Protection against the H1N1 influenza virus using self-assembled nanoparticles formed by lumazine synthase and bearing the M2e peptide.

There is an urgent need for influenza vaccines that offer broad cross-protection. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) is a promising candidate; however, its low immunogenicity can be addressed. In this study, we developed influenza vaccines using the Lumazine synthase (LS) platform. The primary objective of this study was to determine the protective potential of M2e proteins expressed on Lumazine synthase (LS) nanoparticles. M2e-LS proteins, produced through the E. coli system, spontaneously assemble into nanoparticles. The study investigated the efficacy of the M2e-LS nanoparticle vaccine in mice. Mice immunized with M2e-LS nanoparticles exhibited significantly higher levels of intracellular cytokines than those receiving soluble M2e proteins. The M2e-LS protein exhibited robust immunogenicity and provided 100% protection against cross-clade influenza.

Evaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants.

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.

Structure of Aquifex aeolicus lumazine synthase by cryo-electron microscopy to 1.42†Šresolution.

Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5 Šor better, which so far has only been achieved for the iron carrier ferritin. Using state-of-the-art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42 Šmap of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48 h microscope session. In addition to water molecules and ligands involved in the function of AaLS, we can observe positive density for ∼50% of the hydrogen atoms. A small improvement in the resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5 Šfor a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.

Protein Nanoparticles as Vaccine Platforms for Human and Zoonotic Viruses.

Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to these subunit vaccines. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present a repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterisation of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs, have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from human viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious viral diseases and enhancing vaccine efficacy and safety.

A rapid and sensitive fluorescent chromatography with cloud system for MPXV point-of-care diagnosis.

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation.

Design and Preclinical Evaluation of a Nanoparticle Vaccine against Respiratory Syncytial Virus Based on the Attachment Protein G.

Human respiratory syncytial virus (RSV) poses a significant human health threat, particularly to infants and the elderly. While efficacious vaccines based on the F protein have recently received market authorization, uncertainties remain regarding the future need for vaccine updates to counteract potential viral drift. The attachment protein G has long been ignored as a vaccine target due to perceived non-essentiality and ineffective neutralization on immortalized cells. Here, we show strong G-based neutralization in fully differentiated human airway epithelial cell (hAEC) cultures that is comparable to F-based neutralization. Next, we designed an RSV vaccine component based on the central conserved domain (CCD) of G fused to self-assembling lumazine synthase (LS) nanoparticles from the thermophile Aquifex aeolicus as a multivalent antigen presentation scaffold. These nanoparticles, characterized by high particle expression and assembly through the introduction of N-linked glycans, showed exceptional thermal and storage stability and elicited potent RSV neutralizing antibodies in a mouse model. In conclusion, our results emphasize the pivotal role of RSV G in the viral lifecycle and culminate in a promising next-generation RSV vaccine candidate characterized by excellent manufacturability and immunogenic properties. This candidate could function independently or synergistically with current F-based vaccines.

A Divalent Chikungunya and Zika Nanovaccine with Thermostable Self-Assembly Multivalent Scaffold LS-SUMO.

The convergence strategies of antigenic subunits and synthetic nanoparticle scaffold platform improve the vaccine production efficiency and enhance vaccine-induced immunogenicity. Selecting the appropriate nanoparticle scaffold is crucial to controlling target antigens immunologically. Lumazine synthase (LS) is an attractive candidate for a vaccine display system due to its thermostability, modification tolerance, and morphological plasticity. Here, the first development of a multivalent thermostable scaffold, LS-SUMO (SUMO, small ubiquitin-likemodifier), and a divalent nanovaccine covalently conjugated with Chikungunya virus E2 and Zika virus EDIII antigens, is reported. Compared with antigen monomers, LS-SUMO nanoparticle vaccines elicit a higher humoral response and neutralizing antibodies against both antigen targets in mouse sera. Mice immunized with LS-SUMO conjugates produce CD4+ T cell-mediated Th2-biased responses and promote humoral immunity. Importantly, LS-SUMO conjugates possess equivalent humoral immunogenicity after heat treatment. Taken together, LS-SUMO is a powerful biotargeting nanoplatform with high-yield production, thermal stability and opens a new avenue for multivalent presentation of various antigens.

Rapid and highly potent humoral responses to mpox nanovaccine candidates adjuvanted by thermostable scaffolds.

