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Examination of putative outer membrane proteins in Moraxella catarrhalis: A genome mining approach for the identification of potential vaccine antigens.

机译：卡他莫拉菌中推定的外膜蛋白的检查：一种用于鉴定潜在疫苗抗原的基因组挖掘方法。

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摘要

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States and affects 24 million Americans. Moraxella catarrhalis is an important cause of respiratory infections in COPD. This organism is also a common cause of otitis media in children. Developing an effective vaccine to M. catarrhalis would reduce the morbidity, mortality, and financial costs associated with COPD and otitis media. Surface proteins from M. catarrhalis are attractive vaccine antigens. An ideal vaccine candidate has several characteristics including surface exposure, conservation among strains, expression during infection, and generation of a protective immune response.;This thesis examined the overall hypothesis that conserved outer membrane proteins (OMPs) from Moraxella catarrhalis could act as vaccine antigens. Specifically the following hypotheses were examined (1) selected genes that encode surface proteins are differentially regulated during growth in a human simulated environment; (2) these antigens induce an antibody response in humans during infection; and (3) these OMPs generate a potentially protective immune response with antibodies directed at surface exposed epitopes.;An unfinished genome sequence of a strain of M. catarrhalis available in GenBank was analyzed and open reading frames predicted to encode potential vaccine candidates were identified. Three genes, encoding proteins of approximately 22kDa, 75kDa, and 78kDa (named Moraxella surface proteins (Msp) msp22, msp75, and msp78, respectively), were identified to be conserved by competitive hybridization using a microarray, polymerase chain reaction (PCR), and sequencing the genes in clinical isolates of M. catarrhalis. The genes were transcribed, translated, and expressed when M. catarrhalis was grown in vitro.;To assess antibody responses in humans, these genes were first amplified by PCR and cloned into E. coli expression vectors. Recombinant proteins were generated and then studied in enzyme-linked immunosorbent assays (ELISAs) with pre-acquisition and post-clearance serum and sputum samples from 31 adults who had a known M. catarrhalis infection. New antibody responses to the three proteins were generated by a small proportion of patients with COPD indicating that these proteins were expressed during human infection.;To examine if antibodies are directed at surface epitopes, immunized animals were tested in a mouse pulmonary clearance model using two routes of administration: subcutaneous and intranasal. Quantitative enzyme-linked immunosorbent assays were performed on sera and bronchoalveolar lavage (BAL) fluids from the immunized mice to characterize systemic and mucosal antibody responses. The antibodies, induced by immunization with recombinant proteins, were analyzed by flow cytometry. This method detected antibodies that bound to the bacterial surface of multiple M. catarrhalis strains.;To evaluate the ability of these proteins to generate a protective effect after immunization, mice were challenged using a pulmonary clearance model. Mice immunized with recombinant Msp22 and Msp75 showed enhanced clearance of M. catarrhalis as compared to control mice. This observation suggests that immunization with either of these two proteins generates an immune response that mediates bacterial clearance from the lungs.;Collectively the conservation among strains, immunogenicity, localization to the outer membrane, and the ability to enhance clearance in vivo indicate that two of the three proteins examined, Msp22 and Msp75, may make good vaccine antigens. These proteins should be studied further to determine if they are capable of inducing a protective response following immunization in humans.
机译：慢性阻塞性肺疾病（COPD）是美国第四大主要死因，影响2400万人。卡他莫拉菌是COPD中呼吸道感染的重要原因。这种生物也是儿童中耳炎的常见原因。开发一种针对卡他莫拉氏菌的有效疫苗将降低与COPD和中耳炎相关的发病率，死亡率和财务成本。卡他莫拉氏菌的表面蛋白是有吸引力的疫苗抗原。理想的候选疫苗具有以下特征：表面暴露，菌株间的保守性，感染过程中的表达以及保护性免疫应答的产生。；本论文研究了从卡他莫拉菌中保守的外膜蛋白（OMP）可以充当疫苗抗原的总体假设。具体来说，检查了以下假设（1）在人类模拟环境中，编码表面蛋白的选定基因在生长过程中受到差异调节； （2）这些抗原在感染过程中在人中诱导抗体反应； （3）这些OMPs针对表面暴露的表位的抗体产生潜在的保护性免疫应答。；分析了GenBank中可得的粘膜炎莫拉氏菌菌株的未完成的基因组序列，并鉴定了预期编码潜在疫苗候选物的开放阅读框。使用微阵列，聚合酶链反应（PCR）通过竞争性杂交确定了三个基因，它们分别编码大约22kDa，75kDa和78kDa的蛋白质（分别命名为莫拉氏菌表面蛋白（Msp）msp22，msp75和msp78）是保守的，卡他莫拉氏菌临床分离株中的基因进行测序和测序。当粘膜炎莫拉氏菌在体外生长时，这些基因被转录，翻译和表达。为了评估人的抗体反应，首先通过PCR扩增这些基因并将其克隆到大肠杆菌表达载体中。产生重组蛋白，然后在酶联免疫吸附测定（ELISAs）中对31名患有卡他莫拉菌感染的成年人的采集前和清除后血清和痰液样品进行研究。一小部分COPD患者产生了对这三种蛋白质的新抗体应答，表明这些蛋白质在人类感染过程中表达。为了检查抗体是否针对表面表位，在小鼠肺部清除模型中使用两种方法测试了免疫动物给药途径：皮下和鼻内。对来自免疫小鼠的血清和支气管肺泡灌洗液（BAL）进行了定量酶联免疫吸附测定，以表征全身和粘膜抗体反应。通过流式细胞术分析通过重组蛋白免疫诱导的抗体。该方法检测了与多种卡他莫拉氏菌菌株细菌表面结合的抗体。为了评估这些蛋白质在免疫后产生保护作用的能力，使用肺部清除模型对小鼠进行了攻击。与对照小鼠相比，用重组Msp22和Msp75免疫的小鼠表现出卡他莫拉氏菌的清除率提高。该观察结果表明，用这两种蛋白质中的任何一种进行免疫都会产生介导细菌从肺中清除的免疫反应。集体认为，菌株之间的保守性，免疫原性，定位于外膜的能力以及体内清除能力的增强表明，其中两种检测的三种蛋白质Msp22和Msp75可能是好的疫苗抗原。这些蛋白质应进一步研究，以确定它们是否能够在人体免疫后诱导保护性应答。

著录项

  • 作者

    Ruckdeschel, Elizabeth Anne.;

  • 作者单位

    State University of New York at Buffalo.;

  • 授予单位 State University of New York at Buffalo.;
  • 学科 Biology Microbiology.;Health Sciences Medicine and Surgery.;Health Sciences Immunology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 157 p.
  • 总页数 157
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 微生物学;预防医学、卫生学;
  • 关键词

  • 入库时间 2022-08-17 11:38:27

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