Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events.

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

Variable blood processing procedures contribute to plasma proteomic variability.

BACKGROUND: Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC-MS/MS proteomic analysis of plasma. METHODS: Initially two batches of patient plasma samples (120 and 204 samples, respectively) were analyzed using LC-MS/MS shotgun proteomics. Follow-up experiments were designed and carried out on healthy donor blood in order to examine the effects of different centrifugation conditions, length of delay until first centrifugation, storage temperature and anticoagulant type on results from shotgun proteomics. RESULTS: Variable levels of intracellular proteins were observed in subsets of patient plasma samples from the initial batches analyzed. This observation correlated strongly with the site of collection, implicating variability in blood processing procedures. Results from the healthy donor blood analysis did not demonstrate a significant impact of centrifugation conditions to plasma proteome variation. The time delay until first centrifugation had a major impact on variability, while storage temperature and anticoagulant showed less pronounced but still significant effects. The intracellular proteins associated with study site effect in patient plasma samples were significantly altered by delayed processing also. CONCLUSIONS: Variable blood processing procedures contribute significantly to plasma proteomic variation and may give rise to increased intracellular proteins in plasma. Accounting for these effects can be important both at study design and data analysis stages. This understanding will be valuable to incorporate in the planning of protein-based biomarker discovery efforts in the future.

Enhancing reproducibility of gene expression analysis with known protein functional relationships: The concept of well-associated protein.

Identification of differentially expressed genes (DEGs) is well recognized to be variable across independent replications of genome-wide transcriptional studies. These are often employed to characterize disease state early in the process of discovery and prioritize novel targets aimed at addressing unmet medical need. Increasing reproducibility of biological findings from these studies could potentially positively impact the success rate of new clinical interventions. This work demonstrates that statistically sound combination of gene expression data with prior knowledge about biology in the form of large protein interaction networks can yield quantitatively more reproducible observations from studies characterizing human disease. The novel concept of Well-Associated Proteins (WAPs) introduced herein-gene products significantly associated on protein interaction networks with the differences in transcript levels between control and disease-does not require choosing a differential expression threshold and can be computed efficiently enough to enable false discovery rate estimation via permutation. Reproducibility of WAPs is shown to be on average superior to that of DEGs under easily-quantifiable conditions suggesting that they can yield a significantly more robust description of disease. Enhanced reproducibility of WAPs versus DEGs is first demonstrated with four independent data sets focused on systemic sclerosis. This finding is then validated over thousands of pairs of data sets obtained by random partitions of large studies in several other diseases. Conditions that individual data sets must satisfy to yield robust WAP scores are examined. Reproducible identification of WAPs can potentially benefit drug target selection and precision medicine studies.

Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG.

An Integrated Approach to Unique NMR Assignment of Methionine Methyl Resonances in Proteins.

The application of methyl nuclear magnetic resonance (NMR) spectroscopy in protein side-chain structural studies offers unique advantages of greater peak sensitivity, even for high-molecular-weight proteins. Traditionally, the utility of methyl NMR has often been limited by the difficulty in assigning the methyl resonances. Herein, a mass spectrometry (MS)-assisted strategy to assign the methyl resonances of methionine residues is presented. The strategy involves partially oxidizing the methionine and quantifying the oxidation level by both NMR and liquid chromatography-mass spectrometry (LC-MS). The NMR assignment of methyl resonances of methionine is made by correlating the quantitative results obtained from both NMR and MS. The method has been successfully demonstrated using the proteins hen egg-white lysozyme (HEWL) and porcine pepsin. The technique described herein can help facilitate the application of methyl NMR as a useful tool to study protein structure, dynamics, and interactions.

An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure.

Heparan sulfate (HS), a glycosaminoglycan present on the surface of cells, has been postulated to have important roles in driving both normal and pathological physiologies. The chemical structure and sulfation pattern (domain structure) of HS is believed to determine its biological function, to vary across tissue types, and to be modified in the context of disease. Characterization of HS requires isolation and purification of cell surface HS as a complex mixture. This process may introduce additional chemical modification of the native residues. In this study, we describe an approach towards thorough characterization of bovine kidney heparan sulfate (BKHS) that utilizes a variety of orthogonal analytical techniques (e.g. NMR, IP-RPHPLC, LC-MS). These techniques are applied to characterize this mixture at various levels including composition, fragment level, and overall chain properties. The combination of these techniques in many instances provides orthogonal views into the fine structure of HS, and in other instances provides overlapping / confirmatory information from different perspectives. Specifically, this approach enables quantitative determination of natural and modified saccharide residues in the HS chains, and identifies unusual structures. Analysis of partially digested HS chains allows for a better understanding of the domain structures within this mixture, and yields specific insights into the non-reducing end and reducing end structures of the chains. This approach outlines a useful framework that can be applied to elucidate HS structure and thereby provides means to advance understanding of its biological role and potential involvement in disease progression. In addition, the techniques described here can be applied to characterization of heparin from different sources.

