Heterologous saRNA Prime – Multivalent Protein Boost Strategy Induces Broad and Durable Immunity Against SARS-CoV-2 and MERS-CoV

Heterologous saRNA Prime – Multivalent Protein Boost Strategy Induces Broad and Durable Immunity Against SARS-CoV-2 and MERS-CoV | 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 Heterologous saRNA Prime – Multivalent Protein Boost Strategy Induces Broad and Durable Immunity Against SARS-CoV-2 and MERS-CoV Dominik Renn, Justine S. McPartlan, Srivinas Banala, Fabian Kießling, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7875217/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The continuing emergence of SARS- and MERS-related coronaviruses underscores the urgent need for pan-SARBECo vaccines capable of eliciting broad and durable protection across divergent lineages. 1 We present a heterologous prime-boost vaccination strategy combining a modified dendrimer nanoparticle (DNP)-encapsulated self-amplifying (saRNA) prime with an alum-adjuvanted multivalent protein booster containing receptor-binding domains (RBDs) from SARS-CoV-2 (Wuhan-Hu-1 and B.1.351) and MERS-CoV. This approach leverages the potent immunogenicity of RNA priming together with the breadth and safety of protein subunit boosting 2 – 3 to expand coronavirus coverage. In preclinical mouse and hamster models, the heterologous RNA-protein regimen elicited robust antibody responses with markedly enhanced magnitude, durability, and cross-variant neutralization compared with homologous RNA or protein vaccination alone. Inclusion of the MERS-CoV RBD in the booster broadened the response without compromising SARS-CoV-2 immunity. These findings establish a versatile and scalable vaccination strategy with potential to inform the development of next-generation, broadly protective vaccines against emerging coronaviruses. Biological sciences/Biotechnology Biological sciences/Immunology Biological sciences/Microbiology COVID-19 vaccine MERS-CoV SARS-CoV-2 Heterologous prime-boost Pan-coronavirus Protein subunit vaccine saRNA vaccine RNA vaccine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Coronaviruses continue to represent one of the most significant ongoing threats among emerging infectious diseases, owing to their capacity for zoonotic spillover 4 – 6 and rapid adaptation in human populations. 7 – 8 The COVID-19 pandemic, caused by SARS-CoV-2, 9–10,11−12 and previous outbreaks of the highly lethal coronaviruses SARS-CoV-1 in 2003 and MERS-CoV in 2012, underscore the urgent need for vaccines capable of protecting against a broad range of emerging coronaviruses. 13 SARS-CoV-1 infected more than 8,000 people across 30 countries with a case fatality rate of roughly 10%, 14 and continues to cause recurrent outbreaks with mortality rates near 35% among diagnosed cases. 15 The ongoing circulation of SARS-CoV-2, its continual evolution into immune-evasive variants, and sporadic MERS-CoV transmission 16 – 17 from dromedary camels as a primary reservoir illustrate the enduring pandemic potential of this viral family. Additionally, recent molecular and historical analyses suggest that the 1889–1894 “Russian flu” pandemic - characterized by considerable global mortality - may have resulted from the original zoonotic spillover of the beta-coronavirus lineage that gave rise to today’s endemic human coronavirus OC43, responsible for up to 30% of cases of the common cold. 18 – 19 Together, these observations highlight the need for vaccine platforms that elicit durable and cross-lineage immunity rather than variant-specific protection. Seven members of the vast zoonotic Coronaviridae family are known to be capable of causing respiratory diseases in humans, with symptoms ranging from mild to life-threatening conditions. 20 Among the four coronavirus genera (Alpha, Beta, Gamma, Delta), beta-coronaviruses stand out because their diversity, tropism to mammals, and history of high-impact spillovers make them the most urgent targets for broadly protective vaccine development. Betacoronaviruses exhibit exceptional genetic diversity and widespread presence in bat and mammalian reservoirs, facilitating zoonotic transmission events. 21 At the genomic and structural level, coronaviruses are enveloped, positive-sense RNA viruses with genomes of 27–32 kb. They encode four structural proteins essential for virion assembly and infectivity: spike (S), envelope (E), membrane (M), and nucleocapsid (N). The N protein is the most abundant and plays a crucial role in RNA synthesis, replication, and assembly. 14 The S protein, which mediates interaction, fusion, and entry into human host cells is divided into S1 and S2 subunits. Within the SARS-CoV-2 genome, the S gene displays the greatest sequence variability, driven by immune pressure and transmission adaptation with a particularly highly sequence variability found for the receptor binding domain (RBD) within the S1 subunit. 22 – 23 Current vaccines aim to elicit antibodies that block the function of the spike protein. Because non-neutralizing antibodies can in some contexts contribute to antibody-dependent enhancement (ADE) of infection, 24–25 inducing neutralizing antibodies directed against the RBD represents a prioritized vaccine target. However, the RBD’s exceptional sequence variability and concomitant susceptibility to immune-escape mutations pose major challenges to vaccine efficacy and durability of protection. 26 Most approved COVID-19 vaccines 27 follow a traditional homologous prime/boost regimen, 28–32 however, heterologous prime-boost vaccination strategies have been explored in several countries 33 – 34 though evidence of safety and robust immunogenicity to support the application of the heterologous regimens was scarce. 35 – 36 It has been suggested that mixing different SARS-CoV-2 vaccine types might lead to more efficacious and longer-lasting humoral protection against breakthrough infections. 33 While both, the homologous 37 – 38 and the heterologous booster 39 – 40 have successfully induced durable immune responses and extended the range of protection to SARS-CoV-2 variants of concern (VOCs), such as Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), direct comparison studies showed that heterologous boosting resulted in more robust immune responses than homologous boosting, and might enhance protection. 39 , 41 Taking into account that most people worldwide have been immunized with mRNA-based vaccines, a protein-based SARS-CoV-2 vaccine booster would be preferable to confer the benefits of a heterologous regimen. In addition to platform heterogeneity, expanding the antigenic diversity of the booster can further increase neutralization breadth. Protein-based boosters incorporating multiple antigens have demonstrated cross-variant immunity: Pavot et al. showed that boosting previously vaccinated primates with a SARS-CoV-2 S protein vaccine markedly increased cross-neutralizing antibody titers against diverse variants. 42 Bruno et al. demonstrated that a bivalent RBD vaccine elicited neutralizing antibodies even against SARS-CoV-2. 2 In this study, we integrate these concepts into a unified vaccination strategy. We prime with a nanoparticle-encapsulated self-amplifying RNA vaccine encoding SARS-CoV-2 S protein, and boost with an alum-adjuvanted combination of RBD proteins from SARS-CoV-2 (Wuhan-Hu-1 and B.1.351) and MERS-CoV. By leveraging an RNA vaccine prime to induce rapid systemic immunity and then a multivalent protein booster to expand breadth, this strategy aims to elicit both robust immediate protection and cross-reactive immunity, thereby offering a potential path toward a novel and cost-effective pan-coronavirus immunization strategy that leverages the immunological insights gained from studying both SARS-CoV-2 and MERS-CoV. RESULTS RNA priming induces solid, yet variant-specific immune responses The sequence design and construct architecture of all self-amplifying mRNA replicon vaccines used in this study, including codon optimization, stabilizing mutations, and variant-specific RBD selection as well as full S-protein constructs, are summarized in Supplementary Figure S1 . Having formulated these constructs in dendrimer nanoparticles (DNPs), we first evaluated the immunogenicity and protective efficacy of the RNA vaccination prime as a reference standard and to enable comparison with published data from other RNA-based vaccine platforms. This assessment provides the experimental and immunological benchmark for subsequent evaluation of heterologous RNA-protein prime-boost strategies. The dendrimer-formulated (saRNA) vaccines induced antigen-specific humoral responses in mice, consistent with titers reported for other RNA and lipid nanoparticle (LNP)-based vaccines in preclinical studies. 43 – 44 The overall magnitude of the antibody response was high across constructs encoding either the full-length S protein or variant-specific RBDs, reaching endpoint titers of approximately 10 2 -10 4 (geometric mean). Antigen specificity followed the encoded sequence precisely: the Alpha RBD construct induced markedly reduced titers, nearly two orders of magnitude lower, against the Beta RBD antigen than did the Beta RBD construct, underscoring that humoral responses were primarily directed toward the homologous variant. This finding reflects the strong influence of amino acid substitutions in the receptor-binding motif (K417N, E484K, and N501Y) on cross-neutralization capacity, as reported previously. 45 In parallel, the MERS-CoV RBD replicon elicited high-titer antibody responses comparable in magnitude to those of the SARS-CoV-2 constructs, confirming efficient antigen expression and potent immunogenicity. However, no measurable cross-reactivity was observed between MERS- and SARS-CoV-2-derived antigens, in line with their phylogenetic divergence within the Betacoronavirus genus. Alum-adjuvanted protein subunit RBD vaccines elicit dose-dependent antibody responses but require high antigen loads To establish a baseline for the subunit vaccine component, we evaluated the immunogenicity of alum-adjuvanted RBD proteins derived from SARS-CoV-2 Wuhan, B.1.351 (Beta), and MERS-CoV in C57BL/6 mice. Animals received intramuscular injections of 0.6, 3, or 15 µg of the respective recombinant RBD proteins, and sera were collected two weeks after vaccination for analysis of anti-RBD IgG titers by direct ELISA (Fig. 2 ). All three RBD vaccines elicited clear, dose-dependent antibody responses. At the highest dose of 15 µg, all three RBD formulations induced robust seroconversion in all animals, reaching endpoint titers of 10 5 (MERS), 10 4 (Wuhan) and 10 3 (Beta) measured against the respective RBD antigen. Responses dropped markedly at 3 µg and were weak and inconsistent at 0.6 µg, indicating that substantial antigen input is required to elicit consistent immunity from a single alum-adjuvanted protein dose. Generally, the MERS RBD induces the strongest response across all tested doses. Interestingly, mice immunized with the Wuhan RBD displayed notable cross-reactivity toward the B.1.351 antigen, which even exceeds the homologous response, whereas animals immunized with the B.1.351 RBD generated lower reciprocal reactivity toward Wuhan (Fig. 2 B). No measurable cross-reactivity was observed between the MERS RBD and either of the SARS-CoV-2 variant RBDs, confirming the antigenic separation between sarbecoviruses and merbecoviruses within the betacoronavirus genus. RNA priming markedly enhances the efficacy of protein subunit boosters To assess the functional effect of RNA priming on booster potency, we compared the dose-response of an alum-adjuvanted RBD protein booster following a self-amplifying RNA (saRNA) prime to that of a homologous RNA-RNA regimen (Fig. 3 ). Six groups of mice were primed with a dendrimer nanoparticle-delivered saRNA encoding the full-length S protein of SARS-CoV-2 B.1.351 (Beta). Four of these groups received a booster immunization three weeks later, consisting either of a second RNA dose or of Wuhan-strain RBD protein formulated on alum at concentrations of 0.6, 3, or 15 µg. Serum was collected immediately prior to boosting and again 21 days post-boost for ELISA quantification of anti-Wuhan RBD IgG titers and semi-quantitative surrogate viral neutralization test (sVNT) 46 analysis. In line with the previous data, animals immunized with either B.1.351 saRNA or Wuhan RBD alone (prime-only or boost-only, respectively) showed robust homologous antibody responses at Day 21 (Fig. 3 B). However, mice that received an RNA prime followed by a protein boost displayed a substantial amplification of the humoral response. The heterologous prime-boost regimen increased anti-RBD IgG titers by up to 69-fold (for the 15 µg booster) relative to the RNA prime-only group, while the 3 µg dose produced nearly equivalent enhancement (≈ 50-fold). Even the 0.6 µg protein boost, which as a prime was largely non-immunogenic, elicited a 27-fold increase in antibody titers when administered following RNA priming. sVNT results mirrored the ELISA data, suggestive of neutralizing activity consistent with the observed binding titers. Heterologous boosting sustains antibody response, and inclusion of MERS RBD