Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals

Plant-made polio type 3 stabilized VLPs—a candidate synthetic polio vaccine

Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized viruslike particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single particle reconstruction reveals a structure almost indistinguishable from wild-type PV3

The Enterovirus 71 A-particle Forms a Gateway to Allow Genome Release: A CryoEM Study of Picornavirus Uncoating

Since its discovery in 1969, enterovirus 71 (EV71) has emerged as a serious worldwide health threat. This human pathogen of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. Upon binding to a host receptor on the cell surface, the virus begins a two-step uncoating process, first forming an expanded, altered "A-particle", which is primed for genome release. In a second step after endocytosis, an unknown trigger leads to RNA expulsion, generating an intact, empty capsid. Cryo-electron microscopy reconstructions of these two capsid states provide insight into the mechanics of genome release. The EV71 A-particle capsid interacts with the genome near the icosahedral two-fold axis of symmetry, which opens to the external environment via a channel ~10 Å in diameter that is lined with patches of negatively charged residues. After the EV71 genome has been released, the two-fold channel shrinks, though the overall capsid dimensions are conserved. These structural characteristics identify the two-fold channel as the site where a gateway forms and regulates the process of genome release. © 2013 Shingler et al

Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line.

The Na+ pump (Na+, K+-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na+ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 +/- 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, 14C-taurine efflux increased by 183 +/- 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a 14C-taurine efflux of 207 +/- 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic alpha isoforms of Na+, K+-ATPase demonstrated high levels of the ubiquitously expressed alpha1 and the neuronal-specific alpha3. Ouabain-binding data showed that CHP-100 cells express approximately 3 x 10(5) pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na+, K+-ATPase in CHP-100 cells

Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line.

The Na+ pump (Na+, K+-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na+ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 +/- 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, 14C-taurine efflux increased by 183 +/- 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a 14C-taurine efflux of 207 +/- 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic alpha isoforms of Na+, K+-ATPase demonstrated high levels of the ubiquitously expressed alpha1 and the neuronal-specific alpha3. Ouabain-binding data showed that CHP-100 cells express approximately 3 x 10(5) pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na+, K+-ATPase in CHP-100 cells