Characterization of heparin-protein interaction by saturation transfer difference (STD) NMR.

The binding affinity and specificity of heparin to proteins is widely recognized to be sulfation-pattern dependent. However, for the majority of heparin-binding proteins (HBPs), it still remains unclear what moieties are involved in the specific binding interaction. Here, we report our study using saturation transfer difference (STD) nuclear magnetic resonance (NMR) to map out the interactions of synthetic heparin oligosaccharides with HBPs, such as basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10), to provide insight into the critical epitopes of heparin ligands involved. The irradiation frequency of STD NMR was carefully chosen to excite the methylene protons so that enhanced sensitivity was obtained for the heparin-protein complex. We believe this approach opens up additional application avenues to further investigate heparin-protein interactions.

Identification of a novel structure in heparin generated by sequential oxidative-reductive treatment.

Unfractionated heparin is isolated from animal organs, predominantly porcine intestinal mucosa, and goes through an extensive process of purification before it can be used for pharmaceutical purposes. While the structural microheterogeneity of heparin is predominantly biosynthetically imprinted in the Golgi, subsequent steps involved in the purification and manufacture of commercial heparin can lead to the introduction of additional modifications. Postheparin crisis of 2008, it has become increasingly important to identify what additional structural diversity is introduced as a function of the purification process and thus can be determined as being heparin-related, as opposed to being an adulterant or contaminant, e.g., oversulfated chondroitin sulfate. Our study focuses on the identification of a previously unreported structure in heparin that arises due to specific steps used in the manufacturing process. This structure was initially observed as a disaccharide peak in a complete enzymatic digest of heparin, but its presence was later identified in the NMR spectra of intact heparin as well. Structural elucidation experiments involved isolation of this structure and analysis based on multidimensional NMR and liquid chromatography coupled with mass spectrometry (LC-MS). Heparin was also subjected to specific chemical reactions to determine which steps in the manufacturing process are responsible for this novel structure. Our results allowed for the definitive assignment of the structure of this novel process-related modification and enabled an identification of the putative steps in the process that give rise to the structure.

Structural elucidation of the tetrasaccharide pool in enoxaparin sodium.

Low-molecular-weight heparins (LMWHs) are produced from heparin by various depolymerization strategies, which result in a reduction of the average molecular weight of the polysaccharide chains, a reduction of the anti-factor IIa activity (and a concomitant increase in the anti-factor Xa/anti-factor IIa ratio), and introduction of process-related structural signatures. Numerous techniques have been developed to characterize LMWHs and to measure the type and extent of structural modifications that are introduced as a function of the depolymerization process. We present here an analysis of the tetrasaccharide pool of enoxaparin sodium, a LMWH produced by chemical ß-elimination of heparin benzyl ester. We identify the predominant sequences present within the tetrasaccharide pool and demonstrate that this pool provides a sensitive, specific readout of the physicochemical process conditions used to generate enoxaparin sodium.

Heparin and heparan sulfate: analyzing structure and microheterogeneity.

The structural microheterogeneity of heparin and heparan sulfate is one of the major reasons for the multifunctionality exhibited by this class of molecules. In a physiological context, these molecules primarily exert their effects extracellularly by mediating key processes of cellular cross-talk and signaling leading to the modulation of a number of different biological activities including development, cell proliferation, and inflammation. This structural diversity is biosynthetically imprinted in a nontemplate-driven manner and may also be dynamically remodeled as cellular function changes. Understanding the structural information encoded in these molecules forms the basis for attempting to understand the complex biology they mediate. This chapter provides an overview of the origin of the structural microheterogeneity observed in heparin and heparan sulfate, and the orthogonal analytical methodologies that are required to help decipher this information.

Mass Spectrometric Methods for the Analysis of Heparin and Heparan Sulfate.

Glycosaminoglycans like heparin and heparan sulfate exhibit a high degree of structural microheterogeneity. This structural heterogeneity results from the biosynthetic process that produces these linear polysaccharides in cells and tissues. Heparin and heparan sulfate play critical roles in normal physiology and pathophysiology, hence it is important to understand how their structural features may influence overall activity. Therefore, high-resolution techniques like mass spectrometry represent a key part of the suite of methodologies available to probe the fine structural details of heparin and heparan sulfate. This chapter outlines the application of techniques like LC-MS and LC-MS/MS to study the composition of these polysaccharides, and techniques like GPC-MS that allow for an analysis of oligosaccharide fragments in these mixtures.

