Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies.

Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

A Fast, Affordable, and Minimally Invasive Diagnostic Test for Cancer of Unknown Primary Using DNA Methylation Profiling.

Currently, we cannot provide a conclusive diagnosis for 3% to 5% of people who are confronted with cancer. These patients have cancer of unknown primary (CUP), ie, a metastasized cancer for which the tissue of origin cannot be determined. Studies have shown that the DNA methylation profile is a unique "fingerprint" that can be used to classify tumors. Here we used cell-free reduced representation bisulfite sequencing (cfRRBS), a technique that allows us to identify the methylation profile starting from minimal amounts of highly fragmented DNA, for CUP diagnosis on formalin-fixed paraffin-embedded (FFPE) tissue and liquid biopsies. We collected 80 primary tumor FFPE samples covering 16 tumor entities together with 15 healthy plasma samples to use as a custom cfRRBS reference data set. Entity-specific methylation regions are defined for each entity to build a classifier based on nonnegative least squares deconvolution. This classification framework was tested on 30 FFPE, 19 plasma, and 40 pleural and peritoneal effusion samples of both known metastatic tumors and clinical CUPs for which pathological investigation finally resulted in a cancer diagnosis. Using this framework, 27 of 30 FFPE (all CUPs) and 16 of 19 plasma samples (10/13 CUPs) obtained an accurate diagnosis, with a minimal DNA input of 400 pg. Diagnosis of the 40 pleural and peritoneal effusion samples is possible in 9 of 27 samples with negative/inconclusive cytology (6/13 CUPs), showing that cell-free DNA (cfDNA) methylation profiling could complement routine cytologic analysis. However, a low "cfDNA - high-molecular weight DNA ratio" has a considerable impact on the prediction accuracy. Moreover, the accuracy improves significantly if the predicted tumor percentage is >7%. This proof-of-concept study shows the feasibility of using DNA methylation profiling on FFPE and liquid biopsy samples such as blood, ascites, and pleural effusions in a fast and affordable way. Our novel RRBS-based technique requires minimal DNA input, can be performed in <1 week, and is highly adaptable to specific diagnostic problems as we only use 5 FFPE references per tumor entity. We believe that cfRRBS methylation profiling could be a valuable addition to the pathologist's toolbox in the diagnosis of CUPs.

Knockout of RSN1, TVP18 or CSC1-2 causes perturbation of Golgi cisternae in Pichia pastoris.

The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.

GlyConnect-Ugi: site-selective, multi-component glycoprotein conjugations through GlycoDelete expressed glycans.

Recently, the GlyConnect-oxime (GC) protein conjugation strategy was developed to provide a site-selective glycan-based conjugation strategy as an extension to the in-house developed GlycoDelete (GD) technology. GD gives access to glycoproteins with single GlcNAc, LacNAc, or LacNAc-Sia type glycans on their N-glycosylation sites. We have previously shown that these glycans provide a unique handle for site-selective conjugation as they provide a short, homogeneous and hydrophilic link to the protein backbone. GC focused on the use of chemical and chemo-enzymatic pathways for conjugation of a single molecule of interest via oxime formation or reductive amination. In the current work, we explore multicomponent reactions (MCR), namely Ugi and Passerini reactions, for GlycoDelete glycan directed, site-specific protein conjugation (MC-GC). The use of the Ugi and Passerini multicomponent reactions holds the potential of introducing multiple groups of interest in a single reaction step while creating a hydrophilic peptide-like linker.

Exploration of Synergistic Action of Cell Wall-Degrading Enzymes against Mycobacterium tuberculosis.

