An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2–neutralizing antibody-like therapeutics.

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource.

Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived transposon insertion libraries.

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.

SapM mutation to improve the BCG vaccine: Genomic, transcriptomic and preclinical safety characterization.

The Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur.

Characterization of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase Produced in Pichia pastoris as Potential Enzyme for Mucopolysaccharidosis IVA Treatment.

Mucopolysaccharidosis IVA (MPS IVA or Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal storage of keratan sulfate and chondroitin-6-sulfate. Currently, enzyme replacement therapy using an enzyme produced in CHO cells represents the main treatment option for MPS IVA patients. As an alternative, we reported the production of an active GALNS enzyme produced in the yeast Pichia pastoris (prGALNS), which showed internalization by cultured cells through a potential receptor-mediated process and similar post-translational processing as human enzyme.

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.

A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes.

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast.

Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man3GlcNAc2 N-glycan core.

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans.

Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man₈GlcNAc₂ and Man₅GlcNAc₂.

Background: Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective.

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate.

Background: Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.

Results: Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter).

Abscisic acid deficiency causes changes in cuticle permeability and pectin composition that influence tomato resistance to Botrytis cinerea.

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato.

Genome sequence of the recombinant protein production host Pichia pastoris.

The methylotrophic yeast Pichia pastoris is widely used for the production of proteins and as a model organism for studying peroxisomal biogenesis and methanol assimilation. P. pastoris strains capable of human-type N-glycosylation are now available, which increases the utility of this organism for biopharmaceutical production.

GlycoFibroTest is a highly performant liver fibrosis biomarker derived from DNA sequencer-based serum protein glycomics.

Liver fibrosis is currently assessed by liver biopsy, a costly and rather cumbersome procedure that is unsuitable for frequent patient monitoring, which drives research into biomarkers for this purpose. To investigate whether the serum N-glycome contains information suitable for this goal, we developed a 96-well plate-based serum N-glycomics sample preparation protocol that only involves fluid transfer steps and incubations in a PCR thermocycler yielding 8-aminopyrene-1,3,6-trisulfonic acid-labeled N-glycans. These N-glycans are then ready for analysis on the capillary electrophoresis-based DNA sequencers that are the current standard in clinical genetics laboratories worldwide.

Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics.

We applied our 'clinical glycomics' technology, based on DNA sequencer/fragment analyzers, to generate profiles of serum protein N-glycans of liver disease patients. This technology yielded a biomarker that distinguished compensated cirrhotic from noncirrhotic chronic liver disease patients, with 79% sensitivity and 86% specificity (100% sensitivity and specificity for decompensated cirrhosis). In combination with the clinical chemistry-based Fibrotest biomarker, compensated cirrhosis was detected with 100% specificity and 75% sensitivity.