Guangchuan Wang

Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions

The development of vaccines against infectious diseases represents one of the most important contributions to medical science. However, vaccine-preventable diseases still cause millions of deaths each year due to the thermal instability and poor efficacy of vaccines. Using the human enterovirus type 71 vaccine strain as a model, we suggest a combined, rational design approach to improve the thermostability and immunogenicity of live vaccines by self-biomineralization. The biomimetic nucleating peptides are rationally integrated onto the capsid of enterovirus type 71 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior. This engineered self-biomineralized virus was characterized in detail for its unique structural, virological, and chemical properties. Analogous to many exteriors, the mineral coating confers some new properties on enclosed vaccines. The self-biomineralized vaccine can be stored at 26 °C for more than 9 d and at 37 °C for approximately 1 wk. Both in vitro and in vivo experiments demonstrate that this engineered vaccine can be used efficiently after heat treatment or ambient temperature storage, which reduces the dependence on a cold chain. Such a combination of genetic technology and biomineralization provides an economic solution for current vaccination programs, especially in developing countries that lack expensive refrigeration infrastructures.

Extracellular Silica Nanocoat Confers Thermotolerance on Individual Cells: A Case Study of Material‐Based Functionalization of Living Cells

During evolution, living organisms have developed various special structures to achieve optimal function, for example, some marine organisms, such as mollusks, arthropods, and diatoms use biominerals as their exterior coats/shells to fulfill many roles including protection against physical stress, toxic substances, natural enemies, and even mediating messages for control of their life cycle. Inspired by the formation of functional shells in nature, the design of novel shells from either natural constituents or synthetic materials can artificially confer new and unique properties on individual cells. Such a material-based chemical strategy for cell modification could meet various needs for more efficient applications of cell biotechnology. In this study, we demonstrate that a biomimetically constructed extracellular silica nanocoat can confer thermotolerance on individual cells. Silica plays an astonishingly large number of diverse roles in various organisms. 8–10] Many plants armor themselves with solid hydrated amorphous silica or opal, incorporated in their cell walls. There is ample evidence for the protection that Si often provides plant species against environmental aggression. 9, 11] A recent study of the presence of silicified structures in leaf epidermal cells of creeping bentgrass (Agrostis palustris) by thermal infrared imaging suggested a reduction of leaf heat-load, providing an effective cooling mechanism and thereby improving plant tolerance to high temperatures. Preservation of cell architecture and function under some potentially harmful environmental changes, such as fluctuations in ambient temperatures, is a basic requirement for many biological processes. Herein, we use a biomimetic strategy 13] to wrap nonsilicon-accumulating yeast cells (Saccharomyces cerevisiae), and demonstrate that silica-coated cells exhibit a significantly higher percent viability than uncoated controls during high-temperature stress. This finding reveals how extracellular silica nanocoating helps cells to withstand heat stress. To some extent, the silica coating serves as an extracellular air-conditioner to cool living cells. Unveiling the roles of extracellular silica not only provides a simple approach for moderating temperature stress for potentially useful cells when environmental temperatures rise, but also suggests that structure–function relationships for silica-accumulating organisms especially higher plants occupying hot or arid habitats reflects evolutionarily conserved mechanisms. Plants are highly sensitive to temperature and can perceive a difference of as little as 1 8C. Because plants cannot relocate to more favorable places, they must adapt when faced with ambient temperature changes due to global warming. Consequently, they are highly attuned to their environment and respond rapidly to change in order to optimize their growth and reproduction. Over the past 100 years, the global average temperature has increased by approximately 0.6 8C, and the biological ecology has already been altered. The global temperature is projected to continue to rise at a rapid rate so that a robust response to warming becomes critically serious for many organisms especially plants and individual cells. Our current study actually shows the importance of silicon accumulation for the improvement of heat-resistance of individual cells ; this can be further developed into a nanomaterial-based technique to functionalize living units to counteract changes in the environment. Due to the chemical structure of the cell wall of S. cerevisiae, which consists mainly of negatively charged polysaccharides, it hardly induces spontaneous mineralization and deposition of silica. Inspired by biosilica shell formation of diatom cell walls catalyzed by polycationic peptides containing polyamine, we modified the layer-by-layer (LBL) method to coat individual yeast cells using two polyelectrolytes (PEs) with opposite charges, poly(diallyldimethylammonium chloride) (PDADMAC) and poly(styrene sulfonate) (PSS, a biocompatible polyelectrolyte with high density of negative charge), to form a typical PE-coated yeast bearing a multilayer structure of (PDADMAC/ PSS)3–PDADMAC. Exposed PDADMAC molecules contain positively charged quaternary amines that adsorb negatively charged silica nanoparticles in suspension culture through electrostatic interactions under near-physiological conditions for pH (7.0), temperature (30 8C), and ionic strength (0.15 m). In addition to bare and PE-coated cells as controls, we exposed the PE-coated cells to a supersaturated calcium phosphate (CaP) solution to generate CaP-coated cells as the third control. Typical scanning electron microscopy (SEM; Figure 1 A and B) and transmission electron microscopy (TEM; Figure 1 C and D) images of S. cerevisiae show the morphological features of native and silica-coated yeast, and demonstrate that the shape of cells remains unchanged following the encapsulation. Prior to silica coating, the cell surface was relatively smooth (Figure 1 A and C), whereas the surface became uneven following the encapsulation treatment (Figure 1 B and D). Energy dispersive X-ray spectrometry (EDX) revealed that the outermost layer of the yeast cell was coated completely by silica nanopar[a] G. Wang, P. Liu, Y. Yan, Prof. Dr. X. Xu , Prof. Dr. R. Tang Center for Biomaterials and Biopathways, Zhejiang University Hangzhou, Zhejiang 310027 (China) [b] Prof. Dr. L. Wang College of Resources and Environment, Huazhong Agricultural University Wuhan, Hubei 430070 (China) Fax: (+ 86) 27-87288095 E-mail : ljwang@mail.hzau.edu.cn [c] Prof. Dr. X. Xu , Prof. Dr. R. Tang State Key Laboratory of Silicon Materials, Zhejiang University Hangzhou, Zhejiang 310027 (China) Fax: (+ 86) 571-87953736 E-mail : xrxu@zju.edu.cn

