Date Published: January 19, 2017
Publisher: Public Library of Science
Author(s): Helen Fox, Sarah Knowlson, Philip D. Minor, Andrew J. Macadam, Bert L. Semler.
While wild type polio has been nearly eradicated there will be a need to continue immunisation programmes for some time because of the possibility of re-emergence and the existence of long term excreters of poliovirus. All vaccines in current use depend on growth of virus and most of the non-replicating (inactivated) vaccines involve wild type viruses known to cause poliomyelitis. The attenuated vaccine strains involved in the eradication programme have been used to develop new inactivated vaccines as production is thought safer. However it is known that the Sabin vaccine strains are genetically unstable and can revert to a virulent transmissible form. A possible solution to the need for virus growth would be to generate empty viral capsids by recombinant technology, but hitherto such particles are so unstable as to be unusable. We report here the genetic manipulation of the virus to generate stable empty capsids for all three serotypes. The particles are shown to be extremely stable and to generate high levels of protective antibodies in animal models.
The Global Polio Eradication Initiative (GPEI) is the largest intervention against a single disease in history. Naturally occurring wild type 2 poliovirus has not been seen globally since 1999, type 3 since 2012 and Afghanistan and Pakistan are the only countries where endemic type 1 virus is still circulating . The situation is complex however ; there are sporadic importations into countries where endogenous circulation had been previously interrupted and the use of the live vaccine is problematic as the strains can revert to virulent transmissible forms (circulating vaccine derived polio viruses, cVDPV) or be excreted over long periods from immune deficient patients exposed to the live attenuated vaccine (immune deficient vaccine derived polioviruses, iVDPVs) . Processes are being put in place to ensure that when polio is eradicated it does not re-emerge and these encompass the containment of work on the live virus and of production of the vaccine needed to ensure that coverage is maintained to guard against possible re-emergence . The issues have been brought into focus by the WHO decision to withdraw the type 2 component from Oral Polio Vaccine (OPV) from mid-2016, and to introduce at least one immunisation with Inactivated Polio Vaccine (IPV). This means that work on all type 2 viruses must be contained at a higher level [4, 5]. This will include IPV production where colossal amounts of virus are grown and subsequently inactivated with formalin; the strains in current use are mainly wild types known to be able to cause poliomyelitis in humans, although two companies have licensed products based on the live attenuated Sabin strains used in OPV. Safe production of IPV is essential and one way to do this is to devise viable production strains that are intrinsically safer [6, 7, 8]. An alternative described here involves production of empty viral particles with the correct antigenic and immunogenic properties which could be expressed by recombinant technology and not involve infectious virus at any stage. The main difficulty with such an approach is that naturally occurring empty particles are extremely unstable. We have therefore engineered empty viral particles based on the three existing IPV production strains to give virus-like particles (VLPs) that are at least as stable as the inactivated virus in IPV, have the same antigenic structures as the native viruses and are at least as immunogenic as IPV. Provided they can be produced at the appropriate scale this platform has major advantages compared to the current procedures in terms of both safety and the properties of the product.
We reported the production of empty capsids by expression of poliovirus proteins in recombinant baculovirus-infected insect cells that did not involve poliovirus growth more than two decades ago but the particles were too unstable to be useful as a vaccine candidate . Immunogenic, but unstable, VLPs were also produced by recombinant expression in yeast . A strategy to address this instability is described here where candidate stabilising mutations were identified then multiple changes introduced into capsid proteins in combination. This approach took advantage of a mutation known to be present in the type 3 Sabin vaccine strain of poliovirus that destabilises capsid assembly intermediates (including empty capsids) without affecting virion stability . Thus revertants from vaccinees and other sources were thought likely to possess mutations that restore assembly and increase empty capsid stability. Stabilising mutations in the type 1 and 2 strains were identified by inserting the destabilising Sabin type 3 mutation, selecting in transfected cells at non permissive temperature and screening for revertant genomes by deep sequencing. The approach could also be used to guide selection for other, non-polio, picornaviruses. In some cases stabilising mutations found for type 3 were already present in the type 1 and type 2 viruses. The work described here was performed as part of a consortium whose members are given under Acknowledgments; the consortium is also following other approaches to the identification of candidate capsid stabilising mutations including prediction of stabilising interactions from the atomic structure and selecting for thermostable virus mutants by limited exposure to high temperatures. The consortium is also developing several recombinant expression systems for virus-free synthesis of stable, immunogenic VLPs.