Novel protein ligase based on dual split intein.

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase.

Real-Time, Non-Invasive Monitoring of Neuronal Differentiation Using Intein-Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell-Based Biosensors.

Real-time and non-invasive monitoring of neuronal differentiation will help increase our understanding of neuronal development and help develop regenerative stem cell therapies for neurodegenerative diseases. Traditionally, reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence (IF) staining have been widely used to investigate stem cell differentiation; however, their limitations include endpoint analysis, invasive nature of monitoring, and lack of single-cell-level resolution. Several limitations hamper current approaches to studying neural stem cell (NSC) differentiation. In particular, fixation and staining procedures can introduce artificial changes in cellular morphology, hindering our ability to accurately monitor the progression of the process and fully understand its functional aspects, particularly those related to cellular connectivity and neural network formation. Herein, we report a novel approach to monitor neuronal differentiation of NSCs non-invasively in real-time using cell-based biosensors (CBBs). Our research efforts focused on utilizing intein-mediated protein engineering to design and construct a highly sensitive biosensor capable of detecting a biomarker of neuronal differentiation, hippocalcin. Hippocalcin is a critical protein involved in neurogenesis, and the CBB functions by translocating a fluorescence signal to report the presence of hippocalcin externally. To construct the hippocalcin sensor proteins, hippocalcin bioreceptors, AP2 and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2), were fused to each split-intein carrying split-nuclear localization signal (NLS) peptides, respectively, and a fluorescent protein was introduced as a reporter. Protein splicing (PS) was triggered in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The stem cell-based biosensor showed fluorescence signal translocation only upon neuronal differentiation. Undifferentiated stem cells or cells that had differentiated into astrocytes or oligodendrocytes did not show fluorescence signal translocation. The number of differentiated neurons was consistent with that measured by conventional IF staining. Furthermore, this approach allowed for the monitoring of neuronal differentiation at an earlier stage than that detected using conventional approaches, and the translocation of fluorescence signal was monitored before the noticeable expression of class III ß-tubulin (TuJ1), an early neuronal differentiation marker. We believe that these novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single-cell level and by providing a tool to evaluate how NSCs efficiently differentiate into specific cell types, particularly neurons.

Hypoxia-Inducible Factor-1α-Activated Protein Switch Based on Allosteric Self-Splicing Reduces Nonspecific Cytotoxicity of Pharmaceutical Drugs.

Protein-based therapeutic agents currently used for targeted tumor therapy exhibit limited penetrability, nonspecific toxicity, and a short circulation half-life. Although targeting cell surface receptors improves cancer selectivity, the receptors are also slightly expressed in normal cells; consequently, the nonspecific toxicity of recombinant protein-based therapeutic agents has not been eliminated. In this study, an allosteric-regulated protein switch was designed that achieved cytoplasmic reorganization of engineered immunotoxins in tumor cells via interactions between allosteric self-splicing elements and cancer markers. It can target the accumulated HIF-1α in hypoxic cancer cells and undergo allosteric activation, and the splicing products were present in hypoxic cancer cells but were absent in normoxic cells, selectively killing tumor cells and reducing nonspecific toxicity to normal cells. The engineered pro-protein provides a platform for targeted therapy of tumors while offering a novel universal strategy for combining the activation of therapeutic functions with specific cancer markers. The allosteric self-splicing element is a powerful tool that significantly reduces the nonspecific cytotoxicity of therapeutic proteins.

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer.

The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively.

Conditional protein splicing triggered by SUMO protease.

Conditional protein splicing is a powerful biotechnological tool that can be used to post-translationally control the activity of target proteins. Here we demonstrated a novel conditional protein splicing approach in which the small ubiquitin-like modifier (SUMO) protease induced the splicing of an atypical split intein. The engineered Ter DnaE-3 S11 split intein which has a small C-intein segment with only 6 amino acids was used in this study. A SUMO tag was fused to the N-terminus of the C-intein to inhibit the protein trans-splicing in vitro. The splicing products could be detected in 15 min with the addition of SUMO protease by western blotting and the splicing efficiency was ∼4-fold higher than the control without SUMO protease for overnight reaction. This engineered Ter DnaE-3 S11 split intein-mediated protein trans-splicing had been further shown to be triggered by SUMO protease in different exteins in vitro. Our study provides new insights into the regulation of protein splicing and is a promising tool for the control of protein structure and function in vitro.