Antigenically shielded universal red blood cells by polydopamine-based cell surface engineering

Blood type mismatching is a critical problem in blood transfusions and it occasionally leads to severe transfusion reactions and even patient death. Inspired by the adhesive proteins secreted by mussels, we suggest a catecholic chemistry-based strategy to shelter antigenic epitopes on red blood cells (RBCs) by using polydopamine (PDA), which can guard against coagulation reaction without other negative effects on the RBC structure, function and viability. Both in vitro and in vivo studies confirm that the PDA-engineered RBCs (PDA-RBCs) can be applied in blood transfusion practices. The systemic assessment using a murine model demonstrates that the modified RBCs have a perfect survival profile even with repeated transfusion and high transfusion rates up to around 60%. It follows that an appropriate biogenic-chemical modification can produce antigenically shielded universal RBCs and provide insight for cell transplantation by using cell surface engineering.

Yolk–Shell Nanostructured Fe3O4@NiSiO3 for Selective Affinity and Magnetic Separation of His-Tagged Proteins

Recent developments of nanotechnology encourage novel materials for facile separations and purifications of recombinant proteins, which are of great importance in disease diagnoses and treatments. We find that Fe3O4@NiSiO3 with yolk-shell nanostructure can be used to specifically purify histidine-tagged (His-tagged) proteins from mixtures of lysed cells with a recyclable process. Each individual nanoparticle composes by a mesoporous nickel silicate shell and a magnetic Fe3O4 core in the hollow inner, which is featured by its great loading efficiency and rapid response toward magnetic fields. The abundant Ni(2+) cations on the shell provide docking sites for selective coordination of histidine and the reversible release is induced by excess imidazole solution. Because of the Fe3O4 cores, the separation, concentration, and recycling of the nanocomposites become feasible under the controls of magnets. These characteristics would be highly beneficial in nanoparticle-based biomedical applications for targeted-drug delivery and biosensors.

Human IgG Subclasses against Enterovirus Type 71: Neutralization versus Antibody Dependent Enhancement of Infection

The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases.

Biomineralization-Based Virus Shell-Engineering: Towards Neutralization Escape and Tropism Expansion

Biomineralization-based virus shell-engineering (BVSE) is a potential surface modification strategy to afford a biocompatible and biodegradable calcium phosphate (CaPi) shell onto single virus, allowing development of Trojan virus with enhanced infection, expanded tropism and neutralization escape, which open up the multiple applications of virus in biomedicines and materials.

