A stabilized MERS-CoV spike ferritin nanoparticle vaccine elicits robust and protective neutralizing antibody responses

Domain: neuroscience
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a human pathogen in 2012 and causes ongoing sporadic infections and outbreak clusters. Despite case fatality rates (CFRs) of over 30% and considerable pandemic potential, a safe and efficacious vaccine has not been developed. Here we report the design, characterization, and preclinical evaluation of MERS-CoV antigens. Our lead candidate comprises a stabilized spike displayed on a self-assembling ferritin nanoparticle that can be produced from a high-expressing, stable cell pool. This vaccine elicits robust MERS-CoV pseudovirus and authentic virus neutralizing antibody titers in BALB/c mice. Immunization of male non-human primates (NHPs) with one dose of Alhydrogel-adjuvanted vaccine elicited a >103 geometric mean titer of pseudovirus neutralizing antibodies that was boosted with a second dose. Sera from these NHPs exhibited cross-reactivity against spike-pseudotyped lentiviruses from MERS-CoV clades A, B, and C as well as a distant pangolin merbecovirus. In human DPP4 transgenic mice, immunization provided dose-dependent protection against MERS-CoV lethal challenge, and in an established alpaca challenge model using female alpacas, immunization fully protected against MERS-CoV infection. This MERS-CoV nanoparticle vaccine is a promising candidate for clinical advancement to protect at-risk individuals and for future use in a potential outbreak setting.

Introduction
The emergence of three human pathogenic betacoronaviruses since 2002 has demonstrated the substantial, continuing threat that coronaviruses pose to the human population. MERS-CoV, the causative agent of Middle East respiratory syndrome, was first identified in 2012 and has since caused 2627 laboratory-confirmed cases and 947 deaths (36% CFR)1. MERS-CoV likely originated in bats and is transmitted from its intermediate host, dromedary camels, to humans2–5. In some instances, MERS-CoV undergoes human-to-human transmission, most notably in healthcare settings, and continues to be an ongoing threat as highlighted by nine recent cases in 2025, two of which were fatal, in the Kingdom of Saudi Arabia1,6. There are currently no prophylactic vaccines or therapeutic interventions approved for use against MERS. The ongoing morbidity and mortality associated with MERS, the high prevalence of MERS-CoV in dromedary camel populations, and the potential for increased human-to-human transmission indicate a pressing need for a safe and effective MERS vaccine. The availability of such a vaccine would be useful as a routine prophylactic immunization for at-risk individuals such as healthcare workers, camel industry workers, and travelers to endemic regions. The vaccination of those at highest risk of MERS, due to occupation or travel, could mitigate the risk of a future pandemic and would provide proof-of-principle for deployment of this vaccine for broad use if it became needed.

MERS-CoV is a single-stranded RNA virus which displays a single surface protein, spike, responsible for host-cell recognition and viral entry7–9. MERS-CoV spike is a heavily glycosylated trimeric class I viral fusion protein that is expressed as a single polypeptide and cleaved via host cell proteases into S1 and S2 subunits, which remain non-covalently assembled following proteolysis8. The MERS-CoV spike receptor binding domain (RBD) in the S1 subunit recognizes dipeptidyl peptidase 4 (DPP4)10. Following binding, the virus is endocytosed, at which time a secondary proteolytic cleavage event occurs at the S2’ site within the S2 subunit to allow viral membrane fusion to proceed8. Fusion can also occur at the viral membrane via TMPRSS2-mediated S2’ cleavage if the virus has been precleaved at the S1/S2 site11.

Antibodies targeting the spike protein following MERS-CoV infection demonstrate neutralizing activity12–15. Thus, the spike protein has been an important target for experimental MERS vaccine candidates16–18. Several vaccine platforms, including viral-based, subunit, and DNA have been evaluated in preclinical studies, some of which elicited MERS-CoV neutralizing antibodies and protected from viral challenge in preclinical models19. A few MERS-CoV vaccine candidates have been tested in early-stage clinical trials; however, no candidates have progressed to late-stage clinical development20–24. Therefore, advancing additional candidates towards clinical evaluation is critical.

Similar to other class I viral fusion proteins, the MERS-CoV spike trimer is a metastable protein poised to convert from the prefusion to the postfusion state upon receptor binding and cell entry8. Prior work has demonstrated that betacoronavirus spike proteins can be stabilized by mutating two key residues between the first heptad repeat (HR1) and second heptad repeat (HR2) to proline25,26. Installation of these two, as well as an additional four proline mutations in the SARS-CoV-2 spike, increased protein expression and led to enhanced immunogenicity, presumably as a result of improved stability of the prefusion conformation26,27. We therefore based our MERS-CoV antigen design on a stabilized spike containing a mutated S1/S2 cleavage site and 3 proline mutations (Fig. 1A). Structural studies of the MERS-CoV spike indicate that the region at the base of the trimer prior to the transmembrane domain (residues 1230-1294) is likely flexible, as it is unresolved via cryo-EM (Fig. 1B)28. We thus hypothesized, based in part on our prior betacoronavirus antigen design29, that removal of this flexible region could facilitate stabilized presentation of the spike in a conformation that may be more conducive to eliciting neutralizing antibodies. Though this region of the S2 domain can be the target of neutralizing antibodies30, there is also evidence that it contains linear epitopes that elicit non-neutralizing antibodies in the context of SARS-CoV-231–33, and we reasoned that it could be in MERS-CoV spike as well, further supporting its removal.

Protein subunit vaccines have been shown in several contexts to have favorable stability profiles and to elicit robust, durable protection in humans34–36. In the absence of potent adjuvants, one limitation of protein subunit vaccines is weak immunogenicity compared to viral-based vaccines35. To overcome this limitation, proteins can be multimerized on nanoparticle platforms to confer benefits including facilitation of B cell cross-linking and enhanced neutralizing antibody responses29,35,37–41. However, recent efforts to develop an nanoparticle-based vaccine displaying the MERS-CoV RBD or spike elicited similar neutralizing antibody responses after two doses relative to the prefusion-stabilized spike18, illustrating an ongoing need to identify optimal platforms and critical design attributes tailored for each specific antigen.

Ferritin is a ubiquitous, naturally occurring, self-assembling protein that forms 24-subunit particles42. It contains a 3-fold axis of symmetry and is therefore an ideal platform for display of trimeric viral antigens like the MERS-CoV spike protein37. We and others have shown that displaying the SARS-CoV-2 spike protein on the surface of H. pylori ferritin presents the antigen in a favorable conformation to elicit robust and cross-reactive neutralizing antibody responses29,43–46. Therefore, we applied these same antigen design principles to the MERS-CoV spike protein to develop a vaccine against MERS. Notably, ferritin nanoparticles have been demonstrated to be an effective and safe antigen multimerization platform in preclinical and clinical studies43,47,48, but to our knowledge have not advanced past Ph2 human trials. This is likely due in part to challenges in scaling their manufacturing and purification. As a step in overcoming this hurdle, here we produce a high-expressing stable CHO cell pool, develop scalable purification methods, and perform forced degradation and accelerated stability studies on our lead vaccine candidate. These data provide unique insight informing the identification of critical quality attributes and methods necessary for scalability and process development for highly complex protein vaccine antigens.

The results presented here comprise the design, biochemical characterization, stability profile, and immunogenicity assessment of MERS-CoV spike vaccine candidates. We demonstrate that our lead candidate, MERS-1227 ferritin nanoparticle (FNP), can be purified to homogeneity and retains antigenicity and nanoparticle structural integrity following heat stress at 37 °C for 14 days. We show that MERS-1227 FNP is robustly immunogenic in BALB/c mice, NHPs, and alpacas as determined by MERS-CoV authentic virus and pseudotyped lentiviral neutralization assays. In transgenic BALB/c mice expressing human DPP4, we observed dose-dependent protection from MERS-CoV lethal challenge following immunization with our lead MERS-CoV vaccine candidate. In an alpaca challenge model, we observed no detectable viral infection in alpacas immunized with Alhydrogel-adjuvanted MERS-CoV nanoparticles whereas 80% of the placebo-treated alpacas shed infectious virus following challenge. The robust stability, immunogenicity, and efficacy profile of this protein-based nanoparticle vaccine demonstrate optimal attributes for clinical development as a safe and efficacious vaccine against MERS.

Results
Design of two ferritin-based MERS-CoV spike vaccine candidates with varied ectodomain lengths
The MERS-CoV spike is a 1353 amino acid polypeptide that is cleaved into S1 and S2 subunits via proteolysis at residue 751 (Fig. 1A)8. The ectodomain of MERS-CoV spike extends to residue 1294. We hypothesized that truncation of the ectodomain would facilitate more optimal presentation of the spike when displayed on the surface of a ferritin nanoparticle, potentially improving stability and immunogenicity. To determine the optimal truncation position for the spike ectodomain, we designed two constructs based on the spike sequence from MERS-CoV clade B England 1 strain28. These constructs extended through either residue 1227 (MERS-1227) or 1236 (MERS-1236) to optimally align the distance between the base of the protomers to the N-terminal residue of the ferritin 3-fold axis (Fig. 1C). Following the ectodomain in both constructs, we included an SGG linker and residues 5-167 from H. pylori ferritin (Fig. 1A) with an N19Q mutation in the ferritin sequence to eliminate a potential N-linked glycosylation site37.

Additionally, we introduced three proline mutations guided by known SARS-CoV-2 2P and 6P variations that stabilize the spike protein25–27. We also mutated the S1/S2 furin cleavage site from RSVR to ASVG to prevent spike cleavage, as described in prior stabilized MERS-CoV constructs25.

MERS-1227 and MERS-1236 can be purified to homogeneity from high-expressing CHO cell pools and form stable nanoparticles with expected antigenicity
To express and characterize large quantities of MERS-1227 and MERS-1236 FNPs, we generated stable, high-expressing cell pools at ATUM utilizing their proprietary Leap-In™ transposase technology and CHOK1-derived mi:CHO-GS™ host cells49. The cell line development process yielded two stable cell pools with protein titers of approximately 2.7 (MERS-1227) and 3.2 (MERS-1236) grams per liter of cell culture in fed-batch supernatant harvested at day 14 as determined by SDS-PAGE quantitation (Fig. S1A). We purified the nanoparticles at a research-level scale to a high degree of homogeneity using anion exchange chromatography in flow-through mode, dialysis, and size-exclusion chromatography as reported previously for a related ferritin nanoparticle vaccine candidate45. Given the simplicity of this process, we utilized materials purified in this manner for all subsequent analyses and immunizations described here. Additionally, we optimized a manufacturing process-applicable method for purification, which involved two bind and elute purification steps using an anion exchange membrane and a hydrophobic interaction chromatography (HIC) resin (Fig. S1B). Both steps result in enrichment of the MERS-CoV FNP, demonstrating the feasibility of future at-scale process development of these complex macromolecules.

We characterized the purity of MERS-1227 and MERS-1236 FNPs from the research-scale purification using SDS-PAGE analysis (Fig. 2A) and observed a pure species for both particles that migrates at the expected molecular weight of a MERS-CoV spike ferritin monomer (~150 kDa). Both proteins exhibited a slight decrease in migration when analyzed under reducing conditions, likely due to disruption of intra-protomer disulfide bonds. We further analyzed the purity and the size of the purified FNPs using size-exclusion chromatography multi-angle light scattering (SEC-MALS). This analysis revealed highly homogenous particles, without notable high or low molecular weight species, and MALS-calculated molecular weights of 4.27 ± 0.05 MDa (MERS-1227) and 4.23 ± 0.09 MDa (MERS-1236) (Fig. 2B). These observed molecular weights are slightly larger than those calculated from the MERS-1227 and MERS-1236 primary amino acid sequences (3.6 and 3.7 MDa, respectively). We hypothesized that this difference was due to glycosylation of the MERS-CoV spike, which has previously been characterized15,50–52. To evaluate glycosylation of our MERS-CoV FNP proteins, we performed mass spectrometry-based peptide mapping to identify specific glycan modifications and to estimate their relative abundances at each glycosite (Supplementary Data 1). We observed extensive N-glycosylation and limited but detectable O-glycosylation throughout the MERS-1227 and MERS-1236 spikes but not within the ferritin nanoparticle core (Fig. S2), consistent with prior analyses of purified MERS-CoV spike proteins50–52.

Additional size characterization via dynamic light scattering (DLS) showed a diameter of 38.1 nm for MERS-1227 and 39.1 nm for MERS-1236 (Fig. 2C), consistent with the expected 10 nm diameter of an H. pylori ferritin nanoparticle with two ~14 nm MERS-CoV spikes projecting radially8. Interestingly, differential scanning fluorimetry (DSF) revealed distinct melting temperatures of 55.4 °C for MERS-1227 and 52.9 °C for MERS-1236, with MERS-1236 also exhibiting a broader melting profile (Fig. 2D). This result suggests that the 9 additional amino acids at the C-terminus of the ectodomain of MERS-1236 influence protein stability. However, the most striking difference between MERS-1227 and MERS-1236 was observed by negative stain transmission electron microscopy (TEM) (Fig. 2E), which showed that MERS-1236 spikes appear to exhibit modulated spike occupancy compared to MERS-1227. Collectively, these data indicate that MERS-1227 is a more stable functionalized nanoparticle, projecting the MERS-CoV spike protein radially from the ferritin core. Based on comparison of MERS-1227 and MERS-1236 via TEM and DSF, we elected to prioritize preclinical characterization of MERS-1227 and included MERS-1236 as a comparator when necessary. The structural and stability advantages of MERS-1227 suggested that it may exhibit fewer liabilities as a candidate for vaccine development.

