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体感染，以测试研究人类免疫力和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，才能故意释放和人类工作。在未来，肺炎、脑膜炎和百日咳等疾病的疫苗将比目前的计划更加复杂，可能会采取滴鼻剂的形式，其中含有在鼻子和喉咙中提供细微免疫的分子。这是导致这些疾病的细菌的关键第一个进入点，新一代疫苗的关键是了解自然免疫如何阻止成功进入。阻止病原体的定殖可以阻止传播给其他人（畜群保护）。我们知道细菌表面的蛋白质帮助它们站稳脚跟。这些蛋白质也可能引发免疫力，可以作为疫苗利用。细菌的“特征”--细菌表面或内部的蛋白质--在细菌家族之间有很大的差异。这种变异使当前的蛋白质疫苗能够有效对抗我们所接触的部分（但不是全部）病原体。为了制造未来的疫苗，我们需要了解更多。我们的大部分知识来自于细胞实验室研究。有一些动物模型，但它们是不完美的。实验性人体挑战是一种强大的技术，人们自愿感染微生物，以研究对整个生物体的免疫反应以及疫苗或治疗的有效性。我们的团队是最早用细菌感染鼻子和喉咙的团队之一;我们使用了一种乳酸奈瑟氏球菌（“友好细菌”），感染了350多名志愿者，并证明它是安全和有用的。然而，如果我们能够控制和比较我们所识别的细菌的特征，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，我们将对内酰胺奈瑟氏球菌进行基因改造。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，以表明该生物体并不比野生型更危险。这将涉及到观察它如何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，它更有可能被遗传改变成更危险的东西）。在我们向当局申请许可之前，我们需要这样做，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进社区。我们联系了DEFRA（与故意释放有关的政府机构），他们告诉我们需要提供哪些信息。获得批准后，我们将招募参与者，他们将被允许进入我们的医院研究机构48小时并接受控制感染。然后，他们将被释放，并明确指示如何防止传染给他人。从志愿者中，将采集咽喉和鼻液以及血液样本，以分离携带的细菌（喉咙中），并检测免疫反应的细胞和可溶性产物，并对其进行表征。从喉咙中收获的细菌将进行基因组测序，以仔细检查细菌在遗传上保持稳定。一旦完成，我们将重复这项研究-这一次是为了发现细菌用来隐藏其特征（相位变化）的自然机制是否会引起不同的免疫反应。了解这一点很重要，因为它告诉我们如何确保未来这种类型的挑战使用正确的工程来解决研究问题。这项研究将是一个真正的开拓者-这项技术将快速发现细菌分子-以及宿主对它们的反应-这对病原体在人类中的定植至关重要。

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.