Enhancing mucosal immunity to Streptococcus pneumoniae by nasal administration of live strains attenuated in virulence

肺炎链球菌（也称为肺炎球菌）是致命细菌感染的最常见原因。肺炎球菌是肺炎的最常见原因，也是与慢性肺病患者病情恶化相关的胸部感染的常见原因，因此是英国疾病的重要原因。目前用于成人和儿童的疫苗在预防血源性肺炎球菌感染方面非常有效;然而，它们在预防成人肺部感染方面无效，并且强烈需要预防肺炎球菌肺炎或胸部感染的新方法。肺炎球菌经常在喉咙后部发现，引起低水平感染，没有任何相关疾病。我们和其他人已经证明，肺炎球菌在喉咙中的定植刺激了宿主的免疫反应，可以预防未来的感染。这表明预防肺炎球菌肺部感染的潜在策略可能是每年在肺炎球菌感染高峰期（冬季）前不久故意用活肺炎球菌喷洒鼻子。要做到这一点，细菌必须进行基因改造，使它们无法预防肺炎或败血症等严重感染。在这项提案中，我们将使用人类模型的肺炎球菌定植，以测试是否管理基因改变的肺炎球菌不能引起严重的感染鼻子预防肺炎球菌感染的人。如果是这样的话，那么我们可以将其作为一种策略来预防那些特别易感的人（如老年人和慢性肺病患者）的肺炎球菌肺部感染。肺炎球菌携带模型已在利物浦安全地使用了5年，没有严重的不良反应，并且是一种经过验证的评估肺炎球菌定植免疫反应的方法。在用于人类定植模型的肺炎球菌菌株中制造突变体，防止其引起严重感染，但允许其定植在喉咙中。这些突变体将在小鼠模型中进行测试，以确认它们使用安全，但在定殖喉咙后仍能刺激显著的免疫反应。然后，我们将选择其中两种突变肺炎球菌菌株用于人类定植模型;年轻健康的志愿者将细菌接种到他们的鼻子中，然后在接下来的几天和几周内收集鼻腔冲洗液和血液样本。每种细菌菌株将给予35名志愿者，另外35名志愿者给予未突变的细菌和35名模拟感染。然后，这些志愿者将通过鼻内接种野生型肺炎球菌进行挑战，以观察对先前毒力降低的肺炎球菌突变体定殖的免疫应答是否会阻止随后的“正常”肺炎球菌定殖。将使用抗体和白色细胞反应的常规试验以及鼻洗液和血液样本评估志愿者对肺炎球菌定植的免疫反应。将比较定植前后的结果。此外，在用肺炎球菌感染小鼠之前，将通过将来自血液样品的血清注射到小鼠体内来测试定殖后抗体的任何增加是否保护免受肺炎球菌感染。这些实验将提供证据，证明用安全的无毒肺炎球菌人工感染咽喉是否能引起强烈的免疫反应，因此可能是预防肺炎球菌肺部感染的策略。如果是这样，下一步将是安全性测试和老年人或肺炎球菌肺部感染高危人群的临床试验。

Streptococcus pneumoniae (also called the pneumococcus) is the commonest cause of fatal bacterial infections. The pneumococcus is the commonest cause of pneumonia and a frequent cause of chest infections that are associated with deteriorations in patients with chronic lung disease, and therefore is an important cause of illness in the UK. The present vaccines used in adults and children are very effective at preventing blood borne pneumococcus infection; however they are not effective at preventing adult lung infections and there is a strong need for new methods of preventing pneumococcal pneumonia or chest infections. The pneumococcus is frequently found at the back of the throat causing a low level infection without any associated illness. We and others have shown that colonisation of the throat with the pneumococcus stimulates the host's immune response that can prevent future infection. This suggests a potential strategy to prevent pneumococcal lung infections could be to deliberately spray the nose with live pneumococcal bacteria on an annual basis shortly before the peak period for pneumococcal infections (the winter). To do so the bacteria would have to be genetically altered so they are unable to prevent serious infections such as pneumonia or septicaemia. In this proposal we will use a human model of pneumococcal colonisation to test whether administering genetically altered pneumococci unable to cause severe infection to the nose prevents pneumococcal infection in humans. If so then we could use this as a strategy to prevent pneumococcal lung infections in those people who are particularly susceptible eg the elderly and those with chronic lung disease. The model of pneumococcal carriage has been used in Liverpool for the past 5 years safely with no serious adverse effects, and is a proven method of assessing the immune response to colonisation with the pneumococcus.Our plan would be to:1. Make mutants in the pneumococcal strain used for the human colonisation model that prevent it from causing severe infection but do allow it to colonise the throat. These mutants will be tested in mouse models to confirm they are safe to use yet retain the ability to stimulate a significant immune response after colonising the throat.2. We will then select two of these mutant pneumococci strains to be used in the human colonisation model; young healthy volunteers will have the bacteria inoculated into their noses and then nasal wash fluid and blood samples collected over the next few days and weeks. 35 volunteers will be given for each strain of bacteria, with an additional 35 volunteers given unmutated bacteria and 35 mock infected. These volunteers will then be challenged by intranasal inoculation of wild type pneumococci to see whether the immune response to previous colonisation with pneumococcal mutants reduced in virulence prevents subsequent colonisation with 'normal' pneumococci.3. The immune response to pneumococcal colonisation in the volunteers will be assessed using conventional tests of antibody and white cell responses and the nasal wash fluid and blood samples. Results will be compared for before and after colonisation. In addition, whether any increases in antibodies after colonisation protects against pneumococcal infection will be tested by injecting the serum from the blood samples into mice before infecting them with pneumococci. These experiments will provide proof of whether artificial infection of the throat with a safe avirulent pneumococci can cause a strong immune response and therefore could be a strategy for preventing pneumococcal lung infection. If so, the next step would be safety testing and a clinical trial of this approach in older individuals or those at high risk of pneumococcal lung infection.