Harnessing Split-Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells.

Biochemical studies of integral membrane proteins are often hampered by low purification yields and technical limitations such as aggregation causing in vitro manipulations to be challenging. The ability of controlling proteins in live cells bypasses these limitations while broadening the scope of accessible questions owing to the proteins being in their native environment. Here we take advantage of the intein biorthogonality to mammalian systems, site specificity, fast kinetics, and auto-processing nature as an attractive option for modifying surface proteins. Using EGFR as a model, we demonstrate that the split-intein pair AvaN /NpuC can be used to efficiently and specifically modify target membrane proteins with a synthetic adduct for downstream live cell application.

Use of the redox-dependent intein system for enhancing production of the cyclic green fluorescent protein.

To expand the reported redox-dependent intein system application, in this work, we used the split intein variant with highly trans-splicing efficiency and minimal extein dependence to cyclize the green fluorescent protein variant reporter in vitro. The CPG residues were introduced adjacent to the intein's catalytic cysteine for reversible formation of a disulfide bond to retard the trans-splicing reaction under the oxidative environment. The cyclized reporter protein in Escherichia coli cells was easily prepared by organic extraction and identified by the exopeptidase digestion. The amounts of extracted cyclized protein reporter in BL21 (DE3) cells were higher than those in hyperoxic SHuffle T7 coexpression system for facilitating the disulfide bond formation. The double His6-tagged precursor was purified for in vitro cyclization of the protein for 3 h. Compared with the purified linear counterpart, the cyclic reporter showed about twofold increase in fluorescence intensity, exhibited thermal and hydrolytic stability, and displayed better folding efficiency in BL21 (DE3) cells at the elevated temperature. Taken together, the developed redox-dependent intein system will be used for producing other cyclic disulfide-free proteins. The cyclic reporter is a potential candidate applied in certain thermophilic aerobes.

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins.

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC ) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (µM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.

Kinetics study of the natural split Npu DnaE intein in the generation of bispecific IgG antibodies.

Rapid and efficient bispecific antibody (BsAb) production for industrial applications is still facing many challenges. We reported a technology platform for generating bispecific IgG antibodies, "Bispecific Antibody by Protein Trans-splicing (BAPTS)." While the "BAPTS" method has shown potential in high-throughput screening of BsAbs, further understanding and optimizing the methodology is desirable. A large number of BsAbs were selected to illustrate the conversion efficiency and kinetics parameters. The temperature of reaction makes no significant influence in conversion efficiency, which can reach more than 70% within 2 h, and CD3 × HER2 BsAb can reach 90%. By fitting trans-splicing reaction to single-component exponential decay curves, the apparent first-order rate constants at a series of temperatures were determined. The rate constant ranges from 0.02 to 0.11 min-1 at 37 °C, which is a high rate reported for the protein trans-splicing reaction (PTS). The reaction process is activated rapidly with activation energy of 8.9 kcal·mol-1 (CD3 × HER2) and 5.2 kcal·mol-1 (CD3 × EGFR). The BsAbs generated by "BAPTS" technology not only had the similar post-translation modifications to the parental antibodies, but also demonstrated excellent in vitro and in vivo bioactivity. The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach. KEY POINTS: • The trans-splicing reaction of Npu DnaE intein in "BAPTS" platform is a rapid process with low reaction activation and high rate. • The BsAb generated by "BAPTS" remained effective in tumor cell killing. • The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach.

Strategies for all-at-once and stepwise selection of cells with multiple genetic manipulations.