Monkeypox (mpox) is a zoonotic disease caused by monkeypox virus (MPXV) of the orthopoxvirus genus. The emergence and global spread of mpox in 2022 was declared as a public health emergency by World Health Organization. This mpox pandemic alarmed us that mpox still threaten global public health. Live vaccines could be used for immunization for this disease with side effects. New alternative vaccines are urgently needed for this re-emerging disease. Specific antibody responses play key roles for protection against MPXV, therefore, vaccines that induce high humoral immunity will be ideal candidates. In the present study, we developed thermostable nanovaccine candidates for mpox by conjugating MPXV antigens with thermostable nanoscafolds. Three MPXV protective antigens, L1, A29, and A33, and the thermostable Aquafex aeolicus lumazine synthase (AaLS), were expressed in E. coli and purified by Ni-NTA methods. The nanovaccines were generated by conjugation of the antigens with AaLS. Thermal stability test results showed that the nanovaccines remained unchanged after one week storage under 37℃ and only partial degradation under 60℃, indicating high thermostability. Very interesting, one dose immunization with the nanovaccine could induce high potent antibody responses, and two dose induced 2-month high titers of antibodes. In vitro virus neutralization test showed that nanovaccine candidates induced significantly higher levels of neutralization antibodies than monomers. These results indicated that the AaLS conjugation nanovaccines of MPXV antigens are highly thermostable in terms of storage and antigenic, being good alternative vaccine candidates for this re-emerging disease.

Potent immune responses against thermostable Foot-and-Mouth disease virus VP1 nanovaccine adjuvanted with polymeric thermostable scaffold.

Foot-and-mouth disease (FMD) is an acute zoonosis causes significant economic losses. Vaccines able to stimulate efficient protective immune responses are urgently needed. In this study, Escherichia coli-derived recombinant VP1 of serotype A and O FMD virus (FMDV) was conjugated to thermostable scaffold lumazine synthase (LS) or Quasibacillus thermotolerans encapsulin (QtEnc) using a robust plug-and-display SpyTag/SpyCatcher system to generate multimeric nanovaccines. These nanovaccines induced highly potent antibody responses in vaccinated mice. On day 14 after the first immunisation, antibody titres were approximately 100 times higher than those of monomer antigens. Both vaccines induced high and long-term IgG antibody production. Moreover, the QtEnc-VP1 nanovaccine induced higher antibody titres than the LS-VP1 nanovaccine. The nanovaccines also induced Th1-biased immune responses and higher levels of neutralising antibodies. These data indicated that FMDV nanovaccines generated by conjugating VP1 with a thermostable scaffold are highly immunogenic and ideal candidates for FMDV control in low-resource areas.

Inhibitors of riboflavin biosynthetic pathway enzymes as potential antibacterial drugs.

Multiple drug resistance is the main obstacle in the treatment of bacterial diseases. Resistance against antibiotics demands the exploration of new antimicrobial drug targets. A variety of in silico and genetic approaches show that the enzymes of the riboflavin biosynthetic pathway are crucial for the survival of bacteria. This pathway is absent in humans thus enzymes of the riboflavin biosynthetic pathway are emerging drug targets for resistant pathogenic bacterial strains. Exploring the structural details, their mechanism of action, intermediate elucidation, and interaction analysis would help in designing suitable inhibitors of these enzymes. The riboflavin biosynthetic pathway consists of seven distinct enzymes, namely, 3,4-dihydroxy-2-butanone 4-phosphate synthase, GTP cyclohydrolase II, pyrimidine deaminase/reductase, phosphatase, lumazine synthase, and riboflavin synthase. The present review summarizes the research work that has been carried out on these enzymes in terms of their structures, active site architectures, and molecular mechanism of catalysis. This review also walks through small molecule inhibitors that have been developed against several of these enzymes.

A self-assembled nanoparticle vaccine based on pseudorabies virus glycoprotein D induces potent protective immunity against pseudorabies virus infection.

