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

粘膜免疫和流感疫苗：活化 B 细胞的表型和作用

• 批准号: 7833729
• 负责人: EUGENE C BUTCHER
• 金额: $ 49.18万
• 依托单位: PALO ALTO VETERANS INSTIT FOR RESEARCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-02 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7833729
• 关键词: Address; Adult; Affect; Affinity; Antibodies; Antibody Formation; Area; Attenuated; B-Lymphocytes; Binding; Biological Assay; Biological Response Modifiers; Blood specimen; Cell Communication; Cells; Characteristics; Child; Clinical Research; Color; Disease; Elderly; Employment; Enzyme-Linked Immunosorbent Assay; Exposure to; Face; Flow Cytometry; General Population; Health; Home environment; Homing; Immune Targeting; Immune response; Immunity; Immunization; Immunoglobulin A; Immunoglobulin G; Immunology; Influenza; Influenza vaccination; Intramuscular Injections; Life; Lymphocyte; Mediating; Messenger RNA; Methods; Morbidity - disease rate; Mucosal Immunity; Nature; Phenotype; Plasma Cells; Plasmablast; Process; Production; Research; Respiratory Mucosa; Respiratory System; Respiratory tract structure; Role; ST14 gene; Sampling; Scientist; Serological; Serum; Sorting - Cell Movement; Study of serum; Testing; Tissues; United States; Vaccination; Vaccine Antigen; Vaccines; Vagina; Viral; Virus; Virus Diseases; age group; antigen binding; base; cross reactivity; cytokine; design; gastrointestinal; improved; influenza epidemic; influenza virus vaccine; influenzavirus; insight; killings; mortality; mucosal site; novel; novel strategies; novel vaccines; pandemic influenza; pathogen; peripheral blood; polyclonal antibody; programs; protective efficacy; public health relevance; receptor; research study; response; trafficking; trivalent influenza vaccine; vaccine efficacy; young adult

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.

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。流感疫苗包括经鼻斯帕伊接种的流感减毒活疫苗（LAIV）和经全身肌肉注射接种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。这些不同类型的疫苗的保护效力的机制还不完全清楚。尽管B细胞应答被认为是流感粘膜免疫的关键组成部分，但流感疫苗接种后B细胞免疫的传统分析在很大程度上限于血清抗体的研究，其不能为LAIV和TIV的功效提供足够或统一的标志物。我们和其他人最近发现，在流感疫苗接种后的狭窄窗口期间，外周血中高度富集的流感特异性浆母细胞池为研究疫苗活化的B细胞的表型和功能提供了独特的机会。利用这一机会，我们提出以下具体目标：目标1。表征LAIV或TIV免疫后活化伊加和IgG B细胞（浆母细胞）的运输受体谱。有效粘膜免疫的一个基本组成部分是将免疫细胞靶向感染病原体进入并复制的粘膜部位。淋巴细胞运输由淋巴细胞运输受体的表达介导，其控制组织内淋巴细胞归巢、趋化性导航和细胞-细胞相互作用的多步骤过程。我们假设这两种疫苗激活的B细胞将表达不同的运输受体，从而导致不同的归巢呼吸道的能力。为了检验这一假设，我们将在LAIV或TIV疫苗接种后7天从健康成人收集外周血样品，并使用多色流式细胞术分析浆母细胞的运输受体表达。目标二。比较LAIV或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，其直接影响疫苗效力。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。由于流感流行的重复性和每年使用新疫苗株进行流感疫苗接种的做法，大多数成人和年龄较大的儿童已经有不同数量的既往流感暴露，导致长寿命的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，即使不是不可能，也很难使用常规血清学测定（ELISA、HAI和血清样品的中和测定）来特异性鉴定和表征由新疫苗接种直接诱导的抗体。为了以避免这种干扰的方式测试我们的假设，我们将通过FACS分选分离浆母细胞的不同子集，基于其表型标记物的表达，包括运输受体（Aim 1）。将离体批量培养分选的浆母细胞以收集分泌的多克隆抗体，将用ELISA测试所述多克隆抗体以确定它们与流感疫苗病毒的结合亲和力。目标3。分析LAIV或TIV免疫后活化B细胞产生的细胞因子。最近关于活化的B细胞产生细胞因子的发现强烈地表明，B细胞作为免疫调节剂，超出了它们作为抗体生产者的常规作用。我们假设LAIV和TIV免疫导致B细胞产生不同的细胞因子组，这些细胞因子差异调节对两种疫苗的免疫应答。为了检验这一假设，我们将利用两种策略来比较LAIV活化的与IV活化的B细胞的细胞因子谱：1）分析由离体培养的FACS分选的浆母细胞亚群产生的细胞因子和2）分析来自FACS分选的浆母细胞亚群的编码精氨酸的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表征用LAIV或TIV直接激活的B细胞的表型或功能。这些研究将增强我们对两种不同流感疫苗引起的保护性粘膜免疫的机制的理解。这对于合理设计用于每年流感流行和面对潜在流感大流行的新型流感疫苗至关重要。我们的实验还有望为保护性免疫提供重要的见解，以对抗感染气道、胃肠道和阴道的其他医学上重要的病毒，并提高我们理解B细胞对免疫的反应的能力。 公共卫生相关性：这项研究探讨了最近被流感疫苗激活的B细胞如何被编程以限制病毒复制。在美国，流感感染每年导致超过35，000人死亡。这些研究将提供新的信息，以提高我们改进流感疫苗的能力。