Synergistic immune augmentation enabled by covalently conjugating TLR4 and NOD2 agonists.

Enhancing the efficacy of subunit vaccines relies significantly on the utilization of potent adjuvants, particularly those capable of triggering multiple immune pathways. To achieve synergistic immune augmentation by Toll-like receptor 4 agonist (TLR4a) and nucleotide-binding oligomerization-domain-containing protein 2 agonist (NOD2a), in this work, we conjugated RC529 (TLR4a) and MDP (NOD2a) to give RC529-MDP, and evaluated its adjuvanticity for OVA antigen. Compared to the unconjugated RC529+MDP, RC529-MDP remarkably enhanced innate immune responses with 6.8-fold increase in IL-6 cytokine, and promoted the maturation of antigen-presenting cells (APCs), possibly because of the conjugation of multiple agonists ensuring their delivery to the same cell and activation of various signaling pathways within that cell. Furthermore, RC529-MDP improved OVA-specific antibody response, T cells response and the memory T cells ratio relative to the unconjugated mixture. Therefore, covalently conjugating TLR4 agonist and NOD2 agonist was an effective strategy to enhance immune responses, providing the potential to design and develop more effective vaccines.

Effects of the covalent conjugation between caffeic acid and peanut allergen protein Ara h1 on the antigenicity and structure of Ara h1.

Ara h1 was the highest content of peanut allergen protein, identified as a biomarker of peanut allergen. In this study, Ara h1 was covalently complexed with caffeic acid (CA) to research the effects of covalent conjugation on the antigenicity and protein structural properties of Ara h1. After the covalent complexing of Ara h1 and CA, the IgG-binding capacity of Ara h1 was reduced compared with that of control Ara h1. Moreover, the structure of Ara h1 changed from ordered to disordered, the number of intermolecular hydrogen bonds decreased, and some hydrophobic groups were exposed or hydrophobic peptides were released. The carboxyl group in CA reacted with the amino group in Ara h1. The digestibility of Ara h1-CA was increased. The antigenicity of Ara h1-CA was undetectable after 30 min of digestion in vitro. These findings can serve as a reference for further research on hypoallergenic peanut products.

A Chemoselective Enrichment Strategy for In-Depth Coverage of the Methyllysine Proteome.

Proteomics is a powerful method to comprehensively understand cellular posttranslational modifications (PTMs). Owing to low abundance, tryptic peptides with PTMs are usually enriched for enhanced coverage by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Affinity chromatography for phosphoproteomes by metal-oxide and pan-specific antibodies for lysine acetylome allow identification of tens of thousands of modification sites. Lysine methylation is a significant PTM; however, only hundreds of methylation sites were identified by available approaches. Herein we report an aryl diazonium based chemoselective strategy that enables enrichment of monomethyllysine (Kme1) peptides through covalent bonds with extraordinary sensitivity. We identified more than 10000 Kme1 peptides from diverse cell lines and mouse tissues, which implied a wide lysine methylation impact on cellular processes. Furthermore, we found a significant amount of methyl marks that were not S-adenosyl methionine (SAM)-dependent by isotope labeling experiments.

How can biomaterial-conjugated antimicrobial peptides fight bacteria and be protected from degradation?

The emergence of antibiotic-resistant bacteria is a serious threat to public health. Antimicrobial peptides (AMP) are a powerful alternative to antibiotics due to their low propensity to induce bacterial resistance. However, cytotoxicity and short half-lives have limited their clinical translation. To overcome these problems, AMP conjugation has gained relevance in the biomaterials field. Nevertheless, few studies describe the influence of conjugation on enzymatic protection, mechanism of action and antimicrobial efficacy. This review addresses this gap by providing a detailed comparison between conjugated and soluble AMP. Additionally, commonly employed chemical reactions and factors to consider when promoting AMP conjugation are reviewed. The overall results suggested that AMP conjugated onto biomaterials are specifically protected from degradation by trypsin and/or pepsin. However, sometimes, their antimicrobial efficacy was reduced. Due to limited conformational freedom in conjugated AMP, compared to their soluble forms, they appear to act initially by creating small protuberances on bacterial membranes that may lead to the alteration of membrane potential and/or formation of holes, triggering cell death. Overall, AMP conjugation onto biomaterials is a promising strategy to fight infection, particularly associated to the use of medical devices. Nonetheless, some details need to be addressed before conjugated AMP reach clinical practice. STATEMENT OF SIGNIFICANCE: Covalent conjugation of antimicrobial peptides (AMP) has been one of the most widely used strategies by bioengineers, in an attempt to not only protect AMP from proteolytic degradation, but also to prolong their residence time at the target tissue. However, an explanation for the mode of action of conjugated AMP is still lacking. This review extensively gathers works on AMP conjugation and puts forward a mechanism of action for AMP when conjugated onto biomaterials. The implications of AMP conjugation on antimicrobial activity, cytotoxicity and resistance to proteases are all discussed. A thorough review of commonly employed chemical reactions for this conjugation is also provided. Finally, details that need to be addressed for conjugated AMP to reach clinical practice are discussed.