Optimizing Antibody Affinity and Developability Using a Framework-CDR Shuffling Approach-Application to an Anti-SARS-CoV-2 Antibody.

The computational methods used for engineering antibodies for clinical development have undergone a transformation from three-dimensional structure-guided approaches to artificial-intelligence- and machine-learning-based approaches that leverage the large sequence data space of hundreds of millions of antibodies generated by next-generation sequencing (NGS) studies. Building on the wealth of available sequence data, we implemented a computational shuffling approach to antibody components, using the complementarity-determining region (CDR) and the framework region (FWR) to optimize an antibody for improved affinity and developability. This approach uses a set of rules to suitably combine the CDRs and FWRs derived from naturally occurring antibody sequences to engineer an antibody with high affinity and specificity. To illustrate this approach, we selected a representative SARS-CoV-2-neutralizing antibody, H4, which was identified and isolated previously based on the predominant germlines that were employed in a human host to target the SARS-CoV-2-human ACE2 receptor interaction. Compared to screening vast CDR libraries for affinity enhancements, our approach identified fewer than 100 antibody framework-CDR combinations, from which we screened and selected an antibody (CB79) that showed a reduced dissociation rate and improved affinity against the SARS-CoV-2 spike protein (7-fold) when compared to H4. The improved affinity also translated into improved neutralization (>75-fold improvement) of SARS-CoV-2. Our rapid and robust approach for optimizing antibodies from parts without the need for tedious structure-guided CDR optimization will have broad utility for biotechnological applications.

Temporal targeting of tumour cells and neovasculature with a nanoscale delivery system.

In the continuing search for effective treatments for cancer, the emerging model is the combination of traditional chemotherapy with anti-angiogenesis agents that inhibit blood vessel growth. However, the implementation of this strategy has faced two major obstacles. First, the long-term shutdown of tumour blood vessels by the anti-angiogenesis agent can prevent the tumour from receiving a therapeutic concentration of the chemotherapy agent. Second, inhibiting blood supply drives the intra-tumoural accumulation of hypoxia-inducible factor-1alpha (HIF1-alpha); overexpression of HIF1-alpha is correlated with increased tumour invasiveness and resistance to chemotherapy. Here we report the disease-driven engineering of a drug delivery system, a 'nanocell', which overcomes these barriers unique to solid tumours. The nanocell comprises a nuclear nanoparticle within an extranuclear pegylated-lipid envelope, and is preferentially taken up by the tumour. The nanocell enables a temporal release of two drugs: the outer envelope first releases an anti-angiogenesis agent, causing a vascular shutdown; the inner nanoparticle, which is trapped inside the tumour, then releases a chemotherapy agent. This focal release within a tumour results in improved therapeutic index with reduced toxicity. The technology can be extended to additional agents, so as to target multiple signalling pathways or distinct tumour compartments, enabling the model of an 'integrative' approach in cancer therapy.

Multiomic study of skin, peripheral blood, and serum: is serum proteome a reflection of disease process at the end-organ level in systemic sclerosis?

BACKGROUND: Serum proteins can be readily assessed during routine clinical care. However, it is unclear to what extent serum proteins reflect the molecular dysregulations of peripheral blood cells (PBCs) or affected end-organs in systemic sclerosis (SSc). We conducted a multiomic comparative analysis of SSc serum profile, PBC, and skin gene expression in concurrently collected samples. METHODS: Global gene expression profiling was carried out in skin and PBC samples obtained from 49 SSc patients enrolled in the GENISOS observational cohort and 25 unaffected controls. Levels of 911 proteins were determined by Olink Proximity Extension Assay in concurrently collected serum samples. RESULTS: Both SSc PBC and skin transcriptomes showed a prominent type I interferon signature. The examination of SSc serum profile revealed an upregulation of proteins involved in pro-fibrotic homing and extravasation, as well as extracellular matrix components/modulators. Notably, several soluble receptor proteins such as EGFR, ERBB2, ERBB3, VEGFR2, TGFBR3, and PDGF-Rα were downregulated. Thirty-nine proteins correlated with severity of SSc skin disease. The differential expression of serum protein in SSc vs. control comparison significantly correlated with the differential expression of corresponding transcripts in skin but not in PBCs. Moreover, the differentially expressed serum proteins were significantly more connected to the Well-Associated-Proteins in the skin than PBC gene expression dataset. The assessment of the concordance of between-sample similarities revealed that the molecular profile of serum proteins and skin gene expression data were significantly concordant in patients with SSc but not in healthy controls. CONCLUSIONS: SSc serum protein profile shows an upregulation of profibrotic cytokines and a downregulation of soluble EGF and other key receptors. Our multilevel comparative analysis indicates that the serum protein profile in SSc correlates more closely with molecular dysregulations of skin than PBCs and might serve as a reflection of disease severity at the end-organ level.

