of mRNA vaccines\u003csup\u003e20,21\u003c/sup\u003e. We recently developed a new library of rationally designed ionizable lipids able to induce high protein expression levels in mice inoculated by the intramuscular route\u003csup\u003e22,23\u003c/sup\u003e, the most common route for administration of mRNA vaccines. However, their applicability as part of mRNA vaccines and their ability to induce immunity has not been addressed yet. In this study we have characterized this new family of ionizable lipids as part of mRNA vaccines against SARS-CoV-2, showing a strong potential for their use in mRNA-LNPs based vaccines.\u003c/p\u003e \u003cp\u003eA critical limitation in the global distribution of current mRNA vaccines is their dependence on cold-chain logistics to preserve mRNA integrity and maintain the physicochemical properties of LNPs\u003csup\u003e24,25\u003c/sup\u003e. The need for ultra-cold storage conditions poses a major barrier to ensure higher coverage in resource limited-settings which is essential for ensuring equitable protection.\u003csup\u003e11\u003c/sup\u003e. To address this challenge, lyophilization, or freeze-drying, has long been utilized to stabilize pharmaceuticals and vaccines, prolonging their shelf life and simplifying storage and distribution logistics. Applying this technique to mRNA vaccines holds immense potential to overcome the cold chain requirements associated with traditional liquid formulations, thereby enabling broader global access to advanced immunization technologies, since cryogenic transport and storage are needed for the current licensed mRNA vaccines\u003csup\u003e26\u003c/sup\u003e. In this study, we developed a lyophilized formulation of our candidate mRNA vaccine, CPVax-CoV, which maintains LNP physicochemical properties and biological activity showing comparable results to the standard liquid formulation.\u003c/p\u003e \u003cp\u003eCOVID-19 crisis underscored the critical need for novel vaccine platforms able to provide rapid and effective solutions in response to future outbreaks and emerging pathogens. Here we have applied innovative optimizations on delivery systems, mRNA sequence design and vaccine stability to develop CPVax-CoV, a novel SARS-CoV-2 mRNA-based vaccine that is thermostable for long-term storage in lyophilized format and elicits robust protective immunity compared to commercially available SARS-CoV-2 vaccines. Importantly, lyophilized CPVax-CoV induced protective immunity in mice challenged with SARS-CoV-2 even after one year of storage at 4 and 25\u0026deg;C, while maintaining intact LNP physicochemical properties. This platform has been validated against ancient and Omicron SARS-CoV-2 variants of concern (VOCs), and in a recently developed mouse model using a mouse adapted SARS-CoV-2 virus that resembles human COVID19\u003csup\u003e27\u003c/sup\u003e. Our findings support the efficacy of CPVax-CoV as a robust and flexible mRNA vaccine platform and provide significant impact on pandemic preparedness, highlighting the potential novel ionizable lipids, UTR sequences and lyophilization technology to be transferred to future mRNA vaccines against infectious diseases.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNovel ionizable lipid-based mRNA vaccines induce robust anti-SARS-CoV-2 immune response\u003c/h2\u003e \u003cp\u003eWe have recently reported the development of an extensive library of novel ionizable lipids proven to be safe and effective for mRNA expression \u003cem\u003ein vivo\u003c/em\u003e, showing enhanced results compared to approved lipid based LNPs\u003csup\u003e22,23\u003c/sup\u003e. Here we have selected the most promising novel ionizable lipids in terms of protein expression after intramuscular administration to evaluate their potential to induce immunity for mRNA vaccine applications (Structures shown in Supplementary Fig.\u0026nbsp;1.).\u003c/p\u003e \u003cp\u003eWe incorporated these lipids into LNP formulations to construct various mRNA vaccine candidates, all using the Spike-coding mRNA sequence from the commercial Comirnaty vaccine. Both the mRNA and LNPs were prepared in-house via \u003cem\u003ein vitro\u003c/em\u003e transcription and microfluidic mixing, respectively. These vaccine candidates were compared to a control LNP formulation, which replicated the Comirnaty vaccine using ALC-0315 as the ionizable lipid. Thus, ALC-0315 was formulated using Comirnaty lipid components at molar lipid ratios of 46.6:9.4:42.7:1.6 (ALC-0315: DSPC: cholesterol: ALC-0159) and the evaluated candidate lipids were formulated at molar ratios of 50:10:38.5:1.5 (ionizable lipid:DOPE:cholesterol:DMG-PEG2000). Physicochemical characterization of the resulting LNPs showed optimal values of diameter and encapsulation efficiency with no significant differences between the various candidates (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo evaluate the immune response elicited by the different vaccine formulations, BALB/c mice were intramuscularly immunized with 1 \u0026micro;g of mRNA/mice following a 21-day prime-boost regimen and specific B and T cell responses were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Production of specific anti-RBD IgG antibodies were detected in serum from all the vaccinated animals and they were significantly enhanced after boost immunization. Mice vaccinated with LNPs formulated with our novel ionizable lipids showed comparable antibody levels to mice vaccinated with the control vaccine using ALC-0315 as ionizable lipid. Importantly, antibody levels in mice vaccinated with LNPs containing CP-LC-0729 lipid were significantly higher than in those vaccinated with control vaccine after boost (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). T-cell response was evaluated 3 weeks post-boost in splenocytes after specific stimulation with SARS-CoV-2 Spike peptide mix. IFNγ-producing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) and cytokine secretion of IFNγ and IL-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) were measured by ELISPOT and ELISA respectively, showing T cell induction in all vaccinated animals with strong IFNγ and low IL-4 response, indicating a Th1 biased response. Although all groups exhibited elevated T cell responses, IFNγ production was notably higher in mice vaccinated with CP-LC-0729, CP-LC-0867, CP-LC-0743 or ALC-0315 compared to those vaccinated with CP-LC-0431 or CP-LC-0474 based vaccines.\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that LNPs formulated with our proprietary novel ionizable lipids exhibit robust induction of both B and T cell responses \u003cem\u003ein vivo\u003c/em\u003e, providing evidence for their potential use in next generation mRNA vaccines against SARS-CoV-2 and, potentially, other pathogens. CP-LC-0729 lipid was chosen to be further characterized in the next assays as it exhibits superior antibody response after boost compared to the evaluated candidates and the control vaccine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003emRNA sequence optimization enhances S-specific B and T-cell responses\u003c/h3\u003e\n\u003cp\u003eDespite their significant impact on therapeutic protein expression, current mRNA vaccines candidates often utilize 5\u0026prime; and 3\u0026prime;UTRs derived from commonly used human genes with minimal optimization. We have previously explored novel UTRs sequences following a semi-rational RNA sequence design approach leading to the discovery of new RNA motifs that enhance mRNA expression \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e16\u003c/sup\u003e. These optimized UTR sequences, when incorporated into mRNA, significantly improve protein production and outperform UTR designs used in commercially approved mRNA vaccines. Herein, we designed a codon-optimized mRNA sequence based on full length Spike protein of the Wuhan SARS-CoV-2 virus incorporating our proprietary UTR sequences and we tested its efficacy as part of a new mRNA vaccine candidate.\u003c/p\u003e \u003cp\u003eFirst, we evaluated Spike \u003cem\u003ein vitro\u003c/em\u003e expression in HEK293T to verify antigen production of our newly developed mRNA Spike sequence based on Wuhan strain (CP-S) by Western Blot and flow cytometry (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Then, we encapsulated Comirnaty or CP-S mRNA in LNPs formulated with CP-LC-0729 lipid at molar lipid ratios of 40.7:34.9:23.3:1.2 (CP-LC-0729:DOPE:cholesterol:DMG-PEG2000). These specific molar ratios were chosen based on our previous optimization studies of the LNP formulation for CP-LC-0729, which demonstrated enhanced intramuscular mRNA expression using these lipid ratios\u003csup\u003e23\u003c/sup\u003e. In order to evaluate vaccine immunity \u003cem\u003ein vivo\u003c/em\u003e, BALB/c mice were immunized in a 21-day prime-boost regimen. Serum antibody responses were analyzed at different time-points and T-cell response was evaluated in spleen 3 weeks post-boost (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAnti-RBD total IgG antibody levels in serum were 2.5-fold higher in the animals vaccinated with LNPs formulated with CP-S mRNA compared to the ones encapsulating Comirnaty mRNA or the control vaccine after the first immunization. After the second immunization, these differences increased significantly, showing 5-fold and 4.3-fold higher titers in the CP-S mRNA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eNeutralizing antibodies were analyzed through a pseudovirus-based assay using GFP-expressing lentivirus pseudotyped with SARS-CoV-2 Spike protein from Wuhan strain. Neutralizing titer 50 (NT50) post-boost was comparable in all the experimental groups and slightly higher, although not significantly, in serum from mice vaccinated with CP-LC-0729 encapsulating CP-S mRNA, in accordance with the results of total anti-RBD binding IgG titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eSpecific T cell response was analyzed in splenocytes at 3 weeks post-boost. Cytokine secretion after specific stimulation showed enhanced production of IFNy in mice vaccinated with CP-LC-0729 LNPs compared with the cell samples from animals immunized with the control vaccine. Notably, this difference was significantly higher in CP-LC-0729 LNPs encapsulating CP-S mRNA, both in terms of IFNy secreting splenocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) and total IFNy secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). T cell populations in spleen were evaluated by flow cytometry, showing higher levels of total CD4 T cells in mice vaccinated with CP-LC-0729 LNPs encapsulating CP-S mRNA. No significant differences were found in total CD8 T cell levels between the evaluated vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eTo further characterize the immune response triggered by these mRNA vaccine candidates, we evaluated early germinal center (GC) B cell and T helper follicular (Thf) cell responses in the draining lymph nodes. Thf are critical for GC formation, which directly impacts B-cell differentiation and long-lived plasma cell expansion. Early Thf and GC responses have been previously related to protective humoral immunity in mRNA vaccines\u003csup\u003e28\u003c/sup\u003e. BALB/c mice were immunized with a single dose of 5 \u0026micro;g mRNA/mouse. One-week post-prime Thf and GC responses were evaluated in the draining lymph nodes by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, significant expansion of GC B cells and Thf cells populations was observed in the vaccinated animals compared to the non-vaccinated controls. This result correlates with a robust antibody specific response previously observed in the serum analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn summary, these data indicate that our optimized mRNA sequence enhances the immune response induced by our novel lipid-based vaccine, underscoring the importance of UTR optimization in the development of more effective mRNA vaccines. In base of these results, we defined our final vaccine candidate, CPVax-CoV, that incorporates novel ionizable lipid CP-LC-0729 and CP-S mRNA sequence. Our results indicate that this novel vaccine candidate induces robust immune B