To evaluate the antigenicity of MERS-1227 and MERS-1236, we utilized a panel of previously characterized monoclonal antibodies (mAbs) that bind to distinct epitopes across the MERS-CoV spike and used biolayer interferometry (BLI) to measure their binding to the nanoparticles. Within this panel, we included 11 S1-targeting antibodies13,53–56, 8 known to bind the RBD and 3 known to bind outside of the RBD, and 1 S2-targeting antibody57 (Fig. 2F and S3, sequences provided in Supplementary Data 2). We observed that the antibodies targeting epitopes in the S1 domain outside of the RBD bind MERS-1227 and MERS-1236 equivalently, whereas RBD-directed antibodies exhibit enhanced binding to MERS-1236. Given that antibodies binding to S1 epitopes outside of the RBD (G2, FIB-H1, and CDC2-A2) bind to MERS-1227 and MERS-1236 equivalently, this suggests that non-RBD epitopes, such as those on the NTD, are presented on both proteins in the same manner. Regarding RBD-targeting epitopes, we hypothesize that the increased binding of RBD antibodies to MERS-1236 is related to the higher degree of flexibility of the spike on the surface of the nanoparticle, as evidenced by our TEM results. This flexibility may impact the degree to which RBDs are displayed in the “up” or “down” conformation on MERS-1236 compared to MERS-1227, which could impact binding of antibodies that target RBD epitopes, or the enhanced flexibility could increase accessibility to IgGs. Alternatively, increased trimer flexibility in MERS-1236 could allow for antibody bridging between adjacent trimers on the same nanoparticle which may not occur on MERS-1227. Furthermore, the S2-directed antibody, 3A3, binds at the trimer interface which is only accessible in an open conformation of SARS-CoV-2 spike that the trimer reversibly samples58. The increased binding of MERS-1236 to 3A3 is therefore consistent with a more flexible trimer and may suggest MERS-1236 samples an “open trimer” conformation more frequently. We confirmed binding specificity by demonstrating that MERS-1227 and MERS-1236 do not bind to a SARS-CoV-2 specific antibody, casirivimab.

MERS-1227 and MERS-1236 are stable over a range of pH and temperature conditions
Low-pH treatment is commonly employed for viral inactivation during downstream process development, and knowledge of pH tolerance is helpful in optimizing chromatography steps. Therefore, we sought to understand the effect of low pH on MERS-1227 and MERS-1236 nanoparticle stability and antigenicity. We formulated purified MERS-1227 and MERS-1236 in pH 7.5 formulation buffer and held them in the pH-adjusted solutions at pH 2, 3, 3.5, and 4 for 2 h. Then, the nanoparticles were dialyzed back into pH 7.5 buffer and evaluated using analytical SEC, DLS, DSF, and BLI (Fig. 3A and S4A-B). These analyses revealed that pH 2 treatment causes irreversible damage to both MERS-1227 and MERS-1236 as observed by all biophysical measurements, suggesting dissociation of the nanoparticle and/or denaturation of the MERS-CoV spike. BLI revealed that binding of an RBD mAb (CDC-C2) and an NTD mAb (G2) is retained following pH 2 treatment, which may suggest that antigenicity of the isolated protomer is retained to some degree following nanoparticle dissociation. Treatments at pH 3, 3.5, and 4 do not notably affect particle structure or antigenicity.

Temperature stability is important in vaccine development with the goal of optimizing a modality and formulation such that cold-chain requirements can be minimized. This is especially relevant to a MERS vaccine, as the endemic regions where it would be distributed regularly experience high ambient temperatures. To understand the thermostability of MERS-1227 and MERS-1236, we formulated purified materials in formulation buffer and stored them at 4, 22, or 37 °C for 14 days. Following these temperature holds, we evaluated the quality and stability of the FNPs using analytical SEC, DLS, DSF, and BLI (Fig. 3B and S4C-D). We confirmed that DLS, DSF, and BLI binding to these mAbs are appropriate metrics of protein integrity, as incubation for 30 min at 90 °C notably perturbed particle size (DLS) and fully ablated the melting curve (DSF) and binding to both mAbs (BLI) for both FNPs (Fig. 3B). By SEC analysis, we observed that both MERS-1227 and MERS-1236 remained stably associated as nanoparticles even after 14 days at 37 °C, albeit with a minor fraction of particles forming an aggregate species as evidenced by a peak at ~7.0 min that elutes prior to the nanoparticle peak at ~8.0 min. DLS showed no major change in the size of particles following heat-treatment, and DSF revealed no change in the primary melting peak following the temperature hold conditions, providing evidence for the conformational stability of the particles. We also found that both MERS-1227 and MERS-1236 retain antigenicity against an RBD-specific (CDC-C2) and an NTD-specific (G2) monoclonal antibody after 14 days at either 22 or 37 °C (Fig. 3B).

MERS-1227 and MERS-1236 are more immunogenic compared to trimer alone in BALB/c mice following one or two doses
We sought to determine the effect of spike multimerization on elicitation of neutralizing antibodies by immunizing BALB/c mice (n = 5 or 10) with MERS-1227, MERS-1236, or the MERS-CoV full-length spike (S) ectodomain trimer (residues 1-1294, containing the 3 proline mutations and mutated furin site used in MERS-1227 and MERS-1236) with a GCN4 trimerization domain59 as a comparator. We immunized the antigens at a total protein dose of 2 or 10 µg with 150 µg Alhydrogel (Fig. 4A) and collected serum at day 21 (following a single dose) and day 42 (following two doses). As shown in Fig. 4B, after a single dose, mice immunized with either 2 or 10 µg of MERS-1227 or MERS-1236 FNPs elicited ~2- to 3-fold higher levels of both anti-spike and anti-RBD IgG antibodies as compared to mice immunized with MERS-CoV S trimer. Following a second dose, mice across the three antigen groups at all doses exhibited similar levels of MERS-CoV spike and RBD IgG (Fig. 4B, right panels).

We quantified neutralizing antibody titers using a luciferase-based assay60 with lentivirus pseudotyped with MERS-CoV spike and HeLa cells stably expressing human DPP4 as the targets of infection. We validated our assay using MERS-CoV specific neutralizing antibodies with known IC50 values13,55. The values obtained in our assay concorded well with those reported in the literature (Fig. S5A). As expected, no neutralization was detected with a negative control SARS-CoV-2 specific mAb, casirivimab. We also used convalescent serum from a camel challenged with MERS-CoV with a known PRNT90 of 1:320 serum dilution as a benchmark for our pseudovirus neutralizing titers (Fig. S5B)61. In our pseudovirus assay, this camel serum sample inhibits 90% of viral entry (IC90) at a serum dilution of 1:6810. This result indicates that our lentivirus-based pseudovirus assay exhibits higher sensitivity than a plaque-reduction neutralization assay using authentic MERS-CoV virus, as reported for other lentivirus-based neutralization assays compared to authentic viral comparators62.

Following a single dose, we observed robust pseudovirus neutralizing titers of GMT ~103 for MERS-1227 and MERS-1236 at all doses tested. Comparatively, MERS-CoV trimer elicited a lower and more heterogeneous antibody response at 2 µg and a more homogenous but still overall lower response at 10 µg. After a second dose, we observed a boost in titers across all antigens and dose groups. Notably, the MERS-CoV S trimer groups show a higher degree of variability, especially at the 10-µg dose level, with a geometric standard deviation of 2.6 as compared to 1.3 and 1.4 for MERS-1227 and MERS-1236, respectively (Fig. 4C, right panel). We additionally tested pools of serum from the MERS-1227 and MERS-1236 groups and confirmed they neutralized authentic MERS-CoV in a plaque-reduction neutralization assay (Table S1). This initial mouse immunogenicity study revealed that the multimerized, stabilized MERS-CoV spike elicited a stronger neutralizing antibody response following one dose and a more homogeneous response following two doses compared to soluble trimer. Subsequently, we performed an additional dose de-escalation study with Alhydrogel-adjuvanted MERS-1227 and found that it elicited a neutralizing antibody response at 0.016 µg, the lowest dose tested, following a prime and a boost (Fig. S6).

MERS-1227 and MERS-1236 elicit robust neutralizing antibody responses in NHPs following a single dose that increase and persist at least several months following a second dose
To evaluate MERS-1227 and MERS-1236 immunogenicity in an NHP model, we immunized cynomolgus macaques (n = 5 per antigen) with 50 µg FNP (total protein dose) adjuvanted with 750 µg Alhydrogel and measured the magnitude, durability, and breadth of the antibody response following one and two doses (Fig. 5 and Table S2). Following a single immunization, we observed pseudovirus neutralizing antibody titers at or greater than reciprocal serum dilution (NT50) of 103 for all animals in both vaccine groups (Fig. 5B). Importantly, we did not observe notable waning of these titers over the three-month period following the prime vaccination. We immunized the NHPs with a homologous boost at study day 93 and observed a robust increase of greater than 10-fold in pseudovirus neutralizing titers at study day 100 that continued to increase to study day 127. At study day 253 (160 days post-boost), titers remained higher than those observed following a single dose. Importantly, the rate of decay observed between days 180 and 253 was decreased compared to that observed between days 127 and 180, which is suggestive of the potential for a durable response as has been reported for other alum-adjuvanted protein-based vaccines63.

To further characterize the response, we used a Luminex panel containing MERS-CoV spike, RBD, and NTD, and evaluated total IgG levels against these antigens. We observed similar spike and RBD IgG levels, with slightly more waning of the spike titers observed between study days 188 and 253. Interestingly, spike and RBD binding titers decrease less quickly than pseudovirus neutralizing titers (Fig. 5, compare B to C). To understand how antibody responses in NHPs compare to those elicited in the context of human infection with MERS-CoV, we used VSV pseudotyped with MERS-CoV EMC/2012 S to evaluate neutralizing antibody titers from NHPs at study days 93 and 127 side-by-side with a panel of MERS patient serum samples. As shown in Fig. 5D (full neutralization curves shown in Fig. S7A), following a single dose (at 93 days), GMTs were approximately 2-fold higher than the convalescent samples (NT50 255 compared to 130 serum dilution) which improve to over 100-fold higher after two doses (NT50 14,058 compared to 130 serum dilution).

To characterize the breadth of the antibody response to known MERS-CoV strains, we established a panel of MERS-CoV spike-pseudotyped lentiviruses comprising clades A, B (currently circulating in humans and the vaccine strain in this study), C, and a distantly related pangolin merbecovirus, MjHKU4r-CoV-164,65. We observed robust neutralization of all MERS-CoV clades in the panel (Fig. 5E), including clade C strains which are known to circulate in camels but not humans64. Remarkably, we also observed neutralization of MjHKU4r-CoV-1, which only shares a 66% amino acid sequence identity with the spike of England 1 MERS-CoV, albeit at reduced potency relative to other strains tested65. Importantly, we did not observe neutralization of the MjHKU4r-CoV-1 pseudotyped lentivirus with pre-immune NHP serum (Fig. S7B). Pseudovirus neutralization, breadth, and total IgG levels were similar between MERS-1227 and MERS-1236 immunized NHPs. Overall, the results of this study demonstrate that adjuvanted MERS-1227 and MERS-1236 elicit a robust, durable, and broad response in NHPs that can be boosted following a second dose administered at a 3-month interval, which is consistent with other effective vaccine regimens66.

To understand cellular immune responses to MERS-1227 and MERS-1236, we collected peripheral blood mononuclear cells (PBMCs) from vaccinated NHPs at study days 0, 21, 93, and 107 (Fig. 5A). We conducted ELISpot analysis to assess Th1 and Th2 (interferon gamma (IFNγ) and IL-4, respectively) responses to both MERS-CoV spike trimer and H. pylori ferritin (Fig. 5F and S7C). IFNγ and IL-4 responses to both spike and ferritin are primed and can be boosted following one and two doses of vaccine. Interestingly, spike and ferritin responses are similar between NHPs immunized with MERS-1227 and MERS-1236 following a single dose, but NHPs vaccinated with MERS-1227 exhibit enhanced IFNγ to both spike and ferritin following two doses. IL-4 responses are similar across the two vaccination groups at all timepoints tested. After boosting, the ratio of the IFNγ to the IL-4 response for both MERS-CoV spike and ferritin is below 1 for MERS-1236 vaccinated animals implying a Th2-biased response, and near 1 in MERS-1227 vaccinated animals, suggesting balanced Th1/Th2 immunity. Of note, preclinical studies with SARS1 vaccine candidates have previously demonstrated that immunization inducing a Th2 dominated response resulted in lung immunopathology following viral challenge67. Therefore, while the response observed in NHPs was robust, the potential for a Th2-biased response to MERS-1236 indicates that evaluation of other adjuvants may be key to balancing high neutralizing antibody responses while reducing potential for Th2-induced immunopathology.

Vaccination with MERS-1227 or MERS-1236 protects from viral shedding in alpacas
Alpacas have been established as a surrogate infection model for camels, the predominant intermediate animal host of MERS-CoV. Unlike camels, alpacas do not develop rhinorrhoea (nasal discharge) upon MERS-CoV infection, but they do shed infectious virus that is detectable in nasal turbinates61,68. We immunized alpacas (n = 5 per group) at two dose levels (20 or 200 µg) of MERS-1227 or MERS-1236 adjuvanted with 500 µg Alhydrogel using a prime-boost regimen (Fig. 6A and Table S2). As a placebo control, an additional group of alpacas (n = 5) were dosed with 500 µg Alhydrogel at the same time points. Three weeks following the boost, (study day 41), all vaccinated alpacas had detectable pseudovirus neutralizing titers in their sera (Fig. 6B). Furthermore, all alpacas vaccinated at both dose levels of MERS-1227 and the 200-µg dose of MERS-1236 had detectable neutralizing titers in an authentic virus assay (Fig. 6C).

On study day 42, alpacas were challenged by intranasal inoculation with live MERS-CoV virus (clade A, EMC/2012) in a BSL3 containment facility, as previously reported68. On days 1-5 and 7 post-challenge, nasal swabs were collected from each nare, and authentic virus neutralization titers were determined using a plaque assay68. Detectable levels of MERS-CoV were observed via nasal swab for at least one time point following challenge in 4/5 alpacas that received the Alhydrogel placebo, while authentic virus was not detected in any (0/20) of the alpacas that received either dose level of Alhydrogel-adjuvanted MERS-1227 or MERS-1236 (Fig. 6D). Taken together, these results demonstrate that immunization with Alhydrogel-adjuvanted MERS-1227 and MERS-1236 elicited protection from infection with authentic MERS-CoV.

