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

Abstract

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.

Keywords: genetic engineering; shell; vaccine design.

Fig. 1.

Design and characterization of engineered EV71 carrying nucleating peptides. (A) EV71 genome and the insertion site of the β-(BC)-loop of VP1. (B) Plaque morphologies of parental EV71 and engineered viruses in RD cells. (C) One-step growth curves of parental EV71 and engineered viruses in RD cells [multiplicity of infection (MOI) = 0.1]. (D) Homology modeling of the mutant viral protein. EV71 capsid proteins VP1, VP2, and VP3 are shown in cyan, yellow, and orange, respectively. The inserted peptides are marked with blue, and 60 copies are uniformly distributed on the surface of the engineered EV71 virion. These peptides may induce in situ biomineralization to form a CaP mineral exterior (gray) for the vaccine.

Fig. 2.

Self-biomineralization of genetically engineered vaccines. (A, Upper) After biomineralization, the viral surface proteins were immunologically detected by dot blot assays to indicate the stealth conditions by the CaP exterior. (A, Lower) Following spontaneous mineralization and centrifugation, the biomineralization efficacy was determined by examining the infectious viral particles in the supernatant and pellet using plaque assays; error bars represent SDs (n ≥ 4, Student’s paired t test, one-tailed, **P < 0.01). (B) SEM images of CaP-mineralized EV71-W6; the inserted energy dispersive X-ray spectroscopy shows the presence of Ca and P on the vaccine surfaces. (Scale bar: 100 nm.) (C) Transmission EM images of biomineralized EV71-W6. (Inset) Image shows that each particle contains a negatively stained vaccine. (Scale bar: 100 nm.) (D) Biomineralization capacity of progeny EV71-W6 was determined by quantifying viral RNA using qRT-PCR assays; error bars represent SDs (n ≥ 3).

Fig. 3.

Biological characterizations. (A) Plaque morphologies of EV71, EV71-W6, and EV71-W6-CaP in RD cells. (B) Indirect immunological fluorescence of recovered virus in Vero cells, RD cells (MOI = 0.1), and THP-1 cells (MOI = 1) at 12 h postinfection; the cell nuclei were stained by DAPI (blue). (Magnification: B, 40×.) (C) EV71-specific serum IgG titers of mice (n ≥ 5) at 4 wk postimmunization. (D) Serum neutralization antibody titers of mice (n ≥ 5) at 4 wk postimmunization were determined by microneutralization assays (Student’s paired t test, one-tailed, *P < 0.05).

Fig. 4.

In vitro tests of virus thermostability. Thermal-inactivation kinetics were determined at 26 °C (A), 37 °C (B), or 42 °C (C). The remaining percentage of infectivity is represented in a logarithmic scale as a function of incubation time (n ≥ 4); error bars represent SDs. The calculated average inactivation rate constants for EV71, EV71-W6, and EV71-W6-CaP, as shown in the figures, were K_EV71 = 0.25246 d⁻¹, K_EV71-W6 = 0.26011 d⁻¹, and K_EV71-W6-CaP = 0.09921 d⁻¹ at 26 °C; K_EV71 = 0.01590 h⁻¹, K_EV71-W6 = 0.01639 h⁻¹, and K_EV71-W6-CaP = 0.00594 h⁻¹ at 37 °C; and K_EV71 = 0.29896 h⁻¹, K_EV71-W6 = 0.31073 h⁻¹, and K_EV71-W6-CaP = 0.11117 h⁻¹ at 42 °C.

Fig. 5.

Animal tests of stored vaccines. EV71-specific IgG titers (A) and neutralizing antibody responses induced in mice by EV71, EV71-W6, and EV71-W6-CaP after 5 d of storage at 37 °C (B). Antibody titers were determined at 4 wk postimmunization (n ≥ 5); error bars represent SDs. (C) Frequencies of EV71-specific INF-γ–secreting splenocytes in the immunized mice before and after storage. Splenocytes were isolated from the immunized mice at 2 wk postimmunization and subjected to enzyme-linked immunospot assay (n ≥ 3); error bars represent SDs (Student’s paired t test, one-tailed, *P < 0.05, **P < 0.01). SFC, spot forming cells.