Mucosal Immunity and Influenza Vaccines: Phenotype and Role of Activated B Cells

描述(由申请人提供)：本申请涉及广泛的挑战领域(04)临床研究和特定的挑战主题，04-AI-101*开发新的方法和解决粘膜免疫学的关键问题。流感病毒是通过呼吸道粘膜感染的呼吸道病原体，在所有年龄段都会导致显著的发病率和死亡率。流感疫苗包括经鼻腔喷洒的减毒活疫苗(LAIV)和系统肌肉注射的三价灭活流感疫苗(TIV)，可有效地保护儿童和健康的年轻人免受流感病毒的感染。这些不同类型疫苗的保护效力的机制还不完全清楚。虽然B细胞反应被认为是流感粘膜免疫的重要组成部分，但传统的对流感疫苗接种后B细胞免疫的分析在很大程度上局限于对血清抗体的研究，这不能为LAIV和TIV的疗效提供足够或统一的标志物。最近，我们和其他人在流感疫苗接种后的一个狭窄窗口内，在外围发现了高度浓缩的流感特异性浆母细胞池，这为研究疫苗激活的B细胞的表型和功能提供了一个独特的机会。利用这一机会，我们提出了以下具体目标：目的1.鉴定LAIV或TIV免疫后激活的IgA和IgGB细胞(浆母细胞)的转运受体的特征。有效的粘膜免疫的一个重要组成部分是将免疫细胞定位于感染病原体进入并复制的粘膜部位。淋巴细胞转运是由淋巴细胞转运受体的表达介导的，它控制着组织内淋巴细胞归巢、趋化导航和细胞-细胞相互作用的多步骤过程。我们假设，由这两种疫苗激活的B细胞将表达不同的贩运受体，导致不同的能力归宿到呼吸道。为了验证这一假设，我们将在接种LAIV或TIV疫苗后7天收集健康成年人的外周血样，并使用多色流式细胞术分析浆母细胞贩运受体的表达。目的：比较LAIV和TIV免疫后成浆细胞产生的抗体。疫苗诱导的抗体与疫苗抗原的亲和力是抗体反应的重要特征，直接影响疫苗的效力。我们假设LAIV和TIV诱导的浆母细胞产生具有不同抗原结合亲和力的抗体。由于流感疫情的反复发生和每年接种新疫苗株的做法，大多数成年人和年龄较大的儿童以前已经接触过不同数量的流感，导致长寿命的浆细胞和循环抗体与当前的流感疫苗具有可变的交叉反应。因此，即使不是不可能，也很难使用传统的血清学分析方法(血清样本的酶联免疫吸附试验、血凝抑制试验和中和试验)来具体识别和表征由新疫苗直接诱导的抗体。为了以避免这种干扰的方式检验我们的假设，我们将通过FACS分类分离不同的浆母细胞亚集，基于它们的表型标记的表达，包括贩运受体(目标1)。分选后的成浆细胞将进行体外批量培养，收集分泌的多克隆抗体，并用ELISA进行检测，以确定其与流感疫苗病毒的结合亲和力。目的3.LAIV或TIV免疫后活化的B细胞产生细胞因子的情况。最近关于激活的B细胞产生细胞因子的研究结果有力地表明，B细胞除了作为抗体产生者的传统角色外，还具有免疫调节器的作用。我们假设LAIV和TIV免疫导致B细胞产生不同的细胞因子，这些细胞因子以不同的方式调节对这两种疫苗的免疫反应。为了验证这一假设，我们将利用两种策略来比较LAIV激活的B细胞和TIV激活的B细胞的细胞因子谱：1)分析体外培养的FACS分选的浆母细胞亚群产生的细胞因子；2)分析FACS分选的浆母细胞亚群中编码细胞因子的mRNA。为了揭示流感B细胞免疫的新方面，这三个目标中的每一个都利用了一种新的方法来描述由LAIV或TIV直接激活的B细胞的表型或功能特征。这些研究将加深我们对两种不同流感疫苗诱导的粘膜保护性免疫机制的理解。这对于为每年的流感流行和面对潜在的流感大流行合理设计新的流感疫苗至关重要。我们的实验还有望为抵御其他感染呼吸道、胃肠道和阴道的重要医学病毒的保护性免疫提供重要见解，并提高我们了解B细胞对免疫的一般反应的能力。 公共卫生相关性：这项研究调查了最近被流感疫苗激活的B细胞是如何被编程来限制病毒复制的。在美国，每年有超过3.5万人死于流感感染。这些研究将为提高我们改进流感疫苗的能力提供新的信息。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (04) Clinical Research and Specific Challenge Topic, 04-AI- 101* Develop novel methods and address key questions in mucosal immunology. Influenza viruses are respiratory tract pathogens that infect through the respiratory mucosa and cause significant morbidity and mortality in all age groups. Influenza vaccines, including live attenuated influenza vaccine (LAIV) given via intranasal spay and trivalent inactivated influenza vaccine (TIV) given by systematic intramuscular injection, are effective in protecting children and healthy young adults against influenza virus infection. The mechanisms for the protective efficacy of these different types of vaccines are not completely understood. Although B cell responses are considered a critical component of the influenza mucosal immunity, traditional analyses of B cell immunity after influenza vaccination have been largely limited to studies of serum antibodies, which do not provide adequate or unified markers for the efficacy of LAIV and TIV. The recent finding by us and others of a highly enriched influenza-specific plasmablast pool in the periphery during a narrow window after influenza vaccination provides a unique opportunity to study the phenotypes and functions of vaccine-activated B cells. Taking advantage of this opportunity, we propose the following specific aims: Aim 1. Characterize the trafficking receptor profiles of activated IgA and IgG B cells (plasmablasts) after immunization with LAIV or TIV. An essential component of effective mucosal immunity is targeting of the immune cells to the mucosal site where the infecting pathogen enters and replicates. Lymphocyte trafficking is mediated by the expression of lymphocyte trafficking receptors, which control the multi-step processes of lymphocyte homing, chemotactic navigation, and cell-cell interactions within tissues. We hypothesize that B cells activated by these two