Versatile Functionalization of Ferritin Nanoparticles by Intein-Mediated

Jun 28, 2019 - Here, we report an intein-mediated trans-splicing approach that ..... Detailed description of the materials, methods, and additional fi...
0 downloads 0 Views 4MB Size
Download PDF
Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/NanoLett

Versatile Functionalization of Ferritin Nanoparticles by InteinMediated Trans-Splicing for Antigen/Adjuvant Co-delivery Shubing Tang,†,‡,§ Zhi Liu,‡,§ Wenjia Xu,‡,§ Qi Li,‡ Tian Han,‡ Deng Pan,‡ Nan Yue,‡ Mangteng Wu,‡ Qingwei Liu,‡ Weiming Yuan,† Zhong Huang,‡ Dongming Zhou,‡ Wei Zhou,*,† and Zhikang Qian*,‡ †

Downloaded via GUILFORD COLG on July 17, 2019 at 23:56:28 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Guangzhou Institute of Pediatrics, Department of Neonatology, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, 510623 Guangzhou, China ‡ Institut Pasteur of Shanghai, Chinese Academy of Sciences, University of the Chinese Academy of Sciences, 200031 Shanghai, China S Supporting Information *

ABSTRACT: Self-assembling protein nanoparticles are extensively and increasingly engineered to integrate adjuvants with antigens to elicit potent and long-term immunity due to uniform architecture, inherent biocompatibility, and excellent plasticity. However, functionalization of nanoparticles by surface tailoring has two common problems: (1) disassembly caused by loaded cargoes; and (2) an adjuvant that is inconvenient to co-deliver with an antigen by genetic fusion. Here, we report an intein-mediated trans-splicing approach that overcomes the detrimental effects of loaded proteins on ferritin nanoparticle stability and allows concurrent display of antigen and adjuvant in a facile, efficient, and site-specific manner. An immunization study with an epitope-based model antigen reveals that antigen and adjuvant co-delivery nanoparticles induce a more potent protective immunity than other formulations do. Our results demonstrate that protein engineering represents an intriguing approach for antigen/adjuvant codelivery to potentiate antigen-associated immune responses. KEYWORDS: Versatile tailoring, ferritin nanoparticles, intein-mediated trans-splicing, antigen/adjuvant co-delivery

V

immunostimulator co-delivery systems utilizing polymer carriers, fullerenol or porous silicon nanoparticles,9−11 there is still no convenient and general co-delivery approach to functionalize self-assembling protein nanoparticles. Genetic fusion and chemical conjugation are two major functionalization approaches to incorporate desired moieties onto nanoparticles; both of which possess pros and cons.12 Genetic fusion is widely utilized due to uniform orientation, simple manipulation, and high loading efficiency. Nonetheless, genetic insertion occasionally disrupts nanoparticle stability.13 Another shortcoming is that it is inconvenient to co-deliver antigens with immunostimulators by genetic fusion due to the limited insertion sites on nanoparticles. Chemical conjugation is versatile and allows simultaneous delivery of TLR ligands and antigens on nanoparticles. However, coupling efficiency is still a limiting factor, and intricate procedures hinder its general application.14 Low specificity of chemical conjugation is unsuitable to tackle protein-based molecules.15 To address the above problems, efforts have been made to develop a

accine development carries an inherent dilemma of maximizing immunogenicity and minimizing safety concerns.1 Although subunit vaccines offer significantly improved safety, they are insufficient to stimulate adequate immune responses.2 Particulate nanoparticles give recombinant subunit vaccines a viral feature that provides a solution to this long-lasting problem.3 Particulate nanoparticle vaccines adopt virus mimicry by size, shape, and repetitive display of surface antigens that facilitates antigen presentation.4 Proteinbased nanoparticles are widely used as particulate antigen carrier owing to a safe profile, easy manipulation, and defined architecture. Among them, ferritin nanoparticles are one of the most intensively explored antigen carriers for their substantially thermal and chemical stability.5 In addition to antigen delivery, nanoparticles are used to co-deliver moieties that target the immune system to enhance innate and adaptive immune responses.6 Toll-like receptor (TLR) ligands help with activation of antigen-presenting cells (APCs), interaction with B cells, and IgG isotype drift to induce long-term humoral responses and have been investigated extensively as immunostimulators.7 Free TLR ligands can have an unfavorable reactogenicity, including local pain, swelling, systemic fever, and malaise.8 Although these unwanted adverse effects have been overcome by recent advances in antigen and © XXXX American Chemical Society