The major global health threat tuberculosis is caused by Mycobacterium tuberculosis. M. tuberculosis has a complex cell envelope-a partially covalently linked composite of polysaccharides, peptidoglycan, and lipids, including a mycolic acid layer-which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating Gram-positive and -negative infections with cell wall-degrading enzymes, we investigated such an approach for M. tuberculosis. In this study, we aimed to (i) develop an M. tuberculosis microtiter growth inhibition assay that allows undisturbed cell envelope formation to overcome the invalidation of results by typical clumped M. tuberculosis growth in surfactant-free assays, (ii) explore anti-M. tuberculosis potency of cell wall layer-degrading enzymes, and (iii) investigate the concerted action of several such enzymes. We inserted a bacterial luciferase operon in an auxotrophic M. tuberculosis strain to develop a microtiter assay that allows proper evaluation of cell wall-degrading anti-M. tuberculosis enzymes. We assessed growth inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase [LysB], fungal α-amylase, and human and chicken egg white lysozymes) and combinations thereof in the presence or absence of biopharmaceutically acceptable surfactant. Our biosafety level 2 assay identified both LysB and lysozymes as potent M. tuberculosis inhibitors but only in the presence of surfactant. Moreover, the most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase, and polysorbate 80. Synergistically acting cell wall-degrading enzymes are potently inhibiting M. tuberculosis, which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

Protein Glycosylation as a Diagnostic and Prognostic Marker of Chronic Inflammatory Gastrointestinal and Liver Diseases.

Glycans are sequences of carbohydrates that are added to proteins or lipids to modulate their structure and function. Glycans modify proteins required for regulation of immune cells, and alterations have been associated with inflammatory conditions. For example, specific glycans regulate T-cell activation, structures, and functions of immunoglobulins; interactions between microbes and immune and epithelial cells; and malignant transformation in the intestine and liver. We review the effects of protein glycosylation in regulation of gastrointestinal and liver functions, and how alterations in glycosylation serve as diagnostic or prognostic factors, or as targets for therapy.

Tissue N-linked glycosylation as potential prognostic biomarker for biochemical recurrence-free survival.

PURPOSE: Only few biomarkers have been evaluated for their prognostic value following radical prostatectomy. We explored if tissue N-glycosylation shows prognostic properties for biochemical recurrence (BCR)-free survival. MATERIALS AND METHODS: Tissue N-glycosylation profile was determined from 82 prostate cancer (PCa) patients and prognostic features were compared to clinical and biochemical parameters for BCR-free survival. RESULTS: Majority presented with Gleason score 3 + 4 (41%), extensive local disease (62%) and without pelvic lymph nodes invasion (83%). Several parameters (low T stage, low Gleason score, low EAU risk groups for BCR, absence of positive surgical margins, high ratio of fucosylated triantennary structures on total of multiantennary structures [3AFc/MA], low ratio of fucosylated biantennary with core-branched N-acetylglucosamine on total of biantennary structures, and high ratio of triantennary structures on total of multiantennary structures) proved to have a univariate beneficial effect on BCR-free survival. Multivariate analysis proved positive surgical margins and 3AFc/MA to be independent prognosticators. CONCLUSIONS: Tissue N-glycans are a powerful prognostic tool and can be an asset in PCa as the ratio of 3AFc/MA is independently associated with BCR-free survival. This could be of clinical use in guiding patients following radical prostatectomy, e.g. referral to adjuvant radiotherapy. Further elaboration of this biomarker is warranted.

A Potential Role for Fructosamine-3-Kinase in Cataract Treatment.

Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.

Functional exploration of the GH29 fucosidase family.

The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family's large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5 GlcNAc2 modified with a Glcα-1,2-Manß-1,2-Manß-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5 GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

Interference of anti-streptavidin antibodies in immunoassays: a very rare phenomenon or a more common finding?