and T cell immune responses and outperforms Comirnaty vaccine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCPVax-CoV biodistribution and preliminary safety evaluation\u003c/h3\u003e\n\u003cp\u003eSafety is an essential requisite for any newly developed vaccine. LNPs, especially ionizable lipids, are frequently identified as a safe concern in mRNA vaccines, since they exhibit adjuvant properties and are determinant for the pharmacokinetics and biodistribution profile of mRNA vaccines\u003csup\u003e29,30\u003c/sup\u003e. Consequently, special attention needs to be addressed to the tolerability and safety profile of novel ionizable lipids as part of LNPs-based therapies. We have preliminarily evaluated our ionizable lipid candidate CP-LC-0729 in mice after systemic administration of high dose of FLuc mRNA-LNPs by intravenous injection, showing a good safety profile with no signs of hepatic or systemic toxicity\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo further characterize the safety and pharmacokinetics (PK) profile of CPVax-CoV vaccine, we carried out a biodistribution study to track LNPs accumulation and elimination \u003cem\u003ein vivo\u003c/em\u003e. LNPs were labeled with indium-111 oxinate and intramuscularly injected in male and female BALB/c mice. Images were acquired using microSPECT/CT at 1, 3 and 6 h and 1, 2, 3, 4 and 7 days post-injection. The visual analysis of the images showed a strong signal at the injection site, but LNPs signal was also found in the sacral ganglion chain, bone marrow (long bone heads/iliac), and kidneys (elimination pathway) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eMaximum signal intensity at the injection site decreased over time, but persisted for at least 4 days, reflecting a primarily distribution of LNPs at the injection site within the first 24 hours post-administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Draining through the retroperitoneal and medial sacral lymph nodes was observed in all animals starting from 1 h post-administration, with signal intensity peaking at 6 h and gradually decreasing thereafter (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). At 6 h post-administration, the appearance of signal in the bone marrow was visually detected, peaking approximately at 1\u0026ndash;2 days post-injection (Figure S5). \u003cem\u003eEx vivo\u003c/em\u003e analysis on day 7 supported the \u003cem\u003ein vivo\u003c/em\u003e imaging data, showing clear drainage through the lymph nodes when normalized by organ weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eMigration to draining lymph nodes is compatible with the observed Thf cell expansion, GC formation and immune response elicited by vaccination, the signal observed in kidneys is compatible with urine excretion and no accumulation in liver or other organs was detected.\u003c/p\u003e \u003cp\u003eTo corroborate vaccine tolerability, hepatic enzyme levels in serum were evaluated 24 h post-vaccination. No increment in AST and ALT levels were observed compared to the serum analysis data pre-vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These data provide preliminary evidence of vaccine safety and support CPVax-CoV for further evaluation as mRNA vaccine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCPVax-CoV induces protection against SARS-CoV-2 challenge\u003c/h3\u003e\n\u003cp\u003eTo evaluate vaccine-induced protection of CPVax-CoV against SARS-CoV-2, we conducted protection experiments \u003cem\u003ein vivo\u003c/em\u003e and validated the results in two different mouse infection models: one using a mouse adapted SARS-CoV-2 strain and the other using a hACE2-K18 transgenic mice.\u003c/p\u003e \u003cp\u003eFirst, a mouse-adapted SARS-CoV-2 strain (MA20) was used to infect wild type BALB/c mice. MA20 was obtained from a clinical isolate of the SARS-CoV-2 Alpha variant (Pango lineage Nomenclature B.1.1.7) after 20 serial passages in one-year-old C57BL/6 mice. MA20 is able of inducing severe lung pathology in mice and recapitulates key pathological features of COVID-19 in humans including higher severity in males, lymphodepletion and increase in inflammatory cytokine response and acture respiratory distress syndrome (ARDS)\u003csup\u003e27\u003c/sup\u003e. BALB/c female mice were immunized in a 21-day prime-boost regimen with 1 \u0026micro;g of mRNA/animal of CPVax-CoV and the control vaccine. Four weeks post-boost mice were intranasally infected with MA20. Survival, viral load and cytokine profile in lungs were determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). All vaccinated animals survived over a 25-day period after virus challenge (endpoint of the experiment) without showing weight loss or clinical signs of disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). In contrast, unvaccinated control mice showed severe weight loss and were euthanized on day 4 post-infection according to previously established human endpoint criteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). In accordance with survival outcomes, infecting virus were detected by TCID50 assay in lungs from unvaccinated mice but not in lungs from vaccinated mice at day 2 or 4 post-challenge, except for one mouse in the control vaccine group, where low viral load was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eInflammatory virus-associated cytokine and chemokine markers were analyzed in lung homogenates using a multiplex assay as a complementary indicator of vaccine-induced protection. These cytokines are typically associated to virus-induced inflammation as well as lung damage and correlate with disease progression\u003csup\u003e32\u003c/sup\u003e. Cytokine and chemokine levels were significantly incremented in the unvaccinated compared to the vaccinated animals, especially at day 2 post-infection. These cytokines were not upregulated in the lungs from the vaccinated groups in any of the evaluated time-points after virus challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThe results obtained with the virus adapted strain were validated in a transgenic C57BL/6 hACE2-K18 mouse model infected with an Alpha variant SARS-CoV-2 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The K18-hACE2 model of SARS-CoV-2 infection has been previously described as closely mimicking many aspects of severe COVID-19 and has been widely used in the evaluation of vaccines and antiviral therapies\u003csup\u003e33\u003c/sup\u003e. In accordance with the results for the mouse-adapted strain, no weight loss or clinical signs of disease were observed in vaccinated animals during the course of the experiment, in contrast with the non-vaccinated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Lung viral load was not detected in lungs from any of the vaccinated groups at day 4 post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In addition, no significant increment in lung inflammatory cytokines and chemokines was detected in the vaccinated mice, in contrast with the non-vaccinated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eCollectively, these data reveal that CPVax-CoV exhibit a strong vaccine-induced protection against viral SARS-CoV-2 challenge in wild type and ACE-2 humanized mouse models.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCPVax-CoV platform validation for SARS-CoV-2 Variants of Concern\u003c/h3\u003e\n\u003cp\u003eDespite the remarkable efficacy demonstrated by SARS-CoV-2 vaccines, the emergence of novel variants poses a persistent challenge, compromising the level of protection conferred by current immunization strategies. As the virus continues to evolve, these variants exhibit diverse genetic mutations that may impact vaccine effectiveness. This underscores the need for continuous surveillance and adaptation of vaccination strategies to address the evolving challenge posed by these variants. To validate the versatility of CPVax-CoV vaccine platform to be adapted to emerging variants of concern, we developed a vaccine candidate against Omicron XBB1.5 variant using CPVax-CoV technology. We chose XBB lineage since it was one of the most recently emerged and prevalent variants at the time these experiments were conducted\u003csup\u003e34,35\u003c/sup\u003e. XBB variants exhibited superior growth advantages over most omicron mutants and have shown a strong evasive ability against the neutralization of plasma and serum from vaccinated or convalescent individuals\u003csup\u003e36,37\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe first verified antigen expression \u003cem\u003ein vitro\u003c/em\u003e of the new XBB mRNA after HEK293T transfection by Western Blot and Flow Cytometry and we confirmed that Spike XBB mRNA was correctly translated in eukaryotic cells (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Then, XBB and Wuhan mRNAs were formulated into LNPs with CPVax-CoV lipid composition resulting in CPVax-CoV-XBB vaccine, which exhibited comparable physicochemical properties to that of CPVax-CoV (Table SX).\u003c/p\u003e \u003cp\u003eImmunity of these vaccines against ancient strain and XBB variant was evaluated in BALB/c mice following a 21-day prime-boost vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To evaluate variant-specific antibody neutralizing ability, an XBB Spike pseudovirus expressing GFP was constructed. Infection ability of XBB-Pseudovirus was confirmed in Vero E6-ACE2-TMPRSS2 cells (Figure S3). Serum neutralization was determined after boost immunization against both Wuhan and XBB variants and neutralization titers were found to be variant specific. No neutralization was observed in serum from CPVax-CoV-XBB against the ancient Wuhan variant. Conversely, NT50 of Wuhan Spike-based vaccines were not detected against XBB variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Spleen specific T-cell response was also evaluated at week 6 post prime. Variant selectivity was lower for specific-T cell response than that observed for antibody-response. IFN-y secreting cells were detected in splenocytes from all vaccinated animals after stimulation with specific peptides mixes of Wuhan or XBB Spike variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). A strong cross-reactive T cell response against various SARS-CoV-2 VOCs has been previously reported, highlighting the importance of robust T-cell induction in SARS-CoV-2 immunization strategies\u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, we evaluated vaccine-induced protection of our XBB based-vaccine candidate. Infection kinetics of XBB strain in hACE2-k18 and BALB/c mouse models was first evaluated to choose the optimal infection model for the vaccine efficacy study. In contrast to ancient strains, no weight loss or clinical signs of disease were detected over the first week of infection. However, infecting viral particles were present in lungs of infected mice in both mouse models, although virus titers were higher in hACE2-K18 mice (Fig S5), which was selected as infection model for XBB strain. These pathological outcomes are in accordance with previous results observed in rodent infection models of SARS-CoV-2 for XBB.1 stain\u003csup\u003e39,40\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eK18 mice were immunized with CPVax-CoV and CPVax-CoV-XBB following a 21-day prime-boost regimen (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Serum neutralization titers against XBB-PSV were analyzed before infection. Neutralization against XBB Pseudovirus was not detected for the CPVax-CoV (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), showing comparable results to those obtained in the BALB/c mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Protective efficacy was evaluated by determination of viral load in lungs. Despite the differences observed between Wuhan and XBB Spike based vaccines in serum neutralizing ability, no infecting viruses were detected in lungs from any vaccinated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Accordingly, no weight loss was observed in any of the vaccinated groups after challenge (Figure S5).