Received: May 14, 2019 Revised: June 20, 2019 Published: June 28, 2019 A

DOI: 10.1021/acs.nanolett.9b01974 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 1. Generation of different nanodevices by TS. A universal adaptor intC was placed at the N terminus of HFT, which extended to the exterior of the nanoparticle. A soluble gb1 tag was introduced to the intC-HFT N-terminus and cargo-intN C-terminus to improve recombinant proteins property and yields. Following incubation with their cargo-intN-gb1 partners, the split inteins and gb1 were expunged as byproducts (d) via TS and formed a rearrangement of cargo-HFT (a−c). A neutralizing epitope of EV71, designated as SP70, was used as epitope-based model antigen. Rhizavidin was introduced to equip biotinylated CpG, a TLR9 agonist. Nanovaccine (a) or nanoadjuvant (b) was separately manufactured when the cargo was SP70 or rhizavidin. Combination of SP70-intN-gb1 and rhizavidin-intN-gb1 with gb1-intC-HFT led to the generation of co-delivery nanoparticles (c). The ratio of SP70-intN-gb1 to rhizavidin-intN-gb1 was 5:1. A 3D model of SP70 (red), split intN and intC (cyan), gb1 (blue), rhizavidin (green), CpG (pink), HFT (gray), and HFT nanoparticle (4Y08) was generated and viewed by UCSF chimera.

gb1 tag and intC on the HFT surface were replaced by cargo after trans-splicing (TS), forming a reconstitution of cargoHFT. The newly developed approach possesses inherent advantages. One is that the removable intein provides benefits for molecular design. For instance, soluble tags like gb1 can be introduced to improve protein properties and are then expunged completely as byproducts. Another is that variable substrates of cargo-based protein allow us to generate different functional nanodevices for vaccine design, including nanovaccines, nanoadjuvants, and antigen and immunostimulator co-delivery nanoparticles (Figure 1a−c). The SP70 epitope that induced neutralizing antibodies and provided protective immunity against enterovirus 71 (EV71), a major cause of human foot and hand diseases, was used as a model antigen.29 Target product SP70-HFT was designed as a nanovaccine in the following study (Figure 1a). The extensively explored TLR9 ligand CpG was intended as an immunostimulator,30 and this oligonucleotide was biotinylated and loaded by biotin−avidin interaction. The monovalent streptavidin, rhizavidin, was implemented as a mediator for CpG attachment to avoid interparticle cross-linking,31 and CpG-HFT served as a nanoadjuvant (Figure 1b). Simultaneous delivery of SP70 and CpG onto HFT, SP70/CpG-HFT, was used as a codelivery nanoparticle (Figure 1c). First, we engineered nanoparticles to introduce the inteinbased technology. We found that direct fusion of intC to ferritin formed inclusion bodies (Figure 2a,b). An additional gb1 tag dramatically restored recombinant protein solubility from 7% to 94% (Figure 2a−c). The recombinant protein fraction was enriched from 70% to 94% by ammonium sulfate precipitation (Figure 2d,e). A cryo-electron microscopy (cryoEM) image showed that recombinant protein sustained sphere nanoparticles with protrusions on the particle surface (Figure 2f, Figure S1a,b). Comparing with HFT, the diameter of the nanoparticles was increased from 11.8 to 14.8 nm by gb1-intC (Figure 2g). In accordance with cryo-EM results, a twodimensional (2D) class average validated that the introduced gb1-intC was distributed on the exterior of the nanoparticles (Figure 2h, Figure S1c,d). A three-dimensional (3D) model also revealed that gb1-intC was present on the nanoparticle surface (Figure 2i, Figure S1e,f). After rational molecular

