Hemagglutination by Binding to H Histo-Blood Group Antigens
Departments of Molecular Virology and Microbiology,1 Medicine,2 Immunology, Baylor College of Medicine, Houston, Texas 7703038xm, http://www.100md.com
Received 5 July 2002/ Accepted 24 September 20028xm, http://www.100md.com
ABSTRACT8xm, http://www.100md.com
Noroviruses are a major cause of epidemic acute nonbacterial gastroenteritis worldwide. Here we report our discovery that recombinant Norwalk virus virus-like particles (rNV VLPs) agglutinate red blood cells (RBCs). Since histo-blood group antigens are expressed on gut mucosa as well as RBCs, we used rNV VLP hemagglutination (HA) as a model system for studying NV attachment to cells in order to help identify a potential NV receptor(s). rNV VLP HA is dependent on low temperature (4°C) and acidic pH. Of the 13 species of RBCs tested, rNV VLPs hemagglutinated only chimpanzee and human RBCs. The rNV VLPs hemagglutinated all human type O (11 of 11), A (9 of 9), and AB (4 of 4) RBCs; however, few human type B RBC samples (4 of 14) were hemagglutinated. HA with periodate- and neuraminidase-treated RBCs indicated that rNV VLP binding was carbohydrate dependent and did not require sialic acid. The rNV VLPs did not hemagglutinate Bombay RBCs (zero of seven) that lack H type 2 antigen, and an anti-H type 2 antibody inhibited rNV VLP HA of human type O RBCs. These data indicated that the H type 2 antigen functions as the rNV VLP HA receptor on human type O RBCs. The rNV VLP HA was also inhibited by rNV VLP-specific monoclonal antibody 8812, an antibody that inhibits VLP binding to Caco-2 cells. Convalescent-phase sera from NV-infected individuals showed increased rNV VLP HA inhibition titers compared to prechallenge sera. In carbohydrate binding assays, the rNV VLPs bound to synthetic Lewis d (Led), Leb, H type 2, and Ley antigens, and these antigens also inhibited rNV VLP HA of human type O RBCs. Overall, our results indicate that carbohydrate antigens in the gut are a previously unrecognized factor in NV pathogenesis.
INTRODUCTIONjj3|k, 百拇医药
Norwalk virus (NV) was first isolated from an outbreak of winter vomiting disease, or acute gastroenteritis, at an elementary school in Norwalk, Ohio, in 1968 (1). Subsequent adult volunteer challenge studies with infectious stool filtrate from the Norwalk outbreak demonstrated that NV readily infects susceptible people, causing an acute illness associated with diarrhea, vomiting, myalgia, nausea, and fever within 15 to 24 h after exposure and lasting 24 to 72 h (16). Although these initial studies demonstrated that most people are highly susceptible to NV, presently no cell culture systems or animal models of infection exist, which limits the study of NV replication and pathogenesis.jj3|k, 百拇医药
NV is the prototype nonenveloped, positive-stranded RNA human virus in the genus Norovirus of the family Caliciviridae. Noroviruses are a major cause of acute gastroenteritis throughout the world and cause virtually all outbreaks of nonbacterial gastroenteritis in adults in the United States (18, 39). Furthermore, there are an increasing number of reports of gastroenteritis cases and outbreaks in children and the elderly caused by noroviruses, in part due to increased surveillance and better detection methods (15, 26, 39).
NV virions are detected in low numbers in infected stool. The virions are 27 to 38 nm in diameter, including 4.5-nm radial protrusions extending from the capsid shell that create the calix, or cup-like structures, apparent by electron microscopy (33, 44). The NV capsid proteins (open reading frames 2 and 3, which produce the structural viral proteins VP1 and VP2, respectively) spontaneously self-assemble into virus-like particles (VLPs) when synthesized in a recombinant baculovirus expression system. These recombinant NV VLPs (rNV VLPs) are structurally and antigenically similar to the capsids of native NV and are useful in modeling virus-cell interactions (27, 53).'f, 百拇医药
Hemagglutination (HA) is one method that has been helpful in identifying cell-binding receptors for many viruses, such as influenza A virus and parvovirus B19 (7, 45). The VLPs from human parvovirus B19, JC human polyomavirus, and SA11 simian rotavirus have HA properties that are similar to those of their virions (8, 14, 42).
