Writing in the Journal of Biotechnology, the team identifies the HMO lacto-N-fucopentaose I (LNFP I) in combination with simple fucosylated structures as more likely to prevent norovirus infections.
“Complex oligosaccharides such as HMOs can mimic the structure of the virus’s natural receptor on human cells, coating the virus and preventing it from interacting with its targets,” says Dr Katja Parschat, Co-Head of R&D at Jennewein Biotechnologie.
“We demonstrated this ability for 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL), in our previous study. But the development of more complex oligosaccharides, which mimic the virus receptors even more closely, allows us to significantly improve this protective effect.”
Noroviruses are positive-sense single-stranded RNA viruses that cause most cases of non-bacterial acute gastroenteritis in all age groups.
Symptoms of norovirus infectious are characterised by diarrhoea and/or vomiting and are generally self-limiting. However, infection can be more severe in children, the immunocompromised and the elderly.
Human noroviruses recognise histo-blood group antigens (HBGAs) as receptors or co-receptors. HBGAs are carbohydrates that exist as soluble molecules in the blood, gastric fluid and breastmilk.
Interactions between noroviruses and HBGAs could be influenced by HMOs due to similarities to HBGA structure and abundance in human breastmilk, providing benefits to breastfed infants including a lower susceptibility to viral diarrhoea.
In the paper, the team began describing a large-scale fermentation process that yielded several kilograms (kg) of LNFP I synthesized using genetically modified Escherichia coli BL21(DE3) cells.
Two strains of the norovirus genogroup GII were also introduced into the study: GII.4 (Sydney, 2012 JX459908) and GII.17 (Kawasaki308, 2015 LC037415).
Tests to determine IC50 values, a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function, were also carried out.
Here, the binding of the norovirus strain GII.17 Kawasaki308 VP1 (50 μg ml−1) to different HMOs (0.08−40 mM) was evaluated using surface plasmon resonance (SPR) spectroscopy.
HMOs used here includes LNFP I, lacto-N-neofucopentaose V (LNnFP V) 3FL; LNnFP I, lacto-N-neotetraose (LNnT), LNT, lactose, fucose and 2′FL.
Virus-Like Particle (VLP) inhibition assays for GII.4 and GII.17 were performed on immobilised human gastric mucins from pooled gastric juice (10μg ml−1) or on immobilised human mucins from amniotic fluid (40μg ml−1).
Finally, SPR spectroscopy was used to investigate the ability of lactose, fucose and various HMOs to inhibit the binding of norovirus VLPs to chip surfaces functionalised with blood group antigen trisaccharides A (TriA) and B (TriB) conjugated to Bovine Serum Albumin (BSA).
The team found increasing concentrations of the non-fucosylated sugars lactose, LNT and LNnT did not inhibit the binding of the norovirus VLPs to TriA or TriB, thus no IC50 values could be determined.
When the team tested the fucosylated sugars, similar IC50 values were observed on surfaces functionalised with TriA and TriB.
Fucose achieved the lowest inhibitory effect (IC50 = 10.6 and 10.5 mM for the inhibition of TriA and TriB binding, respectively) followed by 2′FL (IC50 = 8.9 and 8.4 mM, respectively).
The team found that 3FL and the pentaoses (LNnFP V, LNnFP I and LNFP I) achieved lower IC50 values on both chips (5.9, 5.8, 5.2 and 5.0 mM, respectively).
LNFP I vs 2′FL and 3FL
“We found that LNFP I inhibited the binding of GII.17 VLPs to TriA and TriB more effectively than 2′FL and 3FL, but that all fucosylated structures were more effective than any non-fucosylated structures,” says the team, who also hail from the Fraunhofer Institute for Molecular Biology and Applied Ecology, University of Cologne and University Children’s Hospital Mannheim.
“We also tested the ability of HMOs to inhibit the binding of GII.17 and GII.4 VLPs to human mucins and in this case found that the simpler fucosylated HMOs (2′FL and 3FL) were more effective than complex molecules such as LNFP I, with non-fucosylated structures again showing no inhibition.
“We have confirmed that complex fucosylated HMOs such as LNFP I can be produced by large-scale fermentation without addition of L-fucose,” the team adds.
“Further studies are required to determine the best combination of simple and complex fucosylated HMOs for the prevention of norovirus infections.”
Dr Stefan Jennewein, CEO of Jennewein Biotechnologie adds, “With more than 600 million infections and 200 000 deaths per year and no treatment available, norovirus is definitely a global health problem, and a particular threat to infants, small children and the elderly.
“Our company has been working for many years on the development of HMOs and other complex carbohydrates that bind to norovirus and prevent infections.”
Source: Journal of Biotechnology
Published online ahead of print: doi.org/10.1016/j.jbiotec.2020.05.001
“Biotechnologically produced fucosylated oligosaccharides inhibit the binding of human noroviruses to their natural receptors.”
Authors: Sami Deryaa et al.