modular nanoplatform. Previous studies, including our own work, have succeeded in efficient coupling of different functional units onto protein nanoparticles by sortase.16−18 Nevertheless, this reversible reaction needs high concentrations of oligoglycine reactants that constrain its application potential. Although SpyCatcher/SpyTag is an appealing plugand-display decoration approach to deliver functional moieties onto nanoparticles,19−21 this approach showed moderate or low efficiency of 22−48% in conjugation Acinetobacter phage AP205 virus-like particles (VLPs) with various molecules.22 In general, it remains a major challenge to co-deliver proteins onto the same nanoparticle. Intein-based technologies offer site-specific, efficient, and covalent bioconjugation of two different proteins to achieve various functionalities that are intensively used in many fields.23 Although intein-based technologies have shown promise in modifying superparamagnetic iron oxide nanoparticles,24 they have not been used to decorate protein-based nanoparticles. In this study, we implement this approach to tailor self-assembling protein nanoparticles to obviate instability raised by incorporated cargoes and co-deliver antigen with adjuvants. The rationale of human heavy chain of ferritin (HFT) nanoparticle surface functionalization was illustrated as follows (Figure 1). The 24 monomers of HFT were symmetrically arranged in an octahedral cage that permitted easy and accurate gene insertion25 and were used as a nanoscaffold. The crystal structure (4Y08) of HFT demonstrated that the N terminus of ferritin extended to the exterior of the nanoparticles.26 We introduced the nature split gp41-1 inteinC (intC) that catalyzed unprecedentedly fast and efficient selfexcision to the N-terminal of ferritin.27 Intein was split into inteinN (intN) and intC. IntC was fused to the N-terminus of HFT nanocarrier that served as an adaptor. IntN was placed at the C-terminus of the cargo protein. The gb1 tag, B1 domain of streptococcal protein G,28 was introduced to intC-HFT Nterminus and cargo-intN C-terminus to improve recombinant protein solubility and yields. When gb1-intC-HFT nanoscaffold was incubated with its complementary reactant cargo-intN-gb1, cargo and HFT were covalently linked (Figure 1a−c), leaving intein proteins with gb1 tags as byproducts (Figure 1d). The B

DOI: 10.1021/acs.nanolett.9b01974 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 2. Constructs, purification, and cryo-EM assay of nanoparticle scaffolds. (a) The split gp41-1 inteinC (intC) was directly fused to HFT. A gb1 tag (B1 domain of streptococcal protein G) was placed at the recombinant protein N terminus to enhance solubility. Constructs were numbered from 1 to 2. (b) Direct fusion of intC to HFT resulted in recombinant protein aggregation. Introduction of gb1 dramatically improved intC-HFT solubility. All: crude lysate; up: supernatant of crude lysate after centrifugation at 12,000 rpm, 4 °C for 10 min. (c) Soluble fraction of recombinant protein was significantly increased from 7% to 94%. Solubility percentage was calculated by ImageJ as follows: (up/all) × 100. Statistical analysis (p < 0.001) was performed by unpaired two-tailed t test. (d) The supernatant of new constructs was enriched by ammonium sulfate precipitation (p < 0.001). Up: supernatant of crude lysate; pellet: resuspension of the products by ammonium sulfate precipitation. (e) Fraction percentage of gb1-intC-HFT was calculated by ImageJ as follows: (intensity of gb1-intC-HFT band/intensity of whole lane protein bands) × 100. Fraction difference (p < 0.001) was compared by unpaired two-tailed t test. (f) Cryo-EM image of gb1-intC-HFT. The white scale bar represents 20 nm. (g) Comparison of diameters of HFT and gb1-intC-HFT. Diameters of 20 particles were measured by ImageJ, and the difference in diameters (p < 0.001) was analyzed by unpaired two-tailed t test. (h) A 2D class average of nanoparticles. (i) A 3D model with resolution of 15 Å was generated by cisTEM which showed that the introduced gb1-intC was distributed on the particle surface (blue).