This is the first report of HA by VLPs from a human calicivirus. Our data demonstrate that the H type 2 histo-blood group antigen is the rNV VLP HA receptor on human type O red blood cells (RBCs) and that the rNV VLPs also bind to synthetic H and structurally related Lewis carbohydrate antigens.kh9v, 百拇医药
MATERIALS AND METHODSkh9v, 百拇医药
rNV VLP purification. rNV VLPs were synthesized and purified by using methods described previously (53). Briefly, spinner flasks containing 3.5 x 106 Sf9 insect cells per 200 ml of Grace's insect cell medium were infected with pVL-NV ORF(2 + 3) recombinant baculovirus at a multiplicity of infection of 5. At 7 days postinfection the rNV VLPs were harvested from supernatants of spinner flask cultures. Cells were pelleted and discarded. The VLPs in the supernatant were pelleted by ultracentrifugation through a 30% sucrose cushion made with 0.2 M phosphate-buffered saline (PBS) for VLPs (PBS-V) (0.2 M sodium phosphate, 0.1 M NaCl, pH 6.0) in an SW28 rotor for 3 h at 120,000 x g and 4°C. The pellets were suspended in 0.39 g of CsCl (Invitrogen, Grand Island, N.Y.) per ml of PBS-V and subjected to isopycnic gradient centrifugation in an SW55 rotor for 18 to 24 h at 150,000 x g and 4°C. The visible band of rNV VLPs in the CsCl gradient was collected by micropipetting. CsCl was removed by diluting the VLPs in PBS-V and sedimenting by ultracentrifugation in an SW28 rotor for 3 h at 120,000 x g and 4°C. The VLP pellets were suspended in PBS-V containing protease inhibitors (aprotinin, leupeptin, and pepstatin [Sigma, St. Louis, Mo.] at 1 µg/ml each) and stored at 4°C. The rNV VLP protein concentrations were determined by the DC protein assay (Bio-Rad, Hercules, Calif.) microtiter plate method without detergent, and the rNV VLP structural integrity was ascertained by electron microscopic inspection of negatively stained (1.0% ammonium molybdate [Sigma], pH 5.0) VLPs on carbon-coated grids (Ted Pella, Reading, Calif.).
HA assay. The HA assay methods were based on those described by the Centers for Disease Control and Prevention (9). Whole blood was collected in 2 volumes of Alsever's solution (2.05% [wt/vol] glucose [Sigma], 0.8% sodium citrate [Fisher, Fair Lawn, N.J.], 0.055% citric acid [Fisher], 0.42% NaCl [Fisher], pH 6.1) and stored at 4°C. Individual human RBC samples were graciously provided by volunteer donors at the Baylor College of Medicine's Influenza Research Center and the Methodist Hospital Blood Donor Center (Houston, Tex.). Pooled human RBC reagents from at least five donors were purchased from Immucor (Atlanta, Ga.). Human cord RBC samples were kindly provided by the Children's Nutrition Research Center's Leukocyte Biology Laboratory (Houston, Tex.). Human RBC samples with rare phenotypes were generously donated by Immucor/Gamma Biologicals (Houston, Tex.). Chimpanzee, baboon, spider monkey, and rhesus RBC samples were graciously provided by the Southwest Foundation for Biomedical Research (San Antonio, Tex.). Chicken, guinea pig, canine, and feline blood samples were provided by the Center for Comparative Medicine at Baylor College of Medicine (Houston, Tex.). Murine RBC samples were obtained during routine blood draws from normal control BALB/c mice. Bovine, porcine, and lapine blood samples were purchased from Lampire (Pipersville, Pa.). The RBCs were packed by diluting the cells in 0.01 M PBS (without Ca2+ or Mg2+; pH 7.2) (Invitrogen, Carlsbad, Calif.) and centrifuging for 15 min at 500 x g. The HA activity of rNV VLPs was tested in untreated 96-well V-bottom plates (Nunc, Naperville, Ill.). Equal volumes (50 µl each) of 0.5% packed human RBCs in 0.85% saline (pH 6.2) and rNV VLPs serially diluted in 0.01 M PBS for HA (PBS-H) (0.01 M sodium phosphate [EM Science, Gibbstown, N.J.], 0.15 M NaCl, pH 5.5; filtered with a 0.2-µm-pore-size filter) were combined at 4°C. The plates were covered with adhesive film (Excel Scientific, Wrightwood, Calif.) and incubated for 75 to 120 min in a cold room (at 4 to 8°C), unless otherwise indicated. Incubations at other temperatures were performed at room temperature (22 to 25°C) or in a tissue culture incubator (36 to 37°C). The HA titer was the reciprocal of the greatest rNV VLP dilution that did not allow sedimentation of the RBCs compared to negative control wells that contained only buffer. The positive control for HA was an influenza A virus (H1N1, A/Texas/36/91), grown in eggs, for which the titer had been previously determined. For each animal RBC sample, the percentage of packed RBCs in saline used for each HA assay was determined by the minimum concentration of RBCs able to sediment in negative control wells (PBS-H) within 6 h of incubation at 4°C.
