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

描述（由申请人提供）：该申请涉及广泛的挑战领域（04）临床研究和特定挑战主题，04- ai - 101*开发新方法并解决粘膜免疫学中的关键问题。流感病毒是通过呼吸道粘膜感染的呼吸道病原体，在所有年龄组中引起显著的发病率和死亡率。流感疫苗，包括通过鼻内喷剂给予的减毒活疫苗（LAIV）和通过全身肌肉注射给予的三价灭活流感疫苗（TIV），可有效保护儿童和健康青年免受流感病毒感染。这些不同类型疫苗的保护作用机制尚不完全清楚。虽然B细胞应答被认为是流感粘膜免疫的关键组成部分，但传统的流感疫苗接种后B细胞免疫分析主要局限于血清抗体的研究，这并不能为LAIV和TIV的疗效提供充分或统一的标记。我们和其他人最近发现，在流感疫苗接种后的一个狭窄窗口内，外周有一个高度富集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。借此机会，我们提出以下具体目标：目标一。描述LAIV或TIV免疫后活化的IgA和IgG B细胞（浆母细胞）的运输受体谱。有效粘膜免疫的一个重要组成部分是免疫细胞靶向感染病原体进入和复制的粘膜部位。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。我们假设被这两种疫苗激活的B细胞将表达不同的运输受体，导致不同的能力回到呼吸道。为了验证这一假设，我们将在接种LAIV或TIV疫苗7天后收集健康成年人的外周血样本，并使用多色流式细胞术分析血浆中运输受体的表达。目标2。比较LAIV和TIV免疫后浆母细胞产生的抗体。疫苗诱导的抗体对疫苗抗原的亲和力是抗体应答的重要特征，直接影响疫苗的效力。我们假设LAIV和v - v诱导的质母细胞产生具有不同抗原结合亲和力的抗体。由于流感流行的反复发生性质和每年使用新疫苗株接种流感疫苗的做法，大多数成年人和年龄较大的儿童以前已经有不同数量的流感暴露，导致长寿命浆细胞和循环抗体对当前流感疫苗具有不同的交叉反应性。因此，使用传统的血清学测定（ELISA、HAI和血清样品中和测定）来特异性鉴定和表征由新疫苗直接诱导的抗体是非常困难的，如果不是不可能的话。为了以避免这种干扰的方式验证我们的假设，我们将根据表型标记（包括运输受体）的表达，通过FACS分选分离不同的质母细胞亚群（目的1）。筛选后的质母细胞将在体外大量培养以收集分泌的多克隆抗体，并用ELISA检测其与流感疫苗病毒的结合亲和力。目标3。免疫LAIV或TIV后活化B细胞产生的细胞因子。最近关于活化B细胞产生细胞因子的研究结果强烈表明，B细胞在其传统的抗体产生作用之外，还具有免疫调节作用。我们假设LAIV和TIV免疫导致B细胞产生不同的细胞因子组，不同地调节对两种疫苗的免疫反应。为了验证这一假设，我们将采用两种策略来比较LAIV激活与tiv激活的B细胞的细胞因子谱：1)分析体外培养的facs分类质母细胞亚群产生的细胞因子；2)分析facs分类质母细胞亚群产生的细胞因子编码mRNA。旨在揭示流感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直接被LAIV或TIV激活的B细胞的表型或功能表征。这些研究将增强我们对两种不同流感疫苗引起保护性粘膜免疫机制的理解。这对于合理设计新的流感疫苗以应对每年的流感流行和面对潜在的流感大流行至关重要。我们的实验也有望为保护免疫提供重要的见解，防止其他医学上重要的病毒感染呼吸道、胃肠道和阴道，以及提高我们理解B细胞对免疫反应的能力。

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.