extends protection beyond SARS-CoV-2 Building on the observed dose-sparing and recall efficiency of RNA priming, we next investigated how antigenic composition influences the immune response in homologous and heterologous prime-boost regimens. Using saRNA and alum-adjuvanted RBD protein vaccines, we compared single- and two-dose schedules incorporating monovalent and trivalent antigen formulations to assess whether multivalent boosting could broaden and prolong β-coronavirus immunity (Fig. 4 A). These animal data also provided an opportunity to evaluate the immunogenicity of a protein-only regimen and to determine how inclusion of the MERS-CoV component contributes to cross-lineage antibody reactivity and potential pan-β-coronavirus protection. The priming data for both the trivalent RNA and trivalent RBD-protein formulations closely paralleled the trends observed in the single-antigen experiments. RNA priming induced consistent antibody responses against both SARS-CoV-2 (Fig. 4 B) and MERS-CoV (Fig. 4 C), reaching endpoint IgG titers of approximately 10³ by Day 21 with peak responses observed at Day 35 and a gradual decline thereafter. In contrast, a 3 µg RBD-protein prime generated no measurable SARS-CoV-2-specific responses, which represents a reduction in response compared to those elicited by the corresponding monovalent RBD vaccines, however, surprisingly strong anti-MERS titers exceeded those of the RNA-primed group by roughly 1.5 log. Following the initial prime, animals received either 1 µg or 3 µg of the RBD-protein booster, or a 6 µg RNA booster. All three booster regimens produced comparable enhancements in antibody titers, with higher SARS-CoV-2 responses for protein-boosted mice compared with RNA-boosted mice. The effect was independent of whether the protein booster contained three RBDs (trivalent) or two (divalent, e.g., Beta + MERS or Delta + MERS). Interestingly, antibody titers declined more slowly for RBD-protein on alum boosted animals compared to those receiving the RNA boost, which is consistent with the known kinetic behavior of depot-forming adjuvant systems. Aluminum hydroxide has been shown to reduce antigen degradation and prolong antigen presentation by dendritic cells in vitro , 47 and in vivo comparisons have shown slower but more sustained antibody kinetics when antigen is formulated with depot adjuvants compared to fast-disseminating formulations. 48 For RBD-primed animals, a second 3 µg trivalent RBD dose was sufficient to induce anti-SARS-CoV-2 titers comparable to those achieved with a single RNA dose, albeit with higher inter-individual variability. In contrast, anti-MERS antibody titers, which were already high after priming, rose further upon boosting, reaching 10 6 and remaining stable or slightly increasing through Day 49. Inflammatory signature after vaccine boost: transient vs sustained inflammation responses Current mRNA-based COVID-19 vaccines induce inflammatory responses that may be dose-limiting, and can lead to several days of fatigue, pain, chills, and fever after administration. To compare the systemic inflammatory responses elicited by saRNA/DNP and alum-adjuvanted protein vaccines, serum concentrations of interleukin-6 (IL-6), chemokine (C-X-C motif) ligand 1 (CXCL1), and RANTES (CCL5) were measured at 6 and 24 hours after vaccination in BALB/c mice (Fig. 5 ). These markers were chosen as representative indicators of innate reactogenicity and leukocyte recruitment. IL-6 is a central mediator of early innate responses, regulating acute-phase signaling, fever induction, and adaptive immune activation. 49 CXCL1 is a key chemoattractant for neutrophils and non-hematopoietic cells, mediating local tissue inflammation at injection sites. 50 RANTES is a chemokine that promotes leukocyte recruitment at later stages of inflammation and serves as a surrogate marker of systemic immune activation. 51 As expected, saRNA vaccination induced rapid and strong systemic cytokine responses, with CXCL1 and RANTES markers sharply elevated at 6 hours and persisting at 24 hours post-injection. 52 CXCL1 levels reached up to 16x of the 100 pg/mL TBS baseline at 6 hrs and remained at 7x at 24 hrs, while RANTES levels continued ramping up until the 24 hrs mark (3.5x at 6hrs and 8x at 24 hrs). In contrast, mice boosted with alum-adjuvanted RBD protein exhibited a more transient inflammatory signature. IL-6 levels rose substantially at 6 hours - up to 22x (15 µg RBD-protein per injection) or 7x (3 µg RBD-protein per injection) higher serum concentration compared to the typical mouse serum baseline of 110 pg/ml - consistent with the expected short-term innate activation required for efficient antigen priming but declined to baseline levels by 24 hours. CXCL1 followed a similar kinetic pattern across all vaccine formulations, though residual elevation at 24 hours was highest in the saRNA/LNP group (formulated using the commercially available ionizable lipid DLin-MC3-DMA). RANTES levels remained low or undetectable in protein-boosted mice at 6h and 24 h measurements, but were markedly elevated for the RNA immunizations, particularly for LNP immunization, indicating stronger systemic leukocyte recruitment following RNA administration. Heterologous RNA-protein vaccination maintains long-term immunity and extends protection to new variants in hamsters To confirm the protective potential of the heterologous RNA-protein vaccination strategy in a second species and to assess long-term immune durability, we conducted a prime-boost-boost refresh study in Syrian golden hamsters (Fig. 6 ). Animals received either homologous or heterologous prime-boost regimens using saRNA encoding the SARS-CoV-2 Beta spike or alum-adjuvanted RBD proteins derived from the Beta and Delta variants (𝛽/𝛿) either in a high-dose (RNA: 9 µg, protein 15 µg) or a low-dose (RNA: 3 µg, protein 5 µg). A subsequent second booster administered 78 days after the first boost consisted of a Delta/Omicron (𝛿/o) RBD mixture (Fig. 6 A), enabling both evaluation of booster longevity and introduction of a new, antigenically distinct RBD to test cross-variant adaptability. As observed previously in mice, RNA priming generated substantially higher initial titers than protein priming at all dose levels (Day 21). The most pronounced titer increase after the first boost (Fig. 6 B-D, Day 42) occurred in the RBD/RBD protein-protein regimen (up to 25-fold enhancement), followed by RNA/RNA (7 to 17-fold) and the RNA/RBD heterologous regimens (2–6-fold), with similar trends across high- and low-dose groups. Anti-RBD protein antibody levels were found to be over one order of magnitude lower for the Omicron variant across all groups and dose levels, with a non-detectable prime response for the RBD-prime groups. Notably, despite lower early post-boost titers, the heterologous RNA/RBD group continued to exhibit rising antibody levels during the 57-day rest period (Days 42–99), whereas titers in both homologous groups declined by roughly one order of magnitude. Following the 78-day rest, all animals received a single second boost on Day 99 with the bivalent Beta/Omicron RBD formulation. By Day 120, antibody titers had risen sharply across all pre-immunized groups, confirming preserved immune memory and effective recall. The largest enhancement (up to 65-fold) occurred in animals that had previously received heterologous RNA/RBD regimens, whereas homologous RNA/RNA animals achieved a more modest up to 7-fold rise. Across all groups, responses were strongest against the Wuhan RBD, followed by Delta, and lowest against Omicron, yet detectable titers against Omicron confirmed successful recognition of this antigen despite its extensive immune-evasive mutations. Importantly, this demonstrates that the heterologous regimen remains adaptable-capable of being refocused toward new variant antigens introduced at later time points. All animals survived the three immunizations until scheduled necropsy on Day 121 without adverse clinical signs, body-weight loss, or injection-site reactions, supporting the safety of repeated dosing and of the alum-formulated RBD boosters in this model. DISCUSSION The emergence of three highly pathogenic coronaviruses within two decades - SARS-CoV, MERS-CoV, and SARS-CoV-2 - underscores both the inevitability of future zoonotic spillovers and the limitations of reactive vaccine development. 53 – 54 Current vaccine platforms, though transformative during the COVID-19 pandemic, remain largely optimized for homologous boosting against a single pathogen. There is an ongoing need for vaccination strategies that combine the potency of RNA delivery, the breadth of multivalent antigen design, and the safety and scalability of protein subunit technologies. 55 – 56 In this study, we explored such a strategy by combining RNA priming followed by a multivalent alum-adjuvanted protein boost. Across two species and multiple antigen combinations, the heterologous regimen consistently enhanced immunization quality in three terms compared with homologous RNA-RNA or protein-protein schedules: i) recall efficiency, ii) antigenic breadth, and iii) durability. First, RNA priming enabled potent antibody recall even at sub-microgram protein doses in mice (robust secondary responses at 0.6 RBD protein), and recall titers plateaued between 3 and 15 µg, suggesting that memory B-cell availability rather than antigen dose limits the booster response. 57 Second, the multivalent protein component broadened recognition across SARS-CoV-2 variants, and inclusion of MERS-CoV RBD extended cross-lineage reactivity beyond sarbecoviruses without compromising SARS-CoV-2 responses. Third, in hamsters, the heterologous boost led to sustained titers after a 78-day rest interval and potentiated responses to a second booster immunization that included a newly introduced strain (omicron). The results are consistent with a model in which RNA priming establishes a high-quality memory foundation via germinal center induction and T follicular helper (Tfh) cell engagement 58 that is efficiently re-engaged by a protein recall, even at low antigen dose, to produce large secondary responses. The observation that 3 µg and 15 µg protein boosts achieved similar titers implies a recall plateau, congruent with limited memory B cell clonal expansion rather than simple antigen-limited priming. Similar recall saturation has been observed in heterologous vector-protein regimens for COVID-19. 59–60 The multivalent RBD booster appeared to preferentially expand cross-reactive lineages that recognize conserved RBD epitopes, 61–62 while preserving potent homologous responses, a pattern previously reported in mixed-antigen nanoparticle vaccines. 63 – 64 Two findings deserve emphasis with respect to the role of MERS RBD protein and cross-lineage breadth. First, MERS RBD protein on alum formulations was intrinsically immunogenic even without RNA priming, yielding stable antibody levels that declined little over time. Second, adding MERS RBD did not erode SARS-CoV-2 responses, indicating no detrimental immunodominance in this setting. This suggests that antigens from distinct β-coronavirus clades of sufficient sequence divergence can be co-delivered to expand immune breadth beyond sarbecoviruses. Prior studies have similarly shown that heterologous or mosaic RBD vaccines can redirect immune recognition toward conserved protein surfaces. 55 While alum adsorption chemistry likely influences the presentation efficiency of individual RBDs, 65 the current results indicate that MERS inclusion is both feasible and beneficial for broad coronavirus coverage. The biomarker analysis (IL-6, CXCL1, CCL5/RANTES) indicates qualitatively different innate kinetics for the RNA and alum-adjuvanted protein vaccines tested. Self-amplifying RNA vaccination elicited systemic chemokine elevations (notably CXCL1 and CCL5) to 24 h, hallmarks of LNP-mediated innate sensing and type-I interferon signaling, 66–67 whereas protein/alum boosters induced a short-lived innate burst. IL-6 peaked at 6 h post-immunization and resolved by 24 h, a pattern typical of transient inflammasome-linked alum activation. 