Possible Action of Transition Divalent Metal Ions at the Inter-Pentameric Interface of Inactivated Foot-and-Mouth Disease Virus Provide A Simple but Effective Approach to Enhance Stability

Abstract

The structural instability of inactivated foot-and-mouth disease virus (FMDV) hinders the development of vaccine industry. Here we found that some transition metal ions like Cu²⁺ and Ni²⁺ could specifically bind to FMDV capsids at capacities about 7089 and 3448 metal ions per capsid, respectively. These values are about 33- and 16-folds of the binding capacity of non-transition metal ion Ca²⁺ (about 214 per capsid). Further thermodynamic studies indicated that all these three metal ions bound to the capsids in spontaneous enthalpy driving manners (ΔG<0, ΔH<0, ΔS<0), and the Cu²⁺ binding had the highest affinity. The binding of Cu²⁺ and Ni²⁺ could enhance both the thermostability and acid-resistant stability of capsids, while the binding of Ca²⁺ was helpful only to the thermostability of the capsids. Animal experiments showed that the immunization of FMDV bound with Cu²⁺ induced the highest specific antibody titers in mice. Coincidently, the FMDV bound with Cu²⁺ exhibited significantly enhanced affinities to integrin β6 and heparin sulfate, both of which are important cell surface receptors for FMDV attaching. Finally, the specific interaction between capsids and Cu²⁺ or Ni²⁺ was applied to direct purification of FMDV from crude cell culture feedstock by the immobilized metal affinity chromatography. Based on our new findings and structural analysis of the FMDV capsid, a "transition metal ion bridges" mechanism that describes linkage between adjacent histidine and other amino acids at the inter-pentameric interface of the capsids by transition metal ions coordination action was proposed to explain their stabilizing effect imposed on the capsid.IMPORTANCE How to stabilize the inactivated FMDV without affecting virus infectivity and immunogenicity is a big challenge in vaccine industry. The electrostatic repulsion induced by protonation of a large amount of histidine residues at the inter-pentameric interface of viral capsids is one of the major mechanisms causing the dissociation of capsids. In the present work, this structural disadvantage inspired us to stabilize the capsids through coordinating transition metal ions with the adjacent histidine residues in FMDV capsid, instead of removing or substituting them. This approach was proved effective to enhance not only the stability of FMDV, but also enhance the specific antibody responses; thus, providing a new guideline for designing an easy-to-use strategy suitable for large-scale production of FMDV vaccine antigen.

FIG 1

(a) Structure of O serotype FMDV capsids (PDB accession number 1FOD). VP1, green; VP2, cyan; VP3, magenta; VP4, yellow. Two asymmetrical protomers located on two adjacent pentamers are marked with black solid lines. (b) Enlargement of two asymmetrical protomers colored as in panel a, VP1 to VP4 are shown in the image. GH-loop on VP1 and α-helix on VP2 are colored in red and blue, respectively. The RDG motif on the GH-loop is depicted with a red surface, and histidine residues adjacent to the interpentameric interface are depicted with a blue surface.

FIG 2

Thermodynamic studies of the binding of Cu²⁺, Ni²⁺, and Ca²⁺ to FMDV capsids. (a) Binding affinities measured by MST. (b) ITC titration of Cu²⁺, Ni²⁺, and Ca²⁺ into FMDV capsids at 25°C in 20 mM Tris buffer, pH 7.8. Solid red line is the best fit of the data using the one-site model. (c) ΔH and ΔS contribution to the ΔG of the binding process measured by ITC. For each sample, the average value obtained from 3 independent experiments and the corresponding error bars (standard deviation) are indicated.

FIG 3

Structural characterizations of FMDV after metal ion binding. (a) Negative-stain TEM images measuring the changes of morphology of FMDV capsids after binding Ni²⁺, Cu²⁺, or Ca²⁺ at their high binding levels. CD spectra (b) and fluorescence spectra (c) measuring the changes of size distribution, tertiary structure, and secondary structure of FMDV capsids after binding metal ions at their different binding levels. (d) SDS denaturation test performed by MST to analyze the mechanism for the decrease of fluorescence intensity in FMDV capsids after Cu²⁺ binding.

FIG 4

Stability of FMDV capsids after binding different metal ions. (a) T_m values of FMDV capsids after binding different levels of metal ions measured by DSF. (b) The change of T_m values of FMDV capsids after removing metal ions by EDTA. (c) Acid-resistant stability of FMDV after binding Ni²⁺, Cu²⁺, or Ca²⁺. All samples were separately stored at various pH solutions for 8 min and subsequently analyzed for the contents of remaining intact capsids by HPSEC. The pH value that induced 50% dissociation of the intact capsids was defined as pH₅₀. For each sample above, the average value obtained from 3 independent experiments and the corresponding error bars (standard deviation) are indicated.

FIG 5

(a) FMDV-specific IgG antibody titers in mouse serum. Mice were subcutaneously injected with FMDV samples at 0 and 14 days. Blood samples were assayed at 14 and 28 days. (b) The affinities of FMDV capsids to integrin β6 receptor and heparin sulfate receptor determined by MST analyses. For each sample, the average value was obtained from 5 (IgG titers) or 3 (affinities) independent experiments, and the corresponding error bars (standard deviation) are indicated. Asterisks (*) denote statistically significant differences.

FIG 6

HPSEC analysis of crude inactivated FMDV solution (black line), purified FMDV by one-step Ni²⁺ chelating (red line) or Cu²⁺ chelating (blue line) chromatographic purification.

FIG 7

(a) Structure of three adjacent pentameric subunits of FMDV capsids. Subunits are shown in spheres and colored as in Fig. 1. The interpentameric interface is marked with a black dashed line. Ca²⁺ binding sites and transition metal ion binding areas on capsids are circled in red and blue, respectively. (b) Binding modes of Ca²⁺ at icosahedral 3-fold axes of capsids as reported by Acharya et al. (18). (c) Putative binding sites and modes of Ni²⁺ at the interpentameric interface of capsids. (d) Putative binding sites and modes of Cu²⁺ at the interpentameric interface of capsids. Amino acids involved in binding with metal ions are depicted with sticks. Ca²⁺, Ni²⁺, and Cu²⁺ are depicted with green balls, red balls, and blue balls. Coordination bonds are depicted with black dash lines in panels b, c, and d.