Multivalent proteins for the prophylaxis of influenza

The influenza virus remains a major health challenge. The virus can evade our immune system as a result of its segmented genome, its ability to reassort in various species (e.g. man, pig, duck), its high mutation rate, its ecological niche in nearly all aquatic birds and its ability to subvert various aspects of our innate immunity. Groups at risk from the virus include the very young and very old, pregnant women, the immunocompromised and those with a chronic underlying cardio-pulmonary disease. Vaccines remain the cornerstone of protection, although have only 30-50% efficacy in the elderly. Seasonal vaccination affords reasonable protection (although recent vaccines have reported effectiveness of 25% or less against circulating H3N2 viruses), but would have no effect on a new pandemic strain if one was to emerge - and history tells us this is more likely than not. New vaccines take several months to develop and to generate sufficient stocks to treat whole populations.

The emergence and threat of the avian H5N1 virus in 2005 led several governments to invest huge funds (particularly in the US where President Bush committed >$7billion) to develop new vaccine technologies, new therapeutics and to stockpile influenza neuraminidase inhibitors, particularly oseltamivir (Tamiflu, Roche), although zanamivir (Relenza, GSK) was also stockpiled by several countries: the UK spent £500M on Tamiflu. Overuse of drugs can elicit resistance, and this was clearly evident in the 2008-2009 season when nearly all circulating H1N1 seasonal strains became resistant to Tamiflu. The currently circulating H3N2 and swine H1N1 viruses appear susceptible to both Tamiflu and Relenza, but resistance could easily arise in the future. There are now great efforts to develop novel vaccines, some of which are claimed to be 'universal' against all strains, and to develop novel therapeutics against several proteins of the influenza virus. The ever-changing virus may become resistant to these new therapeutics in time.

Our approach is to target the host rather than the virus. The influenza virus binds to the carbohydrate sialic acid that decorates the surface of most mammalian cells. We have used genetic engineering to create proteins that contain several copies of a small protein domain (CBM) that binds to sialic acid. Multivalency enables us to achieve high binding affinity, such that a protein with six CBM modules binds 10,000 times more tightly than a single CBM module. We have shown that these multivalent proteins can be administered intranasally in a single dose to mice and protect them from a lethal challenge with swine H1N1 influenza virus. The treated mice are healthy, gain weight and show no clinical symptoms. Most importantly, as well as surviving a lethal challenge with virus, the mice mount an immune response to the virus meaning that they would be able to face a subsequent challenge from the same virus. We therefore believe we have developed a novel therapeutic (or protein biologic) that has potential as a prophylactic against any strain of the influenza virus that may emerge, as the virus is unlikely to shift the receptor it uses to gain access to cells.

Our studies will continue to involve mouse experiments at the Roslin Institute, Edinburgh and at St Jude Children's Research Hospital, Memphis. St Jude has one of the World Health Organisation's collaborating laboratories on influenza, headed by Robert Webster FRS, one of the world's top influenza experts. We are now focusing on our two lead biologics that both contain six copies of the sialic acid-binding CBM. The aims and objectives of this new application, a 1 year extension to our current 2 year MRC DPFS grant, is to examine several aspects of the two lead biologics through in vivo studies in mice and ferrets, and through in vitro studies using a cilliated cell line derived from the human epithelial airway.

The influenza virus continues to be a major health threat with the appearance of new assortants (e.g. H1N1 "swine flu" and H5N1 & H7N9 "bird flu"), difficulties in providing fast and effective vaccination and an increasing resistance to licenced antivirals, particularly oseltamivir (Tamiflu). Our approach targets the host rather than the virus, overcoming drug resistance that is a major problem with the rapidly evolving influenza virus with its potential to mutate and genetically reassort. We have engineered a series of multivalent proteins, or biologics, that bind with high affinity to the terminal sialic acids of cell surface glycoconjugates, thereby masking the receptor of the influenza virus and other respiratory pathogens. As this recognition is linkage-independent, the biologics mask sialic acids receptors with (a2,3) and (a2,6) linkages recognized by avian and human viruses, respectively, and these different receptors have been found at varying locations in the human respiratory tract.

In our current DPFS grant we have studied a panel of engineered multivalent proteins that differ in both their valency and the origin of their sialic acid binding modules. Our two lead candidates, when administered intranasally as a single dose, protect mice from a subsequent lethal challenge with influenza virus. Significantly, our current candidate is effective even when given as a single 1ug dose 7 days before virus.

In the 12 month extension, we are seeking to determine an optimal prophylactic dosage regimen in mice and to initiate studies in ferrets, the preferred animal model for influenza studies, against both H5N1 and H7N9 viruses. Critically, immunogenicity assessment of the lead candidates will be considered through evaluation of the immune response to the biologics upon repeat dosing. Deimmunisation strategies will be addressed. The efficacy of delivery by nebulizer will also be explored.

The beneficiaries of this research could potentially be whole populations that require protection from a new pandemic strain of the influenza virus in a scenario where antivirals are ineffective (because of viral resistance or ineffective prophylactic use), where seasonal vaccination affords no protection and where something is needed to bridge the gap until a new vaccine can be formulated and distributed to the whole population. The US alone administers 145 million vaccinations each year, providing cover to less than half of their population. A less extreme scenario would be routine use each season by health workers and care workers, and the elderly who are susceptible to the seasonal influenza virus and in whom the seasonal vaccination is generally only 30-50% effective. Indeed seasonal vaccination with vaccines against H3N2 show only 25% effectiveness in broad sections of the population. In 2008-9 most circulating H1N1 viruses became totally resistant to Tamiflu, one of the front-line antivirals that has been stockpiled by several countries, including the UK. Our studies to date in mice indicate that when used prophylactically, our biologics protect against the virus allowing healthy growth of the mice and limiting any clinical symptoms, yet they allow an immune response to develop to the virus providing long term protection in the event of re-exposure to the same virus.

Potential beneficiaries beyond the health benefits to the general population would be companies that engage in the further stages of development through toxicology studies and phase I, II and III clinical trials. This may be a UK based pharmaceutical company in the longer term, but could potentially be the development of a spin-out company from St Andrews in the short to medium term. An interesting parallel is Biota, the Australian SME that first developed zanamavir (Relenza) that was invented by Professor Mark von Itzstein's Monash laboratory and was subsequently licensed to GSK. More recently, Biota has developed laninamivir, a long-lasting zanamivir analogue. Biota has an agreement to receive up to $US231M from BARDA (Biomedical Advanced Research and Development Authority), part of the US Department of Health and Human Services, to develop the drug for licensing in the US. Such an arrangement is of obvious benefit to the Australian economy both in terms of the employment during the development and the downstream profit sharing. We are exploring the option of a spin-out verses licensing to a pharmaceutical company as the exit point at the end of this requested 1 year extension to our DPFS grant. The former may provide employment benefits to the local Fife economy, but both may in the long term provide income to the University to invest in future education and research.

The team involved in the DPFS-funded studies at St Andrews continue to be skill beneficiaries of the research project. At the start of the project in 2010, the team was largely a structural biology group. Over the 2 years of the current DPFS grant, the team has developed a broad range of skills in immunology, tissue culture, imaging, in vitro viral infectivity and toxicity assays, cytokine analyses, a variety of ELISAs to study antibody responses, as well as professional project development and management skills in coordinating a complex set of data analyses, coordination of outsourced animal studies and the monthly project management meetings and reporting. Additional skills have arisen from presentations to, and discussions with, third parties who have a potential interest, such as pharmaceutical companies and investors.