68 CXCL1 and CCL5 remained close to baseline in the protein groups, supporting a more localized, depot-like activation profile. This shorter inflammatory signature complements the booster’s role: sufficient to trigger a recall response without prolonged systemic inflammation. Together, these data highlight the tolerability and reactogenicity advantages of the RNA-protein sequence, particularly relevant for populations sensitive to reactogenicity and repeat dosing or large-scale immunization programs. The Syrian golden hamster model confirmed the immunogenic trends observed in mice, while demonstrating that the heterologous regimen preserved and could strengthen antibody responses over an extended interval. Following a long, 78-day rest after the first boost, antibody titers in heterologous cohorts were maintained or increased, while those in homologous groups declined compared to the peak measured 3 weeks post-boost, in some cases by nearly an order of magnitude. The second, delayed booster at day 99, which contained a newly introduced β/Omicron RBD formulation, induced a strong recall across all groups, with the largest fold-increases in heterologous cohorts. Despite the known immune evasiveness of Omicron, detectable anti-Omicron titers were achieved after the second boost. These findings emphasize the adaptability of the heterologous approach, and mirror clinical observations that heterologous booster regimens produce broader and more sustained humoral immunity than homologous ones. 69 – 70 They also support the idea that heterologous priming enhances the longevity and flexibility of memory B-cell responses, allowing efficient re-focusing towards novel emerging antigens without rebuilding the regimen from scratch. From a translational standpoint, the heterologous RNA-protein strategy leverages complementary technological strengths: the speed and potency of RNA priming for rapid, potent priming and the safety, stability, and affordability of protein boosting. RNA vaccines can be rapidly updated and induce potent primary immunity, while alum-adjuvanted protein boosters are compatible with existing cold-chain logistics and regulatory experience. 55 , 71 The pronounced dose-sparing effect observed with a robust recall when boosting even at 3 µg has direct manufacturing implications, potentially lowering costs and extending vaccine supply. Alum’s established regulatory track record and broad availability facilitate deployment at scale, including in settings where MERS remains a regional threat. The ability to add or swap RBDs in the booster makes the platform variant-responsive without disrupting established immunity and highlights the adaptability of this regimen to evolving pandemic threats. Limitations and outlook The study is preclinical, and neutralization assays were limited to surrogate formats; future work should employ pseudo- or live-virus neutralization across representative SARS-CoV-2 and MERS strains. Comprehensive profiling of CD4⁺, Tfh, and CD8⁺ responses is also warranted to define cellular contributions to the observed durability. Optimization of alum formulation, particularly adsorption efficiency and antigen spacing, may further refine cross-clade breadth. Commercial mRNA vaccines incorporate N-1-methylpseudouridine modification, which may exhibit different booster effects than saRNA; it will be valuable to study the impact of the modified mRNA modality on protein-based heterologous immunization. Nonetheless, the data provide a strong preclinical rationale for clinical exploration of heterologous RNA-protein regimens as boosters in previously RNA-primed human populations targeting a heterologous, multivalent, and adaptable strategy toward broader coronavirus preparedness. CONCLUSIONS The heterologous RNA-prime/multivalent-protein-boost approach described here delivered (i) high-magnitude recall with clear dose sparing of protein, (ii) broadened reactivity across SARS-CoV-2 variants with extension to MERS, and (iii) durable, recall-responsive immunity across a long inter-boost interval, including successful retargeting to Omicron RBD. By combining the rapid immunogenicity of RNA priming with the durable, broad recall of alum-adjuvanted multivalent RBD proteins, this strategy achieves strong and adaptable protection in two animal models. Protein/alum boosts produced a short-lived innate signature that resolved by 24 h. The regimen shows favorable tolerability, clear dose-sparing potential, and the ability to integrate new antigens without compromising prior immunity. Together, these features provide a practical, immunologically grounded framework and point to a scalable path forward toward broadly protective, pandemic-responsive coronavirus vaccines. METHODS Design and synthesis of saRNA Plasmid DNA encoding the SEAP or SARS-CoV-2/MERS-CoV proteins were cloned using standard molecular biological methods, using fragments encoding the protein ORFs from Thermo Fisher Scientific (Waltham, MA). A poly(A) tract of ~ 100 bp was installed at the end of the protein reading frame followed by a BspQI restriction site. The VEEV replicon encodes the complete VEEV genome minus the subgenomic ORF (replaced by the above sequences), and is available in the Supplementary Information. For RNA synthesis, plasmids were linearized by digestion with BspQI (New England Biolabs, Ipswich, MA), and purified using E.Z.N.A. Cycle Pure Kits (Omega Biotek, Norcross, GA). Linearized plasmid resuspended in RNase-free H 2 O was used as a template for in vitro transcription using T7 enzyme and subsequent 5’ end capping as described previously 15 . Translational fidelity was confirmed by immunoblot or by SEAP measurement by luminescent assay using the Phospha-Light reporter gene assay system (Thermo Fisher Scientific, Waltham, MA). RNA Formulation Purified saRNAs were formulated with modified poly(amidoamine)-based dendrimer nanoparticles (MDNPs) from Tiba Biotech’s RNABL™ platform. The dendrimers carried surface tertiary amines and biodegradable ester linkages, enabling efficient electrostatic complexation and endosomal release. RNA payloads for formulation were prepared first by resuspension of the capped RNA in citrate buffer (Teknova, Hollister, CA) to approximately 150 mg/L. The dendrimer/lipid phase for mixing with the RNA solution was prepared in 100% ethanol, at a volume 3x that of the aqueous RNA phase. This organic phase contained a proprietary blend of excipient lipids and the candidate dendrimer compound at a final concentration of approximately 3.5–5.5 mg of total dendrimer + lipid mass per ml. The aqueous and ethanol phases were combined by in-line mixing using the NanoAssemblr preclinical microfluidics platform (Vancouver, Canada). The resulting turbid nanoparticle suspension was dialyzed extensively at room temperature against PBS to remove residual ethanol and sterilized by filtration across a 0.2 µm membrane (Pall Corporation, New York, NY). All RNA vaccines were diluted in PBS before injection. Characterization of Nanoparticles (NPs) The size and polydispersity index(PDI)of all RNA nanoparticle test articles were determined using ZetaSizer Ultra (Malvern Panalytical, Malvern, UK) in triplicate. The mRNA encapsulation efficiency was analyzed using the Quant-iT RiboGreen RNA kit (Thermo Fisher Scientific, MA, USA) as described previously. Briefly, RNA-LNPs were lysed with 0.5% Triton-X or left untreated, followed by treatment with RiboGreen reagent following the manufacturer’s instructions. The quantity of RNA in the samples was measured using a microplate reader (Spark®, TECAN, Mannedorf, Switzerland). The calculated encapsulation efficiency of RNA was > 80%. RBD Plasmid construction, expression, and purification Plasmid DNA encoding SARS-CoV-2 or MERS-CoV proteins were synthesized and cloned by Proteogenix, Schiltigheim, France or Twist Bioscience. SARS-CoV-2 or MERS-CoV proteins were either produced using transient plasmid transfection of a serum-free by Proteogenix, Schiltigheim, France using transient plasmid transfection of a serum-free suspension of Chinese hamster ovary cells (XtenCHO) cells or in-house as previously described in E. coli . 72 – 73 In brief, the selected RBD protein sequences were codon optimized for expression XtenCHO and synthesized by artificial gene synthesis and cloned into the pTAX1 plasmid for expression in mammalian cells. Purified plasmids from the respective antigen expression cassettes was transfected into the XtenCHO cell line. The cells were monitored for cell viability, cell density and cultured for up to 13 days (312 hours) while the protein antigens were secreted into the culture media. The Spike RBD antigens were purified through ion exchange chromatography (IEX) followed by affinity chromatography using @RBD antibodies developed by ProteoGenix. The research grade antigen was then buffer exchanged into the formulation buffer. The identity of expressed proteins was confirmed by Western blot. Purity of recombinant protein produced by Proteogenix was higher than 90%. Purity evaluation was made on SDS-PAGE gel using the GelAnalyzer software by Proteogenix. Host cell protein and residual host cell DNA analysis The host cell protein was determined via a CHO HCP ELISA Kit (CYG-F550-1, Cygnus Technologies), and the residual host cell DNA was analyzed by a CHO Host Cell DNA Kit (CYG-D550T/ CYG-D555T, Cygnus Technologies) in triplicates and as per manufactures instructions and recommendations. Surface plasmon resonance analysis Recombinant SARS-CoV-2 S proteins (His-tagged) were purchased from Acro Biosystems Inc. (Newark, USA). The purity of recombinant proteins was documented by SDS-PAGE analysis. SPR measurements were performed on a Biacore 3000 instrument (GE Healthcare) by NBS-C BioScience, Vienna Austria as previously described. 74 RBD protein formulation The recombinant RBD proteins described above were formulated with 2.2% aluminum hydroxide gel (Alum) adjuvant. Briefly, the required dosage RBD antigen in pH 7.4 saline buffer was mixed with 70 µg/dose (corresponding to the max. approved Al 3+ -content of 850 µg per dose) Alhydrogel (InvivoGen) and incubated under gentle shaking for 30 min at room-temperature to allow adsorption, followed by storage at 2–8°C until use. All protein/alum formulations were diluted in sterile saline before use in the non-clinical studies. Mouse experiments and approvals Female C57BL/6J (B6) and or BALB/c mice were purchased from the Jackson Laboratory and maintained under specific pathogen-free conditions at the University of Minnesota according to the Institutional Animal Care and Use Committee guidelines (IACUC). All B6 mice used in the experiments were female and 6 to 9 weeks of age at the time point of the first immunization. For inflammatory response testing, BALB/c mice (6 to 10 weeks of age at the time of experimentation) were purchased from the Jackson Laboratory and maintained according to Tiba Biotech’s IACUC guidelines. C57BL/6 or BALB/c mice were immunized by bilateral intramuscular (i.m.) injection into the quadriceps muscles using insulin syringes. Each dose consisted of 50 µL per leg (total 100 µL). For prime-boost regimens, a second injection was administered on day 21 unless otherwise indicated. Control animals received formulation buffer or alum alone. Serum samples were collected from the submandibular vein at baseline and at indicated post-immunization time points (typically days 21, 35, 49, 99). Antibody titers against SARS-CoV-2 (Wuhan-Hu-1, Beta, Delta, and Omicron RBDs) and MERS-CoV RBD were determined by enzyme-linked immunosorbent assay (ELISA). To assess local and systemic inflammatory responses following vaccination, serum samples were collected at 6 h and 24 h post-immunization from representative animals in each treatment group. Concentrations of IL-6, CXCL1 (KC/GROα), and CCL5 (RANTES) were quantified using multiplex bead-based immunoassays (Luminex®) according to the manufacturer’s instructions. These markers were selected as representative indicators of acute-phase cytokine induction, neutrophil-associated chemotaxis, and leukocyte recruitment, respectively. Data were analyzed as fold-change over pre-immune baseline, TBS-buffer groups serving as controls to distinguish adjuvant-driven from RNA-induced responses. Enzyme-linked immunosorbent assay (ELISA) Endpoint antibody titers against SARS-CoV-2 and MERS-CoV RBDs were quantified by direct ELISA following optimized protocols. High-binding 96-well plates (Greiner Bio-One #655061) were coated overnight at 4°C with 1 µg/mL of recombinant RBD protein (Beta lot #15195-101921-C01; Delta lot #15195-101921-B01; MERS lot #15195-100921-D01; ProteoGenix) in PBS (pH 7.4). Plates were washed with Phosphat Buffer Saline – Tween (PBST, VWR Cat# RLMB-075-1000) and blocked for 1 h at room temperature with 0.5% BSA in PBS. Eleven serial three-fold serum dilutions (1:25 − 1:1,462,250) were prepared in PBS/0.5% BSA and added to the plates for 1 h at RT. Following washing, goat anti-mouse IgG (HRP-conjugated; Invitrogen #31430, 1:20 000 dilution) was applied for 45 min at RT. After final washes, plates were developed with TMB substrate (BD #555214) for 2.5 min and stopped with 0.5 M H₂SO₄. Absorbance was read at 450 nm with 570 nm background correction using a Tecan Infinite M Plex. A sigmoidal 4P curve-fit model was applied to the absorbance readings, and endpoint titers were defined as the OD₄₅₀ value of that curve exceeding the mean of negative controls by three standard deviations. Data analysis was performed using GraphPad Prism (v10.0). Antibody titers and surrogate neutralization values were log₁₀-transformed prior to analysis, and group averages were expressed as geometric mean titers (GMT) with 95% confidence intervals. Hamster experiments and approvals 24 Male and 36 female Syrian Golden hamsters were purchased by Attentive Science for this study and maintained under specific pathogen-free conditions. All animals were group housed by sex and dose group in clean solid bottom cages in environmentally controlled housing. The cages contained appropriate bedding material, which were changed at least once weekly. Animals were 4–7 weeks old and had a bodyweight between 120 and 293 g of at the start of the study. Male and female Syrian golden