The target product was purified by gel filtration and confirmed by CB staining and WB, which maintained nanoparticle structure (Figure S3e−h). In vitro splicing experiments demonstrated that a highly efficient nanoparticle tailoring strategy was established. Then we verified whether this approach could be used in vaccine development. The defined SP70 epitope was used as an antigen prototype.29 In vitro TS assays demonstrated that SP70 was covalently linked to HFT within 30 min using 10 μM SP70-intN-gb1 as a reactant, and the spliced product SP70-HFT achieved a yield of >90% (Figure 3a,b and Figure S4). SP70-HFT was recovered by gel filtration after reaction and was present in the nanoparticles (Figure 3c−e). 2D class average showed that the target product retained a spherical shape (Figure 3f), and the 3D model showed that the nanoparticles had a three-fold axis (Figure 3g). When the gb1-intC protein was replaced by a SP70 epitope, nanoparticles became remarkably less spiky than the gb1-intC-HFT particles (Figures 2i and 3g). We also integrated adjuvants onto nanoparticles to heighten pathogenassociated immune responses. TLR9 ligand CpG is a strong

design, we completed the initial step to implement inteinmediated trans-splicing (TS) technology to functionalize the HFT nanoparticles. Next, we explored whether the designed intein-mediated TS was feasible to modify HFT nanoparticles, using a strep2 tag (WSHPQFEK) and GFP as model molecules. TS was efficiently completed within 30 min at room temperature (RT) (Figure S2a). However, reactivity was dramatically reduced with ice incubation, and the reaction was completed within 4 h (Figure S2a,b). All subsequent reactions were performed at RT. Strep2 rejoined HFT with a high yield >90% according to the decreased percentage of gb1-intC-HFT as the reactant (Figure S2a−d). The reconstituted product was recovered from the reaction mixture by gel filtration (Figure S2e,f). Purified products were confirmed by coomassie blue (CB) staining and Western blotting (WB) (Figure S2g). Transmission electron microscopy (TEM) showed that strep2HFT sustained the nanoparticle structure (Figure S2i) as HFT did (Figure S2h). Covalent linkage of GFP to HFT was also achieved with >90% efficiency for 1 h at RT (Figure S3a−d). C

DOI: 10.1021/acs.nanolett.9b01974 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 3. Efficient tailoring of HFT nanoparticle surface by TS. (a) Time course analysis of coupling SP70 to HFT. SP70 epitope was coupled to HFT >90% within 30 min. (b) Semiquantification of spliced products percentage by ImageJ. TS efficiency was calculated as follows: (1 − intensity of retained gb1-intC-HFT/intensity of initial gb1-intC-HFT) × 100. (c) Product was efficiently recovered by gel filtration. (d) UV profile of purification of SP70-HFT by gel filtration. Products peak is indicated by a red arrow. (e) Cryo-EM image of SP70-HFT showed that TS did not disrupt nanoparticle stability. White bar represents 20 nm. (f) 2D-class average of SP70-HFT illustrated that the target product existed in a symmetric sphere. (g) 3D model showed that the target product exhibited a three-fold axis. (h) Cryo-EM image of rhizavidin-HFT showed that rhizavidin proteins were distributed on the surface of the nanoparticles (white arrows). (i) Cryo-EM image of SP70/rhizavidin-HFT showed that one average rhizavidin spike was displayed on each HFT nanoparticle surface (100 particles were calculated), which was consistent with the SP70/ rhizavidin molar ratio. Scale bar of cryo-EM represents 20 nm. The large dark spots on cryo-EM images from (h) and (i) were ice crystals formed during vitrification of cryo-EM samples by liquid ethane. (j) Diameters of SP70-HFT, rhizavidin-HFT, and SP70/rhizavidin-HFT were 12.6, 18.5, and 14.2 nm, respectively. Twenty particles were measured for diameter by ImageJ. Difference between rhizavidin-HFT with SP70-HFT or SP70/ rhizavidin-HFT was calculated by unpaired two-tailed t test.