vaccines will express distinct trafficking receptors, resulting in a differential ability to home to the respiratory tract. To test this hypothesis, we will collect peripheral blood samples from health adults seven days after LAIV or TIV vaccination and analyze plasmablasts for expression of trafficking receptors using multi-color flow cytometry. Aim 2. Compare the antibodies produced by plasmablasts after LAIV or TIV immunization. The affinity of vaccine-induced antibodies to the vaccine antigens is an important characteristic of antibody response that directly affects the vaccine efficacy. We hypothesize that LAIV- and TIV-induced plasmablasts produce antibodies with distinct antigen binding affinities. Due to the recurring nature of influenza epidemics and the practice of annual influenza vaccination with new vaccine strains, most adults and older children already have a varying number of previous exposures to influenza, resulting in long-life plasma cells and circulating antibodies with variable cross-reactivity to the current influenza vaccine. Therefore, it has been very difficult, if not impossible, to use the conventional serological assays (ELISA, HAI and neutralization assays of serum samples) to specifically identify and characterize the antibodies directly induced by a new vaccination. To test our hypothesis in a way that avoids this interference, we will isolate different subsets of plasmablasts by FACS sorting, based on their expression of phenotypic markers, including trafficking receptors (Aim 1). The sorted plasmablasts will be bulk-cultured ex vivo to collect secreted polyclonal antibodies, which will be tested with ELISA to determine their binding affinity to the influenza vaccine viruses. Aim 3. Profile cytokines produced by activated B cells after LAIV or TIV immunization. Recent findings on the production of cytokines by activated B cells strongly suggest that B cells serve as immune regulators, beyond their conventional role as antibody-producers. We hypothesize that LAIV and TIV immunizations result in B cells that produce distinct panels of cytokines that differentially modulate the immune responses to the two vaccines. To test this hypothesis we will utilize two strategies to compare the cytokine profiles of LAIV- activated versus TIV-activated B cells: 1) Analyze cytokines produced by ex vivo-cultured FACS-sorted plasmablast subsets and 2) Analyze cytokine-encoding mRNA from FACS-sorted plasmablast subsets. Designed to reveal new aspects of influenza B cell immunity, each of these three aims utilizes a novel approach for the phenotypical or functional characterization of B cells directly activated with either LAIV or TIV. These studies will enhance our understanding of the mechanisms for the protective mucosal immunity elicited by the two different influenza vaccines. This is critical for rational design of new influenza vaccines for the annual influenza epidemics and in the face of a potential influenza pandemic. Our experiments are also expected to provide important insights into protective immunity against other medically important viruses that infect the airways and gastrointestinal and vaginal tracts as well as advancing our ability to understand the B cell response to immunization in general. PUBLIC HEALTH RELEVANCE: This study examines how B cells that are recently activated by influenza vaccination are programmed to restrict viral replication. Influenza infection kills over 35,000 people in the United States each year. These studies will provide new information to enhance our ability to improve the influenza vaccines.