hamsters ( Mesocricetus auratus ; 7–10 weeks of age; n = 5 per group) were immunized by intramuscular injection into the hind limb muscles. A prime-boost schedule was employed with injections on day 1 and day 21, followed by a delayed second boost at day 99 in selected groups. Control animals received formulation buffer only. Blood samples were collected at designated time points for serological analyses. Antibody titers against SARS-CoV-2 (Wuhan-Hu-1, Beta, Delta, and Omicron RBDs) and MERS-CoV RBD were quantified by ELISA (Attentive Science Study Nos. 1121–3495 and 1121–4898). Ethics statement All experimental protocols with animals were conducted in strict accordance with international ethical standards for animal experimentation (Helsinki Declaration and its amendments, Amsterdam Protocol of welfare and animal protection and National Institutes of Health, NIH USA, guidelines). All procedures were approved by the respective Institutional Animal Care and Use Committee (IACUC). Data analysis All calculations and statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, CA). Data in bars diagrams are shown as mean ± SD and differences were considered statistically significant at P < 0.05. Declarations ACKNOWLEDGEMENTS We thank Ram Karan and Sam Mathew for providing help in the initial experiments. The research reported in this publication was supported by the King Abdullah University of Science and Technology (KAUST); Office of Sponsored Research, OSR-NTGC-2021-4587. DATA AVAILABILITY All data generated or analyzed during this study are included in this published article and its supplementary information files. AUTHOR CONTRIBUTIONS Conceptualization: C.W.M., J.E., J.S.C., M.R. Methodology: D.R., J.S.M., F.K., C.W.M., J.E., J.S.C., M.R. Investigation: D.R., J.S.M., F.K., C.W.M., J.E., J.S.C., M.R. Data curation: D.R., J.S.M., S.B., P.T., J.E. Formal analysis and visualization: D.R., J.S.M., J.E., J.S.C. Writing – original draft: D.R., J.E., M.R. Writing – review & editing: All authors Funding acquisition: C.W.M., J.E., J.S.C., M.R. Resources: C.W.M., J.E., J.S.C., M.R. Project administration: D.R., C.W.M., J.E., J.S.C., M.R. Supervision: C.W.M., J.E., J.S.C., M.R. All authors contributed to study design, data interpretation, and manuscript preparation; approved the final version; and agree to be accountable for all aspects of the work in ensuring its accuracy and integrity. COMPETING INTERESTS P.T., J.S.M., C.W.M., and J.S.C. are employees of Tiba Biotech LLC and have equity interests. Tiba Biotech has filed a patent on the MDNP RNA delivery system (patent application PCT/US21/25542), naming P.T. and J.S.C. as inventors. J.S.C. is also an inventor on U.S. Patent no. 10,548,959 entitled “Compositions and Methods for Modified Dendrimer Nanoparticle Delivery.” C.W.M. has worked as a freelance consultant advising commercial and nonprofit organizations working in the fields of vaccines and viral vectors. All other authors declare that they have no competing interests. ADDITIONAL INFORMATION Supplementary information accompanies this paper and provides detailed descriptions of vaccine construct design, replicon architecture, and RBD sequence selection. It includes full RNA and protein antigen sequences, in vitro expression characterization, and experimental procedures for splenocyte restimulation and cytokine assays. 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O., SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates. npj Vaccines 2021, 6 (1), 28. Zuo, X.; Mattern, M. R.; Tan, R.; Li, S.; Hall, J.; Sterner, D. E.; Shoo, J.; Tran, H.; Lim, P.; Sarafianos, S. G.; Kazi, L.; Navas-Martin, S.; Weiss, S. R.; Butt, T. R., Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr Purif 2005, 42 (1), 100–10. Chen, J.; Miao, L.; Li, J. M.; Li, Y. Y.; Zhu, Q. Y.; Zhou, C. L.; Fang, H. Q.; Chen, H. P., Receptor-binding domain of SARS-Cov spike protein: soluble expression in E. coli, purification and functional characterization. World J Gastroenterol 2005, 11 (39), 6159–64. Monteil, V.; Eaton, B.; Postnikova, E.; Murphy, M.; Braunsfeld, B.; Crozier, I.; Kricek, F.; Niederhöfer, J.; Schwarzböck, A.; Breid, H.; Devignot, S.; Klingström, J.; Thålin, C.; Kellner, M. J.; Christ, W.; Havervall, S.; Mereiter, S.; Knapp, S.; Sanchez Jimenez, A.; Bugajska-Schretter, A.; Dohnal, A.; Ruf, C.; Gugenberger, R.; Hagelkruys, A.; Montserrat, N.; Kozieradzki, I.; Hasan Ali, O.; Stadlmann, J.; Holbrook, M. R.; Schmaljohn, C.; Oostenbrink, C.; Shoemaker, R. H.; Mirazimi, A.; Wirnsberger, G.; Penninger, J. M., Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants. EMBO Molecular Medicine 2022, 14 (8), e15230. Additional Declarations Competing interest reported. P.T., J.S.M., C.W.M., and J.S.C. are employees of Tiba Biotech LLC and have equity interests. Tiba Biotech has filed a patent on the MDNP RNA delivery system (patent application PCT/US21/25542), naming P.T. and J.S.C. as inventors. J.S.C. is also an inventor on U.S. Patent no. 10,548,959 entitled “Compositions and Methods for Modified Dendrimer Nanoparticle Delivery.” C.W.M. has worked as a freelance consultant advising commercial and nonprofit organizations working in the fields of vaccines and viral vectors. All other authors declare that they have no competing interests. Supplementary Files 02HeterologousprimeboostSI.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":165213,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral immunogenicity of self-amplifying replicon RNA (saRNA) vaccines encapsulated in DNPs. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic overview of the study design used to assess the immunogenicity of dendrimer-formulated saRNA vaccines in C57BL/6 mice. Four- to six-week-old female C57BL/6 mice (n = 5) were immunized intramuscularly with 5 µg of the indicated vaccine formulation, and sera were collected 14 days later. The @ symbolizes the encapsulation of RNA inside of the DNPs. (\u003cstrong\u003eB\u003c/strong\u003e) Humoral responses against SARS-CoV-2- and MERS-derived saRNA constructs measured by ELISA. Symbols below each bar indicate which RNA sequence was present (+) or absent (–) in the formulation. Direct mouse IgG ELISA was performed using recombinant SARS-CoV-2 B.1.351 (Beta) or MERS-CoV RBD proteins. Left panel (green): endpoint titers against SARS-CoV-2 Beta RBD; right panel (yellow): endpoint titers against MERS-CoV RBD. Bars represent mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/2d1e500bacb96fa4e16a0cc9.jpeg"},{"id":93742001,"identity":"bca6dc9b-27b7-45db-805d-ec77ad1aec16","added_by":"auto","created_at":"2025-10-17 05:27:58","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":207176,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral immunogenicity of alum-adjuvanted RBD proteins. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic overview of the study design used to assess immunogenicity of alum-formulated recombinant RBD vaccines in C57BL/6 mice. Four- to six-week-old female mice (n = 5 per group) were immunized intramuscularly with the indicated concentrations (0.6, 3, or 15 µg) of RBD antigen formulated with aluminum hydroxide (Alhydrogel®). Serum was collected 14 days later for ELISA analysis. The @ symbolizes the grafting of the protein subunits onto the alum surface. (\u003cstrong\u003eB\u003c/strong\u003e) Humoral responses against SARS-CoV-2 variants and MERS RBD induced by different protein antigens measured by ELISA. Direct mouse IgG ELISA was performed on recombinant B.1.351 or MERS RBD protein; left (blue): antibody titers against Wuhan-SARS-CoV-2 RBD, middle (green): antibody titers against Beta-SARS-CoV-2 RBD C57BL/6, right (yellow): antibody titers against MERS-CoV RBD. Bars represent mean ± SD. Dashed bars indicate cross-protective responses.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/e1b9b64c08b732a4cdef59cf.jpeg"},{"id":93742002,"identity":"f4f307ab-4eb9-4354-bb2e-38fc664401b8","added_by":"auto","created_at":"2025-10-17 05:27:58","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":129215,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral immunogenicity of homologous and heterologous prime-boost regimens combining saRNA and alum-adjuvanted RBD vaccines. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic overview of the study design. Six groups (n = 3) of female C57BL/6 mice were immunized intramuscularly on Day 0 with either 15 µg Wuhan RBD protein on alum (one group) or 5 µg saRNA encoding the SARS-CoV-2 B.1.351 S protein (five groups). Serum was collected 21 days later for baseline ELISA analysis. Four RNA-primed groups then received booster immunizations with 0.6, 3, or 15 µg RBD protein on alum or a second RNA dose. Sera were collected again 28 days post-boost (Day 49). \u003cstrong\u003e(B)\u003c/strong\u003e Anti-Wuhan RBD IgG titers measured by ELISA on Days 21 (blue) and 49 (orange). Geometric means are shown, with dashed lines indicating titers from RNA prime-only animals. Values within bars indicate average sVNT% inhibition; numbers above bars for Day 49 denote fold-increase relative to Day 21. PBS controls not shown. Bars represent mean ± SD.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/f5597f46c76ef2b794982b09.jpeg"},{"id":93742012,"identity":"68b40ad6-a00d-417b-af4e-6363b2eeb02c","added_by":"auto","created_at":"2025-10-17 05:27:58","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":419484,"visible":true,"origin":"","legend":"\u003cp\u003eHeterologous and homologous prime-boost regimens combining trivalent saRNA and multi-antigen RBD-protein vaccines. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic overview of the study design. Groups of female C57BL/6 mice (n = 6) were immunized intramuscularly on Day 0 with either 3 µg (low-dose RNA prime; two groups) or 6 µg (high-dose RNA prime; five groups) of a trivalent saRNA vaccine encoding the SARS-CoV-2 B.1.351 S protein, the SARS-CoV-2 B.1.617.2 RBD, and the MERS-CoV RBD, or with 3 µg of a trivalent RBD-protein formulation (B.1.351 + B.1.617.2 + MERS) on an alhydrogel adjuvant (alum or alphos, as specified in Figure 1A). Serum was collected 21 days later for baseline ELISA analysis. Booster immunizations were administered on Day 21 as indicated, and sera were collected on Days 35 and 49.\u003cstrong\u003e (B,C)\u003c/strong\u003e Anti-RBD IgG titers measured by ELISA at Days 21 (blue), 35 (orange), and 49 (grey). Geometric means are shown, with solid-filled bars indicating groups that received a protein boost. PBS controls not shown. Bars represent mean ± SD. \u003cstrong\u003e(B) \u003c/strong\u003eanti-SARS-CoV-2 B. 1.351 RBD titers. \u003cstrong\u003e(C) \u003c/strong\u003eanti-MERS-CoV RBD titers.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/5d31b2ab2eb1035ccd48e9c2.jpeg"},{"id":93742011,"identity":"04e86c2e-ed21-41d6-b455-3ce77096b474","added_by":"auto","created_at":"2025-10-17 05:27:58","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":170126,"visible":true,"origin":"","legend":"\u003cp\u003eSystemic inflammatory cytokine responses following administration of saRNA formulations (encoding the SARS-CoV-2 B.1.351 S protein) and alum-adjuvanted RBD-protein vaccines. Mice (n = 6 per group) were immunized intramuscularly with 5 µg RNA formulations (RNA@DNP or RNA@LNP), an equivalent amount of DNP only or with 15 µg or 3 µg recombinant RBD proteins adsorbed on alum. Serum was collected 6 hours (blue) and 24 hours (orange) post-immunization. TBS served as a negative control. Dashed line indicates assay background. Bars represent geometric mean ± SD. (\u003cstrong\u003eA\u003c/strong\u003e) IL-6, (\u003cstrong\u003eB\u003c/strong\u003e) CXCL1, and (\u003cstrong\u003eC\u003c/strong\u003e) CCL5/RANTES.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/0b140cdab85294fa2200d118.jpeg"},{"id":93742008,"identity":"69021000-95a7-42ec-b9e9-c469e0c86b75","added_by":"auto","created_at":"2025-10-17 05:27:58","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1475241,"visible":true,"origin":"","legend":"\u003cp\u003eLong-interval heterologous boosting in Syrian golden hamsters expands protection to new variants. (\u003cstrong\u003eA\u003c/strong\u003e) Study design: animals (n = 5 per group) were primed on Day 0 with either saRNA (β-Spike, 3 µg or 9 µg) formulated with DNPs or RBD protein (β/𝛿: physical 1:1 mixture of Beta-RBD and Delta-RBD, total protein: 5 µg or 15 µg) on alum. First boosts were given on Day 21 with corresponding homologous or heterologous vaccines. After a 78-day rest, all animals received a refresh booster (Day 99) using the β/o (physical 1:1 mixture of Beta-RBD and Omicron-RBD, total protein: 20 µg) on alum (Al-content per dose 69 µg for any protein formulation). Direct hamster IgG ELISA was performed on recombinant SARS-CoV-2 Wuhan (\u003cstrong\u003eB\u003c/strong\u003e), Delta (\u003cstrong\u003eC\u003c/strong\u003e) or Omicron (\u003cstrong\u003eD\u003c/strong\u003e) RBD protein. Bars show geometric mean ± SD. Bar color indicates prime boost regimen. Black: RNA prime, RNA boost, blue: RNA prime RBD boost, green: RBD prime, RBD boost; dashed bars denote pre-boost titers. Numbers below the time axis indicate amount of injected pharmacologically active ingredient for prime / boost 1 / boost 2. No significant titers were observed in samples drawn before the first vaccination (data not shown).