Hydrated Silica Exterior Produced by Biomimetic Silicification Confers Viral Vaccine Heat-Resistance

Heat-lability is a key roadblock that strangles the widespread applications of many biological products. In nature, archaeal and extremophilic organisms utilize amorphous silica as a protective biomineral and exhibit considerable thermal tolerance. Here we present a bioinspired approach to generate thermostable virus by introducing an artificial hydrated silica exterior on individual virion. Similar to thermophiles, silicified viruses can survive longer at high temperature than their wild-type relatives. Virus inactivation assays showed that silica hydration exterior of the modified virus effectively prolonged infectivity of viruses by ∼ 10-fold at room temperature, achieving a similar result as that obtained by storing native ones at 4 °C. Mechanistic studies indicate that amorphous silica nanoclusters stabilize the inner virion structure by forming a layer that restricts molecular mobility, acting as physiochemical nanoanchors. Notably, we further evaluate the potential application of this biomimetic strategy in stabilizing clinically approved vaccine, and the silicified polio vaccine that can retain 90% potency after the storage at room temperature for 35 days was generated by this biosilicification approach and validated with in vivo experiments. This approach not only biomimetically connects inorganic material and living virus but also provides an innovative resolution to improve the thermal stability of biological agents using nanomaterials.

Calcium phosphate nanoparticles primarily induce cell necrosis through lysosomal rupture: the origination of material cytotoxicity

The application of nanotechnology for in medicine is developing rapidly, thereby increasing human exposure to nanomaterials and significantly so. A rising question is the biosecurity of nanoparticles (NPs). Although calcium phosphate (CaP) phase is biocompatible and biodegradable, many in vitro experiments have demonstrated that its NPs have significant cytotoxicity. This toxicity is due to that the released Ca2+ ions from the internalized CaP NPs within cells initiate apoptosis. Different from such an understanding, we reveal that the internalized CaP NPs actually result in lysosomal ruptures caused by the fast dissolution of CaP under acidic conditions. The suddenly released ions disturb the osmotic pressure balance across the lysosomal membranes destroying the lysosomes, and excessive lysosomal ruptures lead to cell necrosis. We find that the necrosis process can be regulated by intracellular environments. For examples, the lysosomal ruptures can be inhibited by increasing either cytoplasmic osmotic pressure or lysosomal pH (reduce the dissolution rates of CaP). These changes can significantly decrease the cytotoxicity of CaP NPs. It follows that lysosomal rupture prevention is important in the biomedical applications of CaP NPs. More generally, the study suggests that control of material degradation in lysosomes and cytoplasm osmotic pressure may improve the biosecurity of nanomaterials, which is of special importance to biomimetic nanomaterials.

Crystal structures of enterovirus 71 (EV71) recombinant virus particles provide insights into vaccine design.

Background: No HFMD vaccine is available. Results: Structures of EV71 recombinant virus particles were determined showing that the inserted foreign peptide is exposed on the particle surface without capsid structural changes and that virus uncoating is not affected. Conclusion: VP1 BC loop is suitable for foreign peptide insertion for the generation of recombinant EV71 viruses. Significance: The results provide insights into vaccine development. Hand-foot-and-mouth disease (HFMD) remains a major health concern in the Asia-Pacific regions, and its major causative agents include human enterovirus 71 (EV71) and coxsackievirus A16. A desirable vaccine against HFMD would be multivalent and able to elicit protective responses against multiple HFMD causative agents. Previously, we have demonstrated that a thermostable recombinant EV71 vaccine candidate can be produced by the insertion of a foreign peptide into the BC loop of VP1 without affecting viral replication. Here we present crystal structures of two different naturally occurring empty particles, one from a clinical C4 strain EV71 and the other from its recombinant virus containing an insertion in the VP1 BC loop. Crystal structure analysis demonstrated that the inserted foreign peptide is well exposed on the particle surface without significant structural changes in the capsid. Importantly, such insertions do not seem to affect the virus uncoating process as illustrated by the conformational similarity between an uncoating intermediate of another recombinant virus and that of EV71. Especially, at least 18 residues from the N terminus of VP1 are transiently externalized. Altogether, our study provides insights into vaccine development against HFMD.

Nanomodification of living organisms by biomimetic mineralization

In nature, a few living organisms such as diatoms, magnetotactic bacteria, and eggs have developed specific mineral structures, which can provide extensive protection or unique functions. However, most organisms do not have such structured materials due to their lack of biomineralization ability. The artificial introduction of biomimetic-constructed nanominerals is challenging but holds great promise. In this overview, we highlight two typical types of mineral-living complex systems. One involves biological surface-induced nanomaterials, which produces artificial living-mineral core-shell structures such as the mineralencapsulated yeast, cyanobacteria, bacteria and viruses. The other involves internal nanominerals that could endow organisms with unique structures and properties. The applications of these biomimetic generated nanominerals are further discussed, mainly in four potential areas: storage, protection, “stealth” and delivery. Since biomineralization combines chemical, nano and biological technologies, we suggest that nanobiomimetic mineralization may open up another window for interdisciplinary research. Specifically, this is a novel material-based biological regulation strategy and the integration of living organisms with functional nanomaterials can create “super” or intelligent nanoscale living complexes for biotechnological practices.