Pathfinder:Experimental Human Challenge with Genetically Modified Commensals to Investigate Respiratory Tract Mucosal Immunity and Colonisation

我们将对一种细菌进行基因改造，然后进行两次试点控制的人类感染，以在道路上测试这项研究人类免疫力以及发现和测试新疫苗的技术。这项提议很复杂--我们必须获得监管部门的批准，才能故意释放和人工操作。未来，针对肺炎、脑膜炎和百日咳等疾病的疫苗将比目前计划中的疫苗更加复杂，可能会采取滴鼻剂的形式，这种滴鼻剂含有在鼻子和喉咙中提供微妙免疫的分子。这是导致这些疾病的细菌的关键第一进入点，而新一代疫苗的关键是了解自然免疫如何阻止成功进入。阻止病原体的殖民可以阻止向其他人传播(羊群保护)。我们知道细菌表面的蛋白质帮助它们站稳脚跟。这些蛋白质还可能激发免疫力，可作为疫苗加以利用。细菌的“特征”--细菌上或细菌内的蛋白质--在细菌家族之间差异很大。这种变异使目前的蛋白质疫苗对我们接触到的一些病原体有效，但不是全部。为了制造未来的疫苗，我们需要更多地了解。我们的大部分知识来自于对细胞的实验室研究。有一些动物模型，但它们并不完美。实验性人类挑战是一种强大的技术，人们自愿感染微生物，以研究对整个有机体的免疫反应，以及疫苗或治疗的效果。我们的团队是第一批用细菌感染鼻子和喉咙的人之一；我们使用了一种共生的乳酸奈瑟菌(友好细菌)，并感染了350多名人类志愿者，证明了它是安全的和有意义的。然而，如果我们能够控制和比较我们接种的细菌的特征，实验医学所需的精确度将随之而来。这需要基因改造。在项目的第一部分，我们将对乳链球菌进行基因改造。这应该很简单，因为我们已经做了一个原型。然而，我们将需要广泛的实验室测试来证明这种有机体并不比野生类型更危险。这将包括观察它有多容易被抗生素和人类血液杀死，以及它的基因组有多稳定(即当它在鼻子里时，它是否更有可能从基因上变成更危险的东西)。我们需要这样做，然后我们才能向当局寻求许可，允许那些受到挑战的人在鼻子里仍然携带细菌的情况下走出社区。我们联系了DEFRA(负责故意释放的政府机构)，他们告诉我们需要提供哪些信息。一旦获得批准，我们将招募参与者，他们将被允许进入我们的医院研究设施48小时，并接受受控感染。然后，他们将被释放，并得到明确的指示，如何防止传播给其他人。志愿者将从咽喉和鼻液以及血液中提取样本，以分离(喉咙中)携带的细菌，并检测免疫反应的细胞和可溶性产物，这将是特征。从喉咙中采集的细菌将进行基因组测序，以仔细检查细菌在基因上保持稳定。一旦完成这一点，我们将重复这项研究--这一次是为了发现细菌用来隐藏其签名的自然机制(阶段变化)是否会引起不同的免疫反应。知道这一点很重要，因为它告诉我们如何确保这种类型的未来挑战使用正确的工程来解决研究问题。这项研究将是一项真正的探索者--这项技术将快速发现细菌分子--以及宿主对它们的反应--这些分子对病原体对人类的定居至关重要。

We will genetically modify a bacterium and then perform two pilot controlled human infections to road test the technique for investigating human immunity and discovering and testing new vaccines. The proposal is complex - we have to gain regulatory approval both for deliberate release and human work. In the future, vaccines for such diseases as pneumonia, meningitis and whooping cough will be more sophisticated than those in the current schedule, and will likely take the form of nose drops that contain molecules providing nuanced immunity in the nose and throat. This is the critical first entry point of bacteria that cause these diseases, and the key to new generations of vaccines is to understand how natural immunity blocks successful entry. Stopping colonisation by pathogens stops transmission to other people (herd protection). We know that proteins on the surface of bacteria help them gain a foothold. These proteins may also elicit immunity that could be harnessed as vaccines. The `signature` of the bacteria - the proteins on or within the bacterium - vary widely between bacterial families. This variation renders current protein vaccines effective against some, but not all, the pathogens to which we are exposed. To make the future vaccines we need to understand more. Most of our knowledge is from laboratory studies with cells. There are some animal models but they are imperfect. Experimental human challenge is a powerful technique in which people volunteer to be infected with microorganisms in order to study the immune response to the whole organism, and the efficacy of vaccines or treatments. Our group was one of the first to infect the nose and throat with bacteria; we used a commensal Neisseria lactamica (`friendly bacteria`) and have infected over 350 human volunteers and shown it is safe and informative. However, the precision required for experimental medicine will follow if we can control and compare the signature of bacteria that we inoculate. This requires genetic modification.In the first part of the project we will genetically modify N.lactamica. This should be straightforward because we have made a prototype. However we will then need extensive laboratory testing to show that the organism is not more hazardous than the wild type. This will involve seeing how easily it can be killed by antibiotics and human blood, and also how `stable` its genome is (ie is it more likely to be changed genetically into something more dangerous when it is inside the nose). We will need to do this before we approach authorities for permission to allow those people who are challenged to walk out into the community whilst they are still carrying the bacteria in their noses. We contacted DEFRA (the government body concerned with deliberate release) and they have told us what information we will need to provide.On approval, we will enrol participants who will be admitted to our hospital research facility for 48 hours and undergo controlled infections. They will then be discharged with clear instructions how to prevent transmission to others. From the volunteers, samples will be taken of throat and nose fluid, and blood, to isolate carried bacteria (in the throat) and detect the cells and soluble products of the immune response, which will be characterised. Bacteria harvested from the throats will be genome sequenced to check carefully that the bacteria remain stable genetically. Once this is done we will repeat the study - this time to discover whether the natural mechanisms bacteria employ to conceal their signatures (phase variation) cause a different immune response. This is important to know because it informs how to make sure future challenges of this type use the right engineering to address research questions. This study will be a true pathfinder - this technique will fast-track discovery of the bacterial molecules - and the host response to them - that are critical for colonisation of humans by pathogens.