Background Anti-streptavidin antibodies (ASA) may cause analytical interference on certain immunoassay platforms. Streptavidin is purified from the non-pathogenic Streptomyces avidinii soil bacterium. In contrast to interference with biotin, ASA interference is supposed to be much rarer. In-depth studies on this topic are lacking. Therefore, we carried out an analysis toward the prevalence and the possible underlying cause of this interference. Methods Anti-streptavidin (AS)-immunoglobulin G (IgG) and AS-IgM concentrations were determined on multiple samples from two patients with ASA interference and on 500 random samples. On a subset of 100 samples, thyroid-stimulating hormone (TSH) was measured on a Cobas analyzer before and after performing a neutralization protocol which removes ASA. The relationship between the ratio of TSH after neutralization/TSH before neutralization and the ASA concentration was evaluated. Subsequently, an extract of S. avidinii colonies was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results A positive correlation between AS-IgM concentrations and TSH ratio was obtained. Eight samples out of 500 exceeded the calculated AS-IgM cut-off value. In comparison to the AS-IgM concentrations in the population, titers from the two described cases clearly stood out. The isolated cases represent the end of a broader spectrum as there is a continuum of AS-IgM reactivity in the general population. We could not observe any differences in the immunoblot patterns between the cases and controls, which may indicate the general presence of ASA in the population. Conclusions Interference due to ASA is more prevalent than initially thought and is caused by IgM antibodies.

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-ß-N-acetylglucosaminidase 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.

Quality of blood samples collected at home does not affect clinical decision making for the administration of systemic cancer treatment.

The aim of this exploratory clinical study was to evaluate whether the preanalytical quality of blood samples subjected to delayed centrifugation and transport - as a result of home-sampling - is affected in a way it alters the clinical decision-making for patients under systemic cancer therapy. This evaluation is part of a comprehensive investigation of the opportunities for oncological home-hospitalization. Forty-nine patients with cancer donated two additional blood samples during their ambulatory hospital visit. Fifteen blood analytes were compared between routine blood samples and samples that were subjected to transport and delayed centrifugation in order to mimic a locally implemented model for oncological home-hospitalisation. Deviations were analysed by means of Deming regression. For those analytes showing statistically significant intercepts and/or slopes, the mean deviations were compared to the desirable analytical bias; and the intra-individual differences were compared with the limits for clinical decision-making. Statistically significant intercepts and/or slopes were observed for haematocrit (HCT), mean cellular volume (MCV), platelets count (PLT) and C-reactive protein (CRP). Differences exceeding the allowable margins of desirable analytical bias were observed for HCT and MCV. Risk of different clinical decision-making couldn't be observed for any of the analytes showing statistically significant differences. These results demonstrate that home-collection of blood samples, transported at room temperature and centrifuged within a mean time of five hours after sampling, has no effect on clinical decision-making with regards to systemic cancer therapy. However, attention should be paid to the potential occurrence of haemolysis during the preanalytical phase, which can negatively influence haemolysis-dependent variables.

Capturing the 'ome': the expanding molecular toolbox for RNA and DNA library construction.

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application.

Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine.

BACKGROUND: Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172-1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172-1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains. RESULTS: By combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains. CONCLUSIONS: We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization.

Glycome Patterns of Perfusate in Livers Before Transplantation Associate With Primary Nonfunction.

BACKGROUND & AIMS: Primary nonfunction (PNF) is a rare complication after liver transplantation that requires urgent retransplantation. PNF is associated with livers from extended criteria donors. Clinical and biochemical factors have not been identified that reliably associate with graft function after liver transplantation. Serum patterns of N-glycans associate with changes in the liver. We analyzed perfusate from grafted liver to identify protein glycosylation patterns associated with PNF. METHODS: We performed a prospective study of consecutive patients who underwent liver transplantation (66 patients, from 1 center, in the derivation set, and 56 patients, from 2 centers, in the validation set) in Belgium, from October 1, 2011, through April 30, 2017. All donor grafts were transported using cold static storage, and perfusate samples were collected from the livers by flushing of hepatic veins before transplantation. Protein-linked N-glycans were isolated from perfusate samples and analyzed with a multicapillary electrophoresis-based ABI3130 sequencer. We compared glycan patterns between patients with vs without PNF of transplanted livers. PNF was defined as the need for urgent retransplantation when a graft had no evidence of function, after exclusion of other causes, such as hepatic artery thrombosis or acute cellular rejection. RESULTS: The relative abundance of a single glycan, agalacto core-alpha-1,6-fucosylated biantennary glycan (NGA2F) was significantly increased in perfusate of livers given to 4 patients who developed PNF after liver transplantation compared with livers given to patients who did not develop PNF. Level of NGA2F identified patients with PNF with 100% accuracy. This glycomarker was the only factor associated with PNF in multivariate analysis in the derivation and the validation sets (P < .0001). CONCLUSIONS: In an analysis of patients who underwent liver transplantation, we associated graft perfusate level of glycan NGA2F present on perfusate proteins with development of PNF with 100% accuracy, and validated this finding in a separate cohort of patients. This biomarker might be used to assess grafts before transplantation, especially when high-risk organs are under consideration.