Periodate treatment of RBCs. Human type O RBCs were treated with freshly prepared 10.0 mM KIO4 (Sigma) for 20 min at room temperature and then rinsed and packed as described above. To test the RBCs' ability to be agglutinated, 40 µl of treated or control 2.0% RBCs was mixed with 10 µl of rNV VLPs (0.1 mg/ml), anti-D or anti-H monoclonal antibodies (MAbs) (Immucor) (0.1 mg/ml), or PBS-H on an ice-cold glass plate and incubated for 10 min at 4°C. The degree of HA was determined by visual comparison to controls and verified by an independent observer.|\1, http://www.100md.com
Enzyme treatment of RBCs. All enzyme reactions were performed with 50 µl of packed RBCs in a total enzyme reaction volume of 125 µl. Control reaction mixtures were incubated without enzyme. Neuraminidase from Arthrobacter ureafaciens (6 µU; Sigma) in Dulbecco's PBS (D-PBS) (Invitrogen) was combined with the RBCs and incubated for 60 min at 37°C. L-(Toslylamido-2-phenyl)ethyl chloromethyl ketone-treated trypsin (1.25 ng; Sigma) in D-PBS was added to the RBCs and incubated for 30 min at 37°C. Trypsin activity was stopped by adding 0.5 µg of soybean trypsin inhibitor (Sigma) to the reaction mixture. After enzyme treatment, the RBCs were rinsed three times with 6 ml of PBS and packed by centrifugation as described above. After removal of the PBS supernatant, the packed RBCs were diluted with cold 0.85% saline (pH 6.2) to make a 0.5% RBC suspension for HA assays.
Enzyme-linked immunosorbent assay (ELISA)-based carbohydrate microtiter plate assay (CMA). Multivalent carbohydrate-biotin reagents conjugated to polyacrylamide (CHO-PAA-biotin; Glycotech, Rockville, Md.) were immobilized onto streptavidin-coated plates as suggested by the manufacturer, with all reagents added in volumes of 100 µl/well, unless otherwise specified. Briefly, PVC microtiter flat-bottom plates (Dynex Technologies, Chantilly, Va.) were coated with 10 µg of streptavidin (Sigma) per ml in PBS at 4°C overnight, blocked by addition of 1% fatty acid-free bovine serum albumin (faf-BSA) (Sigma) in PBS at room temperature for 4 h, and then rinsed three times with 200 µl of PBS per well. Next, 0.1 µg of biotin per ml or 10 µg of CHO-PAA-biotin conjugate per ml in PBS with 0.25% faf-BSA was incubated at room temperature for 1 h and rinsed as described above. The rNV VLP-carbohydrate binding was optimized at 10 µg of rNV VLPs per ml in PBS (pH 7.5) with 0.25% faf-BSA and incubation at room temperature for 1 h before rinsing as described above. rNV VLPs bound to carbohydrate were detected by incubation with rabbit anti-rNV VLP serum (1:5,000) in PBS with 0.25% faf-BSA for 1 h at room temperature; rinsing as described above; addition of goat anti-rabbit immunoglobulin A (IgA), IgM, and IgG conjugated to horseradish peroxidase (ICN Biomedicals, Philadelphia, Pa.) (1:50,000) in PBS with 0.25% faf-BSA; incubation for 1 h at room temperature; and rinsing. Plates were developed with the TMB microwell peroxidase endpoint assay (Kirkegaard & Perry Laboratories, Gaithersburg, Md.), and absorbance was read at 450 nm (Spectromax 190; Molecular Devices, Sunnyvale, Calif.). Carbohydrate competition assays for rNV VLP binding to the microtiter plate-immobilized CHO-PAA-biotin were performed by preincubating the rNV VLPs with CHO-PAA reagents (without biotin) in PBS (pH 7.2) for 30 min at 4°C before addition to the microtiter plates prepared as described above. The plates were incubated for 1 h at 4°C and then rinsed and detected as described above.
