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.

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 Cu and Ni 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 Ca (about 214 per capsid). Further thermodynamic studies indicated that all these three metal ions bound to the capsids in spontaneous enthalpy driving manners (<0, <0, <0), and the Cu binding had the highest affinity. The binding of Cu and Ni could enhance both the thermostability and acid-resistant stability of capsids, while the binding of Ca was helpful only to the thermostability of the capsids. Animal experiments showed that the immunization of FMDV bound with Cu induced the highest specific antibody titers in mice. Coincidently, the FMDV bound with Cu 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 Cu or Ni 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. 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.