The genetic manipulation of cells followed by their selection is indispensable for cell biological research. Although antibiotics-resistant genes are commonly used as selection markers, optimization of the condition for each selective agent is required. Here we utilized split-inteins and the drug-selectable marker puromycin N-acetyltransferase (PAC) to develop a system that enables the selection of cells simultaneously or sequentially transfected with multiple genetic constructs, using only puromycin. The active PAC enzyme was reconstituted by intein-mediated trans-splicing at several inherent or engineered serine/cysteine residues. Multiple splitting and reconstitution of active PAC was readily achieved by selecting optimum division sites based on the cellular tolerance to various puromycin concentrations. To achieve the stepwise selection method, PAC-intein fragments were transduced into cells using a virus-like particle (VLP) composed of HIV-1 gag-pol and VSV-G. The PAC-intein-VLP successfully conferred sufficient PAC activity for puromycin selection, which was quickly diminished in the absence of the VLP. Our findings demonstrate a versatile strategy for establishing markers for all-at-once or stepwise selection of multiple genetic manipulations, which will be useful in many fields of biology.

Treatment of a Mouse Model of ALS by In Vivo Base Editing.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Although ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. In this study, we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function, and up to 40% fewer SOD1 immunoreactive inclusions at end-stage mice compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.

Engineering of a Peptide α-N-Methyltransferase to Methylate Non-Proteinogenic Amino Acids.

Introduction of α-N-methylated non-proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre-methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α-N-automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C-terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non-proteinogenic amino acids has not been addressed. An engineered OphMA, intein-mediated protein ligation and solid-phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non-natural amides. This approach may have application in the biotechnological production of therapeutic peptides.

Therapeutic applications of trans-splicing.

BACKGROUND: RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein. SOURCES OF DATA: Comprehensive literature search of published research papers and reviews using Pubmed. AREAS OF AGREEMENT: Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction. AREAS OF CONTROVERSY: Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge. GROWING POINTS: Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies. AREAS TIMELY FOR DEVELOPING RESEARCH: Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.

Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies.

High product purity, preserving natural IgG architecture, and excellent production efficiency are highly desirable in bispecific antibody manufacturing. We have reported a platform called Bispecific Antibody by Protein Trans-Splicing (BAPTS) to synthesize BsAbs with natural human IgG structure and no chain mispairing. In the method, two antibody fragments carrying different target-specificities are separately expressed in mammalian cells and subsequently fused to form BsAbs by utilizing the trans-splicing property of the split intein Npu DnaE. The hinge region of antibody, a region with less functional impact, is selected for conjugating the two fragments. The method involves the following steps: (i) constructing five plasmids coding antibody components; (ii) separately expressing and purifying two antibody fragments A and B. Fragment A contains one Fab, "Knobs-into-Holes" mutations in the CH3 domain and NPU DnaEC. Fragment B contains another Fab and NPU DnaEN; (iii) mixing of fragments A and B under permissive reducing conditions in vitro to enable trans-splicing reaction; (iv) removing the reductant to allow re-oxidation of disulfide bonds; (v) isolating BsAb product from unreacted precursors by affinity chromatography. The method allows correct assembly of two heavy and two light chains to form bispecific IgG antibodies in natural structure with no synthetic linkers. No chain mispairing was observed in the product by UPLC-MASS. In addition, the observed kinetics and low reaction activation energy confirmed that the trans-splicing is thermodynamically favored reaction. The BAPTS technology is feasible for industrial applications.

An off-the-Shelf Approach for the Production of Fc Fusion Proteins by Protein Trans-Splicing towards Generating a Lectibody In Vitro.

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a "lectibody". Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

In Cellulo Protein Semi-Synthesis from Endogenous and Exogenous Fragments Using the Ultra-Fast Split Gp41-1 Intein.

Protein semi-synthesis inside live cells from exogenous and endogenous parts offers unique possibilities for studying proteins in their native context. Split-intein-mediated protein trans-splicing is predestined for such endeavors and has seen some successes, but a much larger variety of established split inteins and associated protocols is urgently needed. We characterized the association and splicing parameters of the Gp41-1 split intein, which favorably revealed a nanomolar affinity between the intein fragments combined with the exceptionally fast splicing rate. Following bead-loading of a chemically modified intein fragment precursor into live mammalian cells, we fluorescently labeled target proteins on their N- and C-termini with short peptide tags, thus ensuring minimal perturbation of their structure and function. In combination with a nuclear-entrapment strategy to minimize cytosolic fluorescence background, we applied our technique for super-resolution imaging and single-particle tracking of the outer mitochondrial protein Tom20 in HeLa cells.