An Advanced Bioconjugation Strategy for Covalent Tethering of TGFß3 with Silk Fibroin Matrices and its Implications in the Chondrogenesis Profile of Human BMSCs and Human Chondrocytes: A Paradigm Shift in Cartilage Tissue Engineering.

The transforming growth factor-ß class of cytokines plays a significant role in articular cartilage formation from mesenchymal condensation to chondrogenic differentiation. However, their exogenous addition to the chondrogenic media makes the protocol expensive. It reduces the bioavailability of the cytokine to the cells owing to their burst release. The present study demonstrates an advanced bioconjugation strategy to conjugate transforming growth factor-ß3 (TGFß3) with silk fibroin matrix covalently via a cyanuric chloride coupling reaction. The tethering and change in secondary conformation are confirmed using various spectroscopic analyses. To assess the functionality of the chemically modified silk matrix, human bone marrow-derived mesenchymal stem cells (hBMSCs) and chondrocytes are cultured for 28 days in a chondrogenic differentiation medium. Gene expression and histological analysis reveal enhanced expression of chondrogenic markers with intense Safranin-O and Alcian Blue staining in TGFß3 conjugated silk matrices than where TGFß3 is exogenously added to the media for both hBMSCs and chondrocytes. Therefore, this study successfully recapitulates the native niche of TGFß3 and the role of the silk as a growth factor stabilizer. When cultured over TGFß3 conjugated silk matrices, hBMSCs display increased proteoglycan secretion and maximum chondrogenic trait with attenuation of chondrocyte hypertrophy over human chondrocytes.

Covalent conjugation with polyphenol reduced the sensitization of walnut and ameliorated allergy by enhancing intestinal epithelial barrier in mice.

In order to reduce the sensitization of walnut protein (WP), the effects of the interaction between WP and (-)-Epigallocatechin gallate (EGCG), quercetin, trans-ferulic acid, and resveratrol were investigated. Covalent and non-covalent conjugations were compared. The results suggested that covalent conjugation reduced the free amino acid content, sulfhydryl content, and surface hydrophobicity. When compared to non-covalent conjugation, covalent modification showed a lower IgE binding capacity, accompanied by changes in protein conformation. Moreover, animal experiments revealed that there were up-regulation of transforming growth factor-ß, T-box expressed in t cells, and forkhead transcription factor Foxp3 mRNA expression, and down-regulation of IL-4, IL-17, GATA binding protein 3 and retinoid-related orphan nuclear receptor γt mRNA expression in the conjugate groups. These results suggested that covalent conjugation of polyphenols, especially EGCG, likely ameliorated allergy by promoting Th1/Th2 and Treg/Th17 balance and alleviating allergy-induced intestinal barrier damage, which might be a support in reducing the allergenicity of WP.

Defined covalent attachment of three cancer drugs to DNA origami increases cytotoxicity at nanomolar concentration.

DNA nanostructures have captured great interest as drug delivery vehicles for cancer therapy. Despite rapid progress in the field, some hurdles, such as low cellular uptake, low tissue specificity or ambiguous drug loading, remain unsolved. Herein, well-known antitumor drugs (doxorubicin, auristatin, and floxuridine) were site-specifically incorporated into DNA nanostructures, demonstrating the potential advantages of covalently linking drug molecules via structural staples instead of incorporating the drugs by noncovalent binding interactions. The covalent strategy avoids critical issues such as an unknown number of drug-DNA binding events and premature drug release. Moreover, covalently modified origami offers the possibility of precisely incorporating several synergetic antitumor drugs into the DNA nanostructure at a predefined molar ratio and to control the exact spatial orientation of drugs into DNA origami. Additionally, DNA-based nanoscaffolds have been reported to have a low intracellular uptake. Thus, two cellular uptake enhancing mechanisms were studied: the introduction of folate units covalently linked to DNA origami and the transfection of DNA origami with Lipofectamine. Importantly, both methods increased the internalization of DNA origami into HTB38 and HCC2998 colorectal cancer cells and produced greater cytotoxic activity when the DNA origami incorporated antiproliferative drugs. The results here present a successful and conceptually distinct approach for the development of DNA-based nanostructures as drug delivery vehicles, which can be considered an important step towards the development of highly precise nanomedicines.

