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Vaccines are one of the greatest achievements of modern medicine. Due to their safer profile, the latest investigations usually focus on subunit vaccines. However, the active component often needs to be coupled with an adjuvant to be effective and properly trigger an immune response. We are developing a new synthetic monosaccharide-based TLR4 agonist, such as glucosamine-derived compounds FP18 and FP20 , as a potential vaccine adjuvant. In this study, we present a new FP20 derivative, FP20Hmp , with a hydroxylated ester linked to the glucosamine core. We show that the modification introduced improves the activity of the adjuvant and its solubility. This study presents the synthesis of FP20Hmp , its in vitro characterization, and in vivo activity while coupled with the ovalbumin antigen or in formulation with an enterococcal antigen. We show that FP20Hmp enables increased production of antigen-specific antibodies that bind to the whole bacterium.

Lipopolysaccharide (LPS) mimicry leading to toll-like receptor 4 (TLR4) active compounds has been so far based mainly on reproducing the lipid A portion of LPS. Our work led to a series of structurally simplified synthetic TLR4 agonists in preclinical development as vaccine adjuvants called FPs. FPs bind MD2/TLR4 similarly to lipid A, inserting the lipid chains in the MD2 lipophilic cavity. A strategy to improve FPs' target affinity is introducing a monosaccharide unit in C6, mimicking the first sugar of the LPS core. We therefore designed a panel of FP derivatives bearing different monosaccharides in C6. We report here the synthesis and optimization of FPs' C6 glycosylation, which presented unique challenges and limitations. The biological activity of glycosylated FP compounds was preliminarily assessed in vitro in HEK-Blue cells. The new molecules showed a higher potency in stimulating TLR4 activation when compared to the parent molecule while maintaining TLR4 selectivity., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands ( FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18 . Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 μM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.

Echinochloa P. Beauv. is an important genus because many of its species are weeds infesting most paddy fields, which can reduce the rice grain production by up to 80%. A controversy exists about the taxonomy of the genus due to the high level of morphological variations found in these species. Cyhalofop-butyl, an aryloxyphenoxy-propionate herbicide, is used to control Echinochloa spp. in paddy fields, although differences in susceptibility were found between different Echinochloa species. E. colona was highly susceptible [ED50= 34 g of active ingredient (ai) ha(-1)]; very similar results were obtained with the remaining species. By contrast, E. oryzicola (170 g of ai ha(-1)) was less sensitive, with the herbicide symptoms appearing later. Because of this differential susceptibility, morphological and molecular studies were carried out. A morphological study, using 21 characters both quantitative and qualitative of spikelets and seedlings, was capable of clearly distinguishing closely related E. crus-galli plants (two populations), E. muricata and E. crus-pavonis, and E. oryzicola, E. utilis, and E. colona species. The resolution of Echinochloa species at the molecular level, based on RAPD analyses, was fairly consistent with morphological analysis results. Among the 60 primers screened, 21 primers exhibited polymorphic bands and produced a total of 136 RAPD markers. Of all the amplified fragments, 90 were found to be polymorphic. E. oryzicola and E. colona were clearly separated, and the RAPD analyses showed that both E. crus-galli populations were 100% related and 51% related to E. utilis, whereas E. crus-pavonis and E. muricata (73% similarity) appeared as being clearly separated from this group.