The VP8* sequences (amino acids 46-231) of a human RV P[11] strain N155 (GenBank accession: EU200796) were synthesized by GenScrip (GenScript USA Inc., Piscataway, NJ), and then sub-cloned into the expression vector PGEX-4T-1(GE Healthcare Life Sciences, Piscataway, NJ) between the BamH I and Sal I sites. After confirmation of the inserts through DNA sequencing, the GST-VP8* fusion protein was expressed in E. coli BL21 with induction of IPTG (0.4 mM) at room temperature (∼22°C) overnight. The GST-VP8* fusion protein was purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare Life Sciences, Piscataway, NJ) according to the manufacturer's protocol. Briefly, bacterial lysates were mixed with the sepharose beads for 2 h at room temperature to allow the GST fused VP8* proteins binding to the beads. After washing the beads with PBS for 4–6 times, the GST fusion VP8* proteins were eluted by glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-Cl, PH 8.0).

Written informed consent was obtained from the parents of the infants. Samples were collected from infants born in Cincinnati, Ohio under IRB-approved protocols (Cincinnati Children's Hospital Medical Center Institutional Review Board study# 2008-0463 and 2008-1131). Whole saliva was collected from preterm infants <33 weeks of age at day 8 of life who were enrolled in two hospital neonatal intensive care units (Novel Biomarkers Cohort). Collection was conducted early in the morning by trained clinical nurses. Two sterile cotton swabs were placed between the check and the gum and held in place until saturated. A similar procedure was used for collection of whole saliva from healthy, term infants enrolled in a longitudinal study of breastfeeding and infant health (GEHM Study, HD13021), at standardized postnatal intervals (weeks of life 2, 4, 13, 26, and 52). For the latter study, samples were collected by two trained research staff between 9 am and noon. Immediately after collection, samples were refrigerated, and then placed in cryogenic freezers in barcoded vials until tested. Infants from both studies were predominantly white, with approximately one-quarter African-American infants.

Saliva samples from adults and infants were tested for potential age-specific ligand in neonates for P[11] RVs by in vitro binding assays . Forty-eight saliva from adults, representing different ABO, secretor and Lewis types described in our previous studies, were included [5], [16]. Seventy-two saliva samples from neonates at day 8 after birth and 79 saliva samples from infants at weeks 4, 13, 26, and 52 after birth were studied. Saliva samples were boiled for 10 min to inactivate potential antibodies against RVs that could interfere with the binding assays. The samples then were diluted 1∶1,000 and coated on 96-well microtiter plates at 4°C overnight. After blocking with 5% nonfat cow milk, 100 µl of GST-VP8* proteins (10 µg/ml) were added and incubated at 37°C for 1 h. The bound GST-VP8* proteins were detected using a rabbit anti-GST serum at 1∶4000 dilution followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ICN, Aurora, OH). The signal intensities were displayed using the TMB kit according to manufacturer (BD Biosciences, San Diego, CA). In each step, the plates were incubated for 1 h at 37°C and washed five times with PBS-Tween 20 using a plate washer. The HBGA types of each saliva sample were determined by binding assay using monoclonal antibodies specific to H-1, A, B, Le^a, Le^b, Le^x and Le^y antigens as previously described [5]. The saliva samples were also tested for binding by lectin Lycopersicon esculentum (LEA) that recognize poly-LacNAc. The biotin-labeled LEA (Sigma-Aldrich Co., St. Louis, MO) in dilution of 1∶1000 was used, which was detected by HRP-conjugated-streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and displayed using the TMB kit (Kierkegaard and Perry Laboratory, Gaithersburg, MD).

The glycan array analysis was performed by the Protein-Glycan Interaction Core at the Emory University under the Consortium for Functional Glycomics. A printed glycan array (version 5.0) consists of 611 glycans in replicates of 6 was used to search for potential carbohydrate ligands for P[11] RVs (http://www.functionalglycomics.org/). The recombinant GST-VP8* proteins were applied to individual glycan arrays at two protein concentrations (20 and 200 µg/ml respectively), and the bound GST-VP8* proteins were detected by using a fluorescent-labeled anti-GST monoclonal antibody. Two sets of relative fluorescent units (RFU) of each glycan at the two concentration were calculated and used to rank the specificity of individual glycans (Table 1) for their interaction with P[11] VP8*.