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/87dd6b2a77b2ed6f36d5e0f6.jpeg"},{"id":96454599,"identity":"fa728c6b-0340-412d-a9d2-cad5402b3d75","added_by":"auto","created_at":"2025-11-21 10:02:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3726115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/35e45218-8025-4c95-b6cb-8f0678a3ce90.pdf"},{"id":93742000,"identity":"5fd61c28-329c-491c-8ab8-e784ccaedb43","added_by":"auto","created_at":"2025-10-17 05:27:58","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":527623,"visible":true,"origin":"","legend":"","description":"","filename":"02HeterologousprimeboostSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7875217/v1/81543bdec8c098f5f75995e3.docx"}],"financialInterests":"Competing interest reported. P.T., J.S.M., C.W.M., and J.S.C. are employees of Tiba Biotech LLC and have equity interests. Tiba Biotech has filed a patent on the MDNP RNA delivery system (patent application PCT/US21/25542), naming P.T. and J.S.C. as inventors. J.S.C. is also an inventor on U.S. Patent no. 10,548,959 entitled “Compositions and Methods for Modified Dendrimer Nanoparticle Delivery.” C.W.M. has worked as a freelance consultant advising commercial and nonprofit organizations working in the fields of vaccines and viral vectors. All other authors declare that they have no competing interests.","formattedTitle":"Heterologous saRNA Prime – Multivalent Protein Boost Strategy Induces Broad and Durable Immunity Against SARS-CoV-2 and MERS-CoV","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCoronaviruses continue to represent one of the most significant ongoing threats among emerging infectious diseases, owing to their capacity for zoonotic spillover\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and rapid adaptation in human populations.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The COVID-19 pandemic, caused by SARS-CoV-2,\u003csup\u003e9\u0026ndash;10,11\u0026minus;12\u003c/sup\u003e and previous outbreaks of the highly lethal coronaviruses SARS-CoV-1 in 2003 and MERS-CoV in 2012, underscore the urgent need for vaccines capable of protecting against a broad range of emerging coronaviruses.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e SARS-CoV-1 infected more than 8,000 people across 30 countries with a case fatality rate of roughly 10%,\u003csup\u003e14\u003c/sup\u003e and continues to cause recurrent outbreaks with mortality rates near 35% among diagnosed cases.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The ongoing circulation of SARS-CoV-2, its continual evolution into immune-evasive variants, and sporadic MERS-CoV transmission\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e from dromedary camels as a primary reservoir illustrate the enduring pandemic potential of this viral family. Additionally, recent molecular and historical analyses suggest that the 1889\u0026ndash;1894 \u0026ldquo;Russian flu\u0026rdquo; pandemic - characterized by considerable global mortality - may have resulted from the original zoonotic spillover of the beta-coronavirus lineage that gave rise to today\u0026rsquo;s endemic human coronavirus OC43, responsible for up to 30% of cases of the common cold.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Together, these observations highlight the need for vaccine platforms that elicit durable and cross-lineage immunity rather than variant-specific protection.\u003c/p\u003e\u003cp\u003eSeven members of the vast zoonotic \u003cem\u003eCoronaviridae\u003c/em\u003e family are known to be capable of causing respiratory diseases in humans, with symptoms ranging from mild to life-threatening conditions.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Among the four coronavirus genera (Alpha, Beta, Gamma, Delta), beta-coronaviruses stand out because their diversity, tropism to mammals, and history of high-impact spillovers make them the most urgent targets for broadly protective vaccine development. Betacoronaviruses exhibit exceptional genetic diversity and widespread presence in bat and mammalian reservoirs, facilitating zoonotic transmission events.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAt the genomic and structural level, coronaviruses are enveloped, positive-sense RNA viruses with genomes of 27\u0026ndash;32 kb. They encode four structural proteins essential for virion assembly and infectivity: spike (S), envelope (E), membrane (M), and nucleocapsid (N). The N protein is the most abundant and plays a crucial role in RNA synthesis, replication, and assembly.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e The S protein, which mediates interaction, fusion, and entry into human host cells is divided into S1 and S2 subunits. Within the SARS-CoV-2 genome, the S gene displays the greatest sequence variability, driven by immune pressure and transmission adaptation with a particularly highly sequence variability found for the receptor binding domain (RBD) within the S1 subunit.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eCurrent vaccines aim to elicit antibodies that block the function of the spike protein. Because non-neutralizing antibodies can in some contexts contribute to antibody-dependent enhancement (ADE) of infection,\u003csup\u003e24\u0026ndash;25\u003c/sup\u003e inducing neutralizing antibodies directed against the RBD represents a prioritized vaccine target. However, the RBD\u0026rsquo;s exceptional sequence variability and concomitant susceptibility to immune-escape mutations pose major challenges to vaccine efficacy and durability of protection.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eMost approved COVID-19 vaccines\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e follow a traditional homologous prime/boost regimen,\u003csup\u003e28\u0026ndash;32\u003c/sup\u003e however, heterologous prime-boost vaccination strategies have been explored in several countries \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e though evidence of safety and robust immunogenicity to support the application of the heterologous regimens was scarce.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e It has been suggested that mixing different SARS-CoV-2 vaccine types might lead to more efficacious and longer-lasting humoral protection against breakthrough infections.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e While both, the homologous\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and the heterologous booster\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e have successfully induced durable immune responses and extended the range of protection to SARS-CoV-2 variants of concern (VOCs), such as Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), direct comparison studies showed that heterologous boosting resulted in more robust immune responses than homologous boosting, and might enhance protection.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Taking into account that most people worldwide have been immunized with mRNA-based vaccines, a protein-based SARS-CoV-2 vaccine booster would be preferable to confer the benefits of a heterologous regimen.\u003c/p\u003e\u003cp\u003eIn addition to platform heterogeneity, expanding the antigenic diversity of the booster can further increase neutralization breadth. Protein-based boosters incorporating multiple antigens have demonstrated cross-variant immunity: Pavot et al. showed that boosting previously vaccinated primates with a SARS-CoV-2 S protein vaccine markedly increased cross-neutralizing antibody titers against diverse variants.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Bruno et al. demonstrated that a bivalent RBD vaccine elicited neutralizing antibodies even against SARS-CoV-2.\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn this study, we integrate these concepts into a unified vaccination strategy. We prime with a nanoparticle-encapsulated self-amplifying RNA vaccine encoding SARS-CoV-2 S protein, and boost with an alum-adjuvanted combination of RBD proteins from SARS-CoV-2 (Wuhan-Hu-1 and B.1.351) and MERS-CoV. By leveraging an RNA vaccine prime to induce rapid systemic immunity and then a multivalent protein booster to expand breadth, this strategy aims to elicit both robust immediate protection and cross-reactive immunity, thereby offering a potential path toward a novel and cost-effective pan-coronavirus immunization strategy that leverages the immunological insights gained from studying both SARS-CoV-2 and MERS-CoV.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eRNA priming induces solid, yet variant-specific immune responses\u003c/h2\u003e\u003cp\u003eThe sequence design and construct architecture of all self-amplifying mRNA replicon vaccines used in this study, including codon optimization, stabilizing mutations, and variant-specific RBD selection as well as full S-protein constructs, are summarized in \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Having formulated these constructs in dendrimer nanoparticles (DNPs), we first evaluated the immunogenicity and protective efficacy of the RNA vaccination prime as a reference standard and to enable comparison with published data from other RNA-based vaccine platforms. This assessment provides the experimental and immunological benchmark for subsequent evaluation of heterologous RNA-protein prime-boost strategies.\u003c/p\u003e\u003cp\u003eThe dendrimer-formulated (saRNA) vaccines induced antigen-specific humoral responses in mice, consistent with titers reported for other RNA and lipid nanoparticle (LNP)-based vaccines in preclinical studies.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e The overall magnitude of the antibody response was high across constructs encoding either the full-length S protein or variant-specific RBDs, reaching endpoint titers of approximately 10\u003csup\u003e2\u003c/sup\u003e-10\u003csup\u003e4\u003c/sup\u003e (geometric mean).\u003c/p\u003e\u003cp\u003e Antigen specificity followed the encoded sequence precisely: the Alpha RBD construct induced markedly reduced titers, nearly two orders of magnitude lower, against the Beta RBD antigen than did the Beta RBD construct, underscoring that humoral responses were primarily directed toward the homologous variant. This finding reflects the strong influence of amino acid substitutions in the receptor-binding motif (K417N, E484K, and N501Y) on cross-neutralization capacity, as reported previously.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e In parallel, the MERS-CoV RBD replicon elicited high-titer antibody responses comparable in magnitude to those of the SARS-CoV-2 constructs, confirming efficient antigen expression and potent immunogenicity. However, no measurable cross-reactivity was observed between MERS- and SARS-CoV-2-derived antigens, in line with their phylogenetic divergence within the Betacoronavirus genus.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAlum-adjuvanted protein subunit RBD vaccines elicit dose-dependent antibody responses but require high antigen loads\u003c/h3\u003e\n\u003cp\u003eTo establish a baseline for the subunit vaccine component, we evaluated the immunogenicity of alum-adjuvanted RBD proteins derived from SARS-CoV-2 Wuhan, B.1.351 (Beta), and MERS-CoV in C57BL/6 mice. Animals received intramuscular injections of 0.6, 3, or 15 \u0026micro;g of the respective recombinant RBD proteins, and sera were collected two weeks after vaccination for analysis of anti-RBD IgG titers by direct ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll three RBD vaccines elicited clear, dose-dependent antibody responses. At the highest dose of 15 \u0026micro;g, all three RBD formulations induced robust seroconversion in all animals, reaching endpoint titers of 10\u003csup\u003e5\u003c/sup\u003e (MERS), 10\u003csup\u003e4\u003c/sup\u003e (Wuhan) and 10\u003csup\u003e3\u003c/sup\u003e (Beta) measured against the respective RBD antigen. Responses dropped markedly at 3 \u0026micro;g and were weak and inconsistent at 0.6 \u0026micro;g, indicating that substantial antigen input is required to elicit consistent immunity from a single alum-adjuvanted protein dose. Generally, the MERS RBD induces the strongest response across all tested doses.\u003c/p\u003e\u003cp\u003eInterestingly, mice immunized with the Wuhan RBD displayed notable cross-reactivity toward the B.1.351 antigen, which even exceeds the homologous response, whereas animals immunized with the B.1.351 RBD generated lower reciprocal reactivity toward Wuhan (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). No measurable cross-reactivity was observed between the MERS RBD and either of the SARS-CoV-2 variant RBDs, confirming the antigenic separation between sarbecoviruses and merbecoviruses within the betacoronavirus genus.