\u003c/p\u003e \u003cp\u003eThese results demonstrate the potential of the CP vaccine platform to be effectively used with different antigen sequences for the development of SARS-CoV-2 vaccines against VOCs. Notably, the CPVax-CoV wild type vaccine also provided significant protection against XBB variant, highlighting its broad efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLyophilized CPVax-CoV exhibits long-term thermostability\u003c/h2\u003e \u003cp\u003eThermostability and storage requirements are major factors limiting universal access to RNA vaccines. The two currently licensed mRNA vaccines, Comirnaty and Spikevax, require cryogenic transport and storage at \u0026minus;\u0026thinsp;80\u0026deg;C to \u0026minus;\u0026thinsp;60\u0026deg;C and \u0026minus;\u0026thinsp;20\u0026deg;C, respectively\u003csup\u003e41\u003c/sup\u003e. Consequently, developing thermostable mRNA vaccines is crucial to ensure accessibility in medium and low-income settings.\u003c/p\u003e \u003cp\u003eTo address this challenge, we have developed a lyophilized formulation for CPVax-CoV vaccine that maintains LNPs physicochemical properties and effectively preserves vaccine induced immune response after long-term storage at 4\u0026deg;C and 25\u0026deg;C. To this end, we employed an optimized lyophilization method based on a previously described protocol​\u003csup\u003e42\u003c/sup\u003e. Furthermore, a long-term stability study was conducted for up to one year, demonstrating that the lyophilization process effectively preserved the characteristics and \u003cem\u003ein vivo\u003c/em\u003e functionality of LNPs under both refrigerated and ambient temperature conditions throughout the study period, without the need of freezing.\u003c/p\u003e \u003cp\u003eTo evaluate the long-term thermostability and biological efficacy of our lyophilized CPVax-CoV formulation, lyophilized CPVax-CoV LNPs from the same production batch were stored at 4 and 25\u0026deg;C for up to one year. Non-lyophilized CPVax-CoV and Comirnaty vaccines stored at -80\u0026deg;C were included as experimental controls as the standard conditions used for the long-term storage of LNP based vaccines\u003csup\u003e43\u003c/sup\u003e. At various time points throughout this time-period, we assessed the physicochemical properties of the LNPs, including particle size, zeta potential, polydispersity index (PDI) and encapsulation efficiency, alongside their \u003cem\u003ein vivo\u003c/em\u003e biological activity. BALB/c mice were immunized via a single intramuscular injection at each indicated time point using the same dosage as in the non-lyophilized vaccine formulation (1 \u0026micro;g of mRNA per animal). Serum samples were collected 21 days post-vaccination for specific antibody quantification. After 12 months of storage, a protective efficacy study was also conducted to assess the vaccine's ability to confer protection against SARS-CoV-2 challenge in the mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, the size of the LNPs increased slightly after lyophilization, a result that is consistent with previous reports in the literature\u003csup\u003e44\u003c/sup\u003e. PDI, RNA encapsulation efficiency, and zeta potential showed no significant deviations between the lyophilized and non-lyophilized LNPs. Over the course of the one-year storage period, these parameters remained largely constant under the tested conditions. Lyophilized CPVax-CoV stored at 4\u0026deg;C maintained their initial particle size for up to one year after lyophilization, while LNPs stored at 25\u0026deg;C exhibited a slight increase in size after six months, which then stabilized and remained consistent for up to one year. In line with the physicochemical characterization results, the lyophilized CPVax-CoV vaccine maintained robust \u003cem\u003ein vivo\u003c/em\u003e biological activity throughout the entire storage period, irrespective of the storage temperature. IgG anti-RBD levels remained consistently high across all experimental groups at each evaluated time point. Importantly, no significant differences were observed between the controls stored at -80\u0026deg;C and the lyophilized CPVax-CoV stored at 4\u0026deg;C or 25\u0026deg;C. Notably, the lyophilized vaccine stored at 25\u0026deg;C successfully induced specific antibody levels that were comparable to those of the \u0026minus;\u0026thinsp;80\u0026deg;C stored controls after one year, demonstrating outstanding preservation of immunogenicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). These results underscore the long-term thermostability of our lyophilized CPVax-CoV formulation, highlighting its potential for effective deployment in diverse temperature conditions without compromising efficacy.\u003c/p\u003e \u003cp\u003eIn addition to assessing the long-term stability of the lyophilized CPVax-CoV, we evaluated the short-term stability of the resuspended lyophilized vaccine, a critical aspect for practical medical applications. To this end, the reconstituted CPVax-CoV was stored at 4\u0026deg;C for up to 96 h, during which we monitored the physicochemical properties of the LNPs and their \u003cem\u003ein vivo\u003c/em\u003e biological activity. Our analysis revealed that the LNP size and encapsulation efficiency remained consistent throughout the storage period, showing no significant deviations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Furthermore, the vaccine's ability to induce specific antibody production in mice was unaffected, with no significant differences observed between the freshly resuspended vaccine and the one stored for 96 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). These findings underscore the potential utility of lyophilized CPVax-CoV in clinical settings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLyophilized CPVax-CoV maintains protective efficacy after one year at 25°C\u003c/h3\u003e\n\u003cp\u003eProtection induced by lyophilized CPVax-CoV after one year storage at 4 and 25\u0026deg;C was evaluated in the mouse model. BALB/c mice were vaccinated in a prime boost regimen and challenged with MA20 mouse adapted SARS-CoV-2 strain\u003csup\u003e27\u003c/sup\u003e. In addition to the one year stored lyophilized and control vaccines, freshly prepared CPVax-CoV and Comirnaty vaccines were included in this study as additional controls. Serum neutralization was evaluated at 3 weeks post-boost and viral load, cytokine production and histopathology were evaluated in lungs at day 3 post-infection.\u003c/p\u003e \u003cp\u003ePrior to virus challenge, anti-RBD IgG antibodies (Figure S8) and serum neutralization were evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea), showing high binding and neutralizing antibody titers with no significant differences between lyophilized vaccines stored at 4 and 25 \u0026ordm;C and freshly formulated LNPs.\u003c/p\u003e \u003cp\u003eNo weight loss or clinical signs of disease progression were observed in any of the vaccinated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Accordingly, infecting virus in lungs were only detected in the non-vaccinated animals in the TCID50 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). These results are consistent with the cytokine profile observed in lung homogenates, since the inflammatory cytokines and chemokines evaluated were significantly higher in lungs from unvaccinated mice and no significant differences in cytokine levels were found between mice vaccinated with controls and 4 and 25\u0026deg;C lyophilized CPVax-CoV (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eFinally, we validated our lyophilization technology to be applied to different mRNA payloads and developed a lyophilized CPVax-CoV-XBB. Physicochemical parameters of the lyophilized CPVax-CoV-XBB were comparable to those obtained with CPVax-CoV (Table S2). Lyophilized CPVax-CoV-XBB and CPVax-CoV protective efficacy was evaluated in the humanized K18 mouse model after infection with XBB or alpha SARS-CoV-2 strains respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef). In line with the results obtained in the mouse-adapted infection model vaccinated with the Wuhan based Spike sequence, no viral load in lungs were detected in K18 mice immunized with lyophilized CPVax-CoV-XBB or CPVax-CoV at day 3 after infection with their respective viral strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that lyophilized CPVax-CoV, stored at both 4\u0026deg;C and 25\u0026deg;C for up to one year, provides robust and consistent protection against SARS-CoV-2 infection in mice. Furthermore, this lyophilization technology proved to effectively maintain the efficacy of our LNP formulations with different mRNA spike sequences, demonstrating its adaptability to other mRNA sequences from SARS-CoV-2 and potentially other pathogens.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe rapid development of mRNA vaccines has proven to be a transformative approach to combating the COVID-19 pandemic\u003csup\u003e45\u003c/sup\u003e. Despite their success, challenges remain in enhancing vaccine stability and optimizing the delivery systems to ensure easily deployment and broader accessibility. This study addresses these challenges by developing an mRNA vaccine platform that incorporates optimized UTR sequences and rationally designed ionizable lipids in a novel LNP formulation that can be successfully lyophilized for long-term storage to overcome the limitations of existing mRNA vaccine formulations.\u003c/p\u003e \u003cp\u003eLNPs are the leading delivery system for mRNA vaccines and the ionizable lipids play a critical role in mRNA transfection efficiency and endosomal escape\u003csup\u003e18\u003c/sup\u003e. Although many ionizable lipids have been already described, very few have been approved for clinical use are currently licensed for mRNA vaccines applications \u003csup\u003e4,5,46\u003c/sup\u003e. We have recently described a new library of ionizable lipids developed using a high-throughput screening using a multicomponent thiolactone-based synthesis platform that allowed us to synthesize many lipid candidates in a cost-effective, scalable and time-efficient manner. This screening process enabled us to establish critical structure-function relationships, particularly related to the hydrophobic tails and functional groups that influence endosomal escape and membrane-disruption activity. As a result, we identified several lipid candidates with enhanced \u003cem\u003ein vivo\u003c/em\u003e mRNA delivery than gold standard ionizable lipids, as evidenced by superior performance in luciferase protein expression after intramuscular injection. Additionally, the use of our thiolactone-based lipids offers significant advantages due to an optimized synthetic method, featuring a one-pot synthesis with short reaction times (2 hours) and room temperature\u003csup\u003e23\u003c/sup\u003e, in contrast to lipids such as SM-102 or ALC-0315\u003csup\u003e47,48\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, we have first evaluated the immunogenic potential of some of our top-performing ionizable lipids in the context of a SARS-CoV-2 vaccination model, hypothesizing that the increased protein expression observed after intramuscular administration would lead to a stronger antigen presentation, thereby enhancing robust immune responses. Our results indicate that all the ionizable lipids tested here efficiently encapsulated the Spike mRNA in a standard LNP formulation and were capable of inducing both humoral and cellular immune responses. Although a strong Th1 polarized immune response was observed across all the experimental groups, lipids CP-LC-0431 and CP-LC-0474 were excluded due to their comparatively lower induction of IFNγ secretion compared to the other lipids and the commercial vaccine formulation. CP-LC-0729 was selected as the lead candidate as it induced one of the strongest immune responses, which correlates with our previous observations for intramuscular protein expression\u003csup\u003e23\u003c/sup\u003e. Furthermore, CP-LC-0729 has been extensively characterized in our previous work and has proven to be safe and well tolerated after systemic injection\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOnce the ionizable lipid CP-LC-0729 was selected, we