Covalent modification of soy protein hydrolysates by EGCG: Improves the emulsifying and antioxidant properties.

In this study, the effect of EGCG conjugation on the emulsifying and antioxidant properties of SPHs was investigated to improve the functional characteristic of soy protein hydrolysates (SPHs) and develop a novel hydrolysates/peptides-EGCG conjugates. Enzymatic hydrolyzed SPHs (DH 5%, 8%, 10%) covalent with 1% EGCG to prepare conjugates at pH 9.0. The free amino group and tryptophan content of SPHs-EGCG conjugates significantly decreased, indicating the successful preparation of SPHs-EGCG conjugates. Additionally, 5% SPHs-EGCG conjugates showed the highest EGCG binding capacity. EGCG conjugation increased the particle sizes and charge of SPHs. Compared with non-covalent SPHs, the covalent modification of EGCG increased the emulsifying and antioxidant capacity, especially for 5% SPHs-EGCG, it exhibited much higher surface hydrophobicity, ESI (emulsifying stability index), EAI (emulsifying activity index), and antioxidant activity than others. This result revealed that SPHs and EGCG played a synergistic effect in improving the emulsifying and antioxidant capacity. Fluorescence spectroscopy analysis showed that the combination of EGCG conjugation significantly decreased the fluorescence intensity and caused maximum emission red-shift. The formation of a covalent bond between SPHs and EGCG was verified through Fourier transform infrared spectroscopy (FTIR), and the results also showed a significant increase in the α-helix and random coil contents of the conjugation, and a significant decrease in the ß-sheet and ß-turn contents. These results indicate that EGCG conjugation with SPHs induced the unfolding and stretching of protein flexibility. Overall, SPHs-EGCG conjugates can be applied as a promising emulsifier to fabricate emulsion systems and would be helpful in designing functional beverages containing polyphenols and peptides with enhanced functional nutritional properties.

Synergistically Enhancing Tumor Chemotherapy Using an Aggregation-Induced Emission Photosensitizer on Covalently Conjugated Molecularly Imprinted Polymer Nanoparticles.

Due to the polygenic and heterogeneous nature of the tumorigenesis process, traditional chemotherapy is far from desirable. Fabricating multifunctional nanoplatforms integrating photodynamic effect can synergistically enhance chemotherapy because they can make the cancer cells much sensitive to chemotherapeutics. However, how to assemble different units in nanoplatforms and minimize side effects caused by chemodrugs and photosensitizers (PSs) still needs to be explored. Herein, a nanoplatform CPP/PS-MIP@DOX is developed using a simultaneously covalently conjugated new aggregation-induced emission (AIE) PS and a cell-penetrating peptide (CPP) on the surface of silica-based molecularly imprinted polymer (MIP) nanoparticles, prepared with doxorubicin (DOX) as the template in the water system via a sol-gel technique. CPP/PS-MIP@DOX has good biocompatibility, high DOX-loading ability, promoted cellular uptake, and sustained and pH-sensitive drug release capability. Furthermore, it can efficiently penetrate into tumor tissue, accurately home to, and accumulate at the tumor site. As a result, a better efficacy with lower cytotoxicity is achieved with a smaller dosage of DOX by utilizing either the photodynamic effect or unique characteristics of the MIP. It is the first nanoplatform fabricated by chemically conjugating AIE PSs directly on the surface of the scaffold via the surface-decorated strategy and successfully applied in cancer therapy. This work provides an effective strategy by constructing AIE PS-based cancer nanomedicines with MIPs as scaffolds.

Dendrimer-siRNA Conjugates for Targeted Intracellular Delivery in Glioblastoma Animal Models.