stimulus that enhances innate and adaptive immune responses.32 We used CpG as an immunostimulator. CpG was biotinylated and then loaded by rhizavidin, a variant of avidin, through biotin−avidin interaction.31 In addition, an extracellular TLR5 ligand named CBLB, which is a derivative of flagellin, was also exploited as an immunostimulator.33 Genetic fusion of rhizavidin or CBLB with HFT was prone to aggregation (Figure S5a,b). Both of them were covalently conjugated to HFT nanoparticles in a convenient and efficient pattern (Figures S5c−h and S7). After conjugation of rhizavidin with a long linker as a spacer to HFT, rhizavidin proteins were attached to the exterior of HFT nanoparticles (Figure 3h). Furthermore, rhizavidin and SP70 were concurrently coupled onto HFT, designated as SP70/ rhizavidin-HFT in a controllable manner (Figure S6a,b). Major target products, fraction 14, were collected, and SP70/ rhizavidin molar ratio was determined as 5:1 (Figure S6c). This fraction was sampled, and SP70/rhizavidin-HFT products were validated by CB staining and WB (Figure S6d). SP70HFT, rhizavidin-HFT, or SP70/rhizavidin-HFT formed complexes that were stable in SDS loading buffer (Figure S6e,f). Consistent with the molar ratio of SP70 and rhizavidin, cryo-EM revealed that one average spike protrusion was present on the nanoparticle surface (100 particles were counted for statistical analysis) (Figure 3i). Biotinylated CpG

was efficiently equipped to the HFT nanoparticle through an avdin−biotin interaction (Figure S6g). Incubation of rhizavidin-HFT with biotinylated CpG created a nanoadjuvant CpGHFT, and mixing SP70/rhizavidin-HFT with biotinylated CpG generated SP70/CpG-HFT co-delivery nanoparticles. CBLBrelated nanodevices were synthesized in the same way, consisting of the co-delivery of the nanoparticles SP70/ CBLB-HFT and nanoadjuvants CBLB-HFT (Figures S7 and S8). Instability caused by rhizavidin or CBLB on ferritin nanoparticles was circumvented by our newly devised approach, and different functional nanodevices were manufactured for the immunization study. To sum up, two polypeptides and three proteins were efficiently conjugated onto the HFT surface to generate different functional nanodevices (Table S1). To optimize vaccination regimens, we categorized the administration formulations into two groups: CpG and CBLB. In the CpG group, four formulations were explored: SP70-HFT as nanovaccine, nanovaccine SP70-HFT with free CpG (SP70-HFT+CpG), nanovaccine SP70-HFT associated with nanoadjuvant CpG-HFT (SP70-HFT+CpG-HFT), and co-delivery SP70/CpG-HFT nanoparticles. In the CBLB group, two formulations were tested: co-delivery SP70/ CBLB-HFT nanoparticles, nanovaccine SP70-HFT, and nanoadjuvant CBLB-HFT mixture (SP70-HFT+CBLB-HFT). A D

DOI: 10.1021/acs.nanolett.9b01974 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 4. Significantly improved immunogenicity by co-delivery nanoparticles. (a) ELISA demonstrated that co-delivery of SP70 with CpG on a homo-HFT nanoparticle stimulated the highest antigen-specific IgG titers. Only SP70/CpG-HFT induced pronouncedly increased anti-SP70 IgG responses than SP70-HFT, while other formulations did not show obvious enhancement effects. (b) SP70/CpG-HFT steered notably up-regulated anti-IgG2a responses than that of SP70-HFT. (c) Neutralizing capability of sera was improved with a remarkable magnitude of 3-fold by co-delivery nanoparticles and a moderate magnitude of 0.5-fold by nanoadjuvant CpG-HFT. Differences between SP70-HFT with SP70-HFT+CpG, SP70HFT+CpG-HFT, or SP70/CpG-HFT in IgG responses and neutralizing titers were compared by unpaired two-tailed t test. (d) In vivo lethal challenge assay showed that co-delivery nanoparticles elicited the most potent protective immunity, followed by SP70-HFT association with CpGHFT. A significant difference was observed between SP70/CpG-HFT and SP70-HFT by a log-rank (Mantel−Cox) test (p < 0.05).

