Rational design of thermostable vaccines by engineered peptide-induced virus self- biomineralization under physiological conditions Guangchuan Wanga,b, Rui-Yuan Caob, Rong Chenc, Lijuan Mod, Jian-Feng Hanb, Xiaoyu Wangb,e, Xurong Xue, Tao Jiangb, Yong-Qiang Dengb, Ke Lyuc, Shun-Ya Zhub, E-De Qinb, Ruikang Tanga,e,1, and Cheng-Feng Qinb,1 aCenter for Biomaterials and Biopathways, and eQiushi Academy for Advanced Studies, Zhejiang University, Hangzhou 310027, China; bDepartment of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China; cKey Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China; and dBiomedical Center, Sir Run Run Shaw Hospital, Hangzhou 310016, China Edited by Bernard Roizman, University of Chicago, Chicago, IL, and approved March 22, 2013 (received for review January 5, 2013) The development of vaccines against infectious diseases represents the efficacy and thermostability of vaccines is of great importance one of the most important contributions to medical science. However, and has been highlighted as a Grand Challenge in Global Health vaccine-preventable diseases still cause millions of deaths each year by the Gates Foundation (www.grandchallenges.org). due to the thermal instability and poor efficacy of vaccines. Using the New advances in genetic and materials sciences have created human enterovirus type 71 vaccine strain as a model, we suggest the possibility of improving and stabilizing vaccine products. a combined, rational design approach to improve the thermostability Stabilizers, such as deuterium oxide, proteins, MgCl2, and non- reducing sugars, have been introduced to produce stabilized and immunogenicity of live vaccines by self-biomineralization. The – biomimetic nucleating peptides are rationally integrated onto the formulations of vaccines (7 9). By using reverse genetic tech- nology, the viral capsid can be rationally engineered to improve its capsid of enterovirus type 71 by reverse genetics so that calcium – phosphate mineralization can be biologically induced onto vaccine physicochemical properties (10 12). In nature, biomineralization is adopted by many organisms to improve their performance in surfaces under physiological conditions, generating a mineral exte- harsh environments. For example, living organisms, such as dia- rior. This engineered self-biomineralized virus was characterized in toms (13), mollusks (14), eggs, and some plants (15), have de- detail for its unique structural, virological, and chemical properties. veloped biomineral exteriors that play a protective role. It should Analogous to many exteriors, the mineral coating confers some new be noted that egg cells with mineral shells are extremely ther- properties on enclosed vaccines. The self-biomineralized vaccine can mostable and can be stored under ambient conditions. Un- be stored at 26 °C for more than 9 d and at 37 °C for approximately fortunately, most living organisms, including vaccines, cannot 1 wk. Both in vitro and in vivo experiments demonstrate that this generate such mineral shells due to the lack of biomineralization- engineered vaccine can be used efficiently after heat treatment or related proteins, which usually play a key role in the control of ambient temperature storage, which reduces the dependence on biomineralization. Among biogenic minerals, calcium phosphate SCIENCES a cold chain. Such a combination of genetic technology and biomi- (CaP), the major component of bones and teeth, is of interest APPLIED BIOLOGICAL neralization provides an economic solution for current vaccination because of its unique biocompatibility (16), and it is now used as programs, especially in developing countries that lack expensive a transfer agent and adjuvant (17). CaP represents an excellent refrigeration infrastructures. mineral shell candidate to stabilize vaccines. Recently, we have shown that a protective CaP shell can be introduced onto some genetic engineering | vaccine design | shell virus surfaces in the presence of a high concentration of calcium ions (18, 19). However, the biomineralization process requires fi CHEMISTRY he development of vaccines against infectious diseases is one speci c treatments and only occurs with selected premade viruses Tof the most successful medical interventions ever developed stocks. A natural virus cannot induce biomineralization by itself by humans (1, 2). Live attenuated viruses provide an effective during its normal life cycle. In contrast, biomineralization in vaccination strategy devised since the beginning of the vacci- organisms is generally controlled using biomacromolecules in nology era, and vaccines against polio, smallpox, measles, ru- a mild biological system, and