HI assay. Inhibition of rNV VLP HA was examined by using the multivalent CHO-PAA-biotin conjugates, human pre- and post-NV challenge sera (24), mouse hyperimmune serum IgG, and anti-rNV VLP MAbs (8812, 3901, and 3912) (30). The human sera were heat inactivated and kaolin treated for the HA inhibition (HI) assays as previously described (9). Mouse IgG from hyperimmune sera and monoclonal hybridoma ascites were purified by using UltraLink protein G columns (Pierce, Rockford, Ill.) according to the manufacturer's protocol and stored at 4°C in PBS with 0.02% NaN3. The HI activities of the carbohydrate conjugates, the treated human sera, and the mouse IgG were tested by serial dilutions of the inhibitors (starting concentrations of 100 µg/ml, 1:10, and 50 µg/ml, respectively) in a volume of 25 µl of PBS-H across 96-well V-bottom plates. An amount of rNV VLPs equal to 4 HA units (HAU) (1 HAU was defined as the amount of antigen present at the HA endpoint dilution) in a volume of 25 µl of PBS-H was added to each well, and the inhibitor was incubated with the rNV VLPs for 30 min at 4°C. (For the mouse IgG HI assays, the PBS-H contained 0.3% BSA fraction V [Calbiochem, San Diego, Calif.].) Next, 50 µl of 0.5% human type O RBCs in 0.85% saline was added to each well, and the plate was incubated as described above. Each inhibitor tested was negative for nonspecific HA of the RBCs at the highest concentration used in the assay. The HI titer was read as the last dilution of inhibitor that prevented HA of the RBCs by rNV VLPs.
RESULTScatx!r', http://www.100md.com
HA by rNV VLPs is temperature and pH dependent. The rNV VLP HA of RBCs was tested at 4, 25, and 37°C in PBS-H (pH 7.2). The rNV VLPs were able to hemagglutinate human type O-positive RBCs at 4°C but not at 25 or 37°C (Fig. 1A). Microscopic inspection of the human type O-positive RBCs and HA of the RBCs by influenza A virus at all three temperatures demonstrated that the cells were intact at these temperatures. The HA of human type O-positive RBCs by rNV VLPs at 4°C was lost within 30 min when plates were warmed to room temperature. The loss of HA at room temperature was reversible. The HA titer was restored to a level equal to or up to fourfold lower than the initially observed titer when the plates were first cooled to 4°C, shaken to suspend the RBCs, and then incubated a second time at 4°C (data not shown). Because several enteric viruses have HA titers that are optimal at pHs of below 7.2 (9), we evaluated the effect of pH on rNV VLP HA of type O-positive RBCs. With pH increments of 0.5, the rNV VLPs hemagglutinated human type O-positive RBCs when diluted in PBS-H at pH 5.0 to 7.0 but not at pH 7.5 and 8.0 (Fig. 1B). HA by influenza A virus was pH independent between pH 5.0 and 8.0. Upon further examination of HA at pHs of between 7.0 and 7.5, rNV VLP HA was observed at pH 7.0 to 7.2 but was lost at pH 7.3 to 7.5 (data not shown). Therefore, for rNV VLP HA of human type O-positive RBCs, an incubation temperature of 4 to 8°C and a pH of between 5.0 and 7.2 were required. At the chosen conditions, i.e., incubation at 4°C and pH 5.5, an HA endpoint of 0.625 ng of rNV VLPs (3.6 x 107 particles) was regularly able to hemagglutinate approximately 3 x 106 human type O-positive RBCs. Thus, to further characterize the HA properties of rNV VLPs, we made our standard HA assay with 400 ng of rNV VLPs per ml as the initial VLP concentration that was serially diluted in PBS-H at pH 5.5 and incubated for 75 to 90 min at 4°C, unless otherwise noted.