Synthetic polyacrylamide polymer (PAA) conjugated oligomers of LacNAc units with variable lengths or fucosyl modifications (Table 2) were used to study their specificity as a ligand for P[11] RVs. Briefly, microtiter plates (Dynex Immulon; Dynatech, Franklin, MA) were coated with recombinant VP8* proteins (10 µg/ml) at 4°C overnight. After blocking with 5% nonfat cow milk, synthetic poly-LacNAc (PAA)-biotin conjugates (Table 2) obtained from the Consortium for Functional Glycomics or from GlycoTech Inc. (Gaithersburg, MD) were added at serial dilutions and incubated at 4°C overnight. The bound oligosaccharides were then detected using HRP-conjugated-streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and displayed using the TMB kit (Kierkegaard and Perry Laboratory, Gaithersburg, MD).

Two mutant cell lines (Lec2 and Lec8) of the Chinese hamster ovary (CHO) cells were studied for their presence (Lec2) or absence (Lec8) of poly-LacNAc on their surfaces [17]. The Lec2 and Lec8 mutant cells that were kindly provided by Dr. Pamela Stanley were grown in monolayer in α-MEM medium supplemented with ribonucleosides, deoxyribonucleosides (Gibco), 10% FBS, 100 unit/mL penicillin, 100 mg/mL streptomycin, and maintained at 37°C with 5% CO₂. The binding of VP8* to the cells was detected by immunofluorescent method. Briefly, the monolayer Lec2 and Lec8 cells were grown on poly-L-lysine treated coverslips to a 90% confluence, and then fixed with 4% paraformaldehyde. After blocking with 3% BSA, GST-VP8* fusion protein (100 µg/mL) was added. The binding signal of VP8* on the cell surface was detected using a rabbit anti-GST antibody followed by the FITC-conjugated goat anti-rabbit second antibody.

Two P[11] RVs, a human strain 116E and a bovine strain B223 [18], were studied for inhibition of replication in cell cultures by the oligosaccharide of PAA- TriLN and by human milk and saliva using procedures described previously [19]. Confluent MA-104 cell monolayers were grown on coverslips in 24-well plates. After rinse twice with serum-free DMEM, 500 µl of serum-free DMEM was added to each well and the plates were incubated at 37°C for 3 h. For blocking of infection, 300 fluorescent forming units (FFU)/10 µl of virus were treated with equal volume of serial dilutions of human milk, LEA positive neonate saliva or PAA-TriLN at room temperature for 30 min. An adult and a LEA negative neonate saliva which did not show bindings with P[11] VP8* were used as controls. Following chilling of all reagents and the 24-well plates on ice, duplicated wells were inoculated with the virus-oligosaccharide or virus-saliva/milk mixtures on ice with continuous rocker platform agitation for 1 h. The inoculum was then removed and the cells were washed twice with ice-cold serum-free DMEM. After replacement with 500 µl serum-free DMEM, the plates were warmed to 37°C and incubated in a 5% CO₂ incubator for 18 to 20 h prior to quantification of infected cells by immunofluorescence with a rabbit anti-rotavirus antibody followed by a FITC-labelled goat anti-rabbit secondary antibody.

Human red blood cells (RBC) of A₁, A₂, B and O type (Immucor, Inc., Norcross, GA) were washed twice in PBS before being tested for hemagglutination with VP8* or authentic RVs. An equal volume of 50 µL of 1% RBC were added to 50 µL of two-fold serial dilutions of either the GST-VP8* fusion proteins or virus on 96-well V-bottom plates. Agglutination was determined after 2 h of incubation at 4°C.

The correlation analysis was conducted using the Pearson analysis, and the binding signal difference of VP8* to neonate saliva between 2–13 weeks and 26–52 weeks was assessed by a two-tailed t test. P values of <0.05 were considered significant.

To explore potential age-specific receptors for P[11] RVs, we performed saliva binding assays using recombinant GST-VP8* fusion protein as described previously [11]. The yields of the fusion proteins were 5–10 mg/L of bacteria culture and high purity of the GST-VP8* protein was obtained for the binding assays (data not shown). Only marginal binding signals (OD<0.35) were detected among the 48 well-characterized adult saliva samples (Fig. 1A). However, strong binding signals were observed when the two sets of neonate/infant saliva samples were tested (60.3% ODs>0.35, N = 151). High binding signals showed no correlation with the ABO, secretor and Lewis types (data not shown). The first set of infant saliva were collected earlier in life (day 8 after birth) and a higher rate (68.1%) of the samples showed a binding signal above 0.35 (Fig. 1B). The second set of infant saliva was collected from a wide age range (weeks 2, 4, 13, 26 and 52 after birth) and an average 53.2% of positive binding (OD>0.35) was observed. Moreover, the binding of P[11] VP8* with the saliva showed a significantly negative correlation with the age of infants (P<0.01). While most of the positive binding occurred in the early development stages (weeks 2, 4 and 13), only sporadic binding was found in saliva collected from infants at weeks 26 and 52 (Fig. 1C).