At this stage of the preclinical development process, we elected to move forward with further characterization of one antigen. While the immunogenicity results in BALB/c mice, NHPs, and alpacas appeared similar between MERS-1227 and MERS-1236, both the decreased melting temperature and increased structural flexibility of MERS-1236 suggested to us that these could be important liabilities during vaccine manufacturing or long-term stability studies. For these reasons, we conducted further evaluations with MERS-1227.

The immune response to MERS-1227 can be tuned using different adjuvant combinations
While alum is the most widely used vaccine adjuvant with a long clinical history and can be readily procured for clinical development programs, alternative adjuvants have potential to enhance the immunogenicity and durability of protein subunit vaccines69. We therefore formulated MERS-1227 with a panel of adjuvants known to engage distinct immune pathways69, including CpG (ODN2395), Quil-A + monophosphoryl lipid A (MPLA), AddaVax, and 3M-052 and probed the resulting immunogenicity in mice. Based on prior preclinical and clinical use of these adjuvants, we evaluated some co-formulated with Alhydrogel (Quil-A+MPLA, CpG, and 3M-052) as well as some without Alhydrogel (AddaVax, Quil-A+MPLA, and 3M-052). We formulated MERS-1227 with the various adjuvants and immunized BALB/c mice with a 0.4 µg dose of FNP protein followed by a homologous boost at day 21 (Fig. 7A).

An adjuvant was required to elicit a robust response, as unadjuvanted MERS-1227 hardly elicited detectable pseudovirus neutralization titers compared to all three doses of Alhydrogel tested (Fig. 7A). Titration of Alhydrogel in the absence of other adjuvants indicated that in mice, the dose response is maximal between 15 and 50 µg. Immunization with MERS-1227 formulated with AddaVax (similar to MF5970 in clinically approved vaccines) did not improve titers compared to the mid- and high-dose Alhydrogel groups following a single dose but did show a ~3-4 fold enhancement in titers following the boost.

The most striking improvement in immunogenicity was observed with Quil-A+MPLA (in the absence of Alhydrogel), which showed a 4-fold enhancement in titers over the 150 µg Alhydrogel group after one dose and a 22-fold enhancement after the second dose. While addition of Quil-A+MPLA to Alhydrogel improved responses compared to the matched 15 µg and 150 µg Alhydrogel-only groups, the strongest response to Quil-A+MPLA was observed in the absence of Alhydrogel. Addition of CpG (ODN2395) only conferred an immunogenicity improvement compared to Alhydrogel alone at the 15 µg Alhydrogel dose, with minimal difference between the 150 µg Alhydrogel +/- CpG groups at both time points. Interestingly, addition of 3M-052, a lipidated TLR7/8 agonist71, did not result in increased neutralizing titers compared to the Alhydrogel only dose groups after a single dose but did show an improved response following a boost, most notably in the presence of either 15 or 150 µg Alhydrogel.

To evaluate the influence of adjuvants on the ratio of Th1/Th2 responses following immunization with MERS-1227, we bound biotinylated MERS-CoV spike trimer to streptavidin Luminex beads and quantified the levels of mouse IgG2a and IgG1 (Fig. S8). In mice, induction of IgG1 is associated with a Th2-biased response and IgG2a is associated with a Th1-biased response72. Mice immunized with MERS-1227 adjuvanted with either 15, 50, or 150 µg Alhydrogel alone elicited an IgG response strongly dominated by IgG1, indicative of a Th2 response, which is well-documented for alum adjuvants73. AddaVax was observed to have a slightly decreased Th2-skewing as compared to Alhydrogel alone. Importantly, we observed that co-formulation of Alhydrogel with either Quil-A+MPLA or 3M-052 generated a Th1 bias characterized by a higher IgG2a/IgG1 ratio in these groups compared to Alhydrogel alone. CpG was observed to also overcome the Alhydrogel-induced Th2 skew at the 15 µg Alhydrogel dose, but not at the 150 µg dose. Taken together, the results of this adjuvant screen demonstrate that modification of the adjuvant formulation of MERS-1227 could be used to enhance the resultant antibody responses and potentially modulate the cellular immune response.

MERS-1227 elicits a comparable response when administered as an adjuvanted protein nanoparticle or an mRNA-encoded ferritin nanoparticle
While protein vaccines exhibit advantages relating to thermostability and lower cost-of-goods, mRNA technology is advancing to meet these challenges and can be produced and modified quickly if needed to respond to a novel or mutated pathogen74. For this reason, we evaluated immunization using mRNAs packaged in SM102-containing lipid nanoparticles (LNPs)75 encoding either MERS-1227 ferritin, a cell-anchored form of the MERS-CoV spike containing a deletion of the ectodomain from residue 1228-1294, or a full-length MERS-CoV spike with no ectodomain deletion (Fig. 7B). All constructs contained 3P stabilizing mutations, a mutated furin cleavage site, and a 21-residue deletion at the C-terminus of the protein to remove the cytoplasmic tail. We evaluated the biophysical parameters of the mRNA/LNPs including encapsulation efficiency, size, and zeta potential (Fig. S9A) as well as their in vitro cellular potency. To assess cellular potency, we treated HeLa cells with the mRNA/LNP complexes at a series of doses and then stained cells with RBD (JC57-14) or NTD (G2) specific antibodies (Fig. S9B). We compared cell-surface expression (no fixation or permeabilization) with total cellular expression (fixation and permeabilization). As expected, the two cell-anchored constructs (MERS-1227 cell-anchored and MERS-FL cell-anchored) are detected in both cell treatment conditions whereas the MERS-1227 ferritin is only detected when cells are fixed and permeabilized.

We immunized naïve BALB/c mice with either 0.1 µg or 1 µg mRNA on day 0 and day 21 and collected serum at days 21 (prime only) and 42 (prime + boost) (Fig. 7B). As a comparator, we included the MERS-1227 FNP protein +/- 150 µg Alhydrogel. Following a single dose, both the Alhydrogel-adjuvanted MERS-1227 FNP protein and MERS-1227 FNP encoded on mRNA (1 µg dose) elicited similar neutralizing antibody responses to each other and that were higher than those of the two cell-anchored forms of the MERS-CoV spike. After boosting, the 1 µg mRNA groups all exhibited similar levels of pseudovirus neutralizing titers, which were comparable to the adjuvanted MERS-1227 protein nanoparticle. Interestingly, the 0.1 µg mRNA dose groups elicited minimal responses after both doses, and the only responders at this dose level were seen in the MERS-1227 ferritin and the MERS-1227 cell-anchored mRNA groups. This suggests that truncation of the ectodomain to remove the flexible region between 1228 and 1294 conferred a benefit in neutralizing response in the mRNA format, which could provide important insight into the design of other mRNA antigens for vaccine delivery. Furthermore, these results show the versatility of the 3P stabilized spike we have designed to be administered as a protein nanoparticle, an mRNA-encoded protein nanoparticle, and as a cell-anchored stabilized spike.

Immunization with MERS-1227 provides dose-dependent protection in hDPP4 BALB/c mice
To evaluate efficacy of MERS-1227 in a small animal model in which we could observe protection from severe disease, we utilized a BALB/c mouse model in which the human DPP4 receptor is expressed in the nasal turbinates, trachea, lungs, and kidney76. We immunized hDPP4 transgenic mice (n = 8 per group) with 0, 0.016, or 0.4 µg MERS-1227 formulated with 150 µg Alhydrogel at days 0 and 21 and collected serum at days 21 and 42 (Fig. 8A and Table S3). MERS-CoV pseudovirus and live-virus neutralization titers (Fig. 8B and C) showed dose-dependence, and pseudovirus neutralizing titers were consistent with our evaluation in wild-type BALB/c mice (Fig. S6).

Three weeks following the booster dose, mice were challenged via intranasal inoculation with 10,000 PFU of the EMC/2012 strain of MERS-CoV. Four mice from each group were sacrificed 3 days post-challenge and live-virus titers of MERS-CoV were quantified from the lungs of these animals using plaque assays (Fig. 8D and Table S3). Three mice in the placebo group had viral titers (≥ 0.9 x 103 PFU/g lung) in the lung as compared to two mice in the 0.016 µg group and no mice in the 0.4 µg group, demonstrating protection from viral replication in the lungs following MERS-1227 vaccination. The remaining four mice in each group were monitored for weight loss (Fig. S10) and survival (Fig. 8E) until day 10 post-challenge. All mice in the placebo group succumbed to challenge by day 9, whereas 1/4 mice in the 0.016 µg group and 3/4 mice in the 0.4 µg groups survived to day 10. Of note, the mouse that died in the 0.4 µg group prior to day 10 was found to be pregnant during necropsy. All mice were necropsied following sacrifice and evaluated for histopathological changes in the lung; minimal changes were observed in both placebo and vaccinated mice as determined by lung scoring metrics (Table S3). Preliminarily, these results suggest that the Alhydrogel-adjuvanted MERS-1227 did not result in vaccine-induced enhanced respiratory disease and lung inflammation following viral challenge. However, future studies involving evaluation of inflammatory infiltrates and characterization of eosinophil and neutrophil content would be necessary to more fully rule out the potential for Th2-type hypersensitivity induced by vaccination prior to challenge67.

Taken together, the lung viral titer and survival results post viral challenge indicate that immunization with Alhydrogel-adjuvanted MERS-1227 was protective against lethal MERS-CoV challenge in hDPP4 mice in a dose-dependent manner. This provides strong evidence that MERS-1227 is protective and is an important demonstration that clinical development of this stabilized MERS-CoV spike ferritin vaccine could be utilized as a prophylactic countermeasure against MERS.

Methods
Construct design and sequences
An England 1 clade B sequence of MERS-CoV spike protein derived from AFY13307.128 was truncated to reflect a similar C-terminal deletion as previously investigated for a SARS-CoV-2 spike29,45 and was stabilized using proline substitutions at residues 889, 1060, and 106125. The S1/S2 furin cleavage site was mutated from the native RSVR to ASVG25. Given the lack of structural resolution at the C-terminal portion of the MERS-CoV spike ectodomain, two truncation positions were tested, one at residue 1227 and one at 1236. An SGG linker was encoded following the truncation site prior to a modified H. pylori ferritin as previously described29. The terminal 3 residues in MERS-1227 encode a potential glycosylation site (NST). An N1225G mutation was made to remove this glycosylation site, as it was hypothesized that a glycan at the base of the trimer immediately preceding the ferritin component could impede nanoparticle formation.

MERS-1227 and MERS-1236 stable CHO cell pool generation, fed-batch culture production, and purification
Stable CHO cell pools were generated as previously described49. Briefly, codon optimized coding sequences were synthesized and cloned into Leap-In™ transposon-based expression constructs. The plasmids were co-transfected with Leap-In™ transposase mRNA into miCHO-GS host cells (ATUM) and the stable pools were established by glutamine-free selection. For fed-batch productions, cells were seeded at 0.75 x 106 per mL in shake flasks at either 25 or 100 mL scale grown at 37 °C with 5% CO2. Growth temperature was adjusted to 30 °C on day 4 and cells were supplemented throughout the fed-batch production using proprietary feed solutions. Cells were harvested on day 14 by spinning at 7000 x g for 10–20 min. Supernatants were filtered using a 0.22 µm filter.

For research-scale purifications, supernatants were diluted with 20 mM Tris pH 8.0, 1 M sodium chloride to a final concentration of 200 mM sodium chloride (not accounting for sodium chloride in the cell culture media). Diluted supernatants were then passed over a Cytiva 5 mL HiTrap Q anion exchange column equilibrated in 20 mM Tris pH 8.0 using an AKTA fast protein liquid chromatography (FPLC) system run with UNICORN software (version 7.8.0.2159). MERS-CoV ferritin nanoparticles were collected from the column flow-through and then dialyzed overnight at 4 °C using cellulose ester 1 MDa molecular weight cutoff dialysis tubing (Spectrum) into 20 mM Tris, pH 7.5, 150 mM sodium chloride. Dialyzed material was concentrated to a final volume of ~0.5-2 mL using an Amicon spin concentrator (100 kDa molecular weight cutoff). Concentrated samples were then filtered using a 0.22-µm filter and injected onto a Cytiva Superose 6 10/300 GL (PN29091596) or Sepax 10x300 mm SRT SEC-1000 (particle size 5 µm, pore size 1000 Å, PN 215950-10030) size-exclusion chromatography column equilibrated in 20 mM Tris pH 7.5, 150 mM sodium chloride using an AKTA FPLC. Protein-containing fractions were assessed by SDS-PAGE analysis, and fractions containing homogenous protein were pooled. Fractions were supplemented with 5% sucrose as a cryoprotectant, frozen in liquid nitrogen, and stored at –80 °C until use. The formulation buffer used throughout the described studies was 20 mM Tris pH 7.5, 150 mM sodium chloride, 5% sucrose.

Alternatively, for bind and elute purifications (Fig. S1B), MERS-1227 14-day fed-batch supernatant was treated with Pierce Universal Nuclease and then diluted 5-fold into 20 mM Tris pH 7.5. Diluted supernatant was injected onto a 1 mL Sartobind Q Nano (Sartorius No. 96IEXQ42DN-11--A) equilibrated in 20 mM Tris-Cl pH 7.5 at 5 mL per min. FNP was eluted using a step elution with 10 column volumes of 14, 30, and 100% 20 mM Tris-Cl pH 7.5, 1 M sodium chloride. FNP-containing fractions were pooled, brought to 1.6 M ammonium sulfate, and loaded onto a 1 mL Cytiva HiTrap Hydrophobic Interaction Capto butyl column equilibrated in 1.8 M ammonium sulfate in 50 mM HEPES pH 7.5. The protein was eluted with a gradient of 50 mM HEPES pH 7.5.

SDS-PAGE
Denaturing SDS-PAGE analysis was performed by diluting proteins with LDS sample buffer (containing 2-mercaptoethanol for reducing conditions) and heating samples for 5 min at 95 °C. Samples were then loaded on a 4-20% Mini PROTEAN TGX precast gel and run for 30 min at 230 V. Gels were stained using GelCode Blue staining reagent and imaged using a Thermo Fisher Scientific imager (iBright CL750 1.8.0).