low dose of free CpG (0.4 μg/mouse) that was formulated with SP70-HFT showed no enhancement compared to the nanovaccine SP70-HFT (Figure 4a). Coordination of CpG with SP70 on a homo-HFT nanoparticle (SP70/CpG-HFT) but not on hetero-nanoparticles (SP70-HFT+CpG-HFT) accounted for a substantial increase of anti-SP70 IgG titer (Figure 4a). It was speculated that the concurrent delivery of immunopotentiator with SP70 on the same nanoparticle was more easy to co-localize in the same APC than other formulations. Hence, the effective concentration of immunopotentiator was increased by the co-delivery approach. In mice, IgG1 is regarded as a T-helper cell type 2 (Th2)-like response, and IgG2a, IgG2b, and IgG3 are associated with Th1 responses.22 Co-delivery SP70/CpG-HFT nanoparticles stimulated a significant increase of anti-IgG2a (Th1) responses (Figure 4b) and a slight elevation of anti-IgG1(Th2) responses (Figure S9a). Th1 biased responses (anti-IgG2a), harboring a higher capacity for complement fixation than IgG1, were driven by co-delivery nanoparticles.22 The neutralizing capability and protective immunity were in accordance with IgG titers (Figure 4c,d). No distinct enhancement effect was monitored when CBLB was used as the adjuvant (Figure S9b).

In general, co-delivery nanoparticles markedly improved antigen immunogenicity, and incorporation of epitope with adjuvant to the same nanoparticle induced the most potent immunity. Nanotechnology is the leading strategy for antigen and adjuvant delivery.34 Genetic fusion is a classic approach for cargo delivery. However, recombinant constructs often aggregate or show no expression.35 Modular decoration of nanoscaffold by SpyTag/SpyCatcher is an appealing approach to liberate labor-intensive trial-and-error optimization required by genetic fusion.36 SpyTag and SpyCatcher are retained after spontaneous isopeptide bond formation.37 However, split intein and soluble tags are removable in our intein-mediated TS platform, which is beneficial for the introduction of soluble tags to facilitate nanoparticle assembly (Figure 2). Reaction of SpyTag/SpyCatcher plug-and-display decoration needs 3 h,38 while intein-mediated TS was achieved in 90%. In vitro assays showed that three proteins and two polypeptides were covalently coupled to ferritin nanoparticles in an efficient and site-specific fashion (Figure 3 and Figures S2−S8). In particular, adverse effects caused by loaded rhizavidin or CBLB on ferritin nanoparticle E

DOI: 10.1021/acs.nanolett.9b01974 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

service. We thank Fulin Yang and Fangkang Meng from SPF Animal Core Facility and Jinze Li and Shuzhu Lin from the biosafety core facility for their assistance with the immunization study. We thank professor Zhong Huang for his gift of EV71 and professor Lanfeng Wang for help with cryo-EM data processing. We also owe our thanks to the International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. Finally, we thank Shanghai Flash Spectrum Biological Technology Co., Ltd. who provided us with the ClearFirst-3000 protein purification demo system for gel filtration.

stability were avoided, and nanoparticle durability was improved by post-translational modification (Figures S5 and S7). Concurrently incorporated immune cell targeting adjuvants with antigenic payloads by particulate nanocarriers can be delivered to the same APCs and trigger an effective immunity.9 In this study, TLR9 ligand CpG was co-delivered with SP70 onto HFT nanoparticles in a controllable manner (Figure 3i and Figure S6) and conferred the most potent antigen-specific immunity (Figure 4). In summary, we devised a complementary approach to known technologies for nanoparticle decoration that overcame instability raised by loaded cargoes and enabled a simultaneous incorporation of adjuvant with epitope to precisely and effectively potentiate antigen-specific immune responses.

■

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b01974. Detailed description of the materials, methods, and additional figures (Figures S1−S9) Table S1 (PDF)

