Identification and characterization of the constituent human serum antibodies elicited by vaccination

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT⁺ serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with K_d ∼ 10⁻⁸–10⁻¹⁰ M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3⁻CD14⁻CD19⁺CD27⁺⁺CD38⁺⁺CD20⁻TT⁺) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the K_d). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.Most approved vaccines confer protection against infectious diseases by the induction of long-lived plasma cells (LLPCs), which secrete antibodies that serve to neutralize and opsonize the pathogen for many years or decades (1–3). Additionally, the generation of memory B cells (mBCs) provides both a mechanism for the rapid synthesis of affinity matured, antigen-specific antibodies following rechallenge and a means to diversify the humoral immune response to confer protection against rapidly evolving viruses or bacteria (4). Although some vaccines elicit antibody titers that remain virtually constant for many decades, for others, including the tetanus toxoid (TT) vaccine, antibody titers wane monotonically over time (5). Booster immunization triggers the rapid expansion and differentiation of cognate B cells, generating antigen-specific plasmablasts that peak in concentration in peripheral blood after 6–7 d and subsequently rapidly decline to nearly undetectable levels (6, 7). Some, but not all, of these peak-wave plasmablasts migrate to specialized niches overwhelmingly located in the bone marrow (BM) and survive as LLPCs (8), which constitute the major source of all classes of Ig in the serum (9).The establishment of serological memory following either primary or booster vaccination is not understood well (10–14). Even though antibody production is the most critical effector function of B-cell immunity, and antigen-specific antibodies in the serum play a key role in protection against pathogen challenge, technical difficulties have precluded direct determination of the identities of the mAbs that comprise the serum antibody response to vaccination. However, recent studies showing that flu vaccination elicits not only neutralizing antibodies but also antibodies that enhance infection by different flu strains (15) underscore the pressing need to develop approaches for delineating the sequences and functionalities of the serum antibodies elicited by vaccination (16).Single-cell cloning has been used to identify neutralizing antibodies encoded by mBCs or plasmablasts in peripheral blood (17). However, although extremely useful for understanding of the structural mechanisms that can lead to the blockade of pathogen infection, the interrogation of single peripheral B cells alone cannot provide information on whether antibodies encoded by single B cells are also produced as secreted IgGs from BM LLPCs, and hence whether they contribute to the serological memory induced by vaccination. A detailed understanding of the diversity of serum antibodies elicited by vaccination, their functionality (e.g., antigen affinity, epitope specificity), and their relative concentrations in the blood can provide key insights toward vaccine evaluation and development.Here, we deployed high-resolution liquid chromatography (LC) tandem MS (MS/MS) (18–20) for the molecular-level analysis of the serum IgG repertoire, combined with deep sequencing of the V gene repertoire of peripheral B lymphocyte subsets (20) and subsequent expression and characterization of representative serum antibodies, to map the dynamics of the human humoral response to vaccination in unprecedented detail. We elected to analyze the response to booster immunization of the TT vaccine because (i) it elicits a highly effective neutralizing response that is protective toward Clostridium tetani challenge; (ii) the vaccine is highly efficacious, and as a result, no deaths from tetanus intoxication have been reported in the United States for individuals who have completed at least primary immunization (21); (iii) TT has been used as a model for analyzing B-cell development following vaccination in humans (6, 22, 23); and (iv) although early serological and mAb studies had pointed to the C-terminal fragment of the toxin heavy chain [recombinant TT fragment C (rTT.C)] as the target for antibody-mediated protection (24), the precise mechanism by which antibodies elicited by the vaccine mediate neutralization has remained unclear.We show that the anti-TT serum IgG repertoire at steady state is composed of a limited number of antibody clonotypes (∼80–100) displaying uniformly high antigen affinity (low nanomolar or subnanomolar), that most of the serum repertoire postboost comprises preexisting (i.e., prevaccination) serum antibody clonotypes, and that there is only partial overlap between the peak-wave plasmablast V gene repertoire and the TT⁺ serum IgG repertoire at steady state after vaccination. We identified several serum monoclonal IgGs that bind to rTT.C, and epitope mapping revealed that all rTT.C-specific antibodies tested bind to an immunodominant linear epitope at the ganglioside-binding site of the toxin that is used for cell entry. Computational antibody docking substantiated that binding of these antibodies to the toxin blocks access to the ganglioside ligand, thus providing a possible mechanistic explanation for how the TT vaccine confers protection. These results highlight the importance of understanding the composition and dynamics of the serum antibody repertoire, together with the V gene repertoire in peripheral B lymphocytes, for the molecular understanding of vaccine function.     

A proteomic evaluation of the effects of the pharmaceuticals diclofenac and gemfibrozil on marine mussels (Mytilus spp.): evidence for chronic sublethal effects on stress‐response proteins

Human pharmaceuticals (e.g. the lipid regulator gemfibrozil and the non‐steroidal anti‐inflammatory drug diclofenac) are an emerging environmental threat in the aquatic environment. This study aimed to evaluate sublethal effects of these two commonly found pharmaceuticals on the protein profiles of marine mussels (Mytilus spp.). Mytilus spp. was exposed to environmentally relevant and elevated concentrations (1 and 1000 µg/l respectively) of both drugs for 14 days. In addition, mussels were maintained for seven days post treatment to examine the potential of blue mussels to recover from such an exposure. Differential protein expression signatures (PES) in the digestive gland of mussels were obtained using two‐dimensional gel electrophoresis after 7, 14, and 21 days of exposure. Twelve spots were significantly increased or decreased by gemfibrozil and/or diclofenac, seven of which were successfully identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis. These proteins were involved in energy metabolism, oxidative stress response, protein folding, and immune responses. Changes in the PES over time suggested that mussels were still experiencing oxidative stress for up to seven days post exposure. In addition, a suite of biomarkers comprising glutathione transferase, lipid peroxidation, and DNA damage were studied. An oxidative stress response was confirmed by biomarker responses. To our knowledge, this is the first investigation using proteomics to assess the potential effects of human pharmaceuticals on a non‐target species in an environmentally‐relevant model. The successful application of this proteomic approach supports its potential use in pollution biomonitoring and highlights its ability to aid in the discovery of new biomarkers. Copyright © 2013 John Wiley & Sons, Ltd.     

