Novel aptamer-based diagnostics and antiviral therapeutics for animal influenza viruses

Avian influenza (AI) infections pose a major threat to animal health and are potential progenitors for human influenza pandemics. A recent example is the emergence of a new pandemic influenza virus (H1N1pdm) from pigs which is currently causing widespread infection and some mortality in humans. Similarly, the continued prevalence of avian influenza viruses of the H9N2 sub-type, and the highly pathogenic H5N1 and H7 (H7N1, H7N3 and H7N7) subtypes in poultry in many countries highlights the need for improved surveillance and control measures. These will require rapid, easy to use AI diagnostics that can differentiate subtypes of these highly prevalent influenza viruses without the need for extensive laboratory resources. Additionally, improved antiviral drugs for treatment and prophylaxis would also help to minimise the impact of these viruses in both humans and animals. Considering these immediate and future requirements, this project will exploit synthetic single stranded nucleic acid molecules known as 'aptamers' to develop the next generation of diagnostic and possibly therapeutic molecules for influenza viruses. Since Influenza A viruses exist in many subtypes this versatile technology can be used as the basis of sensitive and specific diagnostic assays that can detect different subtypes of influenza viruses simultaneously (differential diagnostic assays). In addition the aptamers that show promising binding affinity for influenza viruses can also be examined for their potential as antiviral therapeutics, for example against H1N1 (swine flu) and H5N1(bird flu) influenza viruses. Aptamers will be produced that specifically and selectively bind to selected subtypes of influenza virus surface glycoproteins heamagglutinin (HA) and neuraminidase (NA) (which confer subtype specificity) and the nucleoprotein (NP) and matrix (M1) protein (which is conserved in all AI viruses) and then combined with highly sensitive detection technologies based upon gold nanoparticles. These aptamer conjugates are anticipated to bind to viruses in clinical samples to form core-shell nanostructures. The aptamer-virus nanostructures than can be quantified by either optical or electrical methods. The detection sensitivity of these aptananosensor devices would be such that 100 to 1000 virus particles in a clinical sample and could be detected within 30 minute or less. An aptananosensor should be economical, portable and simple to use for efficient and rapid pen-side diagnosis. Above all it will provide the high sensitivity and specificity necessary for differential diagnosis of influenza viruses infecting both humans and animals. Since the selected aptamers will have a high affinity for viral envelope proteins they could also block virus replication. In this project, we will also explore the therapeutic properties of selected aptamers against influenza virus infection in both in vitro assays and in animal models. This could provide a platform to develop new generation of therapeutics against influenza virus infections in humans or animals. The first step will be to assess the antiviral properties of selected high affinity aptamers in tissue culture against selected H1N1 and H5N1 viruses. The aptamers that show promising results in vitro will then be tested in vivo. The selected mammalian and avian animal models will be infected with selected H1N1 and H5N1 virus strains and the protective effects of the aptamers will be assessed in their ability to prevent infection and also to control infection. The protective efficacy in animals will be assessed by correlating the onset of disease signs and reduction in viral titres compared with infected untreated animals. AI aptamers will provide common molecular entities for both diagnostic and therapeutic platforms in the fight against this ever present, yet ever changing threat.

The expression of a set of Avian Influenza virus proteins comprising haemagglutinin, and neuraminidase glycoproteins as well as the more conserved nucleoprotein (NP) and ion channel (M) proteins will provide target molecules against which DNA/RNA aptamers will be selected. The glycoproteins and proteins will be expressed in Drosophila S2 cells yield well defined functional molecules that will be the targets for screening an aptamer library. Selection and counter-selection methods will be designed to yield molecules that exhibit defined strain and serotype specificity and the affinity and cross-reactivity determined using both surface plasmon resonance and agglutination inhibition assays. Aptamers showing suitable affinity and specificity will then be synthesised as 3' or 5' thiohexyl terminated derivatives for self-assembly onto gold nanoparticles (AuNP's) or gold electrodes. These conjugates will be the foundation for developing point of care diagnostic devices. Optical detection will be based upon the AuNP's forming a shell around the viral envelope with a resultant change in light scattering associated with the different size and structure of the virus coated with nanoparticles and the isolated nanoparticles. Electrochemical sensing will make use of redox labelled aptamers and the change in current associated with virus binding restructuring the aptamer and altering the distance between the label and electrode. High affinity aptamers isolated during library screening will also be evaluated in terms of their antiviral potential. Firstly, the antiviral properties of selected high affinity aptamers against selected H1N1 and H5N1 viruses in vitro through haemagglutination inhibition and neuraminidase inhibition assays and tissue culture virus infectivity assays. Those aptamers that prove most effective in the in vitro assays will then be evaluated for their protective effect in both a mammalian (mouse) and an avian (chicken) model of AI infection.

The proposed research is aimed to exploit an emerging technology to address problems associated with influenza A virus infections (H1N1, H5N1, H9N2 and H7 subtypes) that produce devastating consequences to animal health and pose a threat to public health. The innovative research in aptamer technology leading to the development of a new class of improved diagnostic reagents and serving as a foundation for antiviral therapeutics will facilitate and accelerate the development of novel alternatives and so aid in the reduction of the impact of pandemic and emerging pandemic influenza viruses. The results of the proposed project will be both knowledge and technology. The anticipated diagnostic tests based on aptamers (short synthetic single stranded DNA molecules) coupled to nano biosensors (aptananosensors) will be the basis of a new generation of improved rapid, accurate, highly sensitive and economical tests that can be performed at pen-side at the farm or point of care. They will have an ability to detect and differentiate different subtypes of the influenza A viruses that are widely circulating in poultry, pigs and humans. Early and accurate identification of infectious agent will expedite appropriate control measures. Thus diagnosis of influenza virus infection 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. Therefore, the research into the development of this platform diagnostic technology and the resultant highly sensitive diagnostic tools for influenza virus detection could be a significant contribution to the scientific advancement and cost effective diagnosis of influenza virus at global scale. The allied anticipated outcome, the aptamer based antiviral therapeutics, could in time play an important role lessening the impact of both influenza virus infections in both humans and animals. The knowledge gained from this multidisciplinary proposed research will also enhance the basic understanding of the structure-function relationships between selected aptamers and influenza virus proteins that directly help in advancing this nascent technology to further improve, design and develop new control measures against other infectious disease affecting both humans and animals. We expect that this research will be translated into prototype products of demonstrated effectiveness in terms of efficiency and efficacy during the lifetime of the project. Commercial companies will then have the opportunity to benefit from this research directly by commercialisation and marketing. Large-scale availability of cheap, sensitive, specific and easy to use diagnostic tools will enhance the ability for early detection of influenza virus infections thus enabling effective control and treatment. The reagents and scientific knowledge generated will be publicised in peer-reviewed Journals, on the IAH,Imperial College and NIMR websites and in presentations at meetings of scientists, veterinary and medical and health professionals. The staff and scientists working on this project will benefit enormously by acquiring training in many new emerging scientific disciplines. The project team is multidisciplinary and involves experts in influenza virus molecular virology, bionanotechnology, biosensors and the development of diagnostic tools for human and veterinary medicine. The PIs have experience in interacting with potential beneficiaries outside the immediate academic community around the globe, including both developed and developing countries. These include public government and commercial agencies and medical and veterinary authorities.