Characterization of Functional Components in Bovine Colostrum That Inhibit Norovirus Capsid Protruding Domains Interacting with HBGA Ligands

Abstract

Human noroviruses (huNoVs) cause epidemic acute gastroenteritis with significant mortality and morbidity worldwide. However, there are no commercial vaccines or antivirals against these important pathogens so far. In this study, we found that bovine colostrum (bCM) inhibited huNoV VLPs and their capsid-protruding (P) domains binding to histo-blood group antigens (HBGAs) that are huNoV receptor or attachment factors for infection, suggesting that bCM may function as a natural antiviral against huNoVs. We then characterized the bCM for the functional inhibition components by sequentially separating bCM into multiple fractions through various chromatography approaches, followed by determining their inhibitory abilities against huNoV receptor-binding P protein interacting with HBGAs. The protein components of bCM functional fractions were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Our data suggested that some milk proteins, likely in the form of glycoproteins, contribute to the observed blocking effects of bCM. Our findings lay an important foundation to further develop bCM into a potential natural antiviral against huNoVs.

Bovine natural antibody IgM inhibits the binding of human norovirus protruding domain to its HBGA receptors

“…The P‐GST fusion protein containing the receptor‐binding domain of the GII.4 HuNoV VA378 strain (GenBank access #: GenBank: AY038600.3) was made as described previously [ 21 , 22 ]. This protein has been shown to self‐assemble into large complexes with authentic binding ability to HBGA ligands [ 22 ] and thus suitable to be used in this study.…”

“…This assay was performed as previously described [ 21 ]. Briefly, saliva sample (OH39) with known HBGA types from our lab collection was positive for H type 1 (H1), H2, and Lewis y (Le y ), but negative for Le a and Le x antigens [ 23 ].…”

“…Two components of bCM sample 1 (bCM1) were analyzed by 2D PAGE as described previously [ 21 ]. Briefly, samples containing 0.5–10 μg proteins were loaded to immobilized pH gradient (IPG) strips (7 cm, pH 4–7 or pH 3–10).…”

“…After blocking with 5% nonfat milk in PBST, P‐GST, GST, or other indicated materials were added and incubated at 37 °C for 1 h. Corresponding primary and secondary antibodies were used to detect bound proteins. The OD 450 was measured as previously described [ 21 ].…”

“…Milk, especially bovine colostrum (bCM), is rich in antibodies and immune protective factors, such as immunoglobulins [ 15 ], lactoferrin [ 16 ], complement proteins, oligonucleotides, chitosan, lysozyme, and lactoperoxidase [ 17 ]; thus, bCM has been used to treat acute diarrhea and other gastrointestinal infections [ 18 , 19 , 20 ]. In a previous study [ 21 ], we showed that two components of a bCM sample 1 (bCM1), designated as component 1 and component 2, respectively, after progressive affinity chromatography (to remove IgG, anion exchange chromatography, and gel filtration chromatography, which contained an 84‐kDa protein [isoelectric point (PI) ~ 5.5], could inhibit GII.4 HuNoV P protein–HBGA interactions.…”