Pseudorabies virus (PRV) mainly causes pseudorabies (PR) or Aujeszky's disease in pigs and can infect humans, raising public health concerns about zoonotic and interspecies transmission of PR. With the emergence of PRV variants in 2011, the classic attenuated PRV vaccine strains have failed to protect many swine herds against PR. Herein, we developed a self-assembled nanoparticle vaccine that induces potent protective immunity against PRV infection. PRV glycoprotein D (gD) was expressed using the baculovirus expression system and further presented on the lumazine synthase (LS) 60-meric protein scaffolds via the SpyTag003/SpyCatcher003 covalent coupling system. In mouse and piglet models, LSgD nanoparticles emulsified with the ISA 201VG adjuvant elicited robust humoral and cellular immune responses. Furthermore, LSgD nanoparticles provided effective protection against PRV infection and eliminated pathological symptoms in the brain and lungs. Collectively, the gD-based nanoparticle vaccine design appears to be a promising candidate for potent protection against PRV infection.

Protein-based Nanoparticle Vaccine Approaches Against Infectious Diseases.

The field of vaccine development has seen an increase in the number of rationally designed technologies that increase effectiveness against vaccine-resistant pathogens, while not compromising safety. Yet, there is still an urgent need to expand and further understand these platforms against complex pathogens that often evade protective responses. Nanoscale platforms have been at the center of new studies, especially in the wake of the coronavirus disease 2019 (COVID-19), with the aim of deploying safe and effective vaccines in a short time period. The intrinsic properties of protein-based nanoparticles, such as biocompatibility, flexible physicochemical characteristics, and variety have made them an attractive platform against different infectious disease agents. In the past decade, several studies have tested both lumazine synthase-, ferritin-, and albumin-based nanoplatforms against a wide range of complex pathogens in pre-clinical studies. Owed to their success in pre-clinical studies, several studies are undergoing human clinical trials or are near an initial phase. In this review we highlight the different protein-based platforms, mechanisms of synthesis, and effectiveness of these over the past decade. In addition, some challenges, and future directions to increase their effectiveness are also highlighted. Taken together, protein-based nanoscaffolds have proven to be an effective means to design rationally designed vaccines, especially against complex pathogens and emerging infectious diseases.

Genetically Encoded Self-Assembling Protein Nanoparticles for the Targeted Delivery In Vitro and In Vivo.

Targeted nanoparticles of different origins are considered as new-generation diagnostic and therapeutic tools. However, there are no targeted drug formulations within the composition of nanoparticles approved by the FDA for use in the clinic, which is associated with the insufficient effectiveness of the developed candidates, the difficulties of their biotechnological production, and inadequate batch-to-batch reproducibility. Targeted protein self-assembling nanoparticles circumvent this problem since proteins are encoded in DNA and the final protein product is produced in only one possible way. We believe that the combination of the endless biomedical potential of protein carriers as nanoparticles and the standardized protein purification protocols will make significant progress in "magic bullet" creation possible, bringing modern biomedicine to a new level. In this review, we are focused on the currently existing platforms for targeted self-assembling protein nanoparticles based on transferrin, lactoferrin, casein, lumazine synthase, albumin, ferritin, and encapsulin proteins, as well as on proteins from magnetosomes and virus-like particles. The applications of these self-assembling proteins for targeted delivery in vitro and in vivo are thoroughly discussed, including bioimaging applications and different therapeutic approaches, such as chemotherapy, gene delivery, and photodynamic and photothermal therapy. A critical assessment of these protein platforms' efficacy in biomedicine is provided and possible problems associated with their further development are described.

Improving cross-protection against influenza virus in mice using a nanoparticle vaccine of mini-HA.

This study aimed to investigate the protective effect of mini-hemagglutinin (mini-HA) proteins expressed on lumazine synthase (LS) nanoparticles against influenza. Soluble mini-HA proteins were assembled with LS proteins via SpyTag/SpyCatcher in vitro. The size of mini-HA-LS nanoparticles was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the effect of mini-HA-LS nano-vaccines was explored in mice. The results indicate that the diameter of mini-HA-LS nanoparticles was approximately 60-80 nm. The nanoparticles could induce stronger humoral and cellular immune responses and produce cross-clade protection against influenza in mice.

TRAIL & EGFR affibody dual-display on a protein nanoparticle synergistically suppresses tumor growth.

