Understanding B cell memory in response to diverse virus infections

摘要-项目2，Nussenzweig和水稻 记忆B细胞是免疫记忆和疫苗应答的介质。记忆B细胞是一种 异质细胞群，可以根据表型标记、功能、位置、 抗体同种型表达及抗体基因体细胞突变程度。我们对记忆的理解 B细胞生物学主要基于使用基因修饰小鼠和模型的复杂实验。 抗原相比之下，我们对人类记忆B细胞的了解要少得多，特别是那些发育的细胞。 对特定病原体的反应和自然感染期间。迄今为止，大多数研究都集中在 对来自记忆B细胞的抗体进行测序，而它们生物学的许多基本方面无法被 研究的部分原因是难以识别病原体特异性细胞及其相对缺乏。进展 单细胞技术开始使这些研究成为可能。 在项目2中，Rice和Nussenzweig实验室提议共同研究和比较 B细胞的记忆是为了应对重要的人类病原体：登革热和寨卡黄病毒 (DENV和ZIKV）和B肝炎病毒（HBV）。这些病毒造成相当大的发病率， mortality.超过40%的世界人口生活在DENV和ZIKV黄病毒感染的风险地区 全世界约有2.5亿人感染HBV，尽管存在有效的治疗方法， 乙肝疫苗。对这些病原体的免疫记忆是有趣的，因为它可以是保护性的（例如， HBV疫苗接种）或潜在有害（例如DENV感染的抗体依赖性增强）。我们 假设在选定的个体中由DENV、ZIKV和HBV引起的记忆B细胞是不同的， 其特征在于表达基因，这些基因是通用的或病原体特异性的，并且与 它们所表达的抗体的特征。为了验证这一假设，我们将联合收割机结合抗原特异性B细胞 通过用单细胞转录组学分析的细胞分选进行纯化。我们的方法将利用大型 通过与DENV和ZIKV研究人员的持续合作获得的人类样本队列 巴西和墨西哥的流行区，以及来自自然感染或接种疫苗个体的HBV样本 在洛克菲勒大学医院获得。 来自巴西和墨西哥的具有高DENV和ZIKV中和活性的个体的样品将在 对DENV特异性的鉴定的和单一记忆B细胞将进行单细胞RNA-seq以表征 它们的转录组，并获得配对的抗体重链和轻链序列（目的1）。同样，我们将 分析HBV感染或疫苗接种引起的单个记忆B细胞（目的2），以及记忆转录组 将DENV、ZIKV和HBV的细胞毒性相互比较，并与幼稚B细胞的细胞毒性进行比较。在目标3和4中，我们将 克隆和表征来自相同细胞的抗体，并将此信息与转录组联系起来， 抗体的保护（HBV）或增强感染（DENV）的能力。

ABSTRACT – Project 2, Nussenzweig and Rice Memory B cells are mediators of immunological memory and vaccine responses. Memory B cells are a heterogeneous group of cells, that can be distinguished based on phenotypic markers, function, location, the antibody isotype expressed, and the degree of antibody gene somatic mutation. Our understanding of memory B cell biology is based primarily on sophisticated experiments using genetically modified mice and model antigens. In contrast, we know much less about the human memory B cells, in particular about those that develop in response to a specific pathogen and during natural infection. To date most studies have focused on sequencing the antibodies from the memory B cells, while many basic aspects of their biology could not be studied in part due to the difficulty at identifying pathogen-specific cells and their relative paucity. Advances in single-cell technologies are starting to make these investigations possible. In Project 2, the Rice and Nussenzweig laboratories propose to work together to investigate and compare the B cell memory that develops in response to important human pathogens: the dengue and Zika flaviviruses (DENV and ZIKV) and the hepatitis B virus (HBV). These viruses are responsible for considerable morbidity and mortality. More than 40% of the world population lives in areas at risk for infection by DENV and ZIKV flaviviruses and about 250 million people are living with HBV infection worldwide, despite the existence of an efficacious HBV vaccine. The immune memory to these pathogens is interesting because it can be either protective (e.g. HBV vaccination) or potentially harmful (e.g. antibody dependent enhancement of infection with DENV). We hypothesize that the memory B cells elicited by DENV, ZIKV and HBV in selected individuals are distinct and characterized by the expression of genes that are either general or pathogen-specific and that are linked to features of the antibodies that they express. To test this hypothesis, we will combine antigen-specific B cell purification by cell sorting with single cell transcriptomics analysis. Our approach will take advantage of large cohorts of human samples obtained through ongoing collaborations with investigators in DENV and ZIKV endemic areas of Brazil and Mexico, as well as of HBV samples from naturally infected or vaccinated individuals obtained at the Rockefeller University Hospital. Samples from individuals with high DENV and ZIKV neutralizing activity from Brazil and Mexico will be identified and single memory B cells specific for DENV will be subjected to single cell RNA-seq to characterize their transcriptome and to obtain paired antibody heavy and light chain sequences (Aim 1). Similarly, we will analyze single memory B cells elicited by HBV infection or vaccination (Aim 2), and the memory transcriptomes of DENV, ZIKV and HBV will be compared to each other and to those of naïve B cells. In Aims 3 and 4 we will clone and characterize the antibodies from the same cells, and link this information to the transcriptome and to the antibodies' ability to either protect (HBV) or enhance infection (DENV).