Analytical size-exclusion chromatography multi-angle light scattering
Purified MERS-1227 and MERS-1236 were diluted to 0.125–0.5 mg/mL in 20 mM Tris, pH 7.5, 150 mM sodium chloride, 5% sucrose. For each run, 5–10 µg protein was loaded onto a 4.6 x 300 mm SRT SEC-1000 analytical column (particle size 5 µm, pore size 1000 Å, PN 215950-4630) pre-equilibrated in 20 mM Tris pH 7.5, 150 mM sodium chloride run at 0.35 mL/min on a Waters Arc Premier high-pressure liquid chromatography system run with Empower software (version 7.3.0). Multi-angle light scattering was measured using a Wyatt DAWN and refractive index was measured using a Wyatt Optilab run with ASTRA software (version 8.3.0.132). Molecular weight calculations were performed using Wyatt ASTRA software (version 8.3.0.132) by utilizing the light scattering and refractive index signals from each sample.

Sample preparation for LC-MS/MS peptide mapping
MERS-1227 and -1236 FNP proteins were proteolytically digested using S-Trap columns (Protifi, C02-micro-10) following the manufacturer protocol with minor modifications. All digestions were performed in triplicate. For each replicate, 25 µg protein in formulation buffer was added to an equal volume of 2x lysis buffer (5% SDS, 100 mM Tris, pH 8.5). Proteins were reduced in 5 mM dithiothreitol (DTT) for 30 min at 55 °C and alkylated in 20 mM iodoacetamide for 30 min in the dark at room temperature. Phosphoric acid was added to a final concentration of 2.5% (v/v). Acidified samples were diluted in 10 volumes of 100 mM Tris, pH 7.55 in 90% methanol/10% water and applied to S-Trap columns by centrifugation at 4000 x g. The column was washed in the same buffer. 2.5 µg of trypsin (Promega, #V5113) diluted in 20 µL 50 mM ammonium bicarbonate, pH 8 was applied to the column and allowed to sit overnight at 37 °C. Peptides were eluted in 40 µL ammonium bicarbonate pH 8, followed by 40 µL 0.2% formic acid in water, and finally 40 µL 50% acetonitrile in water. Eluted peptides were dried on a SpeedVac and resuspended in 30 µL 0.1% formic acid in water prior to analysis.

LC-MS/MS peptide mapping method
Peptides were analyzed by LC-MS/MS using a Vanquish Horizon LC directly interfaced to an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific) run with Xcalibur software (version 4.7). 6 µL of resuspended peptides were separated by reversed-phase LC on a Accucore Vanquish C18+ (100 mm length x 2.1 mm ID, 1.5 µm particle size) at 0.3 mL/min using a linear gradient of 5–30% mobile phase B (0.1% formic acid in acetonitrile) over 25 min followed by a ramp to 90% mobile phase B over 3 min, where mobile phase A was 0.1% formic acid in water. Positive electrospray ionization was achieved with a spray voltage of 3500 V, a sheath gas flow rate of 40 arbitrary units, auxiliary gas heated to 250 °C at a flow rate of 15 arbitrary units, and a sweep gas flow rate of 1 arbitrary unit. The ion tube transfer temperature was held at 320 °C and the RF lens was set to 70. MS analysis was carried out in a Top 10 data-dependent acquisition mode. Full MS scans were acquired at a resolution of 60,000 (FWHM @ m/z 200) with an automatic gain control target of “standard”, m/z range of 350–1800, and maximum ion accumulation time of “auto”. Precursor ions with charges 2–6 and a minimum intensity threshold of 1 x 104 were selected with a quadrupole isolation window of 2.0 m/z for HCD fragmentation with a normalized collision energy (NCE) of 27%. MS/MS scans at a resolution of 15,000 (FWHM @ m/z 200) were acquired with an automatic gain control of “standard”, a maximum ion accumulation time of 150 ms, and a fixed first mass of 120 m/z. Precursors selected for fragmentation more than 2 times within a 15 second window were dynamically excluded from additional fragmentation for 10 seconds.

LC-MS/MS peptide mapping data analysis
To identify post-translational modifications of MERS-1227 and -1236 ferritin nanoparticles, raw mass spectrometry data were analyzed using the GlycanFinder module of PEAKS Studio 11 (Bioinformatics Solutions, Inc.). Custom FASTA databases were generated comprising the Chinese hamster (Cricetulus griseus) proteome (downloaded on November 27, 2023), common mass spectrometry contaminant proteins84, and either the MERS-1227 or -1236 FNP protein sequence. The raw MS data were searched against the database with the following modifications: fixed carbamidomethylation on Cys, variable oxidation of Met, and variable deamidation of Gln and Asn, with a maximum of two variable modifications per peptide. Glycans were identified using the default N-linked and O-linked databases within PEAKS GlycanFinder. Semi-specific tryptic peptides with a maximum of two missed cleavages were considered. The allowed mass tolerances were 10 ppm for precursor ions, 0.04 Da for peptide productions, and 20 ppm for glycan diagnostic ions. Hits were filtered to a false discovery rate of 1% using the PEAKS decoy-fusion approach. For estimation of the relative quantities of the glycan compositions at each glycosite, the average integrated precursor peak areas for each detected glycan composition were calculated and reported as the percentage of the total glycan peak areas at a given site. Glycans with quantifiable peak areas in at least 2/3 of the experimental replicates were considered for relative quantitation. Data were analyzed and plotted using ggplot2 (version 3.5.0) and R (version 4.3.3).

Dynamic light scattering and differential scanning fluorimetry
Proteins were diluted to 0.125 - 0.5 mg/mL in formulation buffer (20 mM Tris pH 7.5, 150 mM sodium chloride, 5% sucrose) and loaded into glass capillaries (NanoTemper). Samples were then analyzed on a Nanotemper Prometheus Panta run with PR.Panta Control software (version 1.9) using DLS with a viscosity parameter of 1.149 mPa·s and a refractive index parameter of 1.341 to determine the cumulant radius of the particles and DSF to determine the melting temperature (Tm). Melting temperature runs were performed using a gradient of 1.0 °C per minute from 25 °C to 90 °C. Data was processed using PR.Panta Analysis software (version 1.9).

Transmission electron microscopy
For TEM analysis, purified MERS-1227 and MERS-1236 were diluted using 20 mM Tris pH 7.5, 150 mM sodium chloride. A 3.5 µL drop of diluted sample suspension was applied to a TEM grid overlaid with a 3-4 nm layer of amorphous C (CF200-CU-UL, Electron Microscopy Sciences). Prior to sample application, the carbon-coated grid was plasma-cleaned for 15 seconds via a lab-made device. The 3.5 µL of sample was allowed to incubate on the carbon surface for one minute. After blotting the sample away with filter paper, each grid was twice dipped quickly (~1 second) into separate drops of de-ionized water followed by blotting with filter paper. This double washing and blotting was then repeated with two drops of 1% ammonium molybdate. For all steps, the next dipping was performed before the grid could completely dry. Finally, each grid was dipped into a drop of 1% ammonium molybdate solution. After 15-20 seconds, the stain was blotted away with filter paper, and the grids were allowed to air-dry. To collect hundreds of images rapidly, specimens were imaged on a Thermo Fisher Titan Krios transmission electron microscope equipped with a Gatan Bioquantum K3 energy filter and direct electron detector. The microscope was operated at 300 kV and at liquid nitrogen temperatures, and the program SerialEM (version 4.2.4) was used to collect images85. Images were collected as multiple frames of the same field of view.

After imaging, micrographs were processed to give two-dimensional class averages via the RELION software package (version 5.0.0)86. Frames in the multi-framed images were aligned and summed via the MOTIONCOR2 algorithm87 implemented in RELION. Contract transfer function parameters were determined for each summed micrograph via CTFFIND488, via an interface in RELION. Several hundred particle images were selected manually and used to train the program Topaz (interfaced in RELION) to automatically pick thousands more particle images89. Extracted particle images were then aligned in two dimensions to give 2D class averages via the general Bayesian approach of RELION90.

Biolayer interferometry
Biolayer interferometry analysis of MERS-1227 and MERS-1236 was performed using an Octet R8 instrument run with Octet BLI Discovery software (version 13.0.1.19) with anti-hIgG Fc capture (AHC) biosensors. Antibodies were procured from GenScript with human Fc domains and stored in 1X TBS pH 7.4. VH and VL sequences are found in Supplementary Data 2. Antibodies were diluted to 50 µg/mL and MERS-CoV nanoparticles were diluted to 100 µg/mL in 1X Sartorius kinetics buffer (PBS + 0.1% BSA, 0.02% Tween-20, and an amicrobicide, Kathon) and pipetted into black-walled, black-bottom plates (200 µL per well). AHC tips were dipped into antibody wells and subsequently dipped into nanoparticle wells to assess binding association for 120 s. Dissociation was monitored for 120 s by moving tips into a well containing buffer alone. Data was analyzed using Octet Analysis Studio software (version 13.0.1.35).

pH and temperature stability treatment of MERS-CoV FNPs
To assess the pH stability of MERS-CoV FNP proteins, aliquots of the formulation buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5% sucrose) were adjusted to pHs of 2, 3, 3.5, and 4 (+/- 0.1 pH unit) by dropwise addition of hydrochloric acid. Slide-A-Lyzer MINI Dialysis Devices (15 mL conical format, Thermo Fisher Scientific Cat #88401) were briefly rinsed in these buffer solutions. 350 µL of MERS-1227 or MERS-1236 FNP proteins at 0.25 mg/mL in formulation buffer were added to each dialysis device and dialyzed against 14.5 mL of the pH-adjusted formulation buffers (or pH 7.5 as a control). Proteins were dialyzed at room temperature for 1 h with gentle agitation. After 1 h incubation, 1 µL dialyzed protein was used to confirm the target pH of the solution using pH paper. At this time, dialysis solution was discarded and replaced with a fresh 14.5 mL pH-adjusted formulation buffer and dialysis was allowed to proceed for an additional 1.5 h. Proteins were then dialyzed back to pH 7.5 by replacing dialysis solution with pH 7.5 formulation buffer followed by a 1 h incubation at room temperature. pH 7.5 formulation buffer was then replaced with fresh pH 7.5 buffer and dialysis was allowed to proceed overnight at 4 °C. Proteins were then removed from dialysis devices, transferred to 2 mL polypropylene tubes, snap frozen using liquid nitrogen, and stored at -80 °C until use. For analysis, all samples were thawed at the same time and diluted to 0.125 mg/mL with formulation buffer and evaluated using analytical SEC, DLS, DSF, and BLI as described above.

To assess the thermal stability of MERS-CoV FNP proteins, purified proteins were diluted to 0.5 mg/mL in 20 mM Tris, pH 7.5, 150 mM NaCl, 5% sucrose, and aliquoted in 2 mL polypropylene tubes. Samples were placed at either 4, 22 (ambient temperature), or 37 °C. Control samples were snap-frozen immediately following dilution to 0.5 mg/mL and 90 °C heat-treated controls were heated to 90 °C for 30 min and then snap-frozen and stored at -80 °C until analysis. Aliquots of 4, 22, and 37 °C were taken following 7- and 14-day incubations and snap-frozen at each time-point until analysis. For analysis, all samples were thawed at the same time and assessed at 0.5 mg/mL using analytical SEC, DLS, DSF, and BLI as described above.

Antigen production and purification
MERS-CoV GCN4 trimer, MERS-CoV RBD, and MERS-CoV NTD (amino acid sequences in Supplementary Data 2) were produced in Expi293F cells and purified via His-tag purification. WT H. pylori ferritin was expressed in Expi293F cells and purified as previously described29. Expi293F cells were cultured in Expi293 media (Gibco) at 37 °C, 120 RPM, and 5% CO2. For transfection, cells were grown to a cell density of 3 x 106 cells per mL and transfected with plasmid DNA at 1 µg DNA per mL cell culture using ExpiFectamine 293 transfection reagent (Gibco) according to manufacturer recommendations. Cells were harvested 4–6 days post transfection by centrifugation at 7000 x g followed by filtration using a 0.22-µm filter.

Filtered supernatant was diluted with 10X PBS to a final concentration of 2.5X PBS (pH 7.4) and loaded onto either a HisTrap excel (Cytiva) or a HiTrap TALON crude (Cytiva) column pre-equilibrated in 2.5X PBS pH 7.4. Proteins were eluted with 250 mM imidazole in 2.5X PBS pH 7.4. For Luminex, MERS-CoV GCN4 trimer was buffer exchanged into 20 mM Tris pH 7.5, 150 mM NaCl and biotinylated using a BirA biotinylation kit (Avidity). MERS-CoV RBD and NTD were purified on a Cytiva Superdex 200 Increase 100/300 GL (PN28990944) SEC column pre-equilibrated in 20 mM Tris pH 7.5, 150 mM NaCl. Protein-containing fractions were pooled, snap-frozen, and stored at -80 °C until use.

MERS-CoV FNP formulation for immunization studies
To formulate protein antigens with Alhydrogel adjuvant, the antigens were first diluted to the required concentration in formulation buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5% sucrose). Alhydrogel adjuvant (Invivogen #vac-alu-10, 10 mg/mL) was resuspended by inverting 50 times, and then the required volume was added to the diluted antigen. The combined antigen-adjuvant mixture was then inverted 50 times, maintained at room temperature for 30-60 min to allow for antigen binding to Alhydrogel, and then inverted 50 additional times.

For the adjuvant screen (Fig. 7A), MERS-1227 FNP was first formulated with Alhydrogel adjuvant as described above, except the FNP and Alhydrogel concentration were initially prepared to 2x the required final concentration for injection. For formulations without Alhydrogel, only the protein was included. Additional adjuvants were prepared as follows: ODN2395 (CpG, Invivogen Cat #trtl-2395) was dissolved to 1 mg/mL in sterile water, MPLA-SM (Invivogen Cat. #vac-mpla2) was dissolved to 1 mg/mL in sterile DMSO, and Quil A was dissolved to 1 mg/mL in sterile water. AddaVax (InvivoGen Catt #vac-adx-10) was purchased as a 2x ready-to-use suspension. 3M-052-AF was provided at 0.050 mg/mL by The Access to Advanced Health Institute (AAHI). For each formulation, the appropriate volume of adjuvant plus additional formulation buffer was added to bring the final concentration to that required for injection, and the formulated vaccines were inverted 50 additional times.

