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Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen

02 April 2013

A huge step in foot-and-mouth disease vaccination has been achieved by using the protein shell of the virus (capsids) to create a more stable, safe and less expensive treatment.


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.

Figure 4. Virus neutralising antibody titres pre-challenge.
The group mean virus neutralising antibody titres (Log2) for animals inoculated with A22-wt (black line) or A22-H2093C (grey line) are shown from week 0 to week 34. All animals were vaccinated on week 0 and week 3. Blood samples were taken at weekly intervals until week 6 and then on week 22 and finally on week 34 when challenge with live virus was carried out. Error bars represent the standard error of the mean.


Here we have demonstrated the production of safe, effective FMDV empty capsids that do not require bio-containment during manufacture. Furthermore, enhanced stability of the empty capsids will reduce losses during production, storage and transport whilst maintaining antigenic structure and immunogenicity.

In addition the complete absence of FMDV non-structural proteins from the vaccine formulation will allow the development of diagnostic tests to discriminate between infected and vaccinated animals (DIVA).

Disulphide bonds are used to stabilise many extracellular proteins and also certain virus capsids. Such covalent cross-links are more robust than the non-covalent interactions that generally hold protein assemblies together.

Here we have rationally engineered a disulfide bond by mutating a single histidine residue at position 93 of VP2 located at the icosahedral 2-fold axis between adjacent pentamers. Baculovirus expressed wild-type and stabilised capsids produced equivalent titres of neutralising antibodies, following a standard immunisation regimen, over a 34 week period post immunisation.

These results inform the debate on the effect of increased antigen stability on immunogenicity. Delamarre et al showed that for two proteins with the same T cell and B cell epitopes but with different susceptibilities to lysosomal proteolysis, mediated by single point mutations, less digestible forms induced more efficient T cell priming and antibody responses.

In contrast, recent studies with a model antigen in mice suggested that enhanced conformational stability resulted in reduced antigenicity. Although as yet we do not know if forming a disulphide bridge will be possible for all serotypes, especially in the baculovirus expression system, our results demonstrate that capsid stability can be augmented without compromising immunogenicity and this might be a general tactic for improving vaccine efficacy.

The rational structure-based approach initiated here should in principle allow the tuning of these parameters to match the particular circumstances of different viruses. For instance, enhancing the stability of capsids for highly prevalent FMDV serotype O, which are more labile than those of A22.

Recent work on EV71 has demonstrated that maintaining the proper positions of the 2-fold helices (which harbour the H2093C mutation in FMDV) is essential for maintaining native antigenicity, suggesting that the approach we have demonstrated here may be applicable across a wide range of human and animal picornaviruses, including polioviruses and coxsackieviruses.

Citation: Porta C, Kotecha A, Burman A, Jackson T, Ren J, et al. (2013) Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen. PLoS Pathog 9(3): e1003255. doi:10.1371/journal.ppat.1003255

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April 2013

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