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The emergence of SARS-CoV-2 variants raised questions regarding the durability of immune responses after homologous or heterologous boosters after Ad26.COV2.S-priming. We found that SARS-CoV-2-specific binding antibodies, neutralizing antibodies, and T cells are detectable 5 months after boosting, although waning of antibodies and limited cross-reactivity with Omicron BA.1 was observed.

Pectins are dietary fibers that have been recognized to reduce the incidence of lifestyle related disease. Pectins can contain several chemical structures, including homogalacturonan regions which consist of a galacturonan backbone. These galacturonans can be methyl-esterified causing a difference in degree and distribution of methyl-esters in pectins. It is known that pectins can influence the intestinal immune system, but which specific structures are responsible for these effects is unknown. Therefore, the immunomodulatory role of the degree of methyl-esterification (DM) and the distribution of methyl-esters in homogalacturonan pectins on the intestinal immune system was investigated. Current studies demonstrate that low DM pectins and pectins with an intermediate DM and blockwise distribution of methyl-esters inhibit the activation of Toll-like receptor (TLR) 2-1 and prevent the development of TLR2-mediated inflammation of the small intestine in mice. Besides, administration of a low DM pectin that contains a more blockwise distribution of methyl-esters and intermediate DM pectins influence T cell immunity and the microbiota composition in the intestine of healthy mice. In addition, pectins inhibit the growth of Citrobacter rodentium and prevent inflammation caused by this pathogen in the large intestine. These effects of pectins on C. rodentium were independent of structural differences. In conclusion, homogalacturonan pectins can influence the intestinal immune system through direct effects on TLR2, modulation of intestinal microbiota composition or by inhibiting the growth of enteric pathogens in the intestine. The degree and distribution of methyl-esters of pectins appear to play an important role in these effects.

Galacto-oligosaccharides (GOS) and 2′-fucosyllactose (2′-FL) are non-digestible carbohydrates (NDCs) that are often added to infant formula to replace the functionalities of human milk oligosaccharides (HMOs). It is not known if combining GOS and 2′-FL will affect their fermentation kinetics and subsequent immune-modulatory effects such as AhR-receptor stimulation. Here, we used an in vitro set-up for the fermentation of 2′-FL and GOS, either individually or combined, by fecal microbiota of 8-week-old infants. We found that GOS was fermented two times faster by the infant fecal microbiota when combined with 2′-FL, while the combination of GOS and 2′-FL did not result in a complete degradation of 2′-FL. Fermentation of both GOS and 2′-FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. Digesta of the fermentations influenced dendritic cell cytokine secretion differently under normal conditions and in the presence of the AhR-receptor blocker CH223191. We show that, combining GOS and 2′-FL accelerates GOS fermentation by the infant fecal microbiota of 8-week-old infants. In addition, we show that the fermentation digesta of GOS and 2′-FL, either fermented individually or combined, can attenuate DC cytokine responses in a similar and in an AhR-receptor dependent way.

Scope: Non-digestible carbohydrates (NDCs) such as native chicory inulin and 2′-fucosyllactose (2′-FL) are added to infant formula to mimic some of the human milk oligosaccharide (HMO) functions. It is unknown whether combining inulin and 2′-FL influences their fermentation kinetics and whether the immune-modulatory effects of these NDCs are different under normal and inflammatory-prone Th2-polarizing conditions. Methods and results: We investigated the in vitro fermentation of 2′-FL and native chicory inulin, fermented individually and combined, using fecal inocula of 8-week-old infants. Native inulin was fermented in a size-dependent fashion and expedited the fermentation of 2′-FL. Fermentation of both native inulin and 2′FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. The fermentation digesta of all fermentations differentially influenced both dendritic cell and T-cell cytokine responses under normal culture conditions or in presence of the Th2-polarizing cytokines IL-33 and TSLP, with the most pronounced effect for IL-1β in the presence of TSLP. Conclusions: Our findings show that native inulin can expedite the fermentation of 2′-FL by infant fecal microbiota and that these NDC fermentation digesta have different effects under normal and Th2-polarizing conditions, indicating that infants with different immune backgrounds might benefit from tailored NDC formulations.

Human milk oligosaccharides (hMOs) are unique bioactive components in human milk. 3-Fucosyllactose (3-FL) is an abundantly present hMO that can be produced in sufficient amounts to allow application in infant formula. Lacto-N-triaose II (LNT2) can be obtained by acid hydrolysis of lacto-N-neotetraose (LNnT). Both 3-FL and LNT2 have been shown to have health benefits, but their impact on infant microbiota composition and microbial metabolic products such as short-chain fatty acids (SCFAs) is unknown. To gain more insight in fermentability, we performed in vitro fermentation studies of 3-FL and LNT2 using pooled fecal microbiota from 12-week-old infants. The commonly investigated galacto-oligosaccharides (GOS)/inulin (9 : 1) served as control. Compared to GOS/inulin, we observed a delayed utilization of 3-FL, which was utilized at 60.3% after 36 h of fermentation, and induced the gradual production of acetic acid and lactic acid. 3-FL specifically enriched bacteria of Bacteroides and Enterococcus genus. LNT2 was fermented much faster. After 14 h of fermentation, 90.1% was already utilized, and production of acetic acid, succinic acid, lactic acid and butyric acid was observed. LNT2 specifically increased the abundance of Collinsella, as well as Bifidobacterium. The GOS present in the GOS/inulin mixture was completely fermented after 14 h, while for inulin, only low DP was rapidly utilized after 14 h. To determine whether the fermentation might lead to enhanced colonization of commensal bacteria to gut epithelial cells, we investigated adhesion of the commensal Lactobacillus plantarum WCFS1 to Caco-2 cells. The fermentation digesta of LNT2 collected after 14 h, 24 h, and 36 h, and GOS/inulin after 24 h of fermentation significantly increased the adhesion of L. plantarum WCFS1 to Caco-2 cells, while 3-FL had no such effect. Our findings illustrate that fermentation of hMOs is very structure-dependent and different from the commonly applied GOS/inulin, which might lead t