Small interfering RNAs (siRNAs) are potent weapons for gene silencing, with an opportunity to correct defective genes and stop the production of undesirable proteins, with many applications in central nervous system (CNS) disorders. However, successful delivery of siRNAs to the brain parenchyma faces obstacles such as the blood-brain barrier (BBB), brain tissue penetration, and targeting of specific cells. In addition, siRNAs are unstable under physiological conditions and are susceptible to protein binding and enzymatic degradation, necessitating a higher dosage to remain effective. To address these issues and advance siRNA delivery, we report the development of covalently conjugated hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimer-siRNA conjugates, demonstrated with a siRNA against GFP (siGFP) conjugate (D-siGFP) utilizing glutathione-sensitive linkers. This allows for precise nucleic acid loading, protects the payload from premature degradation, delivers the siRNA cargo into cells, and achieves significant GFP knockdown in vitro (∼40%) and in vivo (∼30%). Compared to commercially available delivery systems such as RNAi Max and Lipofectamine, D-siGFP retains the potency of the siRNA in vitro. In addition, the dendrimer-siGFP conjugate significantly enhances the half-life of siRNA in the presence of plasma and endonucleases and maintains the passive targeting ability of PAMAM dendrimers to reactive microglia. When administered intratumorally to orthotopic glioblastoma multiform tumors (GBM) in CX3CR-1GFP mice, D-siGFP localizes in tumor-associated macrophages (TAMs) within the tumor parenchyma, minimizing off-target effects in other cell populations. The facile conjugation strategy for dendrimer-siRNA conjugates presented here offers a promising approach for targeted, systemic intracellular delivery of siRNA, serving as a potential bridge for the clinical translation of RNAi therapies.

Cell-penetrating peptide for targeted macromolecule delivery into plant chloroplasts.

Reports on chloroplast-targeted protein delivery using cell-penetrating peptides are scarce. In this study, a novel peptide-based macromolecule delivery strategy targeting chloroplasts was successfully developed in wheat mesophyll protoplasts. A peptide derived from the signal sequence of the chloroplast-targeted protein of ferredoxin-thioredoxin reductase catalytic chain of Spinacia oleracea with UniProtKB Id-P41348 exhibits properties of cellular internalization. DNase I was efficiently delivered into the chloroplast using 10 µM cTP with an efficiency of more than 90%. This cell-penetrating peptide-mediated approach offers various advantages over the existing chloroplast targeting methods, such as non-invasiveness, biocompatibility, low-toxicity, and target-specific delivery. The present study shows that peptide-based strategies hold tremendous potential in the field of chloroplast biotechnology. KEY POINTS: • Screening of database of chloroplast targeting peptides in order to develop an efficient cell-penetrating peptide termed as cTP. • cTP efficiently crosses the cell barrier and demonstrated chloroplast-localization. • cTP can be incorporated as a promising strategy for delivering macromolecules for crop improvement.

Selective Delivery of Clinically Approved Tubulin Binding Agents through Covalent Conjugation to an Active Targeting Moiety.

The efficacy and tolerability of tubulin binding agents are hampered by their low specificity for cancer cells like most clinically used anticancer agents. To improve specificity, tubulin binding agents have been covalently conjugated to agents that target cancer cells to give actively targeted drug conjugates. These conjugates are designed to increase uptake of the drug by cancer cells while having limited uptake by normal cells, thereby improving efficacy and tolerability. Approaches used include an attachment to small molecules, polysaccharides, peptides, proteins, and antibodies that exploit the overexpression of receptors for these substances. Antibody targeted strategies have been the most successful to date, with six such examples having gained clinical approval. Many other conjugate types, especially those targeting the folate receptor, have shown promising efficacy and toxicity profiles in pre-clinical models and in early-stage clinical studies. Presented herein is a discussion of the success or otherwise of the recent strategies used to form these actively targeted conjugates.

Diamine Oxidase-Conjugated Multiwalled Carbon Nanotubes to Facilitate Electrode Surface Homogeneity.