incorporated an optimized mRNA sequence encoding the SARS-CoV-2 Spike protein, featuring proprietary UTRs designed to enhance translation efficiency\u003csup\u003e16\u003c/sup\u003e. The incorporation of this optimized sequence significantly improved the immune response associated with the LNP formulated with CP-LC-0729 in terms of B and T cell responses. Notably, the optimization of UTRs can lead to increased protein expression, allowing for a reduction in the required vaccine dose without compromising immunogenicity. For instance, the Comirnaty vaccine uses 30 \u0026micro;g of mRNA per dose, while the Spikevax vaccine requires a higher dose of 100 \u0026micro;g of mRNA per dose. These differences in mRNA dosage likely reflect variations in the efficiency of their respective UTRs and LNP formulations, with Comirnaty\u0026rsquo;s UTRs potentially offering more efficient translation at a lower mRNA concentration\u003csup\u003e21\u003c/sup\u003e. Our findings reinforce the critical role of UTR optimization, along with the synergy between mRNA design and LNP delivery systems, in producing robust and durable immune responses with potentially lower mRNA doses. Based on these data, we defined our final vaccine candidate, CPVax-CoV, which combines the ionizable lipid CP-LC-0729 with our optimized mRNA sequence. CPVax-CoV demonstrated complete protection against SARS-CoV-2 infection in both mouse-adapted and humanized murine infection models, as it was able to fully protect vaccinated mice from productive SARS-CoV-2 infection and virus associated inflammatory response in the lungs. Additionally, the biodistribution study of CPVax-CoV revealed that the vaccine primarily localizes at the injection site and in the draining lymph nodes after intramuscular injection, which is likely contributing to its strong induced immune response. Although more detailed studies are needed to fully characterize the pharmacokinetic profile of CPVax-CoV, renal excretion was observed and no accumulation of the vaccine was detected in major organs after 7 days, preliminarily indicating a favorable tolerability profile.\u003c/p\u003e \u003cp\u003eOne of the most significant achievements of this study is the successful lyophilization of CPVax-CoV. While previous studies on lyophilized mRNA vaccines have demonstrated efficacy with freshly prepared lyophilized formulations and stability for shorter time-periods\u003csup\u003e44,49\u0026ndash;51\u003c/sup\u003e, this is the first report, to our knowledge, that demonstrates vaccine-induced efficacy in a relevant \u003cem\u003ein vivo\u003c/em\u003e infection model after one year of storage at 25\u0026deg;C. We have previously described an optimized lyophilization protocol for LNPs encapsulating luciferase-coding mRNA using SM102 as the ionizable lipid, which successfully preserved LNP physicochemical properties and maintained protein expression \u003cem\u003ein vivo\u003c/em\u003e after extended storage at 4\u0026deg;C, compared to their liquid counterparts\u003csup\u003e42\u003c/sup\u003e. Applying this optimized protocol to CPVax-CoV, we demonstrated that our recently described ionizable lipids are compatible with lyophilization and the LNPs retain their integrity and their ability to induce immune response after prolonged storage.\u003c/p\u003e \u003cp\u003eThe demonstration that the lyophilized LNPs remain stable after reconstitution in liquid buffer for at least 96 h at 4\u0026deg;C further enhances the practicality of this platform in clinical settings, allowing for more flexibility in vaccine administration and in diverse medical environments. It is important to note that this assessment was conducted with freshly prepared lyophilized LNPs, and further studies are needed to evaluate the stability of the reconstituted LNPs after long-term storage and at different temperatures including room temperature. Notably, current SARS-CoV-2 mRNA vaccines have a limited recommended timeframe for use once diluted, with usability restricted to up to 6 hours for Comirnaty and 12 hours for Spikevax\u003csup\u003e52\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results presented here with mRNA encoding the Spike protein of the XBB variant demonstrate that this platform is highly adaptable to other mRNA sequences. Interestingly, despite the lack of detectable neutralizing antibodies against XBB, CPVax-CoV is able to induce protection in lungs. Further supporting this finding and highlighting the potential for broad protection against other VOCs, it should be noted that MA20 strain carries several mutations in the Spike protein and in other viral proteins such as ORF1a and N, as a consequence of mouse adaptation. Despite these genetic changes, our vaccine formulation demonstrated strong protective efficacy. In this regard, immunity studies demonstrated that the vaccine elicited a strong variant-independent T cell response, which likely contributed to the observed protection in mice of CPVax-CoV against XBB challenge. This finding highlights the role of cellular immunity in protecting against viral variants\u003csup\u003e38\u003c/sup\u003e and suggests that CPVax-CoV could provide cross-protection against emerging variants. However, further studies of immunity and other correlates of protection against XBB and different VOCs are required to confirm these preliminary findings and to fully understand the mechanisms of protection conferred by CPVax-CoV against VOCs.\u003c/p\u003e \u003cp\u003eThe findings presented in this study also open up valuable opportunities for future research. First, further investigation into long-term immunity is essential to fully understand the durability of both humoral and cellular responses, particularly the persistence of T cell-mediated protection against variants of concern (VOCs). Exploring long-term immune memory, mucosal immunity, and viral dissemination to other organs, as well as conducting studies in additional relevant animal models, will provide a deeper understanding of CPVax-CoV\u0026rsquo;s full protective potential, PK and safety. Additionally, while the lyophilized formulation of CPVax-CoV has demonstrated stability for one year at 25\u0026deg;C, further research into stability at higher temperatures and under diverse environmental conditions would expand its applicability. This will be especially important for optimizing its widespread use in various climates and global vaccination campaigns, especially in regions where maintaining cold-chain logistics is challenging.\u003c/p\u003e \u003cp\u003eIn summary, CPVax-CoV represents a significant advancement in mRNA vaccine development by addressing key challenges in stability, delivery and efficacy. The combination of novel ionizable lipids and optimized mRNA sequences, along with successful lyophilization, offers a scalable and accessible solution that can overcome the logistical barriers of cold-chain dependency, making it highly suitable for global deployment in future pandemics. Although further research will be crucial to fully unlock its potential and ensure its adaptability across different contexts and pathogens, our findings provide a solid foundation for further research into the use of this technology to broader applications beyond SARS-CoV-2.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDNA template design\u003c/h2\u003e \u003cp\u003eThe DNA template for Comirnaty vaccine was constructed by cloning the publicly available mRNA vaccine sequence\u003csup\u003e53\u003c/sup\u003e into a pUC-based plasmid under the control of a T7 promoter.\u003c/p\u003e \u003cp\u003eFor CPVax-CoV vaccine the coding sequence of the Spike SARS-CoV-2 protein from Wuhan-H1 strain (GISAID\u003csup\u003e54\u003c/sup\u003e accession number EPI_ISL_402124) was codon-optimized for human expression. This sequence included the K986P and V987P (2P) stabilizing mutations and the substitution of the RRAR furin cleavage site at residues 682\u0026ndash;685 with GSAS. For CPVax-CoV -XBB vaccine, the DNA template was generated using the XBB.1.5 strain sequence (GISAID accession number EPI_ISL_15851788). The same 2P and RRAR to GSAS modifications were introduced, and the sequence was similarly codon-optimized for human use.\u003c/p\u003e \u003cp\u003eBoth CPVax-CoV or CPVax-CoV-XBB, optimized sequences were inserted in-frame immediately downstream the 5\u0026acute;UTR into a pUC based plasmid. This plasmid featured the following elements arranged in a 5\u0026prime; to 3\u0026prime; orientation: a T7 RNA polymerase promoter, an optimized 5\u0026prime; UTR from human APOA2\u003csup\u003e16\u003c/sup\u003e, the 3\u0026prime; UTR from human beta-globin (GenBank: NM_000518), a polyadenylation tail consisting of 100 adenines, and a BspQI restriction enzyme site.\u003c/p\u003e \u003cp\u003eGene synthesis, cloning, and plasmid preparations were outsourced to Genscript. The purified plasmids were subsequently used for \u003cem\u003ein vitro\u003c/em\u003e transcription.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etranscription and mRNA purification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach plasmid containing the DNA template sequence of interest was digested with BspQI (ON-124, HONGENE), which cleaved the plasmid immediately after the segment to be transcribed. The linearization reaction was then purified with the Wizard SV Gel and PCR Clean-Up (Promega A7270), in accordance with the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eThe purified linear DNA was subsequently employed for mRNA production by \u003cem\u003ein vitro\u003c/em\u003e transcription using T7 RNA polymerase following manufacturer's instructions. Briefly, transcription reactions were performed at 37\u0026deg;C for 3 h using the following materials:\u003c/p\u003e \u003cp\u003eTemplate linear DNA (50 \u0026micro;g/mL), T7 RNA polymerase (5000 U/mL; HONGENE, ON-004), RNAse inhibitor (1000 U/mL; HONGENE, ON-039), Inorganic Pyrophosphatase (2 U/mL; HONGENE, ON-025), ATP (5 \u0026micro;g /mL; HONGENE, R1331), GTP (5 \u0026micro;g/mL; HONGENE, R2331), CTP (5 \u0026micro;g/mL; HONGENE, R3331), N1-Methylpseudouridine (5 \u0026micro;g/mL, HONGENE, R5-027), CAP AG (4 \u0026micro;g mL, Hongene, ON-134) and RNAse-free double-distilled water.\u003c/p\u003e \u003cp\u003eThe generated mRNA transcripts were initially treated using DNaseI incubation (Hongene, ON-109) according to manufacturer's instructions.\u003c/p\u003e \u003cp\u003eIn order to reduce double-stranded RNA (dsRNA) contaminants, the resulting mRNA was purified through a process involved two sequential chromatography steps, starting with anion exchange chromatography (AEX) followed by affinity chromatography\u003csup\u003e55\u003c/sup\u003e. Initially, the mRNA sample was purified using the CIMmultus PrimaS Chromatography Column (BIA Separations). IVT mixture was diluted once in sample loading/equilibrate buffer A (20 mM Tris, 20 mM BTP, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH\u0026thinsp;=\u0026thinsp;8.0) and loaded onto the column. After unbound IVT components eluted in flow-through, a wash 1 with equilibration buffer was necessary, followed by a high-salt wash with 50 mM Tris, 3.0 M guanidine-HCl, 20 mM EDTA, pH 8.0. And after a wash 3 with buffer A until UV returns to baseline, a step elution was performed with buffer C (20 mM Tris, 20 mM BTP, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH\u0026thinsp;=\u0026thinsp;11.0). Fractions were neutralized immediately after elution. Following, additional purification was carried out through affinity chromatography using POROS Oligo (dT)25 column (ThermoFisher). Specifically, the buffers employed were as follows: Buffer A which contained 50 mM disodium phosphate, 0.5 M NaCl, 5 mM EDTA, pH\u0026thinsp;=\u0026thinsp;7.0 and Buffer B which contained 50 mM sodium dihydrogen phosphate, 5 mM EDTA, pH\u0026thinsp;=\u0026thinsp;7.0. The mRNA samples were initially half-diluted in Buffer A 2x. Following this, the column was equilibrated with 100% Buffer A, loaded with mRNA, washed with Buffer B, and ultimately eluted using double-deionized water. To completely remove Buffer B, mRNA was washed with a 30KDa Amicon\u0026reg; filter and then equilibrated through a one-tenth dilution in citrate buffer 10x with a pH of 6.5.