The ABH and Lewis histo-blood group antigens are carbohydrate epitopes present throughout many tissues of the human body (reviewed in reference 15
). The type 1 and 3 chain ABH histo-blood group antigens are present on mucosal epithelial cell surfaces and in salivary secretions, with variations in the carbohydrate milieu in different individuals based on their secretor status and blood type (Fig. ). Recent observations suggest that NV likely attaches to either H types 1 or 3 present on gastroduodenal epithelial cells (16
). To build upon these observations and to determine if other NLVs attach to ABH histo-blood group antigens, we examined whether NV, SMV, or HV VLPs attach to secreted ABH histo-blood group antigens in saliva. Human saliva samples were obtained and typed for the presence of ABH antigens in relation to blood type and secretor status by using antibodies to Lea
, A antigen, and B antigen (Ortho Clinical Diagnostics, Raritan, N.J.). All samples were from Lewis-positive individuals. Saliva samples were boiled for 5 min prior to binding analysis to denature any potential anti-NLV antibody. Plates were coated with boiled saliva at a 1:500 dilution in carbonate buffer (pH 9.6) for 4 h at ambient temperature and were blocked overnight in 5% milk in Tris-buffered saline (TBS). NV, SMV, or HV VLPs (0.1 μg) were then added to saliva-coated wells in 1% milk-Tween-TBS and were incubated for 1 h at 37°C. VLP binding was detected by using convalescent antiserum from NV-, SMV-, or HV-infected human volunteers and goat anti-human immunoglobulin G (IgG) alkaline phosphatase conjugate. Plates were developed with p
-nitrophenyl phosphate substrate (pNPP) (Sigma-FAST tablets; Sigma, St. Louis, Mo.), and the optical densities at 405 nm (OD405
) were determined.
FIG. 2. Binding of NLV VLPs to saliva components from individuals of different blood types and secretor status. (A) General schematic of the type 1 and 3 chain ABH histo-blood group antigen production pathways adapted from that described in reference 15. Synthetic (more ...)
NV VLPs bound efficiently to saliva components from secretor-positive (Se+
), blood type O, A, and AB individuals but not to saliva from secretor-negative (Se−
) individuals (Fig. ). The fact that NV VLPs bound poorly to saliva containing ABH antigens in blood group B individuals might also account for the observation that blood group B individuals are more resistant to NV challenge (10
). SMV VLPs bound to saliva from Se+
blood type B and AB individuals, suggesting that SMV and NV attach to different ABH histo-blood group carbohydrates. Dose-dependent binding of SMV VLPs based on B antigen expression was also evident (data not shown), suggesting that SMV utilizes B type 1, B type 3, or another downstream carbohydrate for attachment. HV VLPs did not bind to saliva components regardless of secretor phenotype or blood type, suggesting yet a third possible mechanism for NLV attachment. These data suggest that blood group and secretor status may be susceptibility alleles for some, but not all, NLV infections in humans.
To clearly identify which particular ABH histo-blood group antigens are involved in NLV attachment, a microwell-based assay was designed to biochemically detect and quantify levels of synthetic, biotinylated ABH histo-blood group carbohydrate binding to NLV VLPs. High-binding microwells were coated with NLV VLPs or sucrose-purified transmissible gastroenteritis virus virions. The coated wells were incubated with serially diluted, synthetic biotinylated ABH histo-blood group carbohydrates as described in the legend to Fig. . Carbohydrate binding was detected using a streptavidin-alkaline phosphatase conjugate and substrate.
FIG. 3. (A to G) Binding of synthetic ABH histo-blood group antigens with NLV VLPs. Purified NLV VLPs (100 μl at 2 μg/ml in TBS) were added to high-binding enzyme immunoassay plates (Costar, Corning, N.Y.). Sucrose-purified virions of the coronavirus (more ...)
Neither NV, SMV, nor HV VLPs bound synthetic, biotinylated H type 1 precursor (Fig. ), which lacks the FUT2-linked α1-2 fucose in H type 1. However, under identical conditions NV VLPs, but not SMV or HV VLPs, clearly bound H type 1 carbohydrate in a dose-dependent manner (Fig. ), supporting the notion that different NLVs utilize different pathways for docking and entry. NV VLPs also bound H type 3, although not as well as H type 1 (Fig. ). This interaction is specific, as NV VLPs did not bind the H type 3 precursor (Fig. ). SMV and HV VLPs did not detectably bind H type 3 or its precursor (Fig. ). Neither NV, SMV, nor HV VLPs detectably bound Lea
(Fig. ). However, NV VLPs clearly bound synthetic Leb
carbohydrate (Fig. ). Interestingly, this is the first suggestion that Leb
may function as a target for NV attachment. The A, B, and FUT3 enzymes compete for the common H type 1 substrate. Individuals of blood type O do not maintain active A or B enzyme and will likely retain more H type 1 substrate for subsequent FUT3 Lewis enzymatic activity. Therefore, the interaction of NV VLPs with Leb
, but not Lea
, agrees with the findings that saliva from secretor-positive blood type O individuals binds NV VLPs and that individuals of blood type O are most susceptible to NV infection (10
). Finally, we evaluated the ability of the VLPs to attach to H type 2, which is present on red blood cells but is not produced at the surfaces of the gastric mucosa or in salivary secretions. Consistent with the tissue distribution of H type 2, NV, SMV, and HV VLPs did not bind synthetic H type 2 carbohydrate (Fig. ).
To verify the carbohydrate binding results, the binding assays were repeated in microwells coated with 10 μg of each VLP stock/ml, which confirmed that the NV VLPs bound only H type 1, H type 3, and Leb (data not shown). Also, although the initial binding data suggest that HV might bind H type 1 at relatively high carbohydrate concentrations (Fig. ), neither HV nor SMV VLPs coated at 10 μg/ml could detectably bind any of the synthetic carbohydrates tested (data not shown).
To determine if the interaction of NV capsid proteins with H type 1 depends on the ability of the proteins to self-assemble into VLPs, the binding capacity of NV2 protein and heat-denatured VLPs was evaluated. The NV2 capsid construct contains three amino acid mutations from the wild type, which collectively ablate the ability of the protein to self-assemble into VLPs (3
). Neither NV2 nor denatured NV1 VLPs could detectably bind H type 1 (Fig. ). Also, with both the binding assay described above and an NV VLP competition-based assay we were unable to detect any interaction of H type 1 with a series of 15-mer overlapping peptides that span the entire NV capsid protein (data not shown), confirming that H type 1 binding is dependent on VLP assembly.