Mouse immunization studies
Female BALB/c mice (Mus musculus), age 7-8 weeks, were purchased from Charles River Laboratories and acclimated for at least one week in a contract vivarium (Fortis Life Sciences) prior to immunization. Antigens and adjuvants were formulated to deliver the indicated doses in 100 µL volume. Mice were injected intramuscularly with a 50 µL dose of antigen in each hind leg to deliver 100 µL of vaccine formulation per immunization. To obtain blood via retroorbital bleeding, a capillary tube was inserted into the medial canthus. Blood was then transferred to an Eppendorf or BD Microtainer Blood Collection Tube (BD 365967), allowed to clot at room temperature for approximately 2 h, and then spun down at 4000 rpm in a table top centrifuge. The serum layer was transferred to an Eppendorf tube and frozen at −80 °C. Prior to subsequent assays, serum was heat inactivated at 56 °C for 30 min.

Luminex bead conjugation, total IgG binding, and IgG subclass binding assays
To measure the total IgG response and IgG subclasses in mice and NHPs, we used Luminex assays adapted from a previously described method91. Biotinylated MERS-CoV GCN4 trimer (50 µg) was incubated with 2 x 106 MagPlex®-Avidin Microspheres (Luminex #MA-A012-01) for 2 h, then washed and resuspended in 1 mL Luminex wash buffer (0.1% BSA, 0.02% Tween-20 in PBS pH 7.4). For other antigens (MERS-CoV RBD and MERS-CoV NTD), different MagPlex® Microspheres (Luminex #MC10026-01 to MC10029-01 and MC10034-01) were used. Activation involved 1 x 107 microspheres with 50 mg/mL sulfo-NHS (Thermo Fisher Scientific #24510) and EDC (Thermo Fisher Scientific #22980) for 20 min, followed by adding 50 µg of antigen for a 2-h incubation. Conjugated microspheres were resuspended in 1 mL Luminex wash buffer. The post-conjugation antigenicity of each antigen was validated by measuring binding to control mAbs diluted to 20 µg/mL: 3A3 (MERS-CoV GCN4 trimer S2)57, JC57-14 (RBD)13, and G2 (NTD)54.

Serum samples from mice and NHPs were diluted 50-fold in Luminex diluent (1% non-fat milk, 5% FBS, 0.05% Tween-20 in PBS pH 7.4). 25 µL of diluted serum was added to 25 µL of antigen-conjugated microsphere suspension (at least 1000 microspheres/antigen). After a 30-min incubation, microspheres were washed three times with Luminex wash buffer. For total IgG detection, microspheres were incubated with 100 µL of goat anti-mouse IgG R-phycoerythrin (2 µg/mL, Southern Biotech #1030-09) for 30 min. For mouse IgG subclasses, biotinylated secondary antibodies specific to IgG1 and IgG2a (4 µg/mL, Southern Biotech #1070-08, #1080-08) were used, followed by 30 min with streptavidin-PE (5 µg/mL, Thermo Scientific #12-4317-87). Results were read on a Luminex xMAP Intelliflex system using INTELLIFLEX software (version 2.1.1015). For Fig. 4B, statistical analysis was performed in GraphPad Prism (version 10.5.0) using a two-way ANOVA with Tukey’s multiple comparisons.

MERS-CoV spike-pseudotyped lentivirus production and viral neutralization assay
To assess elicitation of neutralizing antibodies by MERS-1227 and MERS-1236, we utilized a MERS-CoV spike-pseudotyped lentivirus neutralization assay. For virus production, HEK293T (ATCC CRL-3216) cells were cultured in cell-growth media (DMEM, 10% fetal bovine serum, 2 mM L-glutamine, 100 Units/mL penicillin, and 100 µg/mL streptomycin) and seeded at a density of 6 million cells in a 10-cm dish. One day following cell seeding, cells were transfected with the following plasmids: 3.4 µg MERS-CoV spike plasmid, 2.2 µg of each lentivirus helper plasmid (pHDM-Hgpm2, pHDM-tat1b, and pRC-CMV-rev1b)60, and 10 µg lentivirus packaging vector (pHAGE-CMV-Luc2-IRES-ZsGreen-W)60 using BioT transfection reagent. Lentivirus production plasmids were obtained through BEI Resources (NR-52948). For all pseudovirus neutralization panels with the exception of Fig. 5C, the MERS-CoV spike plasmid encoded the spike protein (EMC/2012 strain) with the µ-phosphatase signal peptide (sequence in Supplementary Data 2) and an 18-amino acid C-terminal truncation, which has previously been shown to improve viral titers. For viruses used to generate the panel shown in Fig. 5D, native sequences of all viral spike proteins were used, and an 18 amino acid C-terminal truncation was also included (sequences in Supplementary Data 2). The media was removed and replaced with fresh media 18-24 h post transfection. Viruses were harvested 72 h post transfection by spinning at 300 x g for 5 min and filtering through a 0.45-µm PES filter. Viruses were frozen at -80 °C until use.

Infectivity assays with MERS-CoV spike-pseudotyped lentiviruses were performed in HeLa cells expressing human DPP4 (Cellecta RMCOV-CDPP4HE). HeLa/DPP4 cells were plated in white-walled, white-bottom 96-well plates at 5,000 cells per well one day prior to or the day of infection. To quantify neutralizing antibody titers in serum from immunized mice, serum samples were diluted in cell-growth media. Serum dilutions were then mixed with MERS-CoV spike-pseudotyped lentivirus and virus/serum dilutions were incubated at 37 °C for 1 h. Media was either aspirated off of HeLa/DPP4 cells and replaced with virus/serum dilutions, or trypsinized HeLa/DPP4 cells were plated directly into virus/serum dilutions and left to incubate with cells for 72 h at 37 °C. Infectivity in each well was quantified using BriteLite and luciferase signal was readout using a BioTek Synergy H1 microplate reader using Gen5 software (version 3.13.15). Data was analyzed using GraphPad Prism (version 10.5.0). Control wells containing virus only and cells only were averaged, and serum dilution infectivity values were normalized to these averages set at 100% infectivity (virus only) and 0% infectivity (cells only). Normalized values were then fit with a 3-parameter non-linear regression (Y=Bottom + (Top-Bottom)/(1+(X/IC50)). For Fig. 4C, statistical analysis was performed in GraphPad Prism using a two-way ANOVA with Tukey’s multiple comparisons.

NHP immunization studies
Ten male cynomolgus macaques of Mauritian origin (Macaca fascicularis), 6 years of age, were used for immunization studies (Table S2). Animals were maintained at Alpha Genesis, Inc. Alhydrogel-adjuvanted MERS-1227 and -1236 were prepared to a final concentration of 0.1 mg/mL antigen and 1.5 mg/mL Alhydrogel adjuvant. NHPs were injected intramuscularly with 500 µL of vaccine formulation per immunization. To obtain blood, animals were sedated with ketamine HCL 10-20 mg/kg IM and blood was collected from the femoral vein using a 22 G 1.5-inch needle, vacutainer sheath, and collection tube. Blood was allowed to clot at room temperature for approximately 1 h and was then centrifuged at 2000 x g for 20 min. Serum was transferred to Eppendorf tubes, heat inactivated at 56 °C for 30 min, and frozen at -80 °C.

For the collection of PBMCs, blood was carefully layered on top of a Ficoll Paque and then centrifuged at 2,000 x g for 20 min at 21 °C. Plasma was pipetted off into a separate tube. The mononuclear cell layer (buffy coat) was pipetted into conical tubes containing wash buffer (Hanks Balanced Salt Solution + 2% Fetal Bovine Serum) and then filled with wash buffer and mixed by inversion. Samples were then centrifuged at 400 x g for 7 min at 21 °C. Wash buffer was aspirated off and ACK Lysis Buffer was added to lyse red blood cells. Samples were incubated in lysis buffer for 3 min, then tubes were filled with wash buffer to dilute lysis buffer and centrifuged at 400 x g for 5 min at 21 °C. Wash buffer was aspirated off and cell pellets were resuspended in wash buffer. Cells were centrifuged at 400 x g for 5 min at 21 °C and resuspended in freezing buffer (wash buffer + 10% DMSO) and stored in LN2 until use.

VSV pseudovirus production and neutralization assay
VSV pseudotyped with the full-length MERS-CoV EMC/2012 spike protein92 was produced as previously described14,18,93. In brief, 5 x 106 HEK293T cells were split into poly-D-lysine-coated 10 cm2 plates and incubated overnight at 37 °C and 5% CO2 until they reached 80-90% confluency. The cells were then transfected with the construct encoding the MERS-CoV spike protein using Lipofectamine 2000 (Invitrogen) following manufacturer’s recommendations. The cells were incubated at 37 °C and 5% CO2 for 20–24 h after which the cells were washed three times with DMEM (Gibco) and infected with VSVΔG/luc94 for 2 h at 37 °C and 5% CO2. Next, the cells were washed five times with DMEM and incubated in DMEM supplemented with an anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) at 37 °C and 5% CO2. After 20–24 h the supernatant was harvested, clarified by centrifugation, filtered using a 0.45-µm filter, and concentrated with a 100 kDa MWCO centrifugal filter (Amicon). The resulting pseudovirus was frozen at -80 °C until use.

Neutralization assays using VSV pseudotyped with the MERS-CoV EMC/2012 spike were performed as previously described14,18,93. Briefly, Vero-TMPRSS2 cells95 were split into white-walled, clear bottom 96-well plates at a density of 22,000 cells per well and cultured overnight at 37 °C and 5% CO2 until they reached approximately 90-95% confluency. Serum and plasma samples were diluted in DMEM starting at 1:66 (NHP samples) or 1:10 (MERS patient samples) and serially diluted 1:3 thereafter. The diluted sera or plasma were then mixed with pseudovirus diluted 1:50 in DMEM and incubated at room temperature for 45 min. Vero-TMPRSS2 cells were washed three times with DMEM and subsequently the sera/plasma-pseudovirus mixtures were transferred to the cells. The cells were incubated for 2 h at 37 °C and 5% CO2 after which an equal volume of DMEM supplemented with 20% FBS (Gibco) and 2% Pen-Strep (Gibco) was added to each well. After 17-20 h, ONE-Glo EX substrate (Promega) was added to each well and the plates were incubated for 5-10 min in the dark at 37 °C. Luminescence values were recorded using a BioTek Synergy Neo2 plate reader. The data were normalized in GraphPad Prism 10 using the relative light units (RLU) values obtained for uninfected cells to define 0% entry and the RLU values measured for cells infected with pseudovirus only to define 100% entry. NT50 values were determined from the normalized data using a [inhibitor] vs. normalized response – variable slope model using two technical replicates to generate the curves. Plotted NT50 values were obtained by averaging the NT50 values obtained from the two independent biological replicates using two distinct batches of pseudovirus.

IFN-γ and IL-4 ELISpot assays
ELISpot plates (Millipore MSIPS4W10) were activated with 50% ethanol, washed with water, and coated with either IFN-γ or IL-4 monoclonal antibody (Mabtech 3420-2A and 3410-2A) at 15 µg/mL in PBS overnight at 4 °C. Plates were washed with PBS and blocked with complete RPMI for 1 h at 37 °C. Blocking reagent was removed, and 50 µL protein (MERS-CoV spike or H. pylori ferritin) at 20 µg/mL was added to the corresponding wells. PBMCs were then added to wells in a volume of 50 µL at a concentration of 4 x 106 cells/mL, resulting in 2 x 105 cells per well at a final protein concentration of 10 µg/mL. Media and phytohemagglutinin were used for quantitative and qualitative controls, respectively. The plates were placed in a 37 °C incubator with 5% CO2 for 16–20 h. After washing, anti-human IFN-γ biotin or IL-4 biotin detector antibody was added to each well at 1 µg/mL in PBS pH 7.4 + 0.5% BSA. The plates were incubated for approximately 2 h at room temperature. After washing, Streptavidin-ALP was added to each well at 1:1000 in PBS pH 7.4 + 0.5% BSA and plates were incubated for 1 h at room temperature. The plates were washed with PBS and NBT/BCIP chromogen (Thermo Scientific 34042) was added to each well and incubated at room temperature for 10 min. The chromogen was removed and the plate was rinsed thoroughly with tap water. Plates were then dried and removed from direct light for 24 h. Spots were counted by ZellNet using a Zeiss KS ELISpot reader using KSELISPOT software (version 4.10). The median of duplicate well cell counts was calculated, and background subtraction was performed per individual monkey by subtracting the median spot-forming unit (SFU) value of medium-stimulated wells from each median SFU value of the protein-stimulated sample. Values were multiplied by 5 to yield SFU/106 PBMC.

Alpaca challenge study
Twenty-five female alpacas (Vicugna pacos), 5 to 9 years of age, were assigned randomly such that their ages were homogeneously distributed to 5 vaccine groups (Table S2) and identified by thermally sensitive microchip. Alpacas were immunized at study days 0 and 21 by intramuscular injection of Alhydrogel-adjuvanted nanoparticle or Alhydrogel only (placebo group). Two days prior to the challenge on day 42, alpacas were moved into a large animal BSL3 facility. Blood was collected at the time of each immunization and on day 41, and sera were stored frozen until use.

Challenge was conducted as described previously68. Briefly, the alpacas were sedated with xylazine and challenged by intranasal instillation of a total of 5.4 x 105 plaque-forming units (back titrated dose) of the EMC/2012 strain of MERS-CoV, split into 1 mL per nare. Alpacas were evaluated clinically daily for 7 days post-challenge. A deeply inserted nasal swab from each side was collected daily on days 1 to 5 and 7 post-challenge. Swabs were broken off into tubes containing 1 mL of BA1 (Tris-buffered MEM containing 1% bovine albumin) supplemented with 5% fetal bovine serum and antibiotics (gentamycin, penicillin, and streptomycin), then stored frozen until assay. Alpacas were euthanized between 7 and 11 days post-challenge. Virus in nasal swab samples was titrated using a double overlay plaque assay on Vero cells (ATCC CCL-81)61. Titers of neutralizing antibodies to MERS-CoV in sera were determined by plaque reduction neutralization96 and expressed as the reciprocal of the highest dilution resulting in 90, 80, or 50% neutralization of virus. Sera were tested at two-fold dilutions from 1:10 to 1:320.