some polypeptides and macro- molecules have been used to induce biomineralization (20–24). bella, yellow fever, Japanese encephalitis, and influenza, for For example, several peptides have been genetically incorporated example, have been used to immunize billions of children and into the M13 bacteriophage and plant viruses as nucleators to adults worldwide. However, there are still more than 17 million direct nanomaterial synthesis (25, 26) or as specific nucleators deaths caused by infectious diseases every year, accounting for for CaP mineralization in living systems (23, 27–29). Inspired by 25% of all deaths worldwide (3, 4). Most of the deaths are caused this achievement, we propose to design rationally an engineered by vaccine-preventable diseases and the underutilization of vac- virus carrying the selected biomimetic peptides that control the cines. This is especially true for the poorest countries, because biomineralization process. most vaccines are sensitive to heat and the cold chain is very dif- fi Human enterovirus type 71 (EV71) is a typical nonenveloped cult to maintain in countries with a minimal infrastructure. The icosahedral virus belonging to the Picornaviridae family (30). poor efficacy and instability of vaccine products lead to incomplete immunization and a rapid loss of potency during storage and de- livery, which severely limits the coverage of current vaccination ∼ fi Author contributions: G.W., R.T., and C.-F.Q. designed research; G.W., R.-Y.C., L.M., J.-F.H., programs. For example, 50% of vaccine products are nally X.W., Y.-Q.D., and S.-Y.Z. performed research; R.C. contributed new reagents/analytic discarded due to poor thermostability (5). Refrigeration is essential tools; G.W., R.C., L.M., X.X., T.J., K.L., E.-D.Q., R.T., and C.-F.Q. analyzed data; and G.W., for vaccines to maintain their quality. However, keeping vaccines R.T., and C.-F.Q. wrote the paper. at low temperatures is difficult and expensive. The cold chain The authors declare no conflict of interest. consumes ∼80% of the total cost of vaccination programs (3, 6), This article is a PNAS Direct Submission. and it is not always reliable. The situation is even worse in de- 1To whom correspondence may be addressed. E-mail: [email protected] or rtang@zju. veloping countries and in the least-developed countries, where, due edu.cn. to the lack of extensive and reliable refrigeration infrastructure, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. over half of all deaths are caused by infectious diseases. Improving 1073/pnas.1300233110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300233110 PNAS | May 7, 2013 | vol. 110 | no. 19 | 7619–7624 Downloaded by guest on September 24, 2021 Picornaviruses cause acute diseases in humans and animals, in- cluding polio, hepatitis A, and foot-and-mouth disease. Live at- tenuated picornavirus vaccines are widely used with high efficacy. In this study, we protected the EV71 vaccine strain with an egg- shell-like exterior, which is spontaneously formed under physio- logical conditions due to the engineered CaP biomineralization peptide. The biomineralized engineered vaccine exhibits overall improved thermostability and immunogenicity, and it can be used efficiently even after a week’s storage at room temperature. Thus, “vaccines that can be stable in your pocket for 1 wk” can be created by the genetically induced self-biomineralization of the vaccine. Results Genetic Engineering. To endow a virus with the capacity of self- biomineralization, we aimed to display nucleating peptides on the virion surface to enhance its capacity to initiate CaP miner- alization. Among nucleating peptides, two types of representative nucleators, a phosphate chelating agent (N6p) or calcium che- lating agents (NWp and W6p), were selected. N6p is a reported CaP-binding peptide identified through phage display, and its binding effect is thought to be due to phosphate-chelating domains (SVKRGTSVG and VGMKPSP) (31). NWp is derived from the N-terminal 15-residue fragment of salivary statherin, a CaP high-affinity protein, and it has been used in biomimetic mineralization (32). W6p is an acidic analog of the amino-ter- minal 15-residue fragment of salivary statherin and core motifs of dentin matrix protein 1, an acidic protein that can trigger CaP formation in vitro by binding calcium ions (33). The site between amino acids 100 and 101 of the β-(BC)-loop of viral protein 1 (VP1) (Fig. 1A) has been demonstrated to be an appropriate Fig. 1. Design and characterization of engineered EV71 carrying nucleating insertion site for the placement of an engineered peptide on the peptides. (A) EV71 genome and the insertion site of the β-(BC)-loop