Carbon nanomaterials have gained significant interest over recent years in the field of electrochemistry, and they may be limited in their use due to issues with their difficulty in dispersion. Enzymes are prime components for detecting biological molecules and enabling electrochemical interactions, but they may also enhance multiwalled carbon nanotube (MWCNT) dispersion. This study evaluated a MWCNT and diamine oxidase enzyme (DAO)-functionalised screen-printed electrode (SPE) to demonstrate improved methods of MWCNT functionalisation and dispersion. MWCNT morphology and dispersion was determined using UV-Vis spectroscopy (UV-Vis) and scanning electron microscopy (SEM). Carboxyl groups were introduced onto the MWCNT surfaces using acid etching. MWCNT functionalisation was carried out using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS), followed by DAO conjugation and glutaraldehyde (GA) crosslinking. Modified C-MWNCT/EDC-NHS/DAO/GA was drop cast onto SPEs. Modified and unmodified electrodes after MWCNT functionalisation were characterised using optical profilometry (roughness), water contact angle measurements (wettability), Raman spectroscopy and energy dispersive X-ray spectroscopy (EDX) (vibrational modes and elemental composition, respectively). The results demonstrated that the addition of the DAO improved MWCNT homogenous dispersion and the solution demonstrated enhanced stability which remained over two days. Drop casting of C-MWCNT/EDC-NHS/DAO/GA onto carbon screen-printed electrodes increased the surface roughness and wettability. UV-Vis, SEM, Raman and EDX analysis determined the presence of carboxylated MWCNT variants from their non-carboxylated counterparts. Electrochemical analysis demonstrated an efficient electron transfer rate process and a diffusion-controlled redox process. The modification of such electrodes may be utilised for the development of biosensors which could be utilised to support a range of healthcare related fields.

A new method to reduce allergenicity by improving the functional properties of soybean 7S protein through covalent modification with polyphenols.

The 7S fraction contains several major allergens of soybean protein. Here, the effects of covalent modification by chlorogenic acid (CHA) and (-)-epigallo-catechin 3-gallate (EGCG) on the allergenicity and functional properties of soybean 7S protein were investigated. Conjugation with EGCG and CHA resulted in the formation of cross-linked protein polymers and changes to the structures of the protein, which might mask or destroy the epitopes on it. In vitro analysis revealed that modification by polyphenols noticeably reduced IgE binding activity and histamine release. In vivo analysis showed that modification led to milder anaphylactic shock symptoms and minor damage of the intestine in mice, with reducing IgG, IgE, IgG1, mMCP-1, and histamine levels. The allergic response was also suppressed by the repression of IFN-γ, IL-4, and IL-5 and the up-regulation of IL-10 and TGF-ß in the conjugate groups. Furthermore, modification enhanced antioxidant, emulsion, foaming capacity, and foam stability of the protein.

Chitosan Functionalization: Covalent and Non-Covalent Interactions and Their Characterization.

Chitosan (CS) is a natural biopolymer that has gained great interest in many research fields due to its promising biocompatibility, biodegradability, and favorable mechanical properties. The versatility of this low-cost polymer allows for a variety of chemical modifications via covalent conjugation and non-covalent interactions, which are designed to further improve the properties of interest. This review aims at presenting the broad range of functionalization strategies reported over the last five years to reflect the state-of-the art of CS derivatization. We start by describing covalent modifications performed on the CS backbone, followed by non-covalent CS modifications involving small molecules, proteins, and metal adjuvants. An overview of CS-based systems involving both covalent and electrostatic modification patterns is then presented. Finally, a special focus will be given on the characterization techniques commonly used to qualify the composition and physical properties of CS derivatives.

Bioconjugation of Peptides to Hybrid Gold Nanoparticles.

Gold nanoparticles (AuNPs) can be produced by well-assessed synthesis methods and can show a high surface area-to-volume ratio, chemical inertness, high electron density, strong optical absorption as well as low toxicity. AuNPs have been conjugated with many different biomolecules for a wide range of biomedical applications. These applications require an increasingly complex level of surface decoration in order to achieve stability, efficacy, and specific functionalities. This chapter provides detailed instructions about the synthesis of AuNPs and bioconjugation strategies in order to obtain stable hybrid nanomaterials. The described biofunctionalization procedures are based on carbodiimide chemistry and ligand-exchange methods allowing the conjugation of Lys-peptide or Cys-peptide, respectively, to the AuNPs surface.

Structural Deformation of MTX Induced by Nanodrug Conjugation Dictate Intracellular Drug Transport and Drug Efficacy.

BACKGROUND: Understanding structural interactions between the active drug and conjugated nanoparticles is critical for optimizing intracellular drug transport and for increasing nano drug efficacy. In this regard, analyzing the conformational deformation of conjugated drugs surrounding nanoparticles is essential to understand the corresponding nanodrug efficacy. PURPOSE: The objective of this study is to present an optimal synthesis method for efficient drug delivery through a clear structural analysis of nanodrugs according to the type of conjugation. METHODS AND RESULTS: In this study, the structural variation of methotrexate (MTX) surrounding carbon nanotubes, depending on the type of conjugation style, such as covalent and non-covalent (PEGylation) bonds, was investigated. Specifically, covalent bonds of MTX surrounding CNTs induced greater structural deformation compared to non-covalent bonds (ie, PEGylated CNT). CONCLUSION: Greater changes in the structural variations of MTX analyzed by nuclear magnetic resonance (NMR) significantly improved the anti-inflammatory drug efficacy of human fibroblast-like synovial cells (FLS) via stable drug release in the extracellular environment and burst drug release under intracellular conditions.