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eRNA priming markedly enhances the efficacy of protein subunit boosters\u003c/h3\u003e\n\u003cp\u003eTo assess the functional effect of RNA priming on booster potency, we compared the dose-response of an alum-adjuvanted RBD protein booster following a self-amplifying RNA (saRNA) prime to that of a homologous RNA-RNA regimen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Six groups of mice were primed with a dendrimer nanoparticle-delivered saRNA encoding the full-length S protein of SARS-CoV-2 B.1.351 (Beta). Four of these groups received a booster immunization three weeks later, consisting either of a second RNA dose or of Wuhan-strain RBD protein formulated on alum at concentrations of 0.6, 3, or 15 \u0026micro;g. Serum was collected immediately prior to boosting and again 21 days post-boost for ELISA quantification of anti-Wuhan RBD IgG titers and semi-quantitative surrogate viral neutralization test (sVNT)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e analysis.\u003c/p\u003e\u003cp\u003eIn line with the previous data, animals immunized with either B.1.351 saRNA or Wuhan RBD alone (prime-only or boost-only, respectively) showed robust homologous antibody responses at Day 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, mice that received an RNA prime followed by a protein boost displayed a substantial amplification of the humoral response. The heterologous prime-boost regimen increased anti-RBD IgG titers by up to 69-fold (for the 15 \u0026micro;g booster) relative to the RNA prime-only group, while the 3 \u0026micro;g dose produced nearly equivalent enhancement (\u0026asymp;\u0026thinsp;50-fold). Even the 0.6 \u0026micro;g protein boost, which as a prime was largely non-immunogenic, elicited a 27-fold increase in antibody titers when administered following RNA priming. sVNT results mirrored the ELISA data, suggestive of neutralizing activity consistent with the observed binding titers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eHeterologous boosting sustains antibody response, and inclusion of MERS RBD extends protection beyond SARS-CoV-2\u003c/h3\u003e\n\u003cp\u003eBuilding on the observed dose-sparing and recall efficiency of RNA priming, we next investigated how antigenic composition influences the immune response in homologous and heterologous prime-boost regimens. Using saRNA and alum-adjuvanted RBD protein vaccines, we compared single- and two-dose schedules incorporating monovalent and trivalent antigen formulations to assess whether multivalent boosting could broaden and prolong β-coronavirus immunity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These animal data also provided an opportunity to evaluate the immunogenicity of a protein-only regimen and to determine how inclusion of the MERS-CoV component contributes to cross-lineage antibody reactivity and potential pan-β-coronavirus protection.\u003c/p\u003e\u003cp\u003eThe priming data for both the trivalent RNA and trivalent RBD-protein formulations closely paralleled the trends observed in the single-antigen experiments. RNA priming induced consistent antibody responses against both SARS-CoV-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and MERS-CoV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), reaching endpoint IgG titers of approximately 10\u0026sup3; by Day 21 with peak responses observed at Day 35 and a gradual decline thereafter. In contrast, a 3 \u0026micro;g RBD-protein prime generated no measurable SARS-CoV-2-specific responses, which represents a reduction in response compared to those elicited by the corresponding monovalent RBD vaccines, however, surprisingly strong anti-MERS titers exceeded those of the RNA-primed group by roughly 1.5 log.\u003c/p\u003e\u003cp\u003eFollowing the initial prime, animals received either 1 \u0026micro;g or 3 \u0026micro;g of the RBD-protein booster, or a 6 \u0026micro;g RNA booster. All three booster regimens produced comparable enhancements in antibody titers, with higher SARS-CoV-2 responses for protein-boosted mice compared with RNA-boosted mice. The effect was independent of whether the protein booster contained three RBDs (trivalent) or two (divalent, e.g., Beta\u0026thinsp;+\u0026thinsp;MERS or Delta\u0026thinsp;+\u0026thinsp;MERS). Interestingly, antibody titers declined more slowly for RBD-protein on alum boosted animals compared to those receiving the RNA boost, which is consistent with the known kinetic behavior of depot-forming adjuvant systems. Aluminum hydroxide has been shown to reduce antigen degradation and prolong antigen presentation by dendritic cells \u003cem\u003ein vitro\u003c/em\u003e,\u003csup\u003e47\u003c/sup\u003e and \u003cem\u003ein vivo\u003c/em\u003e comparisons have shown slower but more sustained antibody kinetics when antigen is formulated with depot adjuvants compared to fast-disseminating formulations.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eFor RBD-primed animals, a second 3 \u0026micro;g trivalent RBD dose was sufficient to induce anti-SARS-CoV-2 titers comparable to those achieved with a single RNA dose, albeit with higher inter-individual variability. In contrast, anti-MERS antibody titers, which were already high after priming, rose further upon boosting, reaching 10\u003csup\u003e6\u003c/sup\u003e and remaining stable or slightly increasing through Day 49.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eInflammatory signature after vaccine boost: transient vs sustained inflammation responses\u003c/h3\u003e\n\u003cp\u003eCurrent mRNA-based COVID-19 vaccines induce inflammatory responses that may be dose-limiting, and can lead to several days of fatigue, pain, chills, and fever after administration. To compare the systemic inflammatory responses elicited by saRNA/DNP and alum-adjuvanted protein vaccines, serum concentrations of interleukin-6 (IL-6), chemokine (C-X-C motif) ligand 1 (CXCL1), and RANTES (CCL5) were measured at 6 and 24 hours after vaccination in BALB/c mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These markers were chosen as representative indicators of innate reactogenicity and leukocyte recruitment. IL-6 is a central mediator of early innate responses, regulating acute-phase signaling, fever induction, and adaptive immune activation.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e CXCL1 is a key chemoattractant for neutrophils and non-hematopoietic cells, mediating local tissue inflammation at injection sites.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e RANTES is a chemokine that promotes leukocyte recruitment at later stages of inflammation and serves as a surrogate marker of systemic immune activation.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAs expected, saRNA vaccination induced rapid and strong systemic cytokine responses, with CXCL1 and RANTES markers sharply elevated at 6 hours and persisting at 24 hours post-injection.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e CXCL1 levels reached up to 16x of the 100 pg/mL TBS baseline at 6 hrs and remained at 7x at 24 hrs, while RANTES levels continued ramping up until the 24 hrs mark (3.5x at 6hrs and 8x at 24 hrs). In contrast, mice boosted with alum-adjuvanted RBD protein exhibited a more transient inflammatory signature. IL-6 levels rose substantially at 6 hours - up to 22x (15 \u0026micro;g RBD-protein per injection) or 7x (3 \u0026micro;g RBD-protein per injection) higher serum concentration compared to the typical mouse serum baseline of 110 pg/ml - consistent with the expected short-term innate activation required for efficient antigen priming but declined to baseline levels by 24 hours. CXCL1 followed a similar kinetic pattern across all vaccine formulations, though residual elevation at 24 hours was highest in the saRNA/LNP group (formulated using the commercially available ionizable lipid DLin-MC3-DMA). RANTES levels remained low or undetectable in protein-boosted mice at 6h and 24 h measurements, but were markedly elevated for the RNA immunizations, particularly for LNP immunization, indicating stronger systemic leukocyte recruitment following RNA administration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHeterologous RNA-protein vaccination maintains long-term immunity and extends protection to new variants in hamsters\u003c/h2\u003e\u003cp\u003eTo confirm the protective potential of the heterologous RNA-protein vaccination strategy in a second species and to assess long-term immune durability, we conducted a prime-boost-boost refresh study in Syrian golden hamsters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Animals received either homologous or heterologous prime-boost regimens using saRNA encoding the SARS-CoV-2 Beta spike or alum-adjuvanted RBD proteins derived from the Beta and Delta variants (\u0026#120573;/\u0026#120575;) either in a high-dose (RNA: 9 \u0026micro;g, protein 15 \u0026micro;g) or a low-dose (RNA: 3 \u0026micro;g, protein 5 \u0026micro;g). A subsequent second booster administered 78 days after the first boost consisted of a Delta/Omicron (\u0026#120575;/o) RBD mixture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), enabling both evaluation of booster longevity and introduction of a new, antigenically distinct RBD to test cross-variant adaptability.\u003c/p\u003e\u003cp\u003eAs observed previously in mice, RNA priming generated substantially higher initial titers than protein priming at all dose levels (Day 21). The most pronounced titer increase after the first boost (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D, Day 42) occurred in the RBD/RBD protein-protein regimen (up to 25-fold enhancement), followed by RNA/RNA (7 to 17-fold) and the RNA/RBD heterologous regimens (2\u0026ndash;6-fold), with similar trends across high- and low-dose groups. Anti-RBD protein antibody levels were found to be over one order of magnitude lower for the Omicron variant across all groups and dose levels, with a non-detectable prime response for the RBD-prime groups. Notably, despite lower early post-boost titers, the heterologous RNA/RBD group continued to exhibit rising antibody levels during the 57-day rest period (Days 42\u0026ndash;99), whereas titers in both homologous groups declined by roughly one order of magnitude.\u003c/p\u003e\u003cp\u003eFollowing the 78-day rest, all animals received a single second boost on Day 99 with the bivalent Beta/Omicron RBD formulation. By Day 120, antibody titers had risen sharply across all pre-immunized groups, confirming preserved immune memory and effective recall. The largest enhancement (up to 65-fold) occurred in animals that had previously received heterologous RNA/RBD regimens, whereas homologous RNA/RNA animals achieved a more modest up to 7-fold rise. Across all groups, responses were strongest against the Wuhan RBD, followed by Delta, and lowest against Omicron, yet detectable titers against Omicron confirmed successful recognition of this antigen despite its extensive immune-evasive mutations. Importantly, this demonstrates that the heterologous regimen remains adaptable-capable of being refocused toward new variant antigens introduced at later time points.\u003c/p\u003e\u003cp\u003eAll animals survived the three immunizations until scheduled necropsy on Day 121 without adverse clinical signs, body-weight loss, or injection-site reactions, supporting the safety of repeated dosing and of the alum-formulated RBD boosters in this model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe emergence of three highly pathogenic coronaviruses within two decades - SARS-CoV, MERS-CoV, and SARS-CoV-2 - underscores both the inevitability of future zoonotic spillovers and the limitations of reactive vaccine development.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e Current vaccine platforms, though transformative during the COVID-19 pandemic, remain largely optimized for homologous boosting against a single pathogen. There is an ongoing need for vaccination strategies that combine the potency of RNA delivery, the breadth of multivalent antigen design, and the safety and scalability of protein subunit technologies.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn this study, we explored such a strategy by combining RNA priming followed by a multivalent alum-adjuvanted protein boost. Across two species and multiple antigen combinations, the heterologous regimen consistently enhanced immunization quality in three terms compared with homologous RNA-RNA or protein-protein schedules: i) recall efficiency, ii) antigenic breadth, and iii) durability. First, RNA priming enabled potent antibody recall even at sub-microgram protein doses in mice (robust secondary responses at 0.6 RBD protein), and recall titers plateaued between 3 and 15 \u0026micro;g, suggesting that memory B-cell availability rather than antigen dose limits the booster response.\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e Second, the multivalent protein component broadened recognition across SARS-CoV-2 variants, and inclusion of MERS-CoV RBD extended cross-lineage reactivity beyond sarbecoviruses without compromising SARS-CoV-2 responses. Third, in hamsters, the heterologous boost led to sustained titers after a 78-day rest interval and potentiated responses to a second booster immunization that included a newly introduced strain (omicron).