Design and procurement of MERS-1227 mRNA/LNP complexes for immunization
mRNA vaccines encoding MERS-CoV antigens (Fig. 7B) were procured from GenScript. The MERS-1227 ferritin mRNA construct encoded the amino acid sequence described above for the ferritin nanoparticle protein. The MERS-1227 cell-anchored mRNA encoded for a MERS-CoV spike containing the England 1 spike residues 1-1332 with a deletion of residues 1228-1295 to reflect the ectodomain deletion present in the MERS-1227 nanoparticle. This construct also included a GGS linker between residue 1227 and the transmembrane domain. The MERS-CoV FL cell-anchored mRNA contained the England 1 residues 1-1332. Both of the cell-anchored mRNA constructs included the 3 proline mutations described above, a mutated furin cleavage site, and a cytoplasmic tail truncation to remove residues 1332-1353, which encode for a cytoplasmic retention motif97,98. Codons were optimized for mouse expression. RNAs were synthesized using N1-methyl-pseudouridine, capped with a 5’ Cap 1, and stored in 1 mM sodium citrate pH 6.5 buffer until formulation. RNAs were formulated in SM102-containing LNPs at a concentration of 0.1-0.2 mg/mL and stored in PBS pH 7.4, 10% sucrose buffer at -80 °C until use.

mRNA/LNP cellular potency assays
HeLa cells (ATCC) were plated in a 24-well plate at a density of 2.5 x 105 cells per well in 450 µL media (RPMI + 10% FBS + 55 µM β-mercaptoethanol) and allowed to adhere for 4 h. LNPs were then diluted to 10X in PBS and added directly to cells in a volume of 50 µL. Cells were incubated with LNPs for 20 h at which point media was removed and cells were treated with StemPro Accutase (Gibco) for 10 min. Detached cells were transferred in technical quadruplicate to a 96-well V-bottom plate and spun at 500 x g for 5 min. Media was aspirated and cells were washed once with PBS and then treated with a 1:1000 dilution of Live-or-Dye 665/685 (Biotium) in PBS for 15 min at room temperature. After live/dead staining, two wells per condition were treated with flow buffer (PBS + 1% FBS + 1 mM EDTA) for the “cell-surface expression” condition, while two wells per condition were fixed and permeabilized using Cytofix/Cytoperm (BD) according to the manufacturer’s protocol for the “total cell expression” condition. Cells were washed with their respective buffers (flow buffer or 1X Perm/Wash (BD)), then stained with 2.5 µg/mL primary antibodies (JC57-14-hFc and G2-mFc) for 45 min at 4 °C followed by 1 µg/mL of PE-conjugated anti-human secondary antibody (Southern Biotech Cat. No. 2014-09) and Alexa Fluor 405 (AF405)-conjugated anti-mouse secondary antibody (Invitrogen Cat. No. A31553) for 45 min at 4 °C. Cells were then washed in their respective buffers, resuspended in flow buffer, and analyzed on a Cytek Aurora flow cytometer run with Cytek SpectroFlo software. 10,000 events per well were collected and the percentage of positively stained cells in each well was determined relative to a mock-treated condition. Data were analyzed using FlowJo v11 and plotted in GraphPad Prism.

hDPP4 BALB/c mouse challenge study
A DPP4 founder transgenic mice were obtained from Dr. Neeltje van Doremalen at Rocky Mountain Labs (NIH) and used to establish a colony at Colorado State University. Offspring were genotyped as described76. Briefly, tail tips from mice were digested and DNA extracted using a commercial kit (Monarch Sping DNA Extraction kit, NEB) and subjected to conventional PCR using primers that annealed with both the endogenous and transgenic DPP4 alleles (5’-AGCACTTGCTCTCCCAAAGTC) and only the transgenic allele (5’-TCTTCTGTAATCAGCTGCCTTTTA). PCR reactions were electrophoresed on a 2% agarose gel and detection of a 790bp product indicated the mouse was transgenic (homozygous or heterozygous). DPP4 heterozygotes and homozygotes were used in the challenge study and are indicated in Table S3.

Antigens and adjuvants were formulated to deliver the indicated doses in 100 µL volume. Mice were injected intramuscularly with a 50 µL dose of antigen in each hind leg, to deliver 100 µL of vaccine formulation per immunization. Blood was collected at study days 21 and 42, and sera were stored frozen until analysis. For challenge, mice were lightly anesthetized with ketamine-xylazine and challenged by intranasal instillation of 10,000 PFU of the EMC/2012 strain of MERS-CoV. Mice were weighed and scored for clinical signs of disease daily beginning immediately prior to challenge and extending to euthanasia. Half of the mice were euthanized 3 days post-infection and lung collected for virus titering. The remainder of the mice were maintained up to day 10 post-infection to assess changes in body weight and survival.