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer drug candidate because it selectively binds to the proapoptotic death receptors, which are frequently overexpressed in a wide range of cancer cells, subsequently inducing strong apoptosis in these cells. However, the therapeutic benefit of TRAIL has not been clearly proven, mainly because of its poor pharmacokinetic characteristics and frequent resistance to its application caused by the activation of a survival signal via the EGF/epidermal growth factor receptor (EGFR) signaling pathway. Here, a lumazine synthase protein cage nanoparticle isolated from Aquifex aeolicus (AaLS) was used as a multiple ligand-displaying nanoplatform to display polyvalently both TRAIL and EGFR binding affibody molecules (EGFRAfb) via a SpyTag/SpyCatcher protein-ligation system, to form AaLS/TRAIL/EGFRAfb. The dual-ligand-displaying AaLS/TRAIL/EGFRAfb exhibited a dramatically enhanced cytotoxicity on TRAIL-resistant and EGFR-overexpressing A431 cancer cells in vitro, effectively disrupting the EGF-mediated EGFR survival signaling pathway by blocking EGF/EGFR binding as well as strongly activating both the extrinsic and intrinsic apoptotic pathways synergistically. The AaLS/TRAIL/EGFRAfb selectively targeted A431 cancer cells in vitro and actively reached the tumor sites in vivo. The A431 tumor-bearing mice treated with AaLS/TRAIL/EGFRAfb exhibited a significant suppression of the tumor growth without any significant side effects. Collectively, these findings showed that the AaLS/TRAIL/EGFRAfb could be used as an effective protein-based therapeutic for treating EGFR-positive cancers, which are difficult to manage using mono-therapeutic approaches.

Transcription-dependent confined diffusion of enzymes within subcellular spaces of the bacterial cytoplasm.

BACKGROUND: Knowledge on the localization and mobility of enzymes inside bacterial cells is scarce, but important for understanding spatial regulation of metabolism. The four central enzymes (Rib enzymes) of the riboflavin (RF) biosynthesis pathway in the Gram positive model bacterium Bacillus subtilis have been studied extensively in vitro, especially the heavy RF synthase, a large protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which mediates substrate-channeling. However, little is known about the behavior and mobility of these enzymes in vivo. RESULTS: We have investigated the localization and diffusion of the Rib enzymes in the cytoplasm of B. subtilis. By characterizing the diffusion of Rib enzymes in live cells using single particle tracking (SPT) we provide evidence for confined diffusion at the cell poles and otherwise Brownian motion. A majority of RibH particles showed clear nucleoid occlusion and a high degree of confined motion, which is largely abolished after treatment with Rifampicin, revealing that confinement is dependent on active transcription. Contrarily, RibE is mostly diffusive within the cell, showing only 14% encapsulation by RibH nanocompartments. By localizing different diffusive populations within single cells, we find that fast diffusion occurs mostly across the nucleoids located in the cell centers, while the slower, confined subdiffusion occurs at the crowded cell poles. CONCLUSIONS: Our results provide evidence for locally different motion of active enzymes within the bacterial cytoplasm, setting up metabolic compartmentalization mostly at the poles of cells.

Development of a Modular Vaccine Platform for Multimeric Antigen Display Using an Orthobunyavirus Model.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

Comparative evaluation of the immunodominant proteins of Brucella abortus for the diagnosis of cattle brucellosis.

BACKGROUND AND AIM: The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis. MATERIALS AND METHODS: Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done. RESULTS: All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden's index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85). CONCLUSION: BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.

Second career of a biosynthetic enzyme: Lumazine synthase as a virus-like nanoparticle in vaccine development.

Naturally occurring and computationally ab initio designed protein cages can now be considered as extremely suitable materials for new developments in nanotechnology. Via self-assembly from single identical or non-identical protomers large oligomeric particles can be formed. Virus-like particles have today found a number of quite successful applications in the development of new vaccines. Complex chimeric nanoparticles can serve as suitable platforms for the presentation of natural or designed antigens to the immune system of the host. The scaffolds can be cage forming highly symmetric biological macromolecules like lumazine synthase or symmetric self-assembling virus-like particles generated by computational ab initio design. Symmetric nanoparticle carriers display a structurally ordered array of immunogens. This feature can lead to a more favorable interaction with B-cell receptors, in comparison to the administration of single recombinant immunogens. Several pre-clinical animal studies and clinical studies have recently pointed out the efficiency of nanoparticle antigens produced recombinantly in creating strong immune responses against infectious diseases like HIV, Malaria, Borrelia, Influenza.

Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.