Long-lasting cross-protection against influenza A by neuraminidase and M2e-based immunization strategies

There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus

Endoglycosidase S enables a highly simplified clinical chemistry procedure for direct assessment of serum IgG undergalactosylation in chronic inflammatory disease

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohn's and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry because of a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput. We addressed this issue by using endo-beta-N-acetyl-glucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker toward clinical implementation

A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes

A methodology for rapid, high-purity isolation of plasma membranes using superparamagnetic nanoparticles is described. The method is illustrated with high-resolution proteomic, glycomic and lipidomic analyses of presenilin-deficient cells

GlycoHepatoTest: validation and analytical technology development for implementation on microfluidics analyzers

Glycomics research has become a versatile approach to several scientific problems, interfacing multiple disciplines. Profiling the N-glycans derived from total serum protein (N-glycome) has led to the discovery of a biomarker panel, renamed GlycoHepatoTest. The three biomarkers might be valuable over the complete spectrum of chronic liver disease: (i) monitoring liver fibrosis progression (GlycoFibroTest), (ii) diagnosis of early-stage cirrhosis to identify the patients that are at risk to develop decompensated cirrhosis and hepatocellular carcinoma (GlycoCirrhoTest), and (iii) GlycoHCCTest complements α-fetoprotein in the diagnosis of liver cancer in decompensated cirrhosis patients chronically infected with the hepatitis B virus. This novel panel has the potential to, in contrast to the invasive and costly liver biopsy, frequently obtain clinical relevant information during the course of these often long-term chronic conditions. However, the sample preparation protocol in its current format was not suitable for clinical implementation. Moreover, additional well-designed validation studies are required to demonstrate the clinical use and to further elucidate the correlation between N-glycome dynamics and the pathophysiology of chronic liver disease. Therefore, we designed an assay allowing the preparation of glycan samples within three hours that could be analyzed on capillary electrophoresis-based microfluidics platforms. Moreover, we could successfully validate GlycoFibroTest in a multicenter study consisting of 376 chronic hepatitis C patients. Interestingly, we discovered that undergalactosylated N-glycans, exponentially increasing with fibrosis stage, were exclusively present on Immunoglobulin G. This feature is a hallmark of chronic necro-inflammatory diseases, making it an intriguing research question for the future. We further investigated whether N-glycan profiling of the liver produced hemopexin contained information that could not be retrieved from the serum N-glycome profile. Initial results showed that the glycosylation changes on this protein improved the diagnosis of hepatocellular carcinoma in cirrhosis patients chronically infected with the hepatitis C virus. Finally, we examined an old observation that serum antibodies of chronic liver disease patients migrate differently during electrophoresis. We were able to conclude that these electrophoretic migration differences of antibodies correlate with their degree of sialylation. In this way, we could differentiate between healthy individuals and chronic liver disease patients

High-throughput profiling of the serum N-glycome on capillary electrophoresis microfluidics systems

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward

Glycome profiling using modern glycomics technology: technical aspects and applications

Glycomics research has become indispensable in many research fields such as immunity, signal transduction and development. Moreover, changes in the glycosylation of proteins and lipids have been reported in several diseases including cancer. The analysis of a complex post-translational modification such as glycosylation depends on the availability or development of appropriate analytical technologies. The research goal determines the sensitivity, resolution and throughput requirements and guides the choice of a particular technology. This review highlights the evolution of glycan profiling tools in the past 5 years. Here, we focus on capillary electrophoresis, liquid chromatography, mass spectrometry and lectin microarrays

Analysis of γ-globulin mobility on routine clinical CE equipment: exploring its molecular basis and potential clinical utility

A study was conducted on the variability of gamma-globulin mobility in serum protein electrophoresis and its molecular basis. We found that the migration time of gamma-globulins can be reproducibly determined (CV = 1.1%) on clinical CE equipment. Moreover, we found a significant difference (p<0.001) in the migration of gamma-globulins between chronic liver disease patients (n = 98) and a healthy reference group (n = 47). Serum immunoglobulins were purified from these patients' sera using protein L-agarose and their glycosylation was studied using CE on a DNA sequencer. This glycomics approach revealed that several non-sialylated N-glycans show a moderate Pearson correlation coefficient (r = 0.2-0.4) with the migration time of gamma-globulins. Their sialylated structures correlate negatively (r = -0.2 to -0.3). Immunoglobulins are significantly more sialylated in the healthy reference group compared with the patients (p<0.001). We estimated that sialylation heterogeneity contributes about 36% to the molecular variance (carbohydrates and amino-acid composition) that affects the electrophoretic mobility of immunoglobulins. This is the first report on the migration time of gamma-globulins on a clinical CE instrument and its potential clinical value to the routinely analyzed serum protein CE profiles

CE analysis of γ-globulin mobility and potential clinical utility

Serum protein electrophoresis is widely used in clinical laboratories to measure the relative abundance of each obtained fraction. Moreover, we found that the migration time of the g-globulin fraction can be reproducibly determined (CV = 1.1%). Immunoglobulins were purified from serum using protein l-agarose and their N-glycosylation was studied using CE on a DNA sequencer. Liver fibrosis patients showed a lower level of sialylation and this moderately correlates with the migration time of the g-globulins (r = 0.2– 0.4). This allowed us to differentiate healthy individuals from these patients with an acceptable diagnostic accuracy (area under the curve = 0.75). This glycomics approach could become a significant added value to a daily, routine clinical test