Glycosaminoglycan characterization methodologies: probing biomolecular interactions.

Interactions between glycans and proteins are central to many of the regulatory processes within biology. The development of analytical methodologies that enable structural characterization of glycosaminoglycan oligosaccharides has fostered improved understanding of the specificity of these biomolecular interactions. This facilitates an appreciation in understanding how changes in GAG structure can regulate physiology as well as pathology. While there are various techniques for studying the interaction of GAGs with proteins, in this chapter we focus on two approaches. First, an integrated analytical methodology, surface non-covalent affinity mass spectrometry (SNA-MS), is described to isolate, enrich, and sequence tissue-derived GAGs that bind to specific proteins. The broad applicability of this powerful platform offers an insight into how changes in cell-surface and extracellular GAG composition and sequence influences the ability of cells and tissues to dynamically alter responses to signaling molecules. Thus, this approach provides a window into understanding how changes at a molecular level manifest with respect to cellular phenotype. Second, surface plasmon resonance, or SPR, represents an additional platform for the study of protein-polysaccharide interaction, specifically for measuring the binding between GAG chains and proteins.

The structural elucidation of glycosaminoglycans.

There is accumulating evidence of the importance of linear polysaccharides in modulating biological phenomena in both the normal and the diseased states. This layer of regulation results from interactions between polysaccharides and other biomolecules, such as proteins, at the cell-extracellular matrix interface. The specific sequence of chemical modifications within the polymer backbone imparts a potential for interaction with other molecular species, and thus there exists important information within the various sulfation, acetylation, and epimerization states of such complex carbohydrates. A variety of factors have made the deciphering of this chemical code elusive. To this end, this report describes several techniques to elucidate the structural information inherent in glycosaminoglycan species. First, the use of depolymerizing enzymes that cleave polysaccharides at specific sites is described. Then, capillary electrophoretic (CE) techniques are employed to characterize the disaccharide species present in an enzymatically-cleaved polysaccharide sample. Mass spectrometry (MS) procedures can further be used to establish the length of an oligosaccharide chain and the presence of specific functional groups.

Circulating Markers of Inflammation Persist in Children and Adults With Giant Aneurysms After Kawasaki Disease.

BACKGROUND: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients. METHODS: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers. RESULTS: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood. CONCLUSIONS: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.

Molecular profiling of rheumatoid arthritis patients reveals an association between innate and adaptive cell populations and response to anti-tumor necrosis factor.

BACKGROUND: The goal of this study is to use comprehensive molecular profiling to characterize clinical response to anti-TNF therapy in a real-world setting and identify reproducible markers differentiating good responders and non-responders in rheumatoid arthritis (RA). METHODS: Whole-blood mRNA, plasma proteins, and glycopeptides were measured in two cohorts of biologic-naïve RA patients (n = 40 and n = 36) from the Corrona CERTAIN (Comparative Effectiveness Registry to study Therapies for Arthritis and Inflammatory coNditions) registry at baseline and after 3 months of anti-TNF treatment. Response to treatment was categorized by EULAR criteria. A cell type-specific data analysis was conducted to evaluate the involvement of the most common immune cell sub-populations. Findings concordant between the two cohorts were further assessed for reproducibility using selected NCBI-GEO datasets and clinical laboratory measurements available in the CERTAIN database. RESULTS: A treatment-related signature suggesting a reduction in neutrophils, independent of the status of response, was indicated by a high level of correlation (ρ = 0.62; p < 0.01) between the two cohorts. A baseline, response signature of increased innate cell types in responders compared to increased adaptive cell types in non-responders was identified in both cohorts. This result was further assessed by applying the cell type-specific analysis to five other publicly available RA datasets. Evaluation of the neutrophil-to-lymphocyte ratio at baseline in the remaining patients (n = 1962) from the CERTAIN database confirmed the observation (odds ratio of good/moderate response = 1.20 [95% CI = 1.03-1.41, p = 0.02]). CONCLUSION: Differences in innate/adaptive immune cell type composition at baseline may be a major contributor to response to anti-TNF treatment within the first 3 months of therapy.

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics.

The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography-tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the "knowns" are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both "known" and "unknown" site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific "fingerprint" of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.