\u003c/p\u003e \u003cp\u003eThe concentration of mRNA was determined by measuring the optical density at 260 nm, then adjusted to a final concentration of 1 mg/ml, aliquoted and stored at -80\u0026deg;C until needed.\u003c/p\u003e \u003cp\u003eFor quality assurance, all mRNAs underwent analysis through automated electrophoresis (2100 Bioanalyzer G2938B, Agilent). Subsequently, the mRNA samples were aliquoted and stored at -80\u0026deg;C until needed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and characterization of ionizable lipids\u003c/h2\u003e \u003cp\u003eAll intermediate compounds and ionizable lipids were synthesized following protocols based on the Sequential Thiolactone Amine Acrylate Reaction\u003csup\u003e23\u003c/sup\u003e. Briefly, following the synthesis of thiolactone derivatives, the ionizable lipid is obtained via a multicomponent one-pot reaction. Thus, the corresponding thiolactone derivatives (0.15 mmol, 1 equiv.), acrylate (0.15 mmol, 1 equiv.) amine (0.15 mmol, 1 equiv.) were dissolved in 300 \u0026micro;L of tetrahydrofuran (THF) at room temperature. After stirring for two hours, the THF was removed under reduced pressure and the product was purified using a CombiFlash NextGen 300+ (gradient of elution: from 100% dichloromethane to 50% of an 80/20/1 mixture of DCM/MeOH/NH\u003csub\u003e4\u003c/sub\u003eOH (aq)). All synthesized ionizable lipids were characterized by high-performance liquid chromatography coupled with a charged aerosol detector (HPLC-CAD) and mass spectrometry (ThermoFisher ISQ). Noteworthy, in previous publications CP-LC-0743 was also referred to as A4B2C1.\u003c/p\u003e \u003cp\u003eMolecular structures are shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Theoretical and experimental molecular weights (MW):\u003c/p\u003e \u003cp\u003eCP-LC-0729 theoretical [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 740.63, experimental [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 740.85\u003c/p\u003e \u003cp\u003eCP-LC-0867 theoretical [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 684.57, experimental [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 684.74\u003c/p\u003e \u003cp\u003eCP-LC-0431 theoretical [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 766.65, experimental [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 766.85\u003c/p\u003e \u003cp\u003eCP-LC-0474 theoretical [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 768.66, experimental [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 768.76\u003c/p\u003e \u003cp\u003eCP-LC-0743 theoretical [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 766.65, experimental [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e = 766.81\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003emRNA encapsulation into LNPs\u003c/h2\u003e \u003cp\u003eLNPs formulations were prepared following a previously described method with modifications\u003csup\u003e20\u003c/sup\u003e. Briefly, the purified mRNAs were initially diluted in 10 mM sodium citrate buffer at pH\u0026thinsp;=\u0026thinsp;4 reaching a final concentration of 266 \u0026micro;g/ml. Simultaneously, the lipid mixture was prepared in ethanol at the different molar ratios used of 46.6:9.4:42.7:1.6 (Comirnaty), 50:10:38.5:1.5 (CP ionizable lipids formulations in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and 40.7:34.9:23.3:1.2 (CPVax-CoV) for ionizable lipids CP-LC-0729, CP-LC-0867, CP-LC-0431, CP-LC-0743, CP-LC-0474 or ALC-0315 (Merck 586224): helper lipids DSPC (Merck 850365P) or DOPE (Merck 850725P): Cholesterol (Sigma C3045) and DMG-PEG2000 (Cayman 33945-1) or ALC-0159 (Cayman 34336). Aqueous mRNA solution and lipid mixture were combined at a molar N/P ratio of 6:1 (Moderna/Pfizer) or ionizable lipid/RNA weight ratio of 10:1 (CPVax-CoV formulation). Microfluidic technique was used for the synthesis of LNPs, thereby NanoAssemblr\u0026reg; Ignite microfluidic device (Precision Nanosystems) was set at a total flow rate (TFR) of 12 ml/min and a aqueous:ethanol flow rate ratio (FRR) of 3:1. The resulting LNPs were dialyzed (Pur-A-Lyzer\u0026trade; Midi Dialysis Kit) overnight against Tris buffer containing cryoprotectants. Each resulting LNP solution was then collected and adjusted to a final concentration of mRNA of 100 \u0026micro;g/mL, filtered through a 0.22 mm filter and stored at -80\u0026deg;C for further use.\u003c/p\u003e \u003cp\u003eThe average size, polydispersity index (PDI) and zeta potential of LNPs were determined using a Malvern Zetasizer Advance Lab Blue Label (Malvern Instruments Ltd., UK) with a capillary cell (DTS1070) and diluting the sample (typically 1:100) in KCl 10 mM filtrated solution.\u003c/p\u003e \u003cp\u003eThe concentration of mRNA in LNPs was measured using Quant-iT\u0026trade; RiboGreen\u0026trade; RNA Assay Kit from Thermo Fisher Scientific following the manufacturer\u0026rsquo;s protocols. Thus, the % of RNA encapsulated was calculated by comparing the total RNA obtained by the lysis of mRNA-LNPs using 0.5% Triton X-100 and the non-encapsulated RNA obtained when the LNPs are not lysed in absence of detergent. Fluorescence was quantified in a Fluostar Omega microplate reader (BMG Labtech). Agarose gel electrophoresis was additionally used to determine the encapsulation of mRNA in LNPs. The quantification of encapsulated mRNA was determined by band densitometry using ImageJ software. Samples were loaded in a 1% agarose gel including SYBR-Safe, and the electrophoresis was run at 120 V for 30 min. Gels were visualized with a UV transilluminator iBright\u0026trade; CL750 imaging system, using the adequate exposure times to avoid image saturation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLNP lyophilization\u003c/h2\u003e \u003cp\u003eAfter adjusting the concentration of mRNA-LNPs to 100 \u0026micro;g/mL, the mRNA-LNPs suspension was aliquoted into glass vials with a volume of 300 \u0026micro;L per vial. Lyophilization was conducted using a Genesis Pilot Freeze Dryer, following a three-stage process: initial freezing, primary drying, and secondary drying. After lyophilization, vials were backfilled with pure nitrogen, capped, and transferred to various temperatures for stability assessments. The lyophilized mRNA-LNPs were stored at either 4 or 25 \u003csup\u003eo\u003c/sup\u003eC for different times and compared with the non-lyophilized solution stored at -80 \u003csup\u003eo\u003c/sup\u003eC as control. To reconstitute lyophilized samples, 300 \u0026micro;L of RNase-free water were added to each vial and softly mixed until the solution turns into a homogeneous slightly white clear suspension.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eHEK293T cells were obtained from the American Type Culture Collection (ATCC, CRL-3216 and CRL-1586). Vero E6 cells were kindly provided by J\u0026uacute;lia Vergara from the Centro de Investigaci\u0026oacute;n en Sanidad Animal IRTA-CReSA (Barcelona, Spain). HEK293T-ACE2-TMPRSS2 cells were obtained from the National Institute for Biological Standards and Controls (NIBSC, 101008).\u003c/p\u003e \u003cp\u003eCells were cultured on complete DMEM, which consists of high-glucose Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (Merck, D6429) supplemented with 10% Fetal Bovine Serum (Sigma, F7524), 1% Penicillin-Streptomycin Solution (Gibco, 15140122) and 2 mM Glutamax (Fisher, 35050038). Vero E6 cells were additionally supplemented with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperanzineethanesulfonic acid) (Biowest) and HEK293T-ACE2-TMPRSS2 cultures were additionally supplemented with 1 \u0026micro;g/mL puromycin dihydrochloride (Sigma, P8833).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in agreement with European and national directives for protection of experimental animals, and experimental procedures were approved by the Ethics Committee for Animal Experiments of University of Zaragoza (PI59/21 and PI36/22) or University of Navarra (Protocol ref. CEEA026/20). In addition, animal experiments using the SARS-CoV-2 strains were approved by the Biosafety Committee from University of Zaragoza (Refs: 124/20, 156/21 and 184/22) and GMO Interministerial Council (A/ES/20/99).\u003c/p\u003e \u003cp\u003eFor the immunity studies, BALB/cAnNRj mice were purchased from Janvier Labs. For the viral challenge studies, BALB/cOlaHsd and C57BL/6J K18-hACE2 transgenic mice were purchased from Envigo and Charles River (K18-hACE2 JAX Mice Strain), respectively.\u003c/p\u003e \u003cp\u003eMale and female mice aged 8\u0026ndash;10 weeks and weighing 18\u0026ndash;28 g were used for all the experiments. All mice underwent an acclimation period lasting 3\u0026ndash;7 days to adapt to the experimental conditions upon arrival at the research facilities, and were housed and maintained in specific pathogen\u0026ndash;free conditions in the facilities of Centro de Investigaciones Biom\u0026eacute;dicas de Arag\u0026oacute;n (Zaragoza, Spain; reference ES 50 297 0012 011) for immunity studies and Centro de Investigaci\u0026oacute;n de Encefalopat\u0026iacute;as y Enfermedades Transmisibles Emergentes (Zaragoza, Spain; reference ES 50 297 0012 009) for challenge studies. Housing conditions were controlled, maintaining a room temperature of 20\u0026ndash;24\u0026deg;C, humidity levels ranging from 50\u0026ndash;70%, and a light intensity of 60 lux, with a light-dark cycle lasting 12 h. During the studies, all animals were monitored by animal resources center or laboratory staff daily.\u003c/p\u003e \u003cp\u003eFor the procedures requiring anesthesia (SPECT-CT imaging and SARS-CoV-2 challenge) all animals were anesthetized via inhalation using isoflurane (IsoVet) mixed with oxygen at a flow rate of 1 L/min in a rodent-specific anesthesia station. Anesthesia was induced with 5% isoflurane and maintained at 2%. Animals were monitored continuously during anesthesia and observed post-procedure until fully recovered. No anesthesia was used for intramuscular injections, blood collection and weighing. Mice were humanely euthanized with CO\u003csub\u003e2\u003c/sub\u003e asphyxiation followed by cervical dislocation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eVirus strains\u003c/h2\u003e \u003cp\u003eThe SARS-CoV-2, hCoV-19/Sweden/20-53846/2020, (Lineage B.1.1.7; Alpha variant) was provided by The Public Health Agency of Sweden. The mouse adapted strain SARS-CoV-2 MA20 was obtained by serial passaging of SARS-CoV-2, hCoV-19/Sweden/20-53846/2020, (Lineage B.1.1.7; Alpha variant) in mice as described in \u003csup\u003e27\u003c/sup\u003e. The SARS-CoV-2 hCoV19/USA/CA-Stanford-109_S21/2022 (Lineage XBB; Omicron Variant) (GISAID: EPI_ISL_15509864) was isolated from a human on October 10, 2022, in California and was obtained from BEI resources (NR-58925). All procedures involving infectious viruses, including \u003cem\u003ein vivo\u003c/em\u003e experiments, were performed under biosafety level 3 (BSL-3) conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003emRNA\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003etransfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHEK293T cells were seeded in 6-well cell culture plates at a density of 4 x 10\u003csup\u003e5\u003c/sup\u003e cells/well and incubated overnight. The SARS-CoV-2 Spike-coding mRNAs were transfected using Lipofectamine MessengerMax Transfection Reagent (Invitrogen, 15397974) according to manufacturer's protocol. Briefly, a mixture of each mRNA (2.5 \u0026micro;g/well) and Lipofectamine MessengerMAX (5 \u0026micro;L/well) were pre-incubated in OptiMEM media. The mRNA-lipofectamine mixture was added to the corresponding wells in duplicate, resulting in a final mRNA concentration of 2.5 \u0026micro;g/well. The cells were incubated for 24 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFor Western Blot, cells were lysed after incubation with 0,1% TritonX and centrifuged at 13000 x g for 10 min. The cell lysates were run under reducing conditions by SDS-PAGE (SurePAGE, 4\u0026ndash;12% Genscript, M00653) and transferred to a nitrocellulose PVDF membrane (Bio-Rad). The membranes were blocked with 3% BSA in TBST buffer (Tris-buffered saline\u0026thinsp;+\u0026thinsp;0.1% Tween 20) and incubated with a rabbit polyclonal SARS-CoV-2 Spike antibody (SinoBiological, 40591-T62) overnight at 4\u0026deg;C. Membrane was washed and incubated with a goat anti-rabbit Ig, Human ads-HRP (Southern Biotech, 4010-05) as a secondary antibody. Colorimetric detection of the samples was performed with Pierce ECL Western Blotting Substrate (Thermo, 32106) and images were acquired using iBright CL750 Imaging system (Invitrogen).