To determine the ligand for P[11] VP8* in the infant saliva, we performed an array assay consisted of 611 oligosaccharide glycans, and similar binding patterns of the P[11] GST-VP8* fusion protein at both 20 and 200 µg/ml were observed (Fig. 2A–B). A group of glycans with a common feature, linear or branched polymers of the LacNAc disaccharide unit in variable lengths, ranked the highest (Table 1). Strong binding always occurred to the poly-LacNAc oligosaccharides without further terminal modifications. The fucose (α1–2) linked to the terminal Gal of poly-LacNAc did not affect the binding (Table 1). LacNAc polymers containing other terminal modifications, such the sialic acid (Neu5Ac) or GalNAc adding to the terminal galactose resulted in reduction of the binding. LacNAc is a precursor of human HBGAs that express abundantly in neonates and young children. Thus, the glycans of LacNAc polymers could be an age-specific ligand available to P[11] RVs before the maturation of HBGAs of neonates.

Results of the oligosaccharide-based binding assay exhibited a dose-dependent binding to the LacNAc disaccharides, further confirming that LacNAc is a basic unit recognized by P[11] RVs. It was noted that the binding signals of P[11] VP8* increased significantly with the numbers of LacNAc repeats, in which the three repeats (TriLN-PAA) showed the highest signals followed by the two repeats (DiLN-PAA) and then the single copy (LacNAc-PAA). No significant difference of P[11] binding to LacNAc polymers terminated with either a Gal or a GlcNAc residue was observed. A fucose (α1–2) linked to the terminal Gal did not affect the P[11] VP8* binding but a fucose (α1–3) linked to the GlcNAc of a poly-LacNAc totally abolished the binding (Fig. 3).

To further study the role of LacNAc in P[11] RV host range, we performed binding assays of the 151neonate/infant saliva samples with lectin Lycopersicon esculentum (LEA)that recognizes but is not limited to the poly-LacNAc. Interestingly, none of the 48 adult saliva samples demonstrated any binding (Fig. 1A). However, a strong correlation of binding of P[11] with that of the LEA was observed for the neonate/infant saliva (P<0.01, Fig. 1B and C), indicating that it is poly-LacNAc in the neonate/infant saliva responsible for the age-specific binding of P[11]VP8*.

Two Chinese hamster ovary (CHO) cell lines, the Lec2 and Lec8 cells, were chosen for their well-characterized cell surface glycomics to explore the specific binding for P[11] VP8*. The Lec2 cells have an inactive cytidine monophosphate (CMP)-sialic acid Golgi transporter which resulted in a cell surface complex/hybrid N-glycans mainly consist of poly-LacNAc oligosaccharides on the cell surfaces, while the Lec8 cells do not express LacNAc because of a mutant UDP-Gal Golgi transporter gene [17]. Using immune fluorescence staining methods, the P[11] VP8* displayed specific binding to the Lec2 cells but not to the Lec8 cells (Fig. 4A), indicating that P[11] VP8* was selectively attached to the cell-surface poly-LacNAc of the Lec2 cells.

Hemagglutination assays of P[11] VP8* as well as two authentic P[11] RVs (116E and B223) was conducted. A strong agglutination of human RBCs was obtained for both the P[11] VP8* protein and the authentic P[11] RVs, irrespective of the ABO phenotypes, while the VP8* of a P[9] RV, a known type A antigen binder, agglutinated the type A human RBCs only (Fig. 4B). The observed unique hemagglutinating property of P[11] RVs was consistent with the ubiquitous distribution of the poly-LacNAc relevant antigens (group i and I antigens) found on the surface of human erythrocyte cells [20].

To examine the biologic relevance of P[11]VP8* binding to poly-LacNAc, viral replication inhibition assays were performed. The replications of P[11] RV 116E were significantly reduced following incubation of the viruses with a LEA positive neonate saliva (#23) but not with a LEA negative neonate saliva (#45) or with an adult saliva (OH54). A dose-dependent abrogation with up to 76.4% reduction of 116E infectivity by the LEA positive neonate saliva was observed compared to the LEA negative neonate saliva. Moreover the LEA positive saliva (#23) did not inhibit the A-antigen binding RV (Arg720) (Fig. 5A, B). The involvement of poly-LacNAc in RV infection was further demonstrated by a dose-dependent inhibition of 116E incubated with LacNAc polymers (TriLN-PAA) but not with the control oligosaccharide Tri-Lex-PAA (Fig. 5C). Boiled human milk also blocked 116E replication although reductions were also observed for the P[9] Arg720 strain (Fig. 5D).