Introduction: Pectins have anti-inflammatory properties on intestinal immunity through direct interactions on Toll-like receptors (TLRs) in the small intestine or via stimulating microbiota-dependent effects in the large intestine. Both the degree of methyl-esterification (DM) and the distribution of methyl-esters (degree of blockiness; DB) of pectins contribute to this influence on immunity, but whether and how the DB impacts immunity through microbiota-dependent effects in the large intestine is unknown. Therefore, this study tests pectins that structurally differ in DB in a mouse model with Citrobacter rodentium induced colitis and studies the impact on the intestinal microbiota composition and associated attenuation of inflammation. Methods and Results: Both low and high DB pectins induce a more rich and diverse microbiota composition. These pectins also lower the bacterial load of C. rodentium in cecal digesta. Through these effects, both low and high DB pectins attenuate C. rodentium induced colitis resulting in reduced intestinal damage, reduced numbers of Th1-cells, which are increased in case of C. rodentium induced colitis, and reduced levels of GATA3+ Tregs, which are related to tissue inflammation. Conclusion: Pectins prevent C. rodentium induced colonic inflammation by lowering the C. rodentium load in the caecum independently of the DB.

Scope: Intestinal mucositis is a common side effect of the chemotherapeutic agent doxorubicin, which is characterized by severe Toll-like receptor (TLR) 2-mediated inflammation. The dietary fiber pectin is shown to prevent this intestinal inflammation through direct inhibition of TLR2 in a microbiota-independent manner. Recent in vitro studies show that inhibition of TLR2 is determined by the number and distribution of methyl-esters of pectins. Therefore, it is hypothesized that the degree of methyl-esterification (DM) and the degree of blockiness (DB) of pectins determine attenuating efficacy on doxorubicin-induced intestinal mucositis. Methods and Results: Four structurally different pectins that differed in DM and DB are tested on inhibitory effects on murine TLR2 in vitro, and on doxorubicin-induced intestinal mucositis in mice. These data demonstrate that low DM pectins or intermediate DM pectins with high DB have the strongest inhibitory impact on murine TLR2-1 and the strongest attenuating effect on TLR2-induced apoptosis and peritonitis. Intermediate DM pectin with a low DB is, however, also effective in preventing the induction of doxorubicin-induced intestinal damage. Conclusion: These pectin structures with stronger TLR2-inhibiting properties may prevent the development of doxorubicin-induced intestinal damage in patients undergoing chemotherapeutic treatment with doxorubicin.

Dietary fibers have been shown to exert immune effects via interaction with pattern recognition receptors (PRR) such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors. Pectin is a dietary fiber that interacts with PRR depending on its chemical structure. Papaya pectin retains different chemical structures at different ripening stages. How this influence PRR signaling is unknown. The aim of this work was to determine how ripening influences pectin structures and their ability to interact with TLR2, 3, 4, 5 and 9, and NOD1 and 2. It was evaluated the interaction of the water-soluble fractions rich in pectin extracted from unripe to ripe papayas. The pectin extracted from ripe papayas activated all the TLR and, to a lesser extent, the NOD receptors. The pectin extracted from unripe papayas also activated TLR2, 4 and 5 but inhibited the activation of TLR3 and 9. The differences in pectin structures are the higher methyl esterification and smaller galacturonan chains of pectin from ripe papayas. Our finding might lead to selection of ripening stages for tailored modulation of PRR to support or attenuate immunity., This research was financially supported by grants #2012/23970–2, #2013/07914–8 and #2019/11816–8, São Paulo Research Foundation (FAPESP). Research supported by LNNano – Brazilian Nanotechnology National Laboratory, CNPEM/MCTI (Proposal AFM#21087). Scholarship was awarded to SBRP by the National Council for Scientific and Technological Development (CNPq; 167934/2014–7).

Dietary carbohydrate fibers are known to prevent immunological diseases common in Western countries such as allergy and asthma but the underlying mechanisms are largely unknown. Until now beneficial effects of dietary fibers are mainly attributed to fermentation products of the fibers such as anti-inflammatory short-chain fatty acids (SCFAs). Here, we found and present a new mechanism by which dietary fibers can be anti-inflammatory: a commonly consumed fiber, pectin, blocks innate immune receptors. We show that pectin binds and inhibits, toll-like receptor 2 (TLR2) and specifically inhibits the proinflammatory TLR2-TLR1 pathway while the tolerogenic TLR2-TLR6 pathway remains unaltered. This effect is most pronounced with pectins having a low degree of methyl esterification (DM). Low-DM pectin interacts with TLR2 through electrostatic forces between non-esterified galacturonic acids on the pectin and positive charges on the TLR2 ectodomain, as confirmed by testing pectin binding on mutated TLR2. The anti-inflammatory effect of low-DM pectins was first studied in human dendritic cells and mouse macrophages in vitro and was subsequently tested in vivo in TLR2-dependent ileitis in a mouse model. In these mice, ileitis was prevented by pectin administration. Protective effects were shown to be TLR2-TLR1 dependent and independent of the SCFAs produced by the gut microbiota. These data suggest that low-DM pectins as a source of dietary fiber can reduce inflammation through direct interaction with TLR2-TLR1 receptors.