\u003c/p\u003e \u003cp\u003eFor flow cytometry analysis, cells were trypsinized after incubation. Collected cells were incubated with human FcR blocking reagent (Miltenyi, 130-059-901) for 15 min at 4\u0026deg;C. Cells were washed and incubated with fixation buffer (eBioscience, 88-8824-00) for 30 min at 4\u0026deg;C followed by 3 washing steps with permeabilization buffer (eBioscience, 88-8824-00) and incubation with SARS-CoV-2 Spike S1 Subunit Antibody (R\u0026amp;D systems, MAB105403) diluted in permeabilization buffer for 1 h at room temperature. Subsequently, cells were washed and incubated with FITC Goat Anti-Mouse IgG/IgM (BD, 555988) for 1 h at room temperature. After final washing steps, labeled cells were resuspended in PBS buffer and acquisition was performed using Gallios flow cytometer (Beckman Coulter).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eimmunization and blood collection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMice were immunized with LNPs prepared as described above, receiving an intramuscular injection of 1 \u0026micro;g of the specified mRNA-LNP into the right thigh muscle, diluted in Tris buffer containing 15% sucrose in a final volume of 30 \u0026micro;L, and administered using a 30G insulin syringe. An equal booster dose was administered on day 21 following the initial immunization. Blood samples were collected from the submandibular vein on days 0, 21, and 42 post-prime. Blood was allowed to clot and then centrifuged at 6500 g and 4\u0026deg;C for 10 min. The sera were collected and stored at -80\u0026deg;C for antibody analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of antibody levels in serum\u003c/h2\u003e \u003cp\u003eThe serum samples from immunized mice were analyzed for RBD-specific IgG antibodies titers using an indirect enzyme-linked immunosorbent assay (ELISA). 96-well Nunc MaxiSorp microplates (Thermo Scientific) were coated with 50 ng/well of recombinant RBD (Certest Biotec) diluted in carbonate/bicarbonate buffer pH 9.6 and incubated overnight. The next day, plates were washed with phosphate buffer saline \u0026ndash; Tween 20 0.05% (PBST) followed by blocking with 3% BSA (Seqens) in PBST for 1 h. After washing, serially diluted mouse sera were added to the plate and incubated for 1.5 h at 37\u0026deg;C, followed by washing steps and addition of IgG goat anti-mouse horseradish peroxidase (HRP) antibody (Southern Biotech) at a dilution of 1:10.000. The plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Abcam) and 0.2M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (aq) to stop the reaction. Finally, the absorbance was measured at 450 and 630 nm using a FLUOstar Omega microplate reader (BMG Labtech).\u003c/p\u003e \u003cp\u003eThe reciprocal endpoint titer was defined as the highest dilution at which the optical density (OD 450\u0026ndash;630 nm) of the sample reached a predetermined cutoff of 0.1 or greater.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eOrgan single cell suspensions\u003c/h2\u003e \u003cp\u003eOrgans were harvested and placed in RPMI 1640 medium (Fisher, 10379144). Spleens and lymph nodes (LNs) were homogenized using a syringe plunger and filtered through a 70 \u0026micro;m cell strainer. The cell strainer was washed with 10 ml of RPMI and cells were centrifuged at 450 g for 5 min. Red blood cells (RBCs) in spleens were lysed using 1 ml of eBioscience RBC Lysis Buffer (Thermofisher, 00-4300-54) for 1 min. After lysing, the cells were washed with 10 ml of RPMI and centrifuged at 450 g for 5 min.\u003c/p\u003e \u003cp\u003eCells from tissues were resuspended in 1 ml of RPMI 1640 medium supplemented with 10% heat inactivated Fetal Bovine Serum (Sigma F7524), 1% Glutamax (Fisher, 35050038), 1% Penicillin-Streptomycin (Gibco, 15140122) and 0.00035% β-mercaptoethanol (Merck, M3148). The resuspended cells were immediately used for counting, culture, or staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eELISPOT assay\u003c/h2\u003e \u003cp\u003eEnzyme-linked immunospot (ELISPOT) assay was employed to quantify the frequency of cytokine-secreting splenocytes. ELISpot assays were performed using mouse IFN-γ ELISpot kits (CTL Immunospot) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e96-well immunospot plates were coated with 60 \u0026micro;L of murine IFN-γ capture solution and incubated at 4\u0026deg;C overnight. Next day, plates were washed with PBS and 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e splenocytes/well were stimulated with 50 \u0026micro;g/mL of a peptide pool covering the immunodominant sequence domains of the S protein (Miltenyi, 130-126-700). Phorbol-12-myristate-13-acetate (PMA) 50 ng/ml (Merck, 524400) and ionomycin 500 ng/ml (Merck, 407953) were used as positive control while splenocytes without stimulation were utilized as negative control. Samples were incubated for 20 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The following day, plates were washed and 10 \u0026micro;L of anti-murine IFN-γ detection solution was added. After washing, a tertiary solution containing streptavidin-HRP was used followed by the addition of a chromogen substrate for enzyme activity detection. Finally, plates were scanned using an ImmunoSpotS6 Ultra-V analyzer (CTL EUROPE GMBH) and spot numbers were assessed using ImmunoSpot 7.0.34.0 Professional Analyzer DC software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eELISA cytokine assay\u003c/h2\u003e \u003cp\u003e1x10\u003csup\u003e6\u003c/sup\u003e splenocytes/well were seeded in 96-well plates and stimulated with 50 \u0026micro;g/mL of a peptide pool covering the immunodominant sequence domains of the S protein (Miltenyi, 130-126-700). Phorbol-12-myristate-13-acetate (PMA) 50 ng/ml (Merck, 524400) and ionomycin 500 ng/ml (Merck, 407953) were used as positive control while splenocytes without stimulation were utilized as negative control. Samples were incubated for 20 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Supernatants were collected after centrifugation at 1500 g for 5 min, and samples were stored at -80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eCytokine quantification was conducted using Mabtech ELISA Flex (HRP) kits following manufacturer\u0026rsquo;s instructions. Briefly, Nunc MaxiSorp 96-well plates (Thermo Fisher) were coated with capture antibody and incubated at 4\u0026deg;C overnight. Next day, plates were washed with PBST and blocked with 0.1% BSA in PBST buffer for 1 h at room temperature. After washing, samples and standards dilutions were added and incubated for 2 h at room temperature, followed by the addition of detection biotinylated antibody and incubation for 1 h. Streptavidin-HRP was added and incubated for 1 h at RT. Finally, the plates were washed and TMB substrate (Abcam) was added. Reaction was stopped with 0.2M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and the absorbance was measured at 450 and 630 nm using a FLUOstar Omega microplate reader (BMG Labtech).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eLuminex assay\u003c/h2\u003e \u003cp\u003eLung samples were acquired from a weighed portion of murine lung, previously homogenized in 1 ml of DMEM using a GentleMACS Dissociator (Miltenyi), and then clarified by collecting the supernatant after centrifugation at 450 rpm for 5 min. Before analysis, samples were inactivated by the addition of 0.5% Triton X100 and incubated for 30 min at 4\u0026deg;C. Protease inhibitor cocktail (Complete, Roche) was added to prevent protein degradation during incubation. Samples were stored at -80\u0026deg;C until Luminex assay was conducted.\u003c/p\u003e \u003cp\u003eCytokine/Chemokine array analysis was carried out using Luminex Mouse Discovery Assay 11-plex kit (R\u0026amp;D Systems). Samples were centrifuged at 1000 \u0026times; g for 10 min and then diluted 1:2 in calibrator diluent and the assay was conducted following manufacturer\u0026rsquo;s instructions. Measurements were carried out using Luminex LABSCAN 100 and quantified by comparison to a standard curve. Data collection and analysis were performed utilizing LUMINEX 100 IS software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis of Thf and GC B cells\u003c/h2\u003e \u003cp\u003eLymph node single cell suspensions were incubated with mouse FcR blocking reagent (Miltenyi, 130-092-575) for 15 min at 4\u0026deg;C. Cells were washed and incubated with anti-mouse antibodies for surface staining for 15 min at 4 \u0026ordm;C. Cells were washed and fixed with 4% paraformaldehyde for 30 minutes at room temperature and resuspended in PBS buffer. Acquisition was performed using Gallios flow cytometer (Beckman Coulter).\u003c/p\u003e \u003cp\u003eThf cells were defined as CD45R\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e, CD44\u003csup\u003ehigh\u003c/sup\u003e, CXCR5\u003csup\u003e+\u003c/sup\u003e, PD-1\u003csup\u003e+\u003c/sup\u003e and the following antibodies were used for surface staining: CD45R (B220)-APC-Vio770 (Miltenyi, 130-110-849), CD4-VioBright FITC (Miltenyi, 130-118-692), CD44-PerCP-Vio700 (Miltenyi, 130-128-625), CD185 (CXCR5)-APC (Miltenyi, 130-119-129) and CD279 (PD1)-PE (Miltenyi, 130-111-953). GC B cells were defined as CD19\u003csup\u003e+\u003c/sup\u003eGL7\u003csup\u003e+\u003c/sup\u003eCD95\u003csup\u003e+\u003c/sup\u003e and the following antibodies were used for surface staining: CD19-APC (Miltenyi, 130-112-036), CD95 (FAS)-PE (Miltenyi, 130-112-036) and GL7-Alexa Fluor 488 eBioscience (Thermofisher, 53-5902-82).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePseudotyped SARS‑CoV‑2 neutralization assay\u003c/h2\u003e \u003cp\u003eFor the neutralization assay, mouse sera were serially diluted in complete DMEM and added to black 96-well microplates, alongside the previously established volume of pseudovirus. The mixture was then incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 1 h to allow neutralization. After incubation, 2 x 10\u003csup\u003e4\u003c/sup\u003e HEK293T-ACE2-TMPRSS2 cells were added to each well followed by 48 h of further incubation. The cells were then fixed as previously described, and the fluorescent spots were counted using a C.T.L. S6 Ultra-V analyzer with the FluoroSpot-X suite selected. The NT50 titer was defined as the reciprocal of the highest dilution at which a 50% or higher reduction in the number of spots compared to the unnaturalized condition was achieved.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ebiodistribution\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e biodistribution assays were performed using Indium-111 radiolabeled LNPs. Lipid nanoparticle suspensions were incubated with 55 MBq Indium-111 oxine for 15 min at 37\u0026deg;C. Labelled LNPs were afterwards purified using centrifugal concentrators (Vivaspin 500, 10.000 MWCO PES).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e biodistribution assays were performed on BALB/c mice (n\u0026thinsp;=\u0026thinsp;3 male, n\u0026thinsp;=\u0026thinsp;3 female) on the U-SPECT6/E-class (MILabs) single photon emission computed tomography (SPECT) system. Radiolabeled LNPs were administrated via intramuscular injection as described above. At 1, 3, 6, 24, 48, 72, 96 and 168 h post-administration animals were anesthetized using isoflurane (2% in 100% O\u003csub\u003e2\u003c/sub\u003e) and SPECT images were acquired. Computer tomography (CT) scan was performed immediately after adjusting voltage and current to 55 kV and 0,33 mA respectively. After image acquisition these were reconstructed using \u003cem\u003eindium-111\u003c/em\u003e photopeaks with a windo with of 20% and a calibration factor to obtain the exact activity information (MBq/mL).