Molecularly Imprinted Nanoparticles (MIP NPs) as non-animal antibodies substitutes for detection of viruses

Imagine that you create an imprint of your right hand in soft clay and then let the clay harden: as a consequence you will have created a cavity into which your right hand will fit perfectly, unlike your left hand which is similar but not exactly the same. Now think smaller: you can do a similar thing at a very small scale using generic synthetic receptors - molecularly imprinted polymers (MIPs) - produced in the form of very small, one billionth of a metre in size (nano)particles (MIP NPs). Essentially these are "smart" pieces of plastic that can be tailored to bind to a target by the creation of tiny pocket-like cavities (the "imprint") that match the size and shape of the peptide, protein or virus (the "right hand") to be detected. These MIP NPs to date represent the most generic, versatile, scalable and cost-effective approach to the creation of "artificial antibodies", hence the proposed research for this Fellowship would like to determine if MIP NPs can be developed as an alternative and a direct replacement to natural antibodies (which are routinely generated from animal sources such as mice, rabbits and guinea pigs) for the detection of a very important infectious disease, avian influenza, that produces devastating consequences to animal health and poses a threat to public health. Novel MIP NPs will be able to replace natural antibodies in biological detection techniques (bioassays) and significantly reduce or even replace animal use.

Recent developments in the automated synthesis of MIP NPs, pioneered by Prof. Piletsky's group, mean that for the first time a reliable supply of "soluble" synthetic nanoparticles can be made available for testing as potential biological active molecules. MIP NPs have many attributes that surpass current reagents: they can be produced in high yields, are stable unlike the current reagents being used, they are adaptable and versatile presenting themselves for use in many varieties of formats or assay designs. More importantly, substantiating the practicability's of these novel reagents by specific studies will lead to a replacement of animals, both large and small, to raise such specific serological materials. The MIP NPs will have the potential to detect viruses that are widely circulating in farm animals and indeed humans. Early and accurate identification of the infectious agent will expedite appropriate control measures. Thus diagnosis at an early stage of infection of a herd or flock or individual maximises the efficiency with which containment, prevention and possibly treatment strategies can be implemented.

Success of this research could lead to further investigations into which the versatility of MIP NPs could be exploited for animal and human benefit outside veterinary medicine such as pharmaceutical, biotechnology and fine chemicals industry. The research into the development of MIP NPs that are highly specific coupled with diagnostic technology could be a significant contribution to the scientific advancement and cost effective diagnosis at global scale.

This Fellowship proposal aims to determine if artificial antibodies - molecularly imprinted nanoparticles (MIP NPs) - can be developed as new diagnostic/therapeutic entities in veterinary medicine to directly replace bioassays for a very important infectious disease, avian influenza, which possesses an immense potential for harm to poultry, and also a dangerously high chance to spread from poultry and pigs to humans. To date, the molecular imprinting of polymers represents the most generic, versatile, scalable and cost-effective approach to the creation of synthetic molecular receptors. Recent developments in the automated synthesis of MIP NPs using an immobilised template approach pioneered in the group of Prof. Piletsky mean that for the first time a reliable supply of "soluble" synthetic nanoparticles with pre-determined molecular recognition and/or catalytic properties with sub-nanomolar affinities, defined size and surface chemistry can be made available for testing as potential bioactives. These bioactives may have the potential to detect viruses that are widely circulating in farm animals and indeed humans. Early and accurate identification of the infectious agent will expedite appropriate control and even treatment measures. Therefore, the research into the development of MIP NPs with high affinity coupled with diagnostic technology could be a significant contribution to the scientific advancement and cost effective diagnosis at global scale.

In the context of the application, the in vitro antigenic properties of avian influenza virus (AIV) H5N1 are assessed by serological cross-reactivity (HI assay measured by single radial immunodiffusion, SRID) using polyclonal vaccinal sera (PVS) raised against reference vaccine strains. This assay relies on the availability of reference materials for standardisation of vaccine potency, i.e. the haemagglutinin antigen references and corresponding anti-serum. These extremely important reference reagents provide the tools by which expert advice can be given on vaccine strain suitability or, indeed, the need to develop new vaccines. They are updated and distributed by the World Health Organisation (WHO) collaborating centers, taking in general 2-3 months to prepare them from the time when each new strain is recommended. The reliance on these materials for tests such as HI by SRID lead to heavy usage of these important polyclonal anti-sera. Thus, there is a continual need to supplement or recharge these reagents by specifically vaccinating groups of animals of desired immune status, with the prerequisite vaccine strain. Even then, such materials must pass the necessary associated validation in the order for them to be adopted for use in such tests. The use of MIP NPs presents a feasible alternative and a direct replacement to the reliance of reagents such as those raised in animals sera for use in bioassays including those for diagnostic purpose. MIP NPs have many attributes that surpass current biologicals. They can be produced in high yields, they are thermo-stable, adaptable and versatile presenting themselves for use in many varieties of formats or assay design and are consistent in their properties. More importantly, substantiating the practicability's of these novel reagents will lead to a replacement that avoids using animals, both large and small, to raise such specific serological materials. Whilst the actual reduction in the number of animals is hard to measure at the local level, it is not uncommon for an average study which involves the use of these anti-sera to require from 30 to 50 animals (usually chickens, ferrets). In terms of production for reference samples, also larger animals like sheeps are exploited aside of any requirements for similar reagents to be made through the immunisation of mice, rabbits and guinea pigs. If successful, there will be a complete substitution of the antisera animal production with the synthetic counterparts; it is likely that in the early stages of the establishment of the technology it will be mostly a Reduction rather than a complete Replacement (possibly a 50 to 70% Reduction), but once the technology is established the Replacement will be complete.