D lactose

HA-8mer was synthesized as previously described (Kanekiyo et al., 2013 (link); Weaver et al., 2016 (link)). Briefly, plasmids were transiently transfected into FreeStyle 293-F cells in FreeStyle 293 Expression Medium. After 5 days of culturing in conditions described above, cell culture supernatants collected by centrifugation and concentrated using a tangential flow filtration setup with a 30 kDa cutoff. In 100 mL aliquots, the concentrate was mixed with 2 mL of PBS-equilibrated Erythrina cristagalli lectin-immobilized resin (EY Laboratories) at 4°C and incubated overnight with gentle agitation. The resin was then loaded onto a 1.5 × 20 cm glass Econo-Column (Bio-Rad) and washed with five column-volumes of PBS by gravity flow. HA-8mer particles were eluted with two column-volumes of 0.2 M D-lactose (Sigma-Aldrich) in PBS and concentrated in a centrifugal concentrator with a 100 kDa cutoff. Size-exclusion FPLC was then performed using a Superdex 200 10/30 column (GE Healthcare) and purified HA-8mer was again concentrated as before.
Recombinant HPV16 L1 (Abcam ab119880) and recombinant HBsAg AD (Abcam ab193473) were reconstituted following the manufacturer’s guidelines. Nanoparticle formation of the expected size was confirmed by dynamic light scattering.

Carbohydrate coupled Sepharose 6B matrix was prepared according to our previous study (24 (link)). In brief, epoxy-activated Sepharose 6B matrix (GE Healthcare, Sweden) was washed extensively with distilled water, and then mixed with four kinds of carbohydrates, including d-lactose, N-acetyl-d-glucosamine (d-GlcNAc), d-mannose, and l-fucose (Sigma-Aldrich, Buchs, Switzerland), at a final concentration of 200 µM in distilled water (pH 13.0). The mixtures were incubated at 30°C with gentle shaking for 16 h, and 1 M ethanolamine (pH 8.0) was used to block the remaining active groups after TBS washing. The carbohydrate coupled Sepharose 6B matrix was then washed extensively with TBS, and packed into Teflon columns with 100 mm long and 10 mm inner diameter, respectively.

Il10^-/- mice were weaned 21 days after birth and supplemented with a 5 mM solution of either 2-fucosyllactose (2FL), 3-fucosyllactose (3FL), 3-sialyllactose (3SL), 6-sialyllactose (6SL) (Glycom A/S, Hørsholm, Denmark) or D-lactose (Sigma-Aldrich, Buchs, Switzerland) in sterile filtrated water ad libitum for 28 days. For depletion of intestinal microbiota, 4–6 weeks old mice were supplied with 0.5 g/L vancomycin (AppliChem), 1 g/L ampicillin (VWR), 1 g/L neomycin (Fisher Bioreagents) and 0.2% aspartame (Sigma) in drinking water for 6 days. Once per day, mice received intragastric gavage of 250 μL of a 1 g/L metronidazole (Sigma) solution. After antibiotic treatment, mice were treated with 10% PEG 3000 solution (Sigma) in drinking water to flush the remaining bacteria and antibiotic. Neo-colonization with specific bacterial strains was done by intragastric gavage of 250 μL containing 10⁹ CFU of the respective strain in PBS. Fecal pellets were collected to monitor changes of the microbiota.