\u003c/p\u003e \u003cp\u003eImages were analyzed using PMOD v3.2 software (PMOD Technologies, Switzerland). Signal values were corrected using the specific indium-111 radioactive decay correction factor and then transformed to standardized uptake value (SUV) units using the formula:\u003c/p\u003e \u003cp\u003eSUV = [tisular activity concentration (MBq /cm\u003csup\u003e3\u003c/sup\u003e)/dose (MBq)] \u0026times; bodyweight (g).\u003c/p\u003e \u003cp\u003eQuantitative analysis was performed on organs/areas where signal could be correctly detected, setting a volume of interest (VOI) and quantifying during time, calculating the average signal in the VOI (Suvmean). Due to the difficulty in quantifying bone marrow signal, a visual analysis was performed to identify the timing of signal appearance in various bones.\u003c/p\u003e \u003cp\u003eAt 168 h, the animals were euthanized, and the following organs/tissues were dissected: lungs, spleen, liver, kidneys, bone, muscle at the injection site, contralateral muscle, presacral lymph nodes, inguinal lymph nodes, and brain. Tissue activity was measured using a gamma counter (Hidex) to determine counts per minute (cpm) for each tissue. To quantitatively assess the amount of radioactivity in each sample, a ratio was calculated using the brain (background) as the reference tissue, and the values were normalized to tissue weight (g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eSerum biochemical analysis\u003c/h2\u003e \u003cp\u003eSerum samples were collected as specified above. Biochemical analysis of AST and ALT levels was performed using Cobas c-311 automatic analyzer (Roche Diagnostics).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSARS-CoV-2 challenge\u003c/h2\u003e \u003cp\u003eMice were anaesthetized with isoflurane (5% for induction and 2.5% for maintenance) and infected intranasally with the correspondent amount of virus to achieve the indicated doses, which was diluted in PBS in a total volume of 40 \u0026micro;L. After virus challenge, mice were periodically weighed and assessed using a clinical scoring system evaluating various parameters including mouse appearance, level of consciousness, activity, response to stimuli, eye appearance and frequency and quality of respiration\u003csup\u003e56\u003c/sup\u003e. Humane endpoints were established on a weight loss threshold of 25% or greater, alongside clinical scoring criteria.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eViral load determination by TCID50 assay\u003c/h2\u003e \u003cp\u003eLungs were harvested, weighed and homogenized in 1 ml of DMEM using a GentleMACS Dissociator (Miltenyi). Homogenates were centrifuged at 450 g for 5 min and the supernatant was taken for titration.\u003c/p\u003e \u003cp\u003eVirus titration was determined by TCID50 in Vero E6 cells. Vero E6 cells were seeded in 96 well plates at a density of 10\u003csup\u003e4\u003c/sup\u003e cells/well and cultured overnight at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Lung homogenates serial 1-log dilutions were prepared in complete DMEM with only 2% FBS and added to the cultured cells. 72 h after cell infection plates were evaluated for cell death and TCID50 was calculated using Ramakrishan newly proposed method formula \u003csup\u003e57\u003c/sup\u003e and normalized to weight (g) of lung and amount of buffer (ml) used for lung homogenization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn experimental studies, GraphPad Prism 10 software was used for representation and statistical analyses.\u003c/p\u003e \u003cp\u003eWe used ordinary one-way or two-way ANOVA with Tukey\u0026rsquo;s multiple comparison post-test to compare experimental groups. Statistical significances are denoted in the Figures by asterisks, as follows: *P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001 or ****P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. The absence of asterisks indicates non-statistical significance with a P-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05. Outliers were identified and removed using Grubbs' test, applying a significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eThe data supporting the conclusions of this research are accessible within the article and its supplementary files. The source data is included in this paper.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eEM, EB, CM, AS, SA, TA, AL, BA, DDM, JH, JGW, VL, DC, JM and EP are employees at the Certest Pharma Department, Certest Biotec S.L. EB, JH, DDM, JM and JGW are inventors on patents related to this publication\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis study was supported by \u0026ldquo;Ministerio de Ciencia e Innovaci\u0026oacute;n\u0026rdquo; (Spain) through project STABVAC4COV (Desarrollo de vacunas termoestables basadas en mRNA frente a la variante delta del SARS-CoV-2). Work in JP lab is funded by PID2020- 113963RBI00 from AEI (Agencia Estatal de Investigaci\u0026oacute;n), Aragon Government (B29-20R), Postdoctoral Juan de la Cierva Contract (MA); Health National Institute Carlos III (COV20-00308 and CIBERINFEC, CB21/13/ 00087).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: EM, EB, EP, JM; mRNA sequence design: EB; mRNA synthesis: VL, DC; Lipid molecular design, synthesis and characterization: JH, JGW; LNP formulation and characterization: TA, DDM; Biological and immune assays: CM, AS, SA, AL, BA, CG, AC; Biodistribution study: IP, GQ; Establishment of COVID-19 Infection models and in vivo vaccine-induced protection experiments: JP, IU, NP, MA, CM, AS, SA, BM, JB; Supervision: EM, EP, JM; Writing original draft and figure preparation: EM, CM, AS, AL; Manuscript review: all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to extend our sincere gratitude to all the members of Certest Biotec, especially the Certest Pharma group, whose valuable insights and expertise significantly contributed to the success of this research. Authors would like to acknowledge the use of \u0026ldquo;Servicios Cient\u0026iacute;fico T\u0026eacute;cnicos\u0026rdquo; at CIBA (IACS-University of Zaragoza), especially the collaboration of the animal facilities and flow cytometry services, as well as the use of \u0026ldquo;Servicio de an\u0026aacute;lisis Bioqu\u0026iacute;micos\u0026rdquo; at CIMA (University of Navarra).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the conclusions of this research are accessible within the article and its supplementary files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTregoning, J. S., Flight, K. E., Higham, S. L., Wang, Z. \u0026amp; Pierce, B. F. Progress of the COVID-19 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e\u003cstrong\u003e21\u003c/strong\u003e, 626\u0026ndash;636 (2021).\u003c/li\u003e\n\u003cli\u003eVerbeke, R., Lentacker, I., De Smedt, S. C. \u0026amp; Dewitte, H. The dawn of mRNA vaccines: The COVID-19 case. \u003cem\u003eJ. Control. release Off. J. Control. Release Soc.\u003c/em\u003e\u003cstrong\u003e333\u003c/strong\u003e, 511\u0026ndash;520 (2021).\u003c/li\u003e\n\u003cli\u003ePardi, N., Hogan, M. J., Porter, F. W. \u0026amp; Weissman, D. mRNA vaccines \u0026mdash; a new era in vaccinology. \u003cem\u003eNat. Rev. Drug Discov.\u003c/em\u003e\u003cstrong\u003e17\u003c/strong\u003e, 261\u0026ndash;279 (2018).\u003c/li\u003e\n\u003cli\u003ePolack, F. P. \u003cem\u003eet al.\u003c/em\u003e Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. \u003cem\u003eN. Engl. J. Med.\u003c/em\u003e\u003cstrong\u003e383\u003c/strong\u003e, 2603\u0026ndash;2615 (2020).\u003c/li\u003e\n\u003cli\u003eBaden, L. R. \u003cem\u003eet al.\u003c/em\u003e Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. \u003cem\u003eN. Engl. J. Med.\u003c/em\u003e\u003cstrong\u003e384\u003c/strong\u003e, 403\u0026ndash;416 (2021).\u003c/li\u003e\n\u003cli\u003ePawlowski, C. \u003cem\u003eet al.\u003c/em\u003e FDA-authorized mRNA COVID-19 vaccines are effective per real-world evidence synthesized across a multi-state health system. \u003cem\u003eMed (New York, N.Y.)\u003c/em\u003e\u003cstrong\u003e2\u003c/strong\u003e, 979\u0026ndash;992.e8 (2021).\u003c/li\u003e\n\u003cli\u003eScheaffer, S. M. \u003cem\u003eet al.\u003c/em\u003e Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant. \u003cem\u003ebioRxiv : the preprint server for biology\u003c/em\u003e at https://doi.org/10.1101/2022.09.12.507614 (2022).\u003c/li\u003e\n\u003cli\u003eYe, Q. \u003cem\u003eet al.\u003c/em\u003e Rational development of a combined mRNA vaccine against COVID-19 and influenza. \u003cem\u003eNPJ vaccines\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 84 (2022).\u003c/li\u003e\n\u003cli\u003eCheng, L., Chan, W. K., Zhu, L., Chao, M. H. \u0026amp; Wang, Y. Confronting Inequalities and Bridging the Divide: A Retrospective Study Assessment of Country-Level COVID-19 Vaccine Equality with a Cox Regression Model. \u003cem\u003eVaccines\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eAcharya, K. P., Ghimire, T. R. \u0026amp; Subramanya, S. H. Access to and equitable distribution of COVID-19 vaccine in low-income countries. \u003cem\u003enpj Vaccines\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 54 (2021).\u003c/li\u003e\n\u003cli\u003eDuroseau, B., Kipshidze, N. \u0026amp; Limaye, R. J. The impact of delayed access to COVID-19 vaccines in low- and lower-middle-income countries. \u003cem\u003eFront. Public Heal.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eChi, W.-Y. \u003cem\u003eet al.\u003c/em\u003e COVID-19 vaccine update: vaccine effectiveness, SARS-CoV-2 variants, boosters, adverse effects, and immune correlates of protection. \u003cem\u003eJ. Biomed. Sci.\u003c/em\u003e\u003cstrong\u003e29\u003c/strong\u003e, 82 (2022).\u003c/li\u003e\n\u003cli\u003eEchaide, M. \u003cem\u003eet al.\u003c/em\u003e mRNA Vaccines against SARS-CoV-2: Advantages and Caveats. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e\u003cstrong\u003e24\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eKim, S. C. \u003cem\u003eet al.\u003c/em\u003e Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency. \u003cem\u003eMol. Cell. Toxicol.\u003c/em\u003e\u003cstrong\u003e18\u003c/strong\u003e, 1\u0026ndash;8 (2022).\u003c/li\u003e\n\u003cli\u003eLuna-Cerralbo, D. \u003cem\u003eet al.\u003c/em\u003e A statistical-physics approach for codon usage optimisation. \u003cem\u003eComput. Struct. Biotechnol. J.\u003c/em\u003e\u003cstrong\u003e23\u003c/strong\u003e, 3050\u0026ndash;3064 (2024).\u003c/li\u003e\n\u003cli\u003eBroset Blasco, E. \u0026amp; Mart\u0026iacute;nez Oliv\u0026aacute;n, J. E. Artificial polynucleotides for expressing proteins, WO2024110381A1. (2022).\u003c/li\u003e\n\u003cli\u003eRamachandran, S., Satapathy, S. R. \u0026amp; Dutta, T. Delivery Strategies for mRNA Vaccines. \u003cem\u003ePharmaceut. Med.\u003c/em\u003e\u003cstrong\u003e36\u003c/strong\u003e, 11\u0026ndash;20 (2022).\u003c/li\u003e\n\u003cli\u003eHou, X., Zaks, T., Langer, R. \u0026amp; Dong, Y. Lipid nanoparticles for mRNA delivery. \u003cem\u003eNat. Rev. Mater.\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 1078\u0026ndash;1094 (2021).\u003c/li\u003e\n\u003cli\u003eHald Albertsen, C. \u003cem\u003eet al.\u003c/em\u003e The role of lipid components in lipid nanoparticles for vaccines and gene therapy. \u003cem\u003eAdv. Drug Deliv. Rev.\u003c/em\u003e\u003cstrong\u003e188\u003c/strong\u003e, 114416 (2022).\u003c/li\u003e\n\u003cli\u003eHassett, K. J. \u003cem\u003eet al.\u003c/em\u003e Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines. \u003cem\u003eMol. Ther. Nucleic Acids\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 1\u0026ndash;11 (2019).\u003c/li\u003e\n\u003cli\u003eZhang, L. \u003cem\u003eet al.\u003c/em\u003e Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability. \u003cem\u003eNPJ vaccines\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 156 (2023).\u003c/li\u003e\n\u003cli\u003eGimenez-Warren, J. Ionizable lipids and lipid nanoparticles containing thereof, WO2024110381A1. (2022).\u003c/li\u003e\n\u003cli\u003ePena, A. \u003cem\u003eet al.\u003c/em\u003e Multicomponent Thiolactone-Based Ionizable Lipid Screening Platform for an Efficient and Tunable mRNA Delivery to the Lungs. \u003cem\u003ebioRxiv\u003c/em\u003e 2024.10.09.617396 (2024) doi:10.1101/2024.10.09.617396.