Ethics statement
All animal studies were conducted in accordance with protocols approved by an Institutional Animal Care and Use Committee (IACUC). WT BALB/c mouse studies were conducted in accordance with the IACUC of Fortis Life Sciences. NHP studies were conducted at Alpha Genesis, Inc. and approved by the Committee on the Care and Use of Laboratory Animal Resources (IACUC Approval: #23-7). Alpaca and human DPP4 mouse challenge studies were approved by the CSU IACUC protocol 4926.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes two vaccine candidates based on the MERS-CoV spike protein displayed on a self-assembling\nferritin nanoparticle. Overall, design elements such as S protein truncation, proline mutations, furin site mutations, flexible\nlinkers, ferritin nanoparticles, and the CHO stable expression system are relatively common. The key contribution of the\narticle lies in its use of multiple animal models, providing substantial preclinical antibody response data.\nMajor Comments:\n1.BLI analysis: The authors confirmed through 2D class results that MERS-1236 is more flexible on the nanoparticle surface.\nWhile the BLI results for RBD- and S2-specific antibodies seem to align with this interpretation, how should the results for\nthe NTD-specific monoclonal antibody G2 be explained? Additionally, since the article emphasizes the comparison between\nMERS-1236 and MERS-1227, could Figure 3 be presented in a comparative format? Otherwise, results under specific\nconditions, such as stability at 37°C, may be difficult to use as reference indicators for future studies.\n2.Vaccination group settings: Why is there no MERS trimer 0.4 µg group in Figure 4? Why is there no placebo group in\nFigure 5? In Figure 6, why is the MERS-1227 ferritin protein dose set at 0.4 µg, while the mRNA group includes 0.1 and 1 µg\ndoses? Additionally, why is there no MERS-1236 group in Figure 7, as in Figure 8? Does the enhanced flexibility of MERS-\n1236 on the nanoparticle surface improve epitope presentation, thereby inducing a broader range of neutralizing antibodies\nand enhancing protective efficacy?\n3.Adjuvant selection: Not until Figure 6 do the authors begin comparing how different adjuvant choices influence the\nimmune response characteristics mediated by MERS-1227. This raises the question: What was the rationale behind the\nadjuvant selection in Figures 4 and 5? Additionally, do the adjuvant evaluation results in Figure 6 provide meaningful\ninsights for current research? Is Alhydrogel truly the optimal choice? Moreover, the adjuvant dosing strategy seems to lack\nclear logic. Relying solely on the IgG2a/IgG1 ratio to infer cellular immune response characteristics seems insufficient,\nespecially given that the study does not systematically evaluate cellular immunity across its various models.\n4.mRNA vaccine: The evaluation of the mRNA vaccine is insufficiently characterized. While the authors constructed three\nmRNA antigens, the manuscript lacks essential data on the expression of mRNA-LNPs in vivo or in vitro. Additionally, key\nparameters of the mRNA vaccine should be provided, including particle size, zeta potential, and mRNA-LNP encapsulation\nefficiency. Why was MERS-1227 chosen? Additionally, why is there no further challenge protection data?\nMinor Comments:\n1.Please confirm the annotation in Figure 2A. Should the diagram notes \"Non-Reduced\" and \"Reduced\" be exchanged? Is it\nreasonable to run these two types of samples in the same gel?\n2.In Figure 2B, please specify the column used in the SEC-MALS method and provide more details on the methods.\n3.In Figure 2C, please provide the percentage distribution (pd%) in the DLS test results.\n4.In Table S3, it is unusual that the results of PRNT90, PRNT80, and PRNT50 are the same (MERS-1236–Day 42, 0.4 µg\ndose, MERS-1236–Day 42, 10 µg dose).\n5.Add necessary statistical analysis for the related figures.\n6.Please verify the errors in the references, such as Ref 66 and Ref 71, to ensure their accuracy and consistency.", "reviewer_2": "In this manuscript, Abigail et al designed two vaccine candidates, MERS-1227 and MERS-1236, by truncating the\nectodomain of the MERS-CoV spike protein to amino acids 1227 and 1236, respectively. The truncated spike ectodomains\nwere displayed on self-assembled ferritin nanoparticles to improve stability and immunogenicity. In addition, a cytoplasmic\ntail truncation, mutations of three proline sites and a furin cleavage site were also introduced to stabilize the conformation of\nthe spike protein. After construction and purification, the two vaccine candidates were evaluated with comprehensive\ncharacterizations, including size, homogeneity, glycosylation modifications. Even though MERS-1227 displays a more\nstable functionalized nanoparticle, both of MERS-1227 and MERS-1236 are able to maintain antigenicity under pH 7.5 or 37\n°C conditions for 14 days. Both candidates elicit robust neutralizing antibody responses, especially after a second boost\ndose, in mice and non-human primate models, and exhibit obviously protective functions against virus infection in hDPP4\ntransgenic mice and alpacas models. Overall, this work is the first model of MERS nanoparticle vaccine that achieves\ncomplete protection in large animals and has significant clinical development value. The evaluation is systematic and\ncomprehensive, with solid data. The publication is recommended after minor revision: glycosylation is critical for protein\nconformation and stabilization, and also affect the recognition between virus and receptors. Thus, glycosylation\nmodifications on protein are important in vaccine production. It is recommended to add more detailed information of\nglycosylation modifications (e.g., glycan compositions and their abundance distribution at each site) on MERS-1227 and\nMERS-1236. And a minor mistake in line 429 should be addressed: “Figure 7F” should be corrected to “Figure 7E”.", "reviewer_3": "Summary of key results:\nThe manuscript describes the design and production of recombinant MERS vaccine antigen MERS-1227 (and the\nalternative MERS-1236) , a stabilized (truncated) spike ectodomain displayed on a self-assembling ferritin nanoparticle. The\nantigen is evaluated as a vaccine candidate in different animal models. The authors compare its immunogenicity in mice\nusing different adjuvants. They also test the formulation with alum in non-human primates (as a human model) and assess\nefficacy in both mice and alpacas (as a dromedary model).\nValidity and significance:\nOverall, the manuscript presents a substantial amount of high-quality data on the vaccine antigen and its various\nformulations. In my opinion, these findings hold significant value for the scientific community. Moreover, the open publication\nof this data would be particularly valuable if the antigen proceeds to clinical trials, as it would provide important context and\ntransparency for future development.\nClarity and context:\nThe manuscript is well-written, including the methods section, which contains all the necessary details. Results are\nadequately discussed.\nComments and Suggestions:\n- The study seems to focus exclusively on the humoral immune response, specifically the induction of neutralizing\nantibodies. As seen with other coronaviruses, long-term protection, particularly against severe disease, may rely more on\ncellular immune responses. In my opinion, it is relevant to also compare the cellular response (e.g., cytokine release after\nantigen-specific stimulation of T cells). This could easily have been done for the mouse study in the context of adjuvant\nselection (e.g., alum vs. others) and platform comparisons (e.g., recombinant protein vs. mRNA vaccines).\n- The authors appear to favor the alum-adjuvanted MERS-1227 based on the use of the adjuvant in the vaccine challenge\ntest. However, Quil-A + MPLA had a 22-fold better NT50 after 2 doses. Comparing the most immunogenic formulation to\nthe alum formulation in the challenge models would have been interesting.\n- I would further caution that the alum-based Th2-skewed immune response may be less effective in preventing severe\ndisease compared to vaccines that elicit a Th1-biased or more balanced Th1/Th2 response. Although the authors mention,\n“Taken together, the results of this adjuvant screen demonstrate several feasible mechanisms to improve both humoral and\ncell-mediated immune responses to MERS-1227”, the important role of cell-mediated immune reasons is not well discussed\nin the manuscript.\nMinor comments:\n- Fig 6 panel B, replace + /- with the actual dose of alum in ug (0 and 150)\n- The numbering of the references is off by 1; I believe reference 39 is missing in the main text.", "reviewer_4": "In this study, Powell and colleagues described the design, characterization, and preclinical evaluation of two self-\nassembling ferritin nanoparticle-based MERS-CoV Spike vaccine candidates, MERS-1227 and MERS-1236. While ferritin\nnanoparticle vaccines for coronaviruses have been previously explored, the authors enhance the structural uniformity and\nthermodynamic stability of their candidates through optimized manufacturing processes. However, the antigen design lacks\nsufficient novelty, and some critical data of protection effect of the preclinical evaluation of vaccine candidates on several\nanimal model are missing. Moreover, several key issues need to be addressed and clarified.\n1. MERS-1227 exhibits higher structural stability and immunogenicity than MERS-1236. Could the authors provide structural\ninsights (e.g., cryo-EM analysis or molecular dynamics simulations) to explain how the amino acid truncation contributes to\nthese differences?\n2. Cellular immune responses also play a critical role in protection against MERS-CoV infection, it is suggested to evaluate\ncellular immune responses in vaccinated animals.\n3. Additional well-characterized MERS-CoV monoclonal antibodies are recommended to enroll for nanoparticles binding to\nfurther confirm proper antigenic site exposure.\n4. How do these ferritin nanoparticle vaccines compare to other MERS-CoV vaccine candidates (e.g., adenoviral vectors,\nmRNA-LNPs, or subunit vaccines) in terms of neutralizing antibody titers, durability, or cross-reactivity?\n5. The absence of histopathological analysis (e.g., lung tissue scoring for inflammation, viral antigen distribution) in the\nchallenge studies is notable, which will strengthen the correlation between neutralizing antibody levels and actual\nprotection."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript describes two vaccine candidates based on the MERS-CoV spike\nprotein displayed on a self-assembling ferritin nanoparticle. Overall, design elements\nsuch as S protein truncation, proline mutations, furin site mutations, flexible linkers,\nferritin nanoparticles, and the CHO stable expression system are relatively common.\nThe key contribution of the article lies in its use of multiple animal models, providing\nsubstantial preclinical antibody response data.\nMajor Comments:\n1.BLI analysis\nThe authors confirmed through 2D class results that MERS-1236 is more flexible\non the nanoparticle surface. While the BLI results for RBD- and S2-specific\nantibodies seem to align with this interpretation, how should the results for the\nNTD-specific monoclonal antibody G2 be explained? Additionally, since the\narticle emphasizes the comparison between MERS-1236 and MERS-1227, could\nFigure 3 be presented in a comparative format? Otherwise, results under specific\nconditions, such as stability at 37°C, may be difficult to use as reference\nindicators for future studies.\nWe thank the reviewer for this comment regarding the impact of protein flexibility on the\nBLI binding results. To better understand this relationship, we expanded our mAb panel\nfrom 4 to 12. In this expanded panel, we assess 11 antibodies targeting the S1 domain,\n8 which target the RBD, and 3 which target non-RBD epitopes. All 8 RBD antibodies\nshow enhanced binding to MERS-1236, whereas the 3 non-RBD S1 antibodies exhibit\ncomparable MERS-1227 and MERS-1236 binding. This suggests to us that the major\nimpact of this increased flexibility is enhanced dynamics of the RBD sampling the\nknown “up” and “down” conformations. The enhanced binding to 3A3, an S2-targeting\nantibody, could be suggestive of overall increased trimer breathing in the MERS-1236\nconstruct, however without additional available S2 antibodies we are unable to further\nprofile this.\nWe agree with the reviewer that an alternative presentation of the stability data would\nbe helpful and have added a supplemental figure (Figure S4) with the relevant subset of\nthe data presented in a comparative manner between MERS-1227 and MERS-1236.\nWe have also included all raw data in the source data file so that additional\ncomparisons of interest could be plotted.\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\n2. Vaccination group settings\nWe thank the reviewer for this comment regarding the dosing and study design for our\npreclinical immunization experiments. We address these comments by individual point\nbelow and have made clarifying changes to the text, figures, and figure order as noted.\nWhy is there no MERS trimer 0.4 µg group in Figure 4?\nWe agree that it added confusion to include the 0.4 µg dose group in Figure 4 for the\nMERS-1227 and MERS-1236 groups but not the MERS-CoV trimer. At the time of this\nstudy, we did not have the resources to include a 0.4 µg dose group for the trimer and\ntherefore we do not have these data. We have removed the 0.4 µg dose group for\nMERS-1227 and MERS-1236 from Figure 4B-C for increased clarity. We have also\namended the text accordingly.\nWhy is there no placebo group in Figure 5?\nThe inclusion of a placebo group in this experiment would have been prohibitively\nexpensive. However, as a control, we evaluated study day 0 (pre-immune) serum in\npseudovirus neutralization and Luminex binding assays. This serum is from the same\nNHPs as was taken at the later time points, which demonstrates the effects of\nvaccination on the corresponding antibody responses. The pre-immune serum shows\nno quantifiable binding or neutralization signals, indicating that the responses seen at\nlater time points are the result of the immune response to immunization.\nIn Figure 6, why is the MERS-1227 ferritin protein dose set at 0.4 µg, while the\nmRNA group includes 0.1 and 1 µg doses?\nAs the work presented here was primarily focused on characterization of the MERS-\nCoV protein nanoparticles, we conducted a larger set of preclinical analyses using this\nantigen as compared to the mRNA versions of these antigens. We chose 0.4 µg protein\ndose based on our dose de-escalation study shown in Figure S6, which showed that\nbelow 0.4 µg, there was a dose-dependent reduction in antibody responses to the\nprotein. We were unable to perform this thorough dosing evaluation on all antigens. As\nthe mRNA and protein nanoparticles are distinct platforms, we did not expect that\ncomparing corresponding dose levels would necessarily be meaningful. Instead, we\nevaluated the mRNAs at two commonly used dose levels utilized in preclinical mouse\nmRNA studies in the literature (0.1 and 1 µg)1.\nAdditionally, why is there no MERS-1236 group in Figure 7, as in Figure 8?\nThis comment made us recognize that the figure ordering could have improved clarity.\nWhile both MERS-1227 and MERS-1236 have promising attributes and are largely\nequivalent immunogenically, we ultimately down-selected to MERS-1227 as the lead\ndevelopment candidate based on concerns surrounding the flexibility and lower melting\ntemperature of the MERS-1236 construct. As the adjuvant, platform, and DPP4 mouse\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\nchallenge study required extensive resources, we conducted them after this down-\nselection. To make the logic behind this selection clearer, we have added clarifying text\nand moved the alpaca challenge study (the last major study containing both MERS-\n1227 and MERS-1236) to follow the NHP study (Figure 5).\nDoes the enhanced flexibility of MERS-1236 on the nanoparticle surface improve\nepitope presentation, thereby inducing a broader range of neutralizing antibodies\nand enhancing protective efficacy?\nThis is an interesting hypothesis; however, we do not think the neutralization of our\nMERS-CoV viral panel in Figure 5E are conclusive enough to state this in the results.\nWe have added text in the discussion raising this as a possibility.\n3. Adjuvant selection\nNot until Figure 6 do the authors begin comparing how different adjuvant choices\ninfluence the immune response characteristics mediated by MERS-1227. This\nraises the question: What was the rationale behind the adjuvant selection in\nFigures 4 and 5?\nAdditionally, do the adjuvant evaluation results in Figure 6 provide meaningful\ninsights for current research? Is Alhydrogel truly the optimal choice? Moreover,\nthe adjuvant dosing strategy seems to lack clear logic. Relying solely on the\nIgG2a/IgG1 ratio to infer cellular immune response characteristics seems\ninsufficient, especially given that the study does not systematically evaluate\ncellular immunity across its various models.\nWe thank the reviewer for this feedback pertaining to adjuvant usage. The rationale for\nusing alum in the initial immunogenicity studies presented in Figures 4 and 5 stemmed\nfrom its widespread clinical use and the ability to readily procure this adjuvant for a\nclinical development program (lines 388-390). We reasoned that showing strong\nimmunogenicity with a mild adjuvant like alum would provide proof of concept to\npromote further development of these vaccine candidates. The adjuvant screening\nresults are intended to demonstrate that our vaccine is compatible with and can\npotentially be enhanced by alternative adjuvants relative to alum. We hope these results\nmay be further tested in the future, perhaps with a bedside mixing strategy in humans, if\nthe proprietary versions of these adjuvants can be accessed for human use.\nWe agree with the reviewer that the IgG2a/IgG1 ratio does not fully address cellular\nimmune responses. We have now assessed vaccinated samples from our NHP study to\nevaluate IFN and IL-4 responses, presented in Figure 5F and S7C. Since we had not\ncollected mouse spleens, we are unable to retrospectively assess the cellular immune\nresponses in those animals. We have tempered the language around this conclusion\n(lines 427-429), but we do hope the reviewer agrees that we fulfilled the primary goal of\noptimizing neutralizing antibody responses.\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\n4. mRNA vaccine\nThe evaluation of the mRNA vaccine is insufficiently characterized. While the\nauthors constructed three mRNA antigens, the manuscript lacks essential data\non the expression of mRNA-LNPs in vivo or in vitro. Additionally, key parameters\nof the mRNA vaccine should be provided, including particle size, zeta potential,\nand mRNA-LNP encapsulation efficiency. Why was MERS-1227 chosen?\nAdditionally, why is there no further challenge protection data?\nWe thank the reviewer for this comment. We have added both biophysical (Figure S9A)\nand in vitro cellular potency (Figure S9B-C) data to describe these results and added\nassociated discussion in the main text (lines 442-450). The biophysical characterization\nincludes % mRNA encapsulation, size, polydispersity, and zeta potential and the cellular\npotency includes both cell-surface and total cell expression.\nWe added text in the manuscript (lines 380-385) to indicate that following our alpaca\nchallenge results, we down-selected the candidates to focus on MERS-1227 based on\nthe comparable immunogenicity and the more favorable stability profile of MERS-1227.\nThis was largely driven by potential manufacturing concerns due to the flexibility and\nlower melting temperature of MERS-1236. For this reason, when we progressed to the\nadjuvant screen and the mRNA platform evaluation, we solely evaluated MERS-1227.\nMinor Comments:\n1.Please confirm the annotation in Figure 2A. Should the diagram notes \"Non-\nReduced\" and \"Reduced\" be exchanged? Is it reasonable to run these two types\nof samples in the same gel?\nWe thank the reviewer for noting this and confirm that the labels in Figure 2A are\ncorrect. Given the disulfide bonds in the MERS-CoV spike, our interpretation here is that\nreleasing those bonds in the protein lead to an extended conformation that runs more\nslowly via SDS-PAGE (noted in lines 176-177 in the text). Since the reducing agent\n(beta-mercaptoethanol) is added directly to the sample loading buffer (not the gel or the\nrunning buffer), we think it was appropriate to run these on the same gel.\n2.In Figure 2B, please specify the column used in the SEC-MALS method and\nprovide more details on the methods.\nWe have added both the column type and the light scattering detector name to figure\nlegend 2B.\n3.In Figure 2C, please provide the percentage distribution (pd%) in the DLS test\nresults.\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\nThe instrument used to collect these results does not report on the percent distribution\nof the results and thus unfortunately we are unable to add this parameter into our\nreporting.