N-Linked Glycosylation and Near-Infrared Spectroscopy in the Diagnosis of Prostate Cancer.

BACKGROUND: Performing a prostate biopsy is the most robust and reliable way to diagnose prostate cancer (PCa), and to determine the disease grading. As little to no biochemical markers for prostate tissue exist, we explored the possibilities of tissue N-glycosylation and near-infrared spectroscopy (NIR) in PCa diagnosis. METHODS: Tissue specimens from 100 patients (benign prostate hyperplasia (BPH), n = 50; and PCa, n = 50) were obtained. The fresh-frozen tissue was dispersed and a tissue N-glycosylation profile was determined. Consequently, the formalin-fixed paraffin-embedded slides were analyzed using NIR spectroscopy. A comparison was made between the benign and malignant tissue, and between the various Gleason scores. RESULTS: A difference was observed for the tissue of N-glycosylation between the benign and malignant tissue. These differences were located in the fycosylation ratios and the total amount of bi- and tetra-antennary structures (all p < 0.0001). These differences were also present between various Gleason scores. In addition, the NIR spectra revealed changes between the benign and malignant tissue in several regions. Moreover, spectral ranges of 1055⁻1065 nm and 1450⁻1460 nm were significantly different between the Gleason scores (p = 0.0042 and p = 0.0195). CONCLUSIONS: We have demonstrated biochemical changes in the N-glycan profile of prostate tissue, which allows for the distinction between malignant and benign tissue, as well as between various Gleason scores. These changes can be correlated to the changes observed in the NIR spectra. This could possibly further improve the histological assessment of PCa diagnosis, although further method validation is needed.

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g-/-, Fcgr3-/-, and Fcgr1-/-Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies.IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

A pragmatic bottom-up approach to harmonize the units of clinical chemistry tests among Belgian clinical laboratories, focusing on immunoassays.

BACKGROUND: Harmonization of units is an important step to improve the comparability of clinical chemistry results, but few examples exist of successful harmonization efforts. We present the results of a pragmatic approach that was implemented in Belgium from 2012. METHODS: After a large consultation and information of stakeholders, preferred units were proposed for 140 assays, including the 23 immunoassays discussed in more detail here. The change occurred in two phases, first involving assays for which there was no change in the numerical result, then changes involving a change in numerical results. Laboratories were invited to participate in this harmonization on a voluntary basis. The project was based on a bottom-up approach, large consultation and the pragmatic choice of the proposed units, including conventional and SI units. RESULTS: The large heterogeneity of units was drastically reduced; adoption of the preferred units increased from 3% (insulin) - 45% (HCG) to 70% (insulin) - 96% (LH and FSH). Adoption of the preferred units was higher if it involved no change in numerical values (90%) than when there was a change (76%). CONCLUSIONS: We believe that the harmonization effort has reached its goals. Without aiming at implementing SI units for all parameters, our strategy was successful with a large majority of the laboratories switching to the proposed units. Moreover, the harmonization program is still progressing, with additional laboratories converting to the consensus units.