\u003c/li\u003e\n\u003cli\u003eCrommelin, D. J. A., Anchordoquy, T. J., Volkin, D. B., Jiskoot, W. \u0026amp; Mastrobattista, E. Addressing the Cold Reality of mRNA Vaccine Stability. \u003cem\u003eJ. Pharm. Sci.\u003c/em\u003e\u003cstrong\u003e110\u003c/strong\u003e, 997\u0026ndash;1001 (2021).\u003c/li\u003e\n\u003cli\u003eSchoenmaker, L. \u003cem\u003eet al.\u003c/em\u003e mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. \u003cem\u003eInt. J. Pharm.\u003c/em\u003e\u003cstrong\u003e601\u003c/strong\u003e, 120586 (2021).\u003c/li\u003e\n\u003cli\u003eOude Blenke, E. \u003cem\u003eet al.\u003c/em\u003e The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case. \u003cem\u003eJ. Pharm. Sci.\u003c/em\u003e\u003cstrong\u003e112\u003c/strong\u003e, 386\u0026ndash;403 (2023).\u003c/li\u003e\n\u003cli\u003eAlbert, M.-C. \u003cem\u003eet al.\u003c/em\u003e Identification of FasL as a crucial host factor driving COVID-19 pathology and lethality. \u003cem\u003eCell Death Differ.\u003c/em\u003e (2024) doi:10.1038/s41418-024-01278-6.\u003c/li\u003e\n\u003cli\u003eLederer, K. \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation. \u003cem\u003eImmunity\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 1281\u0026ndash;1295.e5 (2020).\u003c/li\u003e\n\u003cli\u003eVerbeke, R., Hogan, M. J., Lor\u0026eacute;, K. \u0026amp; Pardi, N. Innate immune mechanisms of mRNA vaccines. \u003cem\u003eImmunity\u003c/em\u003e\u003cstrong\u003e55\u003c/strong\u003e, 1993\u0026ndash;2005 (2022).\u003c/li\u003e\n\u003cli\u003eSharma, P., Hoorn, D., Aitha, A., Breier, D. \u0026amp; Peer, D. The immunostimulatory nature of mRNA lipid nanoparticles. \u003cem\u003eAdv. Drug Deliv. Rev.\u003c/em\u003e\u003cstrong\u003e205\u003c/strong\u003e, 115175 (2024).\u003c/li\u003e\n\u003cli\u003eChang, H.-D. \u0026amp; Radbruch, A. Maintenance of quiescent immune memory in the bone marrow. \u003cem\u003eEur. J. Immunol.\u003c/em\u003e\u003cstrong\u003e51\u003c/strong\u003e, 1592\u0026ndash;1601 (2021).\u003c/li\u003e\n\u003cli\u003eDubuc, I. \u003cem\u003eet al.\u003c/em\u003e Cytokines and Lipid Mediators of Inflammation in Lungs of SARS-CoV-2 Infected Mice. \u003cem\u003eFront. Immunol.\u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 893792 (2022).\u003c/li\u003e\n\u003cli\u003eWinkler, E. S. \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. \u003cem\u003eNat. Immunol.\u003c/em\u003e\u003cstrong\u003e21\u003c/strong\u003e, 1327\u0026ndash;1335 (2020).\u003c/li\u003e\n\u003cli\u003eTamura, T. \u003cem\u003eet al.\u003c/em\u003e Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants. \u003cem\u003eNat. Commun.\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 2800 (2023).\u003c/li\u003e\n\u003cli\u003eTamura, T. \u003cem\u003eet al.\u003c/em\u003e Virological characteristics of the SARS-CoV-2 Omicron XBB.1.5 variant. \u003cem\u003eNat. Commun.\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 1176 (2024).\u003c/li\u003e\n\u003cli\u003eYue, C. \u003cem\u003eet al.\u003c/em\u003e ACE2 binding and antibody evasion in enhanced transmissibility of XBB.1.5. \u003cem\u003eThe Lancet. Infectious diseases\u003c/em\u003e vol. 23 278\u0026ndash;280 at https://doi.org/10.1016/S1473-3099(23)00010-5 (2023).\u003c/li\u003e\n\u003cli\u003eLewnard, J. A. \u003cem\u003eet al.\u003c/em\u003e Increased vaccine sensitivity of an emerging SARS-CoV-2 variant. \u003cem\u003eNat. Commun.\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 3854 (2023).\u003c/li\u003e\n\u003cli\u003eAzevedo, P. O. \u003cem\u003eet al.\u003c/em\u003e Differential requirement of neutralizing antibodies and T cells on protective immunity to SARS-CoV-2 variants of concern. \u003cem\u003enpj Vaccines\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 15 (2023).\u003c/li\u003e\n\u003cli\u003ePowers, J. M. \u003cem\u003eet al.\u003c/em\u003e Divergent pathogenetic outcomes in BALB/c mice following Omicron subvariant infection. \u003cem\u003eVirus Res.\u003c/em\u003e\u003cstrong\u003e341\u003c/strong\u003e, 199319 (2024).\u003c/li\u003e\n\u003cli\u003eHalfmann, P. J. \u003cem\u003eet al.\u003c/em\u003e Characterization of Omicron BA.4.6, XBB, and BQ.1.1 subvariants in hamsters. \u003cem\u003eCommun. Biol.\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 331 (2024).\u003c/li\u003e\n\u003cli\u003eUddin, M. N. \u0026amp; Roni, M. A. Challenges of Storage and Stability of mRNA-Based COVID-19 Vaccines. \u003cem\u003eVaccines\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eAlejo, T. \u003cem\u003eet al.\u003c/em\u003e Comprehensive Optimization of a Freeze-Drying Process Achieving Enhanced Long-Term Stability and In Vivo Performance of Lyophilized mRNA-LNPs. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e\u003cstrong\u003e25\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eKafetzis, K. N. \u003cem\u003eet al.\u003c/em\u003e The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery. \u003cem\u003eAdv. Healthc. Mater.\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, e2203022 (2023).\u003c/li\u003e\n\u003cli\u003eMuramatsu, H. \u003cem\u003eet al.\u003c/em\u003e Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine. \u003cem\u003eMol. Ther.\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 1941\u0026ndash;1951 (2022).\u003c/li\u003e\n\u003cli\u003eHogan, M. J. \u0026amp; Pardi, N. mRNA Vaccines in the COVID-19 Pandemic and Beyond. \u003cem\u003eAnnu. Rev. Med.\u003c/em\u003e\u003cstrong\u003e73\u003c/strong\u003e, 17\u0026ndash;39 (2022).\u003c/li\u003e\n\u003cli\u003eAkinc, A. \u003cem\u003eet al.\u003c/em\u003e The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. \u003cem\u003eNat. Nanotechnol.\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 1084\u0026ndash;1087 (2019).\u003c/li\u003e\n\u003cli\u003eSabnis, S. \u003cem\u003eet al.\u003c/em\u003e A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates. \u003cem\u003eMol. Ther.\u003c/em\u003e\u003cstrong\u003e26\u003c/strong\u003e, 1509\u0026ndash;1519 (2018).\u003c/li\u003e\n\u003cli\u003eAnsell, S. M.. US 10, 166, 298 B2. (2019).\u003c/li\u003e\n\u003cli\u003eLi, M. \u003cem\u003eet al.\u003c/em\u003e Lyophilization process optimization and molecular dynamics simulation of mRNA-LNPs for SARS-CoV-2 vaccine. \u003cem\u003eNPJ vaccines\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 153 (2023).\u003c/li\u003e\n\u003cli\u003eAi, L. \u003cem\u003eet al.\u003c/em\u003e Lyophilized mRNA-lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2. \u003cem\u003eCell Discov.\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 9 (2023).\u003c/li\u003e\n\u003cli\u003eSuzuki, Y. \u003cem\u003eet al.\u003c/em\u003e Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates. \u003cem\u003eMol. Ther. Nucleic Acids\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 226\u0026ndash;240 (2022).\u003c/li\u003e\n\u003cli\u003eHermosilla, J. \u003cem\u003eet al.\u003c/em\u003e Analysing the In-Use Stability of mRNA-LNP COVID-19 Vaccines Comirnaty\u0026trade; (Pfizer) and Spikevax\u0026trade; (Moderna): A Comparative Study of the Particulate. \u003cem\u003eVaccines\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eWHO. \u003cem\u003eMessenger RNA encoding the full-length SARS‐CoV‐2 spike glycoprotein\u003c/em\u003e. https://berthub.eu/articles/11889.doc (2021).\u003c/li\u003e\n\u003cli\u003eGISAID. https://gisaid.org/wiv04/.\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez, J., Lampaya, V., Larraga, A., Magall\u0026oacute;n, H. \u0026amp; Casabona, D. Purification of linearized template plasmid DNA decreases double-stranded RNA formation during IVT reaction. \u003cem\u003eFront. Mol. Biosci.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eShrum, B. \u003cem\u003eet al.\u003c/em\u003e A robust scoring system to evaluate sepsis severity in an animal model. \u003cem\u003eBMC Res. Notes\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 233 (2014).\u003c/li\u003e\n\u003cli\u003eRamakrishnan, M. A. Determination of 50% endpoint titer using a simple formula. \u003cem\u003eWorld J. Virol.\u003c/em\u003e\u003cstrong\u003e5\u003c/strong\u003e, 85\u0026ndash;86 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5780846/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5780846/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003emRNA vaccines have shown great efficacy against SARS-CoV-2, yet challenges remain in optimizing vaccine components to achieve enhanced immune response and vaccine stability. In this study, we developed CPVax-CoV, a new lyophilized mRNA vaccine that features novel thiolactone-based ionizable lipids and newly designed untranslated regions (UTRs) for enhanced expression. Incorporation of these optimized components into our vaccine candidate CPVax-CoV significantly improved immune responses in mice compared to commercially available mRNA vaccines. Moreover, lyophilized CPVax-CoV has proven to be thermostable, maintaining its biological activity for up to one year at 4\u0026deg;C and 25\u0026deg;C after lyophilization, overcoming the cold-chain limitations of current mRNA vaccines. This vaccine demonstrates protective efficacy against ancestral SARS-CoV-2 and Omicron XBB variant, offering a scalable solution for global distribution and pandemic preparedness. These findings underscore the potential of this platform for future next-generation mRNA vaccine development.\u003c/p\u003e","manuscriptTitle":"Enhanced stability and efficacy of a lyophilized mRNA SARS-CoV-2 vaccine incorporating novel Ionizable lipids after one year storage at 25ºC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-10 17:04:38","doi":"10.21203/rs.3.rs-5780846/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-14T15:25:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T07:56:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-27T01:53:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-26T07:34:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-25T20:57:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-22T06:03:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108895182102399134780953435923206816800","date":"2025-03-19T00:52:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303251883506729571348301754496833809702","date":"2025-03-17T14:12:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129160232623516047352300565545452746190","date":"2025-03-17T06:11:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30304609927984379126036369689146637799","date":"2025-03-17T03:13:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165056663639286495494790351542208406099","date":"2025-03-16T22:52:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245501549045684720522272269271716612977","date":"2025-03-16T21:16:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-16T19:53:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-11T17:30:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-08T17:00:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-01-07T11:22:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00b21b53-ba98-4dfb-8545-c226b3277862","owner":[],"postedDate":"January 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42575145,"name":"Biological sciences/Immunology/Vaccines/Rna vaccines"},{"id":42575146,"name":"Biological sciences/Immunology/Infectious diseases/Viral infection"},{"id":42575147,"name":"Biological sciences/Biotechnology/Applied immunology/Vaccines/Rna vaccines"},{"id":42575148,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"}],"tags":[],"updatedAt":"2025-07-07T16:04:51+00:00","versionOfRecord":{"articleIdentity":"rs-5780846","link":"https://doi.org/10.1038/s41541-025-01201-1","journal":{"identity":"npj-vaccines","isVorOnly":false,"title":"npj Vaccines"},"publishedOn":"2025-07-01 15:58:02","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-01-10 17:04:38","video":"","vorDoi":"10.1038/s41541-025-01201-1","vorDoiUrl":"https://doi.org/10.1038/s41541-025-01201-1","workflowStages":[]},"version":"v1","identity":"rs-5780846","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5780846","identity":"rs-5780846","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}