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

新城疫(ND)是一种影响家禽的高度传染性疾病，具有涉及神经系统、呼吸系统和消化系统的各种症状。死亡率取决于特定的病毒株，从0到100%不等；新城疫病毒(NDV)偶尔会感染人类，出现轻微症状，如结膜炎。这种疾病对家禽养殖户来说是一个重大问题，在世界许多国家都是地方病。针对新城疫病毒的疫苗已经开发出来，通常是基于非强毒(弱毒)形式的病毒。疫苗通过饮用水或气雾剂喷雾给药，以使大群鸡群同时得到保护，或者可以通过滴眼液给药。新城疫病毒是一种特别不稳定的病原体，因为它的遗传物质来自RNA，而不是DNA--这意味着疫苗需要在冻干(干粉)状态下冷藏。其结果是，在世界资源匮乏的国家，许多饲养小群禽类的农民无法获得疫苗，可能会经常失去整个鸡群。疫苗是在装有500-1000剂疫苗的瓶子里生产的，因此在自给自足的农业中使用过于昂贵，这一事实使问题变得更加复杂。对于许多疫苗来说，缺乏热稳定性是一个严重的问题，无论是人类还是动物，因为没有可靠的冷链(冷藏网络)的发展中国家可能无法将活性疫苗运送到偏远地区，疫苗浪费也很严重。该项目是牛津大学和皮尔布赖特研究所的合资项目，两个机构都在开发新疫苗方面经验丰富。我们的目标是开发一种新城疫疫苗的热稳定技术，从长远来看，这种技术可以应用于其他疫苗。冷冻干燥(干燥形成粉末)目前用于稳定许多疫苗，但并不理想，因为i)生产过程导致大量损失，包括冷冻和干燥步骤，这可能对活性产生不利影响，ii)所有冷冻干燥的许可疫苗都需要冷藏。我们计划使用一种名为“糖玻璃”的稳定技术，包括将疫苗与糖的浓缩液混合，然后干燥到纤维膜上，形成惰性糖玻璃，它薄薄地覆盖在膜纤维上。疫苗在储存后，通过在膜上添加缓冲液，可以快速而简单地重新悬浮。这项技术之前已经在实验性疟疾疫苗上进行了测试，但由于缺乏合适的支持基质，进一步的开发受到阻碍。我们的目标是通过等离子体处理对聚丙烯(PP)膜进行改性来解决这个问题。PP在许多方面都是新城疫疫苗的理想基质，但它不是“湿润的”，不经过前处理就不会吸收疫苗。等离子体处理包括使无线电波通过气体以产生带电离子；然后这种“等离子体”被用来处理包括防水聚合物在内的表面，使它们变得可润湿。该方法用于印刷行业对PP膜进行处理，使其附着在油墨上，但尚未应用于生物分子的吸收。该技术是快速、经济高效地生产稳定的新城疫疫苗的理想选择，适合转移到发展中国家的制造商，在发展中国家，PP膜可以切割成适合当地鸡群接种疫苗的大小。在为期18个月的项目中，我们将a)测试我们是否可以在等离子体处理的PP膜上形成含有糖玻璃的新城疫疫苗，b)在不同的温度和不同的时间孵化稳定的疫苗，模拟热带国家的运输和储存条件(4摄氏度至55摄氏度)，以及c)使用各种生物测试方法测试疫苗的回收，包括在鸡体内诱导保护性抗体水平的能力。

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.