Biology subjects

Grootaert, H.

Publications and source records attributed to Grootaert, H..

3 recordsLinked to original sources

bioRxiv2025

ENGINEERING AND PRECLINICAL EVALUATION OF DRIED FOOD-FORMULATED YEAST-SECRETED ANTIBODIES AS PERI-EXPOSURE PROPHYLAXIS AGAINST CLOSTRIDIOIDES DIFFICILE DISEASE.

Clostridioides difficile gastrointestinal infection (CDI) causes significant disease burden, often following gut dysbiosis induced by antibiotic treatment in at-risk individuals. A non-invasive, easy to use targeted prophylaxis in such cases of acute elevated CDI risk, especially in those with a history of recurrent CDI, would prevent much suffering. In this study, we envisioned a simple prophylactic intervention for CDI enabled by the oral administration of yeast-secreted dried food-formulated C. difficile toxin A (TcdA)-and B (TcdB)-neutralizing VHH-based antibodies. We engineered two antibody versions: a previously reported tetravalent VHH (VHH4) comprising dual specificities for TcdA and TcdB, and its murine IgA Fc fusion counterpart (VHH4-mIgA Fc). We explored Generally Recognized as Safe (GRAS) Komagataella phaffii yeast as a low-cost, highly scalable production platform. In a first experiment, we produced the antibodies at small scale in shake flask yeast cultivations and formulated the secreted antibody-containing growth medium into standard-composition dried mouse feed pellets. The antibody food pellet-administration intervention was tested in a C. difficile spore-challenged mouse model in which CDI leads to 50% mortality of untreated animals. We started the antibody-feed prophylaxis dosed at 10 nmol/g pellet one day prior to C. difficile challenge and continued until the infection was cleared with ad libitum feeding. We found that the VHH4 feed almost completely prevented mortality and reduced disease-related weight loss. Despite its higher in vitro potency against TcdA and equipotent potency against TcdB, the VHH4-mIgA Fc fusion did not confer additional in vivo benefit, and further work focused on the simpler VHH4 format. Scalable high-cell-density bioreactor production of VHH4 yielded equally effective prophylactic formulations at the same dose of 10 nmol/g pellet, achieving complete survival and reduced disease burden. Our findings suggest that, with further antibody- and process development, oral prophylaxis with scalable, cost-effective, yeast-produced, dried food-formulated anti-toxin VHHs could offer a simple non-invasive, non-antibiotic prophylaxis option for the high-risk (r)CDI patient population.

molecular biology↗

bioRxiv2022

OPENPichia: building a free-to-operate Komagataella phaffii protein expression toolkit

In the standard toolkit for recombinant protein expression, the yeast known in biotechnology as Pichia pastoris (formally: Komagataella phaffii) takes up the position between E. coli and HEK293 or CHO mammalian cells, and is used by thousands of laboratories both in academia and industry. The organism is eukaryotic yet microbial, and grows to extremely high cell densities while secreting proteins into its fully defined growth medium, using very well established strong inducible or constitutive promoters. Many products made in Pichia are in the clinic and in industrial markets. Pichia is also a favoured host for the rapidly emerging area of precision fermentation for the manufacturing of food proteins. However, the earliest steps in the development of the industrial strain (NRRL Y-11430/CBS 7435) that is used throughout the world were performed prior to 1985 in industry (Phillips Petroleum Company) and are not in the public domain. Moreover, despite the long expiry of associated patents, the patent deposit NRRL Y-11430/CBS 7435 that is the parent to all commonly used industrial strains, is not or no longer made freely available through the resp. culture collections. This situation is far from ideal for what is a major chassis for synthetic biology, as it generates concern that novel applications of the system are still encumbered by licensing requirements of the very basic strains. In the spirit of open science and freedom to operate for what is a key component of biotechnology, we set out to resolve this by using genome sequencing of type strains, reverse engineering where necessary, and comparative protein expression and strain characterisation studies. We find that the industrial strains derive from the K. phaffii type strain lineage deposited as 54-11.239 in the UC Davis Phaff Yeast Strain collection by Herman Phaff in 1954. This type strain has valid equivalent deposits that are replicated/derived from it in other yeast strain collections, incl. in ARS-NRRL NRRL YB-4290 (deposit also made by Herman Phaff) and NRRL Y-7556, CBS 2612 and NCYC 2543. We furthermore discovered that NRRL Y-11430 and its derivatives carry an ORF-truncating mutation in the HOC1 cell wall synthesis gene, and that reverse engineering of a similar mutation in the NCYC 2543 type strain imparts the high transformability that is characteristic of the industrial strains. Uniquely, the NCYC 2543 type strain, which we propose to call OPENPichia henceforth, is freely available from the NCYC culture collection, incl. resale and commercial production licenses at nominal annual licensing fees1. Furthermore, our not-for-profit research institute VIB has also acquired a resale/distribution license from NCYC, which we presently use to openly provide to end-users our genome-sequenced OPENPichia subclone strain and its derivatives, i.e., currently the highly transformable hoc1tr and the his4 auxotrophic mutants. To complement the OPENPichia platform, a fully synthetic modular gene expression vector building toolkit was developed, which is also openly distributed, for any purpose. We invite other researchers to contribute to our open science resource-building effort to establish a new unencumbered standard chassis for Pichia synthetic biology.

molecular biology↗

bioRxiv2018

Clinical assay for direct assessment of IgG galactosylation in serum using endoglycosidase S

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohns 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 due to a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput.\n\nWe addressed this issue by using endo-{beta}-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 towards clinical implementation.

biochemistry↗