\n4.In Table S3, it is unusual that the results of PRNT90, PRNT80, and PRNT50 are\nthe same (MERS-1236–Day 42, 0.4 µg dose, MERS-1236–Day 42, 10 µg dose).\nThis neutralization assay was conducted by limiting dilution, and expressed as the\ndilution point at which 90% (PRNT90), 80% (PRNT80), or 50% (PRNT50) of viral\nplaques were inhibited. Serum was tested from a 1:10 to a 1:320 dilution. We have\nremoved the discussion of the 0.4 µg dose of MERS-1236 to be consistent with\nrevisions made to groups in Figure 4. For the 10 µg dose, the >= 320 indicated that at a\ndilution greater than or equal to 1:320, this serum inhibited 50%, 80%, and 90% of viral\nplaques and thus was at the upper limit of quantitation of this assay. We have adjusted\nthe symbols from >= to  for clarity. We have also added text to the methods section\n(lines 959-962) further describing this live virus neutralization method.\n5.Add necessary statistical analysis for the related figures.\nWe have noted the statistical analyses performed in the figure legends (Figures 4B-C)\nand in the corresponding methods sections (lines 832-833 and 866-867).\n6.Please verify the errors in the references, such as Ref 66 and Ref 71, to ensure\ntheir accuracy and consistency.\nWe thank the reviewer for spotting this error and have corrected these references.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, Abigail et al designed two vaccine candidates, MERS-1227 and\nMERS-1236, by truncating the ectodomain of the MERS-CoV spike protein to amino\nacids 1227 and 1236, respectively. The truncated spike ectodomains were displayed on\nself-assembled ferritin nanoparticles to improve stability and immunogenicity. In\naddition, a cytoplasmic tail truncation, mutations of three proline sites and a furin\ncleavage site were also introduced to stabilize the conformation of the spike protein.\nAfter construction and purification, the two vaccine candidates were evaluated with\ncomprehensive characterizations, including size, homogeneity, glycosylation\nmodifications. Even though MERS-1227 displays a more stable functionalized\nnanoparticle, both of MERS-1227 and MERS-1236 are able to maintain antigenicity\nunder pH 7.5 or 37 °C conditions for 14 days. Both candidates elicit robust neutralizing\nantibody responses, especially after a second boost dose, in mice and non-human\nprimate models, and exhibit obviously protective functions against virus infection in\nhDPP4 transgenic mice and alpacas models. Overall, this work is the first model of\nMERS nanoparticle vaccine that achieves complete protection in large animals and has\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\nsignificant clinical development value. The evaluation is systematic and comprehensive,\nwith solid data.\nThe publication is recommended after minor revision: glycosylation is critical for protein\nconformation and stabilization, and also affect the recognition between virus and\nreceptors. Thus, glycosylation modifications on protein are important in vaccine\nproduction.\nIt is recommended to add more detailed information of glycosylation\nmodifications (e.g., glycan compositions and their abundance distribution at each\nsite) on MERS-1227 and MERS-1236.\nWe agree that analysis of the glycosylation of our antigens could affect their antigenicity\nand that this is an important parameter to monitor and control during vaccine\nproduction. In the original version of the manuscript, we performed LC-MS/MS\nglycopeptide analysis and concluded that both MERS-1227 and MERS-1236 had\nextensive N-glycosylation and limited but detectable O-linked glycosylation (Figure S2\nand Table S1). The glycan compositions were included in Table S1 of the original\nmanuscript, but we did not clearly state this in the main text and thank the reviewer for\nbringing this to our attention. In the revised manuscript, we have more clearly noted this\nanalysis in the Results, lines 183-190. Additionally, we have performed the additional\ndata analysis requested by the reviewer and now report the relative ion abundances of\neach glycan at each site, relative to the total ion abundance at that site. This expanded\nanalysis is also mentioned in the Results (lines 184-187).\nAnd a minor mistake in line 429 should be addressed: “Figure 7F” should be\ncorrected to “Figure 7E”.\nThank you for noting this, we have corrected this in the text.\nReviewer #3 (Remarks to the Author):\nSummary of key results:\nThe manuscript describes the design and production of recombinant MERS vaccine\nantigen MERS-1227 (and the alternative MERS-1236) , a stabilized (truncated) spike\nectodomain displayed on a self-assembling ferritin nanoparticle. The antigen is\nevaluated as a vaccine candidate in different animal models. The authors compare its\nimmunogenicity in mice using different adjuvants. They also test the formulation with\nalum in non-human primates (as a human model) and assess efficacy in both mice and\nalpacas (as a dromedary model).\nValidity and significance:\nOverall, the manuscript presents a substantial amount of high-quality data on the\nvaccine antigen and its various formulations. In my opinion, these findings hold\nsignificant value for the scientific community. Moreover, the open publication of this data\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\nwould be particularly valuable if the antigen proceeds to clinical trials, as it would\nprovide important context and transparency for future development.\nClarity and context:\nThe manuscript is well-written, including the methods section, which contains all the\nnecessary details. Results are adequately discussed.\nComments and Suggestions:\n- The study seems to focus exclusively on the humoral immune response,\nspecifically the induction of neutralizing antibodies. As seen with other\ncoronaviruses, long-term protection, particularly against severe disease, may rely\nmore on cellular immune responses. In my opinion, it is relevant to also compare\nthe cellular response (e.g., cytokine release after antigen-specific stimulation of T\ncells). This could easily have been done for the mouse study in the context of\nadjuvant selection (e.g., alum vs. others) and platform comparisons (e.g.,\nrecombinant protein vs. mRNA vaccines).\nWe thank the reviewer for this comment and agree that cellular immune assessments\ncan provide a more comprehensive evaluation of our vaccine. Since we did not collect\nmouse spleen samples, we were unable to retrospectively evaluate cellular immune\nresponses in these animals. However, we did have PBMC samples from our NHP\nstudy. Thus, we have added both IFN and IL-4 ELISpot results in Figures 5F and S7C\ndemonstrating that both MERS-1227 and -1236 elicit T cell responses against both\nMERS-CoV spike and the H. pylori ferritin scaffold.\n- The authors appear to favor the alum-adjuvanted MERS-1227 based on the use\nof the adjuvant in the vaccine challenge test. However, Quil-A + MPLA had a 22-\nfold better NT50 after 2 doses. Comparing the most immunogenic formulation to\nthe alum formulation in the challenge models would have been interesting.\nWe thank the reviewer for this comment. The basis for our focus on alum is rooted in\nthe availability of alum as a clinical adjuvant as it poses substantially fewer hurdles for\nuse as a clinical development candidate compared to proprietary adjuvants such as\nthose utilized in our adjuvant screen. We agree that future challenge studies with the\nmost immunogenic formulations could be informative, however this was outside the\nscope of this current body of work.\n- I would further caution that the alum-based Th2-skewed immune response may\nbe less effective in preventing severe disease compared to vaccines that elicit a\nTh1-biased or more balanced Th1/Th2 response. Although the authors mention,\n“Taken together, the results of this adjuvant screen demonstrate several feasible\nmechanisms to improve both humoral and cell-mediated immune responses to\nMERS-1227”, the important role of cell-mediated immune reasons is not well\ndiscussed in the manuscript.\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\nWe thank the reviewer for noting this. As stated above, we have added T cell analysis of\nour vaccinated NHP samples into the manuscript. We have also modified the language\nat the end of the adjuvant screen results section to soften the conclusion on cellular\nimmunity to read: “Taken together, the results of this adjuvant screen demonstrate that\nmodification of the adjuvant formulation of MERS-1227 could be used to enhance the\nresultant antibody responses and potentially modulate the cellular immune response.”\n(lines 427-429)\nMinor comments:\n- Fig 6 panel B, replace + /- with the actual dose of alum in ug (0 and 150)\nWe have made this change to this figure (which is now Figure 7).\n- The numbering of the references is off by 1; I believe reference 39 is missing in\nthe main text.\nWe have corrected these errors with the references.\nReviewer #4 (Remarks to the Author):\nIn this study, Powell and colleagues described the design, characterization, and\npreclinical evaluation of two self-assembling ferritin nanoparticle-based MERS-CoV\nSpike vaccine candidates, MERS-1227 and MERS-1236. While ferritin nanoparticle\nvaccines for coronaviruses have been previously explored, the authors enhance the\nstructural uniformity and thermodynamic stability of their candidates through optimized\nmanufacturing processes. However, the antigen design lacks sufficient novelty, and\nsome critical data of protection effect of the preclinical evaluation of vaccine candidates\non several animal model are missing. Moreover, several key issues need to be\naddressed and clarified.\n1. MERS-1227 exhibits higher structural stability and immunogenicity than MERS-\n1236. Could the authors provide structural insights (e.g., cryo-EM analysis or\nmolecular dynamics simulations) to explain how the amino acid truncation\ncontributes to these differences?\nWe thank the reviewer for this comment. We conducted a preliminary follow-up cryo-\nTEM imaging analysis which revealed similar 2D class averaging results to our negative\nstain TEM, albeit with substantial smearing of the spike density surrounding the ferritin\ncore, likely due to the high flexibility of these molecules. For this reason, we concluded\nthat further structural image analysis would be unlikely to reveal additional details about\nthese molecules given their size and flexibility. We currently do not have the\ncomputation resources to run molecular dynamics simulations on these molecules and\nfeel that gaining access to this analysis would be outside the scope of this study.\nHowever, we have provided the amino acid sequences of MERS-1227 and MERS-1236\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\nin Table S2 to allow such MD simulations to be conducted independently by groups with\nthese resources, if desired.\n2. Cellular immune responses also play a critical role in protection against MERS-\nCoV infection, it is suggested to evaluate cellular immune responses in\nvaccinated animals.\nWe thank the reviewer for this comment. We have now evaluated T cell responses in\nthe NHPs vaccinated with alum-adjuvanted MERS-1227 and MERS-1236. These\nresults have been added in Figures 5F and S7C and demonstrate both IFN and IL-4\nresponses to both candidates following both 1 and 2 doses of vaccine.\n3. Additional well-characterized MERS-CoV monoclonal antibodies are\nrecommended to enroll for nanoparticles binding to further confirm proper\nantigenic site exposure.\nWe thank the reviewer for this comment and in response have expanded our mAb panel\nfor characterization from 4 antibodies to 12 antibodies, presented in Figures 2F and S3.\nWith this expanded panel, we can conclude that the differences in binding to MERS-\n1227 and MERS-1236 are specific for the RBD within the S1, given that 3 non-RBD S1-\ntargeting antibodies show equivalent binding between the two antigens.\n4. How do these ferritin nanoparticle vaccines compare to other MERS-CoV\nvaccine candidates (e.g., adenoviral vectors, mRNA-LNPs, or subunit vaccines) in\nterms of neutralizing antibody titers, durability, or cross-reactivity?\nWhile a number of MERS vaccine candidates have been evaluated preclinically, there is\na lack of consistent assay methodology to appropriately compare across different\nstudies. We were unable to find reports of either preclinical or clinical MERS candidates\nthat evaluated either vaccine durability. We found three preclinical vaccine candidates\n(a DNA vaccine, an Adeno-based vaccine, and a protein nanoparticle vaccine) that\nevaluated cross-reactive antibody responses and have added this in the discussion\n(lines 548-550).\nAs an alternative means of benchmarking our reported pseudovirus neutralization titers,\nwe included a new set of neutralization analyses assessing titers from our immunized\nNHPs compared with a set of human convalescent patient serum samples. All samples\nwere analyzed concurrently in a VSV pseudotyped neutralization assay (Figure 5D). We\nwere encouraged that our single-dose titers are comparable to convalescent patient\ntiters and after a boost, we observe titers ~100-fold higher.\n5. The absence of histopathological analysis (e.g., lung tissue scoring for\ninflammation, viral antigen distribution) in the challenge studies is notable, which\nwill strengthen the correlation between neutralizing antibody levels and actual\nprotection.\nReviews received: 15 April 2025\nResponses submitted: 15 July 2025\nWe thank the reviewer for this comment. While we were unable to conduct these\nanalyses for our currently reported challenge studies, we agree that future challenge\nexperiments would benefit from this expanded set of data.\nReferences\n1. Corbett, K. S. et al. SARS-CoV-2 mRNA vaccine design enabled by prototype\npathogen preparedness. Nature 586, 567–571 (2020).\n2. Watanabe, Y. et al. Vulnerabilities in coronavirus glycan shields despite extensive\nglycosylation. Nat Commun 11, 2688 (2020)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed the comments from the first round of peer review and provided satisfactory\nadditional data and clarifications on key aspects such as adjuvant selection, glycosylation profiling, and cellular immune\nresponses. I recommend acceptance pending minor revisions.\nMinor comments:\n1.The statement of Figure S4 is clearer than Figure 3, and it is recommended to add Figure S4 to the main image.\n2.Line 1468, figure 2E, TEM micrographs are missing clear scale.\n3.Line 1493, Figure S3, Figure Note is missing.", "reviewer_3": "The authors did a substantial effort to address most of the reviewer comments. The data of the cellular data of the\nrestimulated PBMC samples from the NHP study is a valuable addition.\nI recommend publication\nOne very minor request can be further addressed\n\nA reviewer asked to provide the percentage distribution (pd%) in the DLS test results, probably referring to the polydispersity\n(PDI). I agree this could be of value to indicate monodispersity and can be added in the figure legend with the size estimate.\nThat number is certainly retrievable from a DLS dataset of the Prometheus Panta.", "reviewer_4": "In this revised manuscript, critical data have been updated with an optimized visual presentation by Powell and colleagues.\nHowever, the revised version has not addressed the key issues raised by the reviewers.\n1. Critical histopathological experiments remain unprovided.\n2. The supplementary T cell response experiments indicate that the vaccine induces a Th2-dominated T cell response,\nraising the question of whether this could lead to more severe inflammation."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have thoroughly addressed the comments from the first round of peer review and\nprovided satisfactory additional data and clarifications on key aspects such as adjuvant\nselection, glycosylation profiling, and cellular immune responses. I recommend acceptance\npending minor revisions.\nMinor comments:\n1.The statement of Figure S4 is clearer than Figure 3, and it is recommended to add\nFigure S4 to the main image.\nWe thank the reviewer for this comment and agree the representation of these results in Figure\nS4 were more cohesive with the remainder of the data presented in the main text. We have\nswitched Figure S4 with Figure 3. All data are available in the Source File if readers want to\nmake any additional comparative analyses.\n2.Line 1468, figure 2E, TEM micrographs are missing clear scale.\nWe thank the reviewer for noting this, we have added the scale in text below the scale bar.\n3.Line 1493, Figure S3, Figure Note is missing.\nWe thank the reviewer for this comment; however, we are not sure what the reviewer is\nrequesting. In our version, the Figure Legend for Figure S3 reads:\n“Figure S3. BLI binding of a panel of MERS-CoV mAbs to MERS-1227 and MERS-1236.\nmAbs were loaded onto anti-human capture sensor tips and dipped into either MERS-1227 or\nMERS-1236 wells at 100 µg/mL FNP. Dashed line indicates the end of the association step.”\nat line 1493.\nReviewer #3 (Remarks to the Author):\nThe authors did a substantial effort to address most of the reviewer comments. The data of the\ncellular data of the restimulated PBMC samples from the NHP study is a valuable addition.\nI recommend publication\nOne very minor request can be further addressed\nReviewer comments received: 20 August 2025\nResponses submitted: 05 November 2025\nA reviewer asked to provide the percentage distribution (pd%) in the DLS test results,\nprobably referring to the polydispersity (PDI). I agree this could be of value to indicate\nmonodispersity and can be added in the figure legend with the size estimate. That\nnumber is certainly retrievable from a DLS dataset of the Prometheus Panta.\nWe thank the reviewer for this comment. We have added a table in Figure 2C including both the\ndiameter and PDI measurements from this DLS analysis.\nReviewer #4 (Remarks to the Author):\nIn this revised manuscript, critical data have been updated with an optimized visual presentation\nby Powell and colleagues. However, the revised version has not addressed the key issues\nraised by the reviewers.\n1. Critical histopathological experiments remain unprovided.\nWhile we are unable to conduct additional challenge experiments at this time, we agree with the\nreviewer that histopathological data, including lung tissue scoring and viral antigen distribution,\nare a key metric in these studies. Upon discussion with our collaborating authors who\nconducted the challenge work, we learned that lung pathology was evaluated for all mice\nincluded in the hDPP4 mouse challenge study. We have added these lung pathology scores\ninto table S5 and described these results in the text. While minimal lung pathology was\nobserved in both the vaccinated and placebo-treated mice, lung inflammation and\nhistopathological changes were also not noted in the publication which established the hDPP4\nmouse model of use1. Though we did not specifically look at viral antigen distribution, our\ncollaborating authors did assess virus levels in the lung using plaque assay. These results are\npresented in Figure 8D and demonstrate that the vaccinated mice have lower levels of\nreplicating virus isolated from lung tissue following the viral challenge. This correlates well with\nproduction of neutralizing antibodies in these animals, as presented in Figure 8B-C. We have\nadded text highlighting these results and have also added the numerical viral titers in Table S5.\nFor the alpaca challenge study, this work was conducted in a specialized large-animal BSL3\nfacility and due to the complexities for this working environment, our collaborating authors were\nunable to conduct extensive necropsy following the virus challenge.\n2. The supplementary T cell response experiments indicate that the vaccine induces a\nTh2-dominated T cell response, raising the question of whether this could lead to more\nsevere inflammation.\nWe thank the reviewer for noting this important point. While we are unable to gather\nexperimental data to confirm or refute whether the Th2-dominated response in our animal\nstudies could lead to severe inflammation, we have noted in the Results when describing the\nReviewer comments received: 20 August 2025\nResponses submitted: 05 November 2025\nNHP PBMC evaluation (lines 357-362) and the hDPP4 mouse challenge study (lines 491-498)\nthe potential for Th2-induced inflammation and concerns surrounding this phenomenon. While\nour hDPP4 mouse lung necropsy results did not suggest vaccine-induced lung inflammation, we\nhave caveated these results in the text indicating that future studies with more in-depth\nhistopathology evaluation would be required to fully rule this out. We also highlighted the\npossibility of immunopathology following a Th2-biased vaccine-induced immune response in the\nDiscussion (lines 570-575) as an area for future evaluation, likely in the context of modified\nadjuvant formulations.\nReferences\n1. van Doremalen, N. et al. Transmission dynamics of MERS-CoV in a transgenic human DPP4\nmouse model. npj Viruses 2, 1–8 (2024)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "No more concerns"}, "author_response": ""}]

Decision letter