一种基于卷积神经网络的DIA数据预处理模型

目的数据非依赖性采集(data independent acquisition,DIA)是目前针对大通量蛋白质组学分析常用的一种数据采集方式。在对DIA数据无目标的分析方式中,由于无法预测肽段出现在DIA数据中的位置,需要对谱中所有的峰进行分析。但谱中含有大量的噪声峰,这些峰会严重影响后续蛋白质定性定量分析的效率与效果,所以在DIA数据的无目标分析过程中先进行预处理以去除噪声峰就成了很重要的一步。为了能充分利用从DIA数据中提取出来的肽段在一级质谱(first stage of mass spectrometry,MS1)和二级质谱(second stage of mass spectrometry,MS2)中的峰信息,提出质谱卷积神经网络(mass spectrometry convolutional neural network,MSCNN)模型。方法不同于传统的方法,本文首先提出适用于MSCNN网络结构的样本提取流程,然后利用MSCNN对样本进行训练和学习,该模型可以最大限度利用肽在MS1和MS2中的特征,最后通过观察模型在测试集中的结果来验证模型的效果。结果和传统算法相比,在保证真峰处理效果大致相同的情况下,MSCNN模型过滤噪声峰的数量提高了约11.2%。结论本文提出的MSCNN模型可以更有效地去除DIA数据中的噪声峰。     

Stimulation of retrotrapezoid nucleus Phox2b‐expressing neurons rescues breathing dysfunction in an experimental Parkinson’s disease rat model

Emerging evidence from multiple studies indicates that Parkinson’s disease (PD) patients suffer from a spectrum of autonomic and respiratory motor deficiencies in addition to the classical motor symptoms attributed to substantia nigra degeneration of dopaminergic neurons. Animal models of PD show a decrease in the resting respiratory rate as well as a decrease in the number of Phox2b‐expressing retrotrapezoid nucleus (RTN) neurons. The aim of this study was to determine the extent to which substantia nigra pars compact (SNc) degeneration induced RTN biomolecular changes and to identify the extent to which RTN pharmacological or optogenetic stimulations rescue respiratory function following PD‐induction. SNc degeneration was achieved in adult male Wistar rats by bilateral striatal 6‐hydroxydopamine injection. For proteomic analysis, laser capture microdissection and pressure catapulting were used to isolate the RTN for subsequent comparative proteomic analysis and Ingenuity Pathway Analysis (IPA). The respiratory parameters were evaluated by whole‐body plethysmography and electromyographic analysis of respiratory muscles. The results confirmed reduction in the number of dopaminergic neurons of SNc and respiratory rate in the PD‐animals. Our proteomic data suggested extensive RTN remodeling, and that pharmacological or optogenetic stimulations of the diseased RTN neurons promoted rescued the respiratory deficiency. Our data indicate that despite neuroanatomical and biomolecular RTN pathologies, that RTN‐directed interventions can rescue respiratory control dysfunction.     

The Staphylococcus aureus proteome

Staphylococcus aureus is a Gram-positive commensal bacterium that is regarded as a major threat for modern health care systems. This relates both to the ability of S. aureus to overcome antibiotic therapy by developing high-level resistance against multiple antibiotics and this bacterium's extensive arsenal of virulence factors. Understanding the mechanisms of resistance and functional studies on stress and starvation responses are the main goals of proteomics in staphylococcal research. This review high-lights recent advances in gel-based and gel-free proteomics analyses of S. aureus and pinpoints the importance of location-specific proteomics studies targeting the cytosol, the membrane, the cell surface and the extracellular milieu in combination with integrated global proteome studies. Emerging hot topics in staphylococcal proteomics are discussed with special focus on in vivo proteomics, membrane vesicles, biofilm formation and the acquisition of absolute proteome data for systems biological modeling approaches.     

大鼠髁突软骨细胞发育中相关信号通路的初步筛查

目的:通过建立的大鼠髁突软骨细胞发育过程中蛋白表达差异谱,分析可能参与的信号通路,为进一步认识髁突软骨生理性及病理性生长改建的信号调控机制提供相关信息。方法:收获出生后1、7、14、28 d共4组SD大鼠髁突软骨细胞,甲苯胺蓝染色、Ⅱ型胶原免疫组化染色鉴定软骨细胞。提取各组软骨细胞总蛋白,采用i TRAQ标记定量蛋白,2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(MALDI-TOF-TOF),获取出生后大鼠髁突软骨细胞发育过程中差异蛋白的表达谱,所得数据用MASCOT软件处理,筛选样本之间有意义的差异蛋白,运用GO法及David软件进行KEGG信号通路分析。结果 :共鉴定137种具有可信度表达的蛋白,有44种蛋白参与至少27条KEGG信号通路,其中ECM-受体相互作用、焦点粘连、actin骨架调节、Ca2+信号通路、血管平滑肌收缩、Gn RH信号通路、肌醇三磷酸代谢、磷脂酰肌醇信号系统以及核糖体等信号通路具有统计学意义。结论:在大鼠髁突软骨发育中,各信号通路蛋白在时间、空间上差异性表达,共同形成复杂的信号传递网络。