■

REFERENCES

(1) Vartak, A.; Sucheck, S. J. Vaccines (Basel, Switz.) 2016, 4 (2), 12. (2) Karch, C. P.; Burkhard, P. Biochem. Pharmacol. 2016, 120, 1−14. (3) Chattopadhyay, S.; Chen, J. Y.; Chen, H. W.; Hu, C. J. Nanotheranostics 2017, 1 (3), 244−260. (4) Bachmann, M. F.; Jennings, G. T. Nat. Rev. Immunol. 2010, 10 (11), 787−96. (5) Lopez-Sagaseta, J.; Malito, E.; Rappuoli, R.; Bottomley, M. J. Comput. Struct. Biotechnol. J. 2016, 14, 58−68. (6) Gomes, A. C.; Mohsen, M.; Bachmann, M. F. Vaccines (Basel, Switz.) 2017, 5 (1), 6. (7) Lanzavecchia, A.; Sallusto, F. Curr. Opin. Immunol. 2007, 19 (3), 268−74. (8) Chauhan, N.; Tiwari, S.; Iype, T.; Jain, U. Expert Rev. Vaccines 2017, 16 (5), 491−502. (9) Cui, J.; De Rose, R.; Best, J. P.; Johnston, A. P.; Alcantara, S.; Liang, K.; Such, G. K.; Kent, S. J.; Caruso, F. Adv. Mater. 2013, 25 (25), 3468−72. (10) Xu, L.; Liu, Y.; Chen, Z.; Li, W.; Liu, Y.; Wang, L.; Ma, L.; Shao, Y.; Zhao, Y.; Chen, C. Adv. Mater. 2013, 25 (41), 5928−36. (11) Gu, L.; Ruff, L. E.; Qin, Z.; Corr, M.; Hedrick, S. M.; Sailor, M. Adv. Mater. 2012, 24 (29), 3981−7. (12) Barcena, J.; Blanco, E. Subcell. Biochem. 2013, 68, 631−65. (13) Karch, C. P.; Li, J.; Kulangara, C.; Paulillo, S. M.; Raman, S. K.; Emadi, S.; Tan, A.; Helal, Z. H.; Fan, Q.; Khan, M. I.; Burkhard, P. Nanomedicine 2017, 13 (1), 241−251. (14) Venter, P. A.; Dirksen, A.; Thomas, D.; Manchester, M.; Dawson, P. E.; Schneemann, A. Biomacromolecules 2011, 12 (6), 2293−301. (15) Kim, H.; Siu, K. H.; Raeeszadeh-Sarmazdeh, M.; Sun, Q.; Chen, Q.; Chen, W. Biotechnol. Bioeng. 2015, 112 (8), 1495−505. (16) Patterson, D.; Schwarz, B.; Avera, J.; Western, B.; Hicks, M.; Krugler, P.; Terra, M.; Uchida, M.; McCoy, K.; Douglas, T. Bioconjug. Chem. 2017, 28 (8), 2114−2124. (17) Sun, Q.; Chen, Q.; Blackstock, D.; Chen, W. ACS Nano 2015, 9 (8), 8554−61. (18) Tang, S.; Xuan, B.; Ye, X.; Huang, Z.; Qian, Z. Sci. Rep. 2016, 6, 25741. (19) Bruun, T. U. J.; Andersson, A. C.; Draper, S. J.; Howarth, M. ACS Nano 2018, 12 (9), 8855−8866. (20) Brune, K. D.; Leneghan, D. B.; Brian, I. J.; Ishizuka, A. S.; Bachmann, M. F.; Draper, S. J.; Biswas, S.; Howarth, M. Sci. Rep. 2016, 6, 19234. (21) Choi, H.; Choi, B.; Kim, G. J.; Kim, H. U.; Kim, H.; Jung, H. S.; Kang, S. Small 2018, 14 (35), No. 1801488. (22) Thrane, S.; Janitzek, C. M.; Matondo, S.; Resende, M.; Gustavsson, T.; de Jongh, W. A.; Clemmensen, S.; Roeffen, W.; van de Vegte-Bolmer, M.; van Gemert, G. J.; Sauerwein, R.; Schiller, J. T.; Nielsen, M. A.; Theander, T. G.; Salanti, A.; Sander, A. F. J. Nanobiotechnol. 2016, 14, 30. (23) Li, Y. Biotechnol. Lett. 2015, 37 (11), 2121−37. (24) Elias, D. R.; Cheng, Z.; Tsourkas, A. Small 2010, 6 (21), 2460− 8. (25) He, D.; Marles-Wright, J. N. New Biotechnol. 2015, 32 (6), 651−7.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Shubing Tang: 0000-0001-6639-0222 Author Contributions §

S.T., Z.L., and W.X. contributed equally to this manuscript. Z.Q. and S.T. conceived and designed the experiments. W.Z., D.Z., and Z.H. gave constructive suggestions. S.T. performed a majority of the experiments and wrote the manuscript. Z.L. prepared cryo-EM samples and collected and processed related data. W.X. revised and organized the figures. Others assisted with the TS assay, immunization study, or data organization. Notes

The authors declare the following competing financial interest(s): S.T., W.Z., and W.Y. are authors on three patent applications in China for tailoring nanoparticles by inteinmediated trans-splicing (application numbers 201910421369.2, 201910421408.9, and 201919421458.0 ).

