Stabilisation of Newcastle Disease vaccine formulated in sugar-glass on polypropylene membranes

Newcastle disease (ND) is a highly contagious disease affecting poultry, with diverse symptoms involving the nervous, respiratory and digestive systems. Mortality rates depend on the particular viral strain and can vary from 0 to 100%; Newcastle Disease Virus (NDV) occasionally infects humans with mild symptoms e.g. conjunctivitis. The disease presents a major issue for poultry farmers, and is endemic in many countries of the world. Vaccines have been developed against NDV, usually based on non-virulent (weakened) forms of the virus. The vaccine is administered in drinking water or by aerosol spray to enable large flocks to be protected simultaneously, or can be dosed by eye drops. NDV is a particularly unstable pathogen because its genetic material is made from RNA, rather than DNA - this means that the vaccine needs to be stored by refrigeration in a lyophilised (dry powder) state. As a result, many farmers with small flocks of birds, typically chickens, in low-resource countries of the world do not have access to vaccine and can lose entire flocks on a regular basis. The problem is further compounded by the fact that the vaccine is manufactured in vials containing 500-1000 doses, and is therefore too expensive for use in subsistence farming. Lack of thermo-stability is a serious problem for many vaccines, both human and animal, as developing countries with no reliable 'cold chain' (network of refrigerated storage) may not be able to deliver active vaccines to remote regions and vaccine wastage is considerable.

This project is a joint venture between the University of Oxford and the Pirbright Institute, both experienced in the development of new vaccines. Our objective is to develop a technology for thermo-stabilisation of NDV vaccine, which in the longer term can be applied to other vaccines. Lyophilisation (drying to form a powder) is currently used to stabilise many vaccines, but is not ideal as i) the manufacturing process results in substantial losses and includes freezing and drying steps which can adversely affect viability, and ii) all lyophilised licensed vaccines require refrigeration. We plan to use a stabilisation technology called 'sugar-glass' formulation which involves mixing vaccines with a concentrated solution of sugar, and then drying onto fibrous membranes to form inert sugar-glass which thinly coats the membrane fibres. Vaccines are quickly and simply resuspended after storage by the addition of buffer to the membrane. The technology has been tested previously with an experimental malarial vaccine but further development was impeded by lack of a suitable support matrix.

We will aim to solve this problem by modifying Polypropylene (PP) membrane using Plasma treatment. PP is in many ways an ideal matrix for NDV vaccine sugar-glass, but is not 'wettable' and will not absorb vaccine without pre-treatment. Plasma treatment involves passing radio waves through a gas in order to produce charged ions; this 'plasma' is then used to treat surfaces including water-repellent polymers to make them wettable. The method is used in the printing industry to treat PP membrane so that it adheres to ink, but has not been applied to absorption of biological molecules. The technology is ideal for quick, cost-effective manufacture of stabilised NDV vaccine and is suitable for transfer to manufacturers in developing countries, where PP membrane can be cut into sizes appropriate for vaccination of local flocks. During the 18 month project we will a) test whether we can form a sugar-glass containing NDV vaccine on plasma treated PP membranes, b) incubate the stabilised vaccine at different temperatures and at various times, mimicking transport and storage conditions in tropical countries (4oC to 55oC), and c) test recovery of the vaccine using a variety of biological assays including the ability to induce a protective level of antibodies against the disease in chickens.

This project will utilise plasma technology to produce a membrane suitable for sugar-glass stabilisation of Newcastle Disease virus (NDV) vaccine. Sugar-glass stabilisation involves formulation of vaccines with trehalose and sucrose, then drying onto fibrous membranes at room temperature for 18-24 hours to form inert sugar-glass which thinly coats the membrane fibres. Whilst used successfully to thermo-stabilise Adenovirus and MVA vectored vaccines, selection of a suitable membrane matrix has proved problematic; polypropylene (PP) membranes have many of the physical properties required, but need to be pre-treated in order to improve hydrophilicity and absorb sugar-vaccine. Plasma (ionised gas) is typically used for industrial applications involving cleaning, adhesion and filtration; we plan to exploit its ability to improve the wettability of hydrophobic surfaces for pre-treatment of PP membranes, using low pressure 13.56 MHz RF glow discharge oxygen gas plasma.

NDV vaccine formulated in trehalose-sucrose will be dried onto plasma-treated PP membranes and incubated for periods of time ranging from 24 hours to a maximum of 6 months in low moisture environments, at temperatures of 4oC, 25oC, 37oC, 45oC and 55oC. Samples will be stored in moisture proof bags to ensure moisture content of the sugar-glass is maintained at levels below 5%. Vaccine recovery will be measured using a) plaque assays in CEF (chicken embryo fibroblast) cells, b) EID50 assay in embryonated SPF eggs, and c) in vivo immunogenicity in chickens, measuring antibody levels with a validated ELISA assay as recommended by OIE standards. Up to 4 different PP membranes will be evaluated, and thermo-stability of NDV vaccine compared to formulation on GF (glass fibre) membrane, and PP membrane treated with a more conventional chemical approach, i.e. soaking in a dilute solution of tween followed by drying for 12 hours. A lyophilised vaccine control will be included at all time points.

Farmers in resource-poor countries would be a major beneficiary of this project, as the over-arching objective is to produce an affordable, thermo-stable vaccine suitable for protection of small flocks of chickens and other fowl. Successful vaccination would prevent loss of birds from NDV outbreaks, with considerable economic benefit to the farmers and the local communities as a whole. Successful vaccination against NDV would support sustainable farming without the disastrous consequences resulting from disease epidemics, and in turn positive impacts on the environment could be achieved, by encouraging sustainable agriculture using the principles of ecology rather than repeated re-stocking of flocks decimated by disease. Farmers and families keeping small numbers of chickens would gain enhanced understanding and knowledge about the benefits of vaccination, improved animal health and wildlife conservation. Village chickens are the most common type of livestock kept by individual households, and make a major contribution to poverty alleviation and the empowerment of women, as poultry are frequently the only livestock under the control of women.

Farmers in developed countries with existing programmes of NDV vaccination would benefit from lower vaccine costs, arising from cheaper formulation costs, increased shelf life and storage at room temperature. The risk of using out-of-date vaccines with reduced potency would also be minimised.

Other economic beneficiaries: companies and/or manufacturing organisations producing NDV vaccine in both the developed world and resource-poor countries would benefit from production of a cheap, stable vaccine with reduced losses from vaccine instability during transport and storage. Additionally, the technology could be applied in the future to stabilise other vaccines and potentially any biomolecule used in the treatment of human and animal disease: pharmaceutical, biotechnology and manufacturing companies could all benefit as a result, by production of biological therapies with greatly improved shelf-lives, without the requirement for storage by refrigeration or freezing.