The human gut microbiome, malnutrition and vaccine responses

描述（由申请人提供）：全球每年有数百万儿童死于营养不良。他们的发病和死亡大多是感染的结果，包括由肠道病原体引起的感染。由于不明原因的疫苗失效，阻止这些感染的努力在一些人群中受到限制。我假设营养不良、肠道微生物组和免疫系统之间存在相互关系，这与疫苗反应性有因果关系，这些相互关系可以通过“人源化”的非生物小鼠进行信息探索。我们的实验室正在对生活在马拉维的1-36个月大的双胞胎进行肠道微生物组的纵向研究，这些双胞胎是健康的，或者是患有严重营养不良（如夸希奥克尔病）的。我们正在解决以下问题：是否存在与严重形式的营养不良相关的可识别的肠道微生物组配置？使用即食治疗性食品（RUTF）干预后，微生物群/微生物组是如何重新配置的？在RUTF停止后，它是否持续存在？如果退出RUTF后配置状态没有持续，这是否意味着在缺乏长期营养支持的情况下，代谢活动的持续异常会导致人类宿主持续的生理异常？我们最近的研究表明，将冷冻的人类粪便样本中的微生物群落有效地移植到无菌小鼠体内，并将移植的微生物群从母体传给后代是可能的。有了这个功能，我为这个NRSA应用程序提出了两个目标。目的1。使用无菌小鼠，在6周龄时定植，从几对患有营养不良（1健康/ 1营养不良）不一致的同卵双胞胎及其母亲的微生物群中收集未鉴定的粪便微生物群。受助人将得到缺乏宏量和微量营养素的马拉维饮食，或营养“充足”的对照饮食。我将使用抗原特异性ELISA跟踪他们对接种霍乱毒素和卵清蛋白（一种模型粘膜佐剂/抗原）的粘膜疫苗的反应。我将通过血清LPS测定、乳果糖/甘露醇吸收测定、肠道形态和免疫细胞群的组织和免疫组织化学测定（探索它们是否具有热带肠病的特征）以及肠道基因表达的功能基因组研究来定义它们的肠道黏膜屏障功能。我将对他们的粪便微生物组进行时间序列宏基因组研究（对细菌16S rRNA基因、总群落DNA和表达mrna进行测序）。目标2。评估母体营养和微生物群“状态”对其后代疫苗反应的作用，方法是将妊娠雌性GF小鼠定植在患有夸希奥克尔病的双胞胎及其母亲的粪便样本上。母亲将在怀孕之前、期间和之后保持马拉维或营养充足的控制饮食；它们的后代将断奶饲喂营养不足或营养充足的饲料，并接受与目标1中描述的相同的疫苗接种方案和分析。这些实验应有助于确定营养不良和疫苗反应性的生物标志物。

DESCRIPTION (provided by applicant): Malnutrition kills millions of children worldwide every year. Much of their morbidity and mortality is a result of infections, including those caused by enteropathogens. Efforts to stop these infections have been limited in some populations by unexplained vaccine failures. I hypothesize that there is an interrelationship between malnutrition, the gut microbiome, and the immune system that is causally linked with vaccine responsiveness and that these interrelationships can be informatively probed using 'humanized' gnotobiotic mice. Our lab is conducting longitudinal studies of the gut microbiomes of twins aged 1-36 months living in Malawi who are healthy, or who are concordant or discordant for severe forms of malnutrition (e.g., kwashiorkor). We are addressing the following questions: Are there identifiable configurations of the gut microbiome associated with severe forms of malnutrition? How is the microbiota/microbiome reconfigured with a Ready to Use Therapeutic Food (RUTF) intervention and does it persist after cessation of RUTF? If the configured state does not persist after withdrawal of RUTF, does this mean that in the absence of longer-term nutritional support, lingering abnormalities in the metabolic activities contribute to persistent physiological abnormalities in the human host? We have recently shown that it is possible to efficiently transplant the microbial community present in frozen human fecal samples into germ-free (GF) mice and to transmit the engrafted microbiota from mothers to their offspring. With this capability, I propose two aims for this NRSA application. Aim 1. Use gnotobiotic mice, colonized at 6 weeks of age with de-identified fecal microbiota already collected from several sets of monozygotic twins discordant for kwashiorkor (1 healthy/ 1 malnourished) as well as their mothers' microbiota. Recipients will be fed a macro- and micronutrient deficient Malawi diet, or a control nutritionally 'sufficient' diet. I will follow their responses to mucosal vaccination with cholera toxin and ovalbumin (a model mucosal adjuvant/antigen) using antigen-specific ELISA. I will define their gut mucosal barrier function by serum LPS assays, lactulose/mannitol absorption assays, histo- and immunohistochemical assays of gut morphology and immune cell populations (exploring whether they have features of tropical enteropathy), plus functional genomic studies of gut gene expression. I will perform time series metagenomic studies on their fecal microbiomes (sequencing bacterial 16S rRNA genes, total community DNA, and expressed mRNAs). Aim 2. Assess the role of maternal nutritional and microbiota 'status' on the vaccine responses of their offspring by colonizing pregnant female GF mice with fecal samples from twins discordant for kwashiorkor and their mothers. Mothers will be maintained on either a Malawi or nutrient- sufficient control diet prior to, during and after pregnancy; their offspring will be weaned onto nutrient- deficient or -sufficient diets and subjected to the same vaccination protocols and analyses as described in Aim 1. These experiments should help identify biomarkers of malnutrition and vaccine responsiveness.