■

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (grants 81371826 and 81572002 to Z.Q., grants 31300148 and 31570169), the Ministry of Science and Technology of China (2016YFA0502101), the Chinese Academy of Sciences (CAS) “100 Talents” program to Z.Q (Y214P1109). This work was also sponsored by the Joint Center for Infection and Immunity project. We would like to thank the National Center for Protein Science Shanghai for our 120 kV TEM and 300 kV cryo-EM analyses of nanoparticles. We are grateful to Qingning Yuan and Yingyi Zhang for their help with the TEM samples and collecting the data. We owe thanks to Liangliang Kong and Fang Wang for their help with the cryo-EM data collection. Our 200 kV TEM images were performed at the core facility of Biological Imaging, Institut Pasteur of Shanghai (IPS), CAS. We thank Chao Wu and the staff at the core facility of Biological Imaging for their excellent F

DOI: 10.1021/acs.nanolett.9b01974 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters (26) Pozzi, C.; Di Pisa, F.; Bernacchioni, C.; Ciambellotti, S.; Turano, P.; Mangani, S. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2015, 71, 1909−20. (27) Carvajal-Vallejos, P.; Pallisse, R.; Mootz, H. D.; Schmidt, S. R. J. Biol. Chem. 2012, 287 (34), 28686−96. (28) Dulal, H. P.; Nagae, M.; Ikeda, A.; Morita-Matsumoto, K.; Adachi, Y.; Ohno, N.; Yamaguchi, Y. Protein Expression Purif. 2016, 123, 97−104. (29) Ye, X.; Ku, Z.; Liu, Q.; Wang, X.; Shi, J.; Zhang, Y.; Kong, L.; Cong, Y.; Huang, Z. J. Virol. 2014, 88 (1), 72−81. (30) Bai, L.; Chen, W.; Chen, J.; Li, W.; Zhou, L.; Niu, C.; Han, W.; Cui, J. J. Transl. Med. 2017, 15 (1), 51. (31) Lee, J. M.; Kim, J. A.; Yen, T. C.; Lee, I. H.; Ahn, B.; Lee, Y.; Hsieh, C. L.; Kim, H. M.; Jung, Y. Angew. Chem., Int. Ed. 2016, 55 (10), 3393−7. (32) Krishnamachari, Y.; Salem, A. K. Adv. Drug Delivery Rev. 2009, 61 (3), 205−17. (33) Burdelya, L. G.; Krivokrysenko, V. I.; Tallant, T. C.; Strom, E.; Gleiberman, A. S.; Gupta, D.; Kurnasov, O. V.; Fort, F. L.; Osterman, A. L.; Didonato, J. A.; Feinstein, E.; Gudkov, A. V. Science 2008, 320 (5873), 226−30. (34) Singh, L.; Kruger, H. G.; Maguire, G. E. M.; Govender, T.; Parboosing, R. Ther. Adv. Infect. Dis. 2017, 4 (4), 105−131. (35) Yassine, H. M.; Boyington, J. C.; McTamney, P. M.; Wei, C. J.; Kanekiyo, M.; Kong, W. P.; Gallagher, J. R.; Wang, L.; Zhang, Y.; Joyce, M. G.; Lingwood, D.; Moin, S. M.; Andersen, H.; Okuno, Y.; Rao, S. S.; Harris, A. K.; Kwong, P. D.; Mascola, J. R.; Nabel, G. J.; Graham, B. S. Nat. Med. 2015, 21 (9), 1065−70. (36) Brune, K. D.; Howarth, M. Front. Immunol. 2018, 9, 1432. (37) Li, L.; Fierer, J. O.; Rapoport, T. A.; Howarth, M. J. J. Mol. Biol. 2014, 426 (2), 309−17. (38) Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; SchwarzLinek, U.; Moy, V. T.; Howarth, M. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (12), E690−7.

G

DOI: 10.1021/acs.nanolett.9b01974 Nano Lett. XXXX, XXX, XXX−XXX