\u003c/p\u003e\u003cp\u003eThe results are consistent with a model in which RNA priming establishes a high-quality memory foundation via germinal center induction and T follicular helper (Tfh) cell engagement\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e that is efficiently re-engaged by a protein recall, even at low antigen dose, to produce large secondary responses. The observation that 3 \u0026micro;g and 15 \u0026micro;g protein boosts achieved similar titers implies a recall plateau, congruent with limited memory B cell clonal expansion rather than simple antigen-limited priming. Similar recall saturation has been observed in heterologous vector-protein regimens for COVID-19.\u003csup\u003e59\u0026ndash;60\u003c/sup\u003e The multivalent RBD booster appeared to preferentially expand cross-reactive lineages that recognize conserved RBD epitopes,\u003csup\u003e61\u0026ndash;62\u003c/sup\u003e while preserving potent homologous responses, a pattern previously reported in mixed-antigen nanoparticle vaccines.\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eTwo findings deserve emphasis with respect to the role of MERS RBD protein and cross-lineage breadth. First, MERS RBD protein on alum formulations was intrinsically immunogenic even without RNA priming, yielding stable antibody levels that declined little over time. Second, adding MERS RBD did not erode SARS-CoV-2 responses, indicating no detrimental immunodominance in this setting. This suggests that antigens from distinct β-coronavirus clades of sufficient sequence divergence can be co-delivered to expand immune breadth beyond sarbecoviruses. Prior studies have similarly shown that heterologous or mosaic RBD vaccines can redirect immune recognition toward conserved protein surfaces.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e While alum adsorption chemistry likely influences the presentation efficiency of individual RBDs,\u003csup\u003e65\u003c/sup\u003e the current results indicate that MERS inclusion is both feasible and beneficial for broad coronavirus coverage.\u003c/p\u003e\u003cp\u003eThe biomarker analysis (IL-6, CXCL1, CCL5/RANTES) indicates qualitatively different innate kinetics for the RNA and alum-adjuvanted protein vaccines tested. Self-amplifying RNA vaccination elicited systemic chemokine elevations (notably CXCL1 and CCL5) to 24 h, hallmarks of LNP-mediated innate sensing and type-I interferon signaling,\u003csup\u003e66\u0026ndash;67\u003c/sup\u003e whereas protein/alum boosters induced a short-lived innate burst. IL-6 peaked at 6 h post-immunization and resolved by 24 h, a pattern typical of transient inflammasome-linked alum activation.\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e CXCL1 and CCL5 remained close to baseline in the protein groups, supporting a more localized, depot-like activation profile. This shorter inflammatory signature complements the booster\u0026rsquo;s role: sufficient to trigger a recall response without prolonged systemic inflammation. Together, these data highlight the tolerability and reactogenicity advantages of the RNA-protein sequence, particularly relevant for populations sensitive to reactogenicity and repeat dosing or large-scale immunization programs.\u003c/p\u003e\u003cp\u003eThe Syrian golden hamster model confirmed the immunogenic trends observed in mice, while demonstrating that the heterologous regimen preserved and could strengthen antibody responses over an extended interval. Following a long, 78-day rest after the first boost, antibody titers in heterologous cohorts were maintained or increased, while those in homologous groups declined compared to the peak measured 3 weeks post-boost, in some cases by nearly an order of magnitude. The second, delayed booster at day 99, which contained a newly introduced β/Omicron RBD formulation, induced a strong recall across all groups, with the largest fold-increases in heterologous cohorts. Despite the known immune evasiveness of Omicron, detectable anti-Omicron titers were achieved after the second boost. These findings emphasize the adaptability of the heterologous approach, and mirror clinical observations that heterologous booster regimens produce broader and more sustained humoral immunity than homologous ones.\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e They also support the idea that heterologous priming enhances the longevity and flexibility of memory B-cell responses, allowing efficient re-focusing towards novel emerging antigens without rebuilding the regimen from scratch.\u003c/p\u003e\u003cp\u003eFrom a translational standpoint, the heterologous RNA-protein strategy leverages complementary technological strengths: the speed and potency of RNA priming for rapid, potent priming and the safety, stability, and affordability of protein boosting. RNA vaccines can be rapidly updated and induce potent primary immunity, while alum-adjuvanted protein boosters are compatible with existing cold-chain logistics and regulatory experience.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e The pronounced dose-sparing effect observed with a robust recall when boosting even at 3 \u0026micro;g has direct manufacturing implications, potentially lowering costs and extending vaccine supply. Alum\u0026rsquo;s established regulatory track record and broad availability facilitate deployment at scale, including in settings where MERS remains a regional threat. The ability to add or swap RBDs in the booster makes the platform variant-responsive without disrupting established immunity and highlights the adaptability of this regimen to evolving pandemic threats.\u003c/p\u003e\n\u003ch3\u003eLimitations and outlook\u003c/h3\u003e\n\u003cp\u003eThe study is preclinical, and neutralization assays were limited to surrogate formats; future work should employ pseudo- or live-virus neutralization across representative SARS-CoV-2 and MERS strains. Comprehensive profiling of CD4⁺, Tfh, and CD8⁺ responses is also warranted to define cellular contributions to the observed durability. Optimization of alum formulation, particularly adsorption efficiency and antigen spacing, may further refine cross-clade breadth. Commercial mRNA vaccines incorporate N-1-methylpseudouridine modification, which may exhibit different booster effects than saRNA; it will be valuable to study the impact of the modified mRNA modality on protein-based heterologous immunization. Nonetheless, the data provide a strong preclinical rationale for clinical exploration of heterologous RNA-protein regimens as boosters in previously RNA-primed human populations targeting a heterologous, multivalent, and adaptable strategy toward broader coronavirus preparedness.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe heterologous RNA-prime/multivalent-protein-boost approach described here delivered (i) high-magnitude recall with clear dose sparing of protein, (ii) broadened reactivity across SARS-CoV-2 variants with extension to MERS, and (iii) durable, recall-responsive immunity across a long inter-boost interval, including successful retargeting to Omicron RBD. By combining the rapid immunogenicity of RNA priming with the durable, broad recall of alum-adjuvanted multivalent RBD proteins, this strategy achieves strong and adaptable protection in two animal models. Protein/alum boosts produced a short-lived innate signature that resolved by 24 h. The regimen shows favorable tolerability, clear dose-sparing potential, and the ability to integrate new antigens without compromising prior immunity. Together, these features provide a practical, immunologically grounded framework and point to a scalable path forward toward broadly protective, pandemic-responsive coronavirus vaccines.\u003c/p\u003e"},{"header":"METHODS","content":"\u003ch2\u003eDesign and synthesis of saRNA\u003c/h2\u003e\u003cp\u003ePlasmid DNA encoding the SEAP or SARS-CoV-2/MERS-CoV proteins were cloned using standard molecular biological methods, using fragments encoding the protein ORFs from Thermo Fisher Scientific (Waltham, MA). A poly(A) tract of ~ 100 bp was installed at the end of the protein reading frame followed by a BspQI restriction site. The VEEV replicon encodes the complete VEEV genome minus the subgenomic ORF (replaced by the above sequences), and is available in the Supplementary Information. For RNA synthesis, plasmids were linearized by digestion with BspQI (New England Biolabs, Ipswich, MA), and purified using E.Z.N.A. Cycle Pure Kits (Omega Biotek, Norcross, GA). Linearized plasmid resuspended in RNase-free H\u003csub\u003e2\u003c/sub\u003eO was used as a template for \u003cem\u003ein vitro\u003c/em\u003e transcription using T7 enzyme and subsequent 5’ end capping as described previously\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Translational fidelity was confirmed by immunoblot or by SEAP measurement by luminescent assay using the Phospha-Light reporter gene assay system (Thermo Fisher Scientific, Waltham, MA).\u003c/p\u003e\u003ch2\u003eRNA Formulation\u003c/h2\u003e\u003cp\u003ePurified saRNAs were formulated with modified poly(amidoamine)-based dendrimer nanoparticles (MDNPs) from Tiba Biotech’s RNABL™ platform. The dendrimers carried surface tertiary amines and biodegradable ester linkages, enabling efficient electrostatic complexation and endosomal release. RNA payloads for formulation were prepared first by resuspension of the capped RNA in citrate buffer (Teknova, Hollister, CA) to approximately 150 mg/L. The dendrimer/lipid phase for mixing with the RNA solution was prepared in 100% ethanol, at a volume 3x that of the aqueous RNA phase. This organic phase contained a proprietary blend of excipient lipids and the candidate dendrimer compound at a final concentration of approximately 3.5–5.5 mg of total dendrimer + lipid mass per ml. The aqueous and ethanol phases were combined by in-line mixing using the NanoAssemblr preclinical microfluidics platform (Vancouver, Canada). The resulting turbid nanoparticle suspension was dialyzed extensively at room temperature against PBS to remove residual ethanol and sterilized by filtration across a 0.2 µm membrane (Pall Corporation, New York, NY). All RNA vaccines were diluted in PBS before injection.\u003c/p\u003e\u003ch2\u003eCharacterization of Nanoparticles (NPs)\u003c/h2\u003e\u003cp\u003eThe size and polydispersity index(PDI)of all RNA nanoparticle test articles were determined using ZetaSizer Ultra (Malvern Panalytical, Malvern, UK) in triplicate. The mRNA encapsulation efficiency was analyzed using the Quant-iT RiboGreen RNA kit (Thermo Fisher Scientific, MA, USA) as described previously. Briefly, RNA-LNPs were lysed with 0.5% Triton-X or left untreated, followed by treatment with RiboGreen reagent following the manufacturer’s instructions. The quantity of RNA in the samples was measured using a microplate reader (Spark®, TECAN, Mannedorf, Switzerland). The calculated encapsulation efficiency of RNA was \u0026gt; 80%.\u003c/p\u003e\u003ch2\u003eRBD Plasmid construction, expression, and purification\u003c/h2\u003e\u003cp\u003ePlasmid DNA encoding SARS-CoV-2 or MERS-CoV proteins were synthesized and cloned by Proteogenix, Schiltigheim, France or Twist Bioscience. SARS-CoV-2 or MERS-CoV proteins were either produced using transient plasmid transfection of a serum-free by Proteogenix, Schiltigheim, France using transient plasmid transfection of a serum-free suspension of Chinese hamster ovary cells (XtenCHO) cells or in-house as previously described in \u003cem\u003eE. coli\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e–\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e In brief, the selected RBD protein sequences were codon optimized for expression XtenCHO and synthesized by artificial gene synthesis and cloned into the pTAX1 plasmid for expression in mammalian cells. Purified plasmids from the respective antigen expression cassettes was transfected into the XtenCHO cell line. The cells were monitored for cell viability, cell density and cultured for up to 13 days (312 hours) while the protein antigens were secreted into the culture media. The Spike RBD antigens were purified through ion exchange chromatography (IEX) followed by affinity chromatography using @RBD antibodies developed by ProteoGenix. The research grade antigen was then buffer exchanged into the formulation buffer. The identity of expressed proteins was confirmed by Western blot. Purity of recombinant protein produced by Proteogenix was higher than 90%. Purity evaluation was made on SDS-PAGE gel using the GelAnalyzer software by Proteogenix.\u003c/p\u003e\u003ch2\u003eHost cell protein and residual host cell DNA analysis\u003c/h2\u003e\u003cp\u003eThe host cell protein was determined via a CHO HCP ELISA Kit (CYG-F550-1, Cygnus Technologies), and the residual host cell DNA was analyzed by a CHO Host Cell DNA Kit (CYG-D550T/ CYG-D555T, Cygnus Technologies) in triplicates and as per manufactures instructions and recommendations.\u003c/p\u003e\u003ch2\u003eSurface plasmon resonance analysis\u003c/h2\u003e\u003cp\u003eRecombinant SARS-CoV-2 S proteins (His-tagged) were purchased from Acro Biosystems Inc. (Newark, USA). The purity of recombinant proteins was documented by SDS-PAGE analysis. SPR measurements were performed on a Biacore 3000 instrument (GE Healthcare) by NBS-C BioScience, Vienna Austria as previously described.