Conjugation of haloalkane dehalogenase DhaA with arabinogalactan to increase its stability.

Haloalkane dehalogenase DhaA can catalyze the hydrolytic cleavage of carbonhalogen bonds, along with production of the corresponding alcohol, a proton and a halide. However, DhaA suffers from poor environmental tolerance, such as sensitivity to high temperature, low pH and hypersaline. Arabinogalactan (AG) is a hydrophilic polysaccharide with highly branched long chains. DhaA was conjugated with AG to improve the environmental stability of DhaA in the present study. Each DhaA was averagely conjugated with 4∼5 AG molecules. Conjugation of AG essentially maintained the enzymatic activity of DhaA (91.4 %) without apparent structural alteration. The hydration layer formed by AG could reduce the solvent accessible area of DhaA and slow the protonation process, thereby improving the pH and high salt stability of DhaA. In particular, the remaining activities of the conjugate (AG-DhaA) were 35.3 % after treatment at pH4.0 for 1 h, and 80.8 % in 1 M NaCl after treatment for 16 h. As compared with DhaA, AG-DhaA showed slightly different kinetic parameters (K M of 1.90 µmol/L and k cat of 2.60 s -1).

Data on the uptake of CpG-loaded amino-dextran nanoparticles by antigen-presenting cells.

Cytosine-phosphate-guanine (CpG) oligonucleotides are commonly-used vaccine adjuvants to promote the activation of antigen-presenting cells (APCs). To mount an effective immune response, CpG needs to be internalized and bind to its endosomal Toll-like receptor 9 (TLR-9) inside the APCs. Using flow cytometry and fluorescence microscopy, this article presents the cellular uptake data of the amino-dextran nanoparticle (aDNP) and aDNP loaded with CpG immobilized on its surface by either electrostatic adsorption or covalent conjugation. The uptake of fluorescently-labelled aDNPs by murine splenic dendritic cells and macrophages was determined by flow cytometry and uptake by murine bone-marrow-derived dendritic cells was evaluated by fluorescence microscopy. The data presented in this paper correlates with the in vitro immune-stimulatory activity observed for the two different CpG loading methods in the research article "Nanoparticle system based on amino-dextran as a drug delivery vehicle: immune-stimulatory CpG-oligonucleotide loading and delivery" (Nguyen et al., 2020) [1]. The data provide experimental evidence for a better understanding how the nanoparticle surface loading method of CpG influences the uptake of these nanoparticles by antigen-presenting cells as a step guide in the design of more effective vaccine formulations.

Rapid Development of SARS-CoV-2 Spike Protein Receptor-Binding Domain Self-Assembled Nanoparticle Vaccine Candidates.

The coronavirus disease pandemic of 2019 (COVID-19) caused by the novel SARS-CoV-2 coronavirus resulted in economic losses and threatened human health worldwide. The pandemic highlights an urgent need for a stable, easily produced, and effective vaccine. SARS-CoV-2 uses the spike protein receptor-binding domain (RBD) to bind its cognate receptor, angiotensin-converting enzyme 2 (ACE2), and initiate membrane fusion. Thus, the RBD is an ideal target for vaccine development. In this study, we designed three different RBD-conjugated nanoparticle vaccine candidates, namely, RBD-Ferritin (24-mer), RBD-mi3 (60-mer), and RBD-I53-50 (120-mer), via covalent conjugation using the SpyTag-SpyCatcher system. When mice were immunized with the RBD-conjugated nanoparticles (NPs) in conjunction with the AddaVax or Sigma Adjuvant System, the resulting antisera exhibited 8- to 120-fold greater neutralizing activity against both a pseudovirus and the authentic virus than those of mice immunized with monomeric RBD. Most importantly, sera from mice immunized with RBD-conjugated NPs more efficiently blocked the binding of RBD to ACE2 in vitro, further corroborating the promising immunization effect. Additionally, the vaccine has distinct advantages in terms of a relatively simple scale-up and flexible assembly. These results illustrate that the SARS-CoV-2 RBD-conjugated nanoparticles developed in this study are a competitive vaccine candidate and that the carrier nanoparticles could be adopted as a universal platform for a future vaccine development.