Correlation of immunogenicity with microarray analysis of vector mutants to improve live recombinant poxvirus vaccines in poultry

我们如何改进更好的新疫苗的开发，以保护家禽(和其他牲畜)免受禽流感等重大疾病威胁？基因操纵(GM)正在对我们的生活产生越来越多的有益影响，特别是在人类和兽医保健方面；尤其是在疫苗方面，许多商业产品已经获得许可，并被放行用于牲畜和同伴动物。这些新疫苗基于可被视为靶向疫苗载体的“载体”，通常基于众所周知的疫苗，如痘病毒，对重要疾病的安全使用历史悠久。最著名的例子是痘苗病毒，它用于全球唯一成功地根除天花病毒疾病，并作为重组病毒用于消除来自比利时和法国的野狐狂犬病。自20世纪20年代以来，鸡痘病毒疫苗接种有效地消除了发达国家温带气候家禽中的鸡痘。它通过叮咬昆虫传播，在热带和亚热带国家仍然是一个主要问题，在这些国家，在孵化场为雏鸟接种疫苗很常见，而且范围很广。利用转基因技术，我们可以将来自不同致病病毒(或病原体)的基因(如禽流感H5N1)整合到载体的“基因组”(或染色体)中，形成“重组载体”。当该基因携带了制造病原体结构蛋白的指令时，用重组载体接种将在接种的动物中诱导针对病原体(和载体)的免疫反应。重组痘病毒已被批准用于兽医对抗西尼罗热、犬瘟、狂犬病和马流感。使用最广泛的是一种商业重组鸡痘载体，该载体含有禽流感的H5表面尖峰。自1995年以来，墨西哥已使用20亿剂疫苗为家禽接种H5禽流感疫苗。在那里，致命的禽流感形式被根除，但一种不那么危险的形式仍在传播。重组疫苗减少了禽流感的脱落和传播，但并不能完全防止禽类感染，可能通过随机突变推动病毒的进化。因此，仍有相当大的改进余地，特别是在清除禽类感染的免疫力方面。这些媒介不仅仅是惰性的递送系统。痘病毒激活免疫系统，必须在宿主的免疫反应存在的情况下生存。为了做到这一点，载体部署了数十种不同的基因产物。其中一些将降低载体作为重组疫苗的有效性。为了改善反应，我们可以使用转基因从载体中移除这些基因，但由于候选基因如此之多，我们的问题是确定哪些基因应该被移除。目前，检验疫苗是否得到改进的唯一方法是在动物身上进行测试。我们建议详细研究我们的鸡痘病毒突变体小组，每个突变体只在载体的250个基因中有1个基因缺陷。当载体进入宿主细胞时，它会增加(或减少)宿主30000个基因中约1,000个基因的蛋白质产量。我们将使用微阵列技术(在实验室的组织培养皿中进行)来观察不同的突变如何影响载体病毒对这些宿主基因的控制。在这项研究中，我们还需要了解每个突变如何影响重组载体诱导免疫反应(针对鸡的H5N1结构蛋白)的能力。然后，我们将寻找对重组载体改善的免疫反应与载体对宿主基因控制的变化之间的相关性。然后，这应该会给我们一个基因控制的轮廓或指纹，我们可以将其与改进的疫苗联系起来。今后，我们将在实验室中寻找这一特征，作为第一步。这将给我们一种方法，在动物试验之前预测哪些新疫苗可能会得到改进。

How can we improve the development of better new vaccines to protect poultry (and other livestock) against major disease threats such as bird flu? Genetic manipulation (GM) is having an increasing beneficial impact on our lives, particularly in human and veterinary health care; nowhere more so than in vaccines, where many commercial products have been licensed and released for use in livestock and companion animals. These new vaccines are based on 'vectors', which can be regarded as carriers for the target vaccine, and are generally based on well-understood vaccines, such as poxviruses, with a long history of safe use against important diseases. The best-known example is Vaccinia virus, used in the only successful global eradication of a virus disease, Smallpox, and as a recombinant in the elimination of feral fox rabies from Belgium and France. Fowlpox virus vaccination since the 1920s has effectively eliminated fowlpox from poultry in developed countries in temperate climates. Spread by biting insects, it remains a major problem in tropical and sub-tropical countries where vaccination of chicks in hatcheries is common and extensive. Using GM, we can incorporate into the 'genome' (or chromosome) of the vector, a gene from a different disease-causing virus (or pathogen), such as bird flu H5N1, making a 'recombinant vector'. When that gene carries the instructions to make a structural protein of the pathogen, vaccination with the recombinant vector will induce an immune response in the vaccinated animal against the pathogen (and vector). Recombinant poxviruses have been licensed for veterinary use against West Nile fever, canine distemper, feral rabies and equine influenza. The most extensively used is a commercial recombinant fowlpox vector incorporating the H5 surface spike of bird flu. Two billion doses have been used to vaccinate poultry against H5 bird flu in Mexico since '95. There, the lethal form of bird flu was eradicated but a less dangerous form remained in circulation. The recombinant vaccine reduces shedding and transmission of bird flu but does not completely prevent infection of birds, possibly driving evolution of the virus by random mutation. There, therefore, remains considerable scope for improvement, particularly in terms of immunity that will clear birds of infection. The vectors are not just inert delivery systems. Poxviruses activate the immune system and have to survive in the presence of the host's immune response. To do so, the vector deploys tens of different gene products. Some of these will reduce the effectiveness of the vector as a recombinant vaccine. To improve the response we can use GM to remove such genes from the vector but, with so many candidates, our problem is identifying those which should be removed. Currently the only way to see if the vaccine has been improved is to test it in animals. We propose to look in detail our panel of fowlpox virus mutants, each defective in just 1 of the 250 genes of the vector. When the vector enters a host cell, it turns up (or down) the production of protein from about 1000 of the host's 30000 genes. We will look to see how the different mutations affect the control of these host genes by the vector virus, using the microarray technique (performed in tissue culture dishes in the laboratory). In this study, we will also need to see how each mutation affects the ability of the recombinant vector to induce an immune response (against structural proteins of H5N1 in chickens). We will then look for correlation between improved immune responses to the recombinant vector and changes in control of the host genes by the vector. This should then give us a profile, or a fingerprint, of gene control that we can associate with improved vaccines. In future, we would look for this profile in the laboratory as a first step. This will give us a way of predicting which new vaccines are likely to be improved, before testing them in animals.