\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003ch2\u003eRBD protein formulation\u003c/h2\u003e\u003cp\u003eThe recombinant RBD proteins described above were formulated with 2.2% aluminum hydroxide gel (Alum) adjuvant. Briefly, the required dosage RBD antigen in pH 7.4 saline buffer was mixed with 70 µg/dose (corresponding to the max. approved Al\u003csup\u003e3+\u003c/sup\u003e-content of 850 µg per dose) Alhydrogel (InvivoGen) and incubated under gentle shaking for 30 min at room-temperature to allow adsorption, followed by storage at 2–8°C until use. All protein/alum formulations were diluted in sterile saline before use in the non-clinical studies.\u003c/p\u003e\u003ch2\u003eMouse experiments and approvals\u003c/h2\u003e\u003cp\u003e Female C57BL/6J (B6) and or BALB/c mice were purchased from the Jackson Laboratory and maintained under specific pathogen-free conditions at the University of Minnesota according to the Institutional Animal Care and Use Committee guidelines (IACUC). All B6 mice used in the experiments were female and 6 to 9 weeks of age at the time point of the first immunization. For inflammatory response testing, BALB/c mice (6 to 10 weeks of age at the time of experimentation) were purchased from the Jackson Laboratory and maintained according to Tiba Biotech’s IACUC guidelines.\u003c/p\u003e\u003cp\u003eC57BL/6 or BALB/c mice were immunized by bilateral intramuscular (i.m.) injection into the quadriceps muscles using insulin syringes. Each dose consisted of 50 µL per leg (total 100 µL). For prime-boost regimens, a second injection was administered on day 21 unless otherwise indicated. Control animals received formulation buffer or alum alone. Serum samples were collected from the submandibular vein at baseline and at indicated post-immunization time points (typically days 21, 35, 49, 99). Antibody titers against SARS-CoV-2 (Wuhan-Hu-1, Beta, Delta, and Omicron RBDs) and MERS-CoV RBD were determined by enzyme-linked immunosorbent assay (ELISA).\u003c/p\u003e\u003cp\u003e To assess local and systemic inflammatory responses following vaccination, serum samples were collected at 6 h and 24 h post-immunization from representative animals in each treatment group. Concentrations of IL-6, CXCL1 (KC/GROα), and CCL5 (RANTES) were quantified using multiplex bead-based immunoassays (Luminex®) according to the manufacturer’s instructions. These markers were selected as representative indicators of acute-phase cytokine induction, neutrophil-associated chemotaxis, and leukocyte recruitment, respectively. Data were analyzed as fold-change over pre-immune baseline, TBS-buffer groups serving as controls to distinguish adjuvant-driven from RNA-induced responses.\u003c/p\u003e\u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\u003cp\u003eEndpoint antibody titers against SARS-CoV-2 and MERS-CoV RBDs were quantified by direct ELISA following optimized protocols. High-binding 96-well plates (Greiner Bio-One #655061) were coated overnight at 4°C with 1 µg/mL of recombinant RBD protein (Beta lot #15195-101921-C01; Delta lot #15195-101921-B01; MERS lot #15195-100921-D01; ProteoGenix) in PBS (pH 7.4). Plates were washed with Phosphat Buffer Saline – Tween (PBST, VWR Cat# RLMB-075-1000) and blocked for 1 h at room temperature with 0.5% BSA in PBS. Eleven serial three-fold serum dilutions (1:25 − 1:1,462,250) were prepared in PBS/0.5% BSA and added to the plates for 1 h at RT. Following washing, goat anti-mouse IgG (HRP-conjugated; Invitrogen #31430, 1:20 000 dilution) was applied for 45 min at RT. After final washes, plates were developed with TMB substrate (BD #555214) for 2.5 min and stopped with 0.5 M H₂SO₄. Absorbance was read at 450 nm with 570 nm background correction using a Tecan Infinite M Plex. A sigmoidal 4P curve-fit model was applied to the absorbance readings, and endpoint titers were defined as the OD₄₅₀ value of that curve exceeding the mean of negative controls by three standard deviations. Data analysis was performed using GraphPad Prism (v10.0). Antibody titers and surrogate neutralization values were log₁₀-transformed prior to analysis, and group averages were expressed as geometric mean titers (GMT) with 95% confidence intervals.\u003c/p\u003e\u003ch2\u003eHamster experiments and approvals\u003c/h2\u003e\u003cp\u003e24 Male and 36 female Syrian Golden hamsters were purchased by Attentive Science for this study and maintained under specific pathogen-free conditions. All animals were group housed by sex and dose group in clean solid bottom cages in environmentally controlled housing. The cages contained appropriate bedding material, which were changed at least once weekly. Animals were 4–7 weeks old and had a bodyweight between 120 and 293 g of at the start of the study.\u003c/p\u003e\u003cp\u003eMale and female Syrian golden hamsters (\u003cem\u003eMesocricetus auratus\u003c/em\u003e; 7–10 weeks of age; n = 5 per group) were immunized by intramuscular injection into the hind limb muscles. A prime-boost schedule was employed with injections on day 1 and day 21, followed by a delayed second boost at day 99 in selected groups. Control animals received formulation buffer only. Blood samples were collected at designated time points for serological analyses. Antibody titers against SARS-CoV-2 (Wuhan-Hu-1, Beta, Delta, and Omicron RBDs) and MERS-CoV RBD were quantified by ELISA (Attentive Science Study Nos. 1121–3495 and 1121–4898).\u003c/p\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e All experimental protocols with animals were conducted in strict accordance with international ethical standards for animal experimentation (Helsinki Declaration and its amendments, Amsterdam Protocol of welfare and animal protection and National Institutes of Health, NIH USA, guidelines). All procedures were approved by the respective Institutional Animal Care and Use Committee (IACUC).\u003c/p\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eAll calculations and statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, CA). Data in bars diagrams are shown as mean ± SD and differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ram Karan and Sam Mathew for providing help in the initial experiments.\u003c/p\u003e\n\u003cp\u003eThe research reported in this publication was supported by the King Abdullah University of Science and Technology (KAUST); Office of Sponsored Research, OSR-NTGC-2021-4587.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: C.W.M., J.E., J.S.C., M.R.\u003c/p\u003e\n\u003cp\u003eMethodology: D.R., J.S.M., F.K., C.W.M., J.E., J.S.C., M.R.\u003c/p\u003e\n\u003cp\u003eInvestigation: D.R., J.S.M., F.K., C.W.M., J.E., J.S.C., M.R.\u003c/p\u003e\n\u003cp\u003eData curation: D.R., J.S.M., S.B., P.T., J.E.\u003c/p\u003e\n\u003cp\u003eFormal analysis and visualization: D.R., J.S.M., J.E., J.S.C.\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: D.R., J.E., M.R.\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: All authors\u003c/p\u003e\n\u003cp\u003eFunding acquisition: C.W.M., J.E., J.S.C., M.R.\u003c/p\u003e\n\u003cp\u003eResources: C.W.M., J.E., J.S.C., M.R.\u003c/p\u003e\n\u003cp\u003eProject administration: D.R., C.W.M., J.E., J.S.C., M.R.\u003c/p\u003e\n\u003cp\u003eSupervision: C.W.M., J.E., J.S.C., M.R.\u003c/p\u003e\n\u003cp\u003eAll authors contributed to study design, data interpretation, and manuscript preparation; approved the final version; and agree to be accountable for all aspects of the work in ensuring its accuracy and integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.T., J.S.M., C.W.M., and J.S.C. are employees of Tiba Biotech LLC and have equity interests. Tiba Biotech has filed a patent on the MDNP RNA delivery system (patent application PCT/US21/25542), naming P.T. and J.S.C. as inventors. J.S.C. is also an inventor on U.S. Patent no. 10,548,959 entitled \u0026ldquo;Compositions and Methods for Modified Dendrimer Nanoparticle Delivery.\u0026rdquo; C.W.M. has worked as a freelance consultant advising commercial and nonprofit organizations working in the fields of vaccines and viral vectors. All other authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e accompanies this paper and provides detailed descriptions of vaccine construct design, replicon architecture, and RBD sequence selection. It includes full RNA and protein antigen sequences, in vitro expression characterization, and experimental procedures for splenocyte restimulation and cytokine assays. Figure S1 summarizes vaccine antigen design and domain boundaries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Christian W. Mandl, J\u0026ouml;rg Eppinger, Jasdave S. Chahal, or Magnus Rueping.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCankat, S.; Demael, M. U.; Swadling, L., In search of a pan-coronavirus vaccine: next-generation vaccine design and immune mechanisms. \u003cem\u003eCellular \u0026amp; Molecular Immunology\u003c/em\u003e 2024, \u003cem\u003e21\u003c/em\u003e (2), 103\u0026ndash;118.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBruno, L. A.; Pueblas Castro, C.; Demar\u0026iacute;a, A.; Prado, L.; Fascetto Cassero, C. G.; Saposnik, L. M.; P\u0026aacute;ez C\u0026oacute;rdoba, F.; Rodriguez, J. M.; Piccini, G.; Antonelli, R.; Lapini, G.; Temperton, N.; Del Priore, S. A.; Hernando Insua, A. C.; Kaufmann, I. G.; Vega, J. 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R., Expression and purification of SARS coronavirus proteins using SUMO-fusions. \u003cem\u003eProtein Expr Purif\u003c/em\u003e 2005, \u003cem\u003e42\u003c/em\u003e (1), 100\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, J.; Miao, L.; Li, J. M.; Li, Y. Y.; Zhu, Q. Y.; Zhou, C. L.; Fang, H. Q.; Chen, H. P., Receptor-binding domain of SARS-Cov spike protein: soluble expression in E. coli, purification and functional characterization. \u003cem\u003eWorld J Gastroenterol\u003c/em\u003e 2005, \u003cem\u003e11\u003c/em\u003e (39), 6159\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonteil, V.; Eaton, B.; Postnikova, E.; Murphy, M.; Braunsfeld, B.; Crozier, I.; Kricek, F.; Niederh\u0026ouml;fer, J.; Schwarzb\u0026ouml;ck, A.; Breid, H.; Devignot, S.; Klingstr\u0026ouml;m, J.; Th\u0026aring;lin, C.; Kellner, M. J.; Christ, W.; Havervall, S.; Mereiter, S.; Knapp, S.; Sanchez Jimenez, A.; Bugajska-Schretter, A.; Dohnal, A.; Ruf, C.; Gugenberger, R.; Hagelkruys, A.; Montserrat, N.; Kozieradzki, I.; Hasan Ali, O.; Stadlmann, J.; Holbrook, M. R.; Schmaljohn, C.; Oostenbrink, C.; Shoemaker, R. H.; Mirazimi, A.; Wirnsberger, G.; Penninger, J. M., Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants. \u003cem\u003eEMBO Molecular Medicine\u003c/em\u003e 2022, \u003cem\u003e14\u003c/em\u003e (8), e15230.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"COVID-19 vaccine, MERS-CoV, SARS-CoV-2, Heterologous prime-boost, Pan-coronavirus, Protein subunit vaccine, saRNA vaccine, RNA vaccine","lastPublishedDoi":"10.21203/rs.3.rs-7875217/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7875217/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe continuing emergence of SARS- and MERS-related coronaviruses underscores the urgent need for pan-SARBECo vaccines capable of eliciting broad and durable protection across divergent lineages.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e We present a heterologous prime-boost vaccination strategy combining a modified dendrimer nanoparticle (DNP)-encapsulated self-amplifying (saRNA) prime with an alum-adjuvanted multivalent protein booster containing receptor-binding domains (RBDs) from SARS-CoV-2 (Wuhan-Hu-1 and B.1.351) and MERS-CoV. This approach leverages the potent immunogenicity of RNA priming together with the breadth and safety of protein subunit boosting\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e to expand coronavirus coverage. In preclinical mouse and hamster models, the heterologous RNA-protein regimen elicited robust antibody responses with markedly enhanced magnitude, durability, and cross-variant neutralization compared with homologous RNA or protein vaccination alone. Inclusion of the MERS-CoV RBD in the booster broadened the response without compromising SARS-CoV-2 immunity. These findings establish a versatile and scalable vaccination strategy with potential to inform the development of next-generation, broadly protective vaccines against emerging coronaviruses.\u003c/p\u003e","manuscriptTitle":"Heterologous saRNA Prime – Multivalent Protein Boost Strategy Induces Broad and Durable Immunity Against SARS-CoV-2 and MERS-CoV","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 05:27:53","doi":"10.21203/rs.3.rs-7875217/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71e0534a-010d-4720-b945-b569d2214682","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56395890,"name":"Biological sciences/Biotechnology"},{"id":56395891,"name":"Biological sciences/Immunology"},{"id":56395892,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-12-22T12:41:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 05:27:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7875217","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7875217","identity":"rs-7875217","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}