In-cell production of a genetically-encoded library based on the θ-defensin RTD-1 using a bacterial expression system.

We report the high-yield heterologous expression of bioactive θ-defensin RTD-1 inside Escherichia coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. RTD-1 is a small backbone-cyclized polypeptide with three disulfide bridges and a natural inhibitor of anthrax lethal factor protease. Recombinant RTD-1 was natively folded and able to inhibit anthrax lethal factor protease. In-cell expression of RTD-1 was very efficient and yielded ≈0.7mg of folded RTD-1 per gram of wet E. coli cells. This approach was used to generate of a genetically-encoded RTD-1-based peptide library in live E. coli cells. These results clearly demonstrate the possibility of using genetically-encoded RTD-1-based peptide libraries in live E. coli cells, which is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide RTD-1asa molecular scaffold.

Supratrigeminal Bilaterally Projecting Neurons Maintain Basal Tone and Enable Bilateral Phasic Activation of Jaw-Closing Muscles.

UNLABELLED: Anatomical studies have identified brainstem neurons that project bilaterally to left and right oromotor pools, which could potentially mediate bilateral muscle coordination. We use retrograde lentiviruses combined with a split-intein-mediated split-Cre-recombinase system in mice to isolate, characterize, and manipulate a population of neurons projecting to both the left and right jaw-closing trigeminal motoneurons. We find that these bilaterally projecting premotor neurons (BPNs) reside primarily in the supratrigeminal nucleus (SupV) and the parvicellular and intermediate reticular regions dorsal to the facial motor nucleus. These BPNs also project to multiple midbrain and brainstem targets implicated in orofacial sensorimotor control, and consist of a mix of glutamatergic, GABAergic, and glycinergic neurons, which can drive both excitatory and inhibitory inputs to trigeminal motoneurons when optogenetically activated in slice. Silencing BPNs with tetanus toxin light chain (TeNT) increases bilateral masseter activation during chewing, an effect driven by the expression of TeNT in SupV BPNs. Acute unilateral optogenetic inhibition of SupV BPNs identifies a group of tonically active neurons that function to lower masseter muscle tone, whereas unilateral optogenetic activation of SupV BPNs is sufficient to induce bilateral masseter activation both during resting state and during chewing. These results provide evidence for SupV BPNs in tonically modulating jaw-closing muscle tone and in mediating bilateral jaw closing. SIGNIFICANCE STATEMENT: We developed a method that combines retrograde lentiviruses with the split-intein-split-Cre system in mice to isolate, characterize, and manipulate neurons that project to both left and right jaw-closing motoneurons. We show that these bilaterally projecting premotor neurons (BPNs) reside primarily in the supratrigeminal nucleus and the rostral parvicellular and intermediate reticular nuclei. BPNs consist of both excitatory and inhibitory populations, and also project to multiple brainstem nuclei implicated in orofacial sensorimotor control. Manipulation of the supratrigeminal BPNs during natural jaw-closing behavior reveals a dual role for these neurons in eliciting phasic muscle activation and in maintaining basal muscle tone. The retrograde lentivirus carrying the split-intein-split-Cre system can be applied to study any neurons with bifurcating axons innervating two brain regions.

Efficient recombinant expression of SFTI-1 in bacterial cells using intein-mediated protein trans-splicing.

We report for the first time the recombinant expression of bioactive wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. SFTI-1 is a small backbone-cyclized polypeptide with a single disulfide bridge and potent trypsin inhibitory activity. Recombinantly produced SFTI-1 was fully characterized by NMR and was observed to actively inhibit trypsin. The in-cell expression of SFTI-1 was very efficient reaching intracellular concentration ≈ 40 µM. This study clearly demonstrates the possibility of generating genetically encoded SFTI-based peptide libraries in live E. coli cells, and is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 818-824, 2016.

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses.

BACKGROUND: Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice. METHODS: Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP. RESULTS: Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection. CONCLUSION: This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.