fig.ommitteedps:pu-, 百拇医药
FIG. 1. HA titers of human type O RBCs by rNV VLPs. Fifty microliters of rNV VLPs (400 ng/ml) influenza A/TX/36/91 virus grown in eggs , or PBS-H was serially diluted in PBS-H and mixed with 50 µl of 0.5% RBCs. (A) The PBS-H was at pH 7.2, and the HA assay incubation times, as determined by sedimentation of RBCs in negative control wells, were 30 min at 37°C, 40 min at 25°C, and 90 min at 4°C. (B) The pH of the PBS-H ranged from 5.0 to 8.0 as indicated, and the HA assay incubation time was 90 min at 4°C.ps:pu-, 百拇医药
rNV VLPs hemagglutinate chimpanzee RBCs. The ability of a virus to agglutinate RBCs depends on the expression of the HA receptor on the cells, and this expression may be limited to a subset of animal species RBCs. Therefore, we tested RBCs from different species to gain insight into the nature and expression pattern of the rNV VLP HA receptor. Additionally, if the rNV VLP HA was a biologically relevant interaction, we hypothesized that the narrow host restriction of NV infection, i.e., to most humans and some chimpanzees (24, 43, 54), would be reflected in the ability of the VLPs to agglutinate RBCs from susceptible species. A panel of RBC samples from 12 nonhuman animal species was chosen based on their susceptibility to other caliciviruses, evolutionary relatedness to humans, availability, and use in other viral HA assays. Of these RBC samples, only the chimpanzee RBCs (0.5%) were hemagglutinated by the rNV VLPs (Fig. 2A). This sample of chimpanzee RBCs also reacted with an anti-A histo-blood group typing reagent (Immucor), indicating the presence of A or A-like histo-blood group antigens on the chimpanzee RBCs (data not shown) (3). The HA titer for the chimpanzee RBCs was 256, similar to the high HA titers exhibited by several human type A RBC samples (Fig. 2B). The baboon (0.5%), spider monkey (0.5%), rhesus macaque (1.5%), chicken (0.5%), guinea pig (1.0%), murine (1.0%), canine (1.0%), bovine (1.0%), porcine (1.0%), feline (1.5%), and lapine (2.0%) RBC samples were not hemagglutinated by 400 ng of rNV VLPs per ml.
fig.ommitteedn7#42, 百拇医药
FIG. 2. HA titers of individual RBC samples by rNV VLPs. Fifty microliters of rNV VLPs (400 ng/ml) serially diluted in PBS-H (pH 5.5) were combined with 50 µl of RBC samples (0.5 to 2.0%) from the indicated 12 animal species (A) and adult human RBC samples (0.5%) (separated by their ABO phenotypes) (B). The RBC concentrations used in HA assays were determined by sedimentation of RBCs in PBS-H (pH 5.5) negative control wells.n7#42, 百拇医药
rNV VLPs hemagglutinate adult human type O, A (A1 and A2), and AB RBCs and a subset of adult human type B RBCs. Many individual RBC samples and pooled human RBC reagent samples were tested for rNV VLP HA to determine the frequency of expression of the rNV VLP HA receptor on human RBCs. For convenience, the human RBC samples were grouped by their ABO and Rh phenotypes. The rNV VLPs hemagglutinated all adult human type O, A, and AB individual RBC samples tested (11, 9, and 4 individual samples, respectively) (Fig. 2B). However, only 4 of the 14 type B individual human RBC samples tested were hemagglutinated by 400 ng of rNV VLPs per ml (Fig. 2B). In HA assays starting at a much higher concentration of rNV VLPs (100 µg/ml), an additional six of the individual human type B RBC samples were hemagglutinated by the VLPs (data not shown). For all human RBC samples tested, the Rh factor did not affect rNV VLP HA of human RBCs. Additional rNV VLP HA assays were performed on pooled human A1, A2, B, and O RBC reagents. In agreement with our individual RBC sample data, 400 ng of rNV VLPs per ml hemagglutinated the pooled type A1, A2, and O RBCs but not the pooled type B RBCs (Table 1).
fig.ommitteedin;n, 百拇医药
TABLE 1. HA titers of untreated and neuraminidase-treated pooled human RBCs by rNV VLPsin;n, 百拇医药
rNV VLP binding to RBCs is carbohydrate dependent. Because the B antigen is a carbohydrate moiety, we tested the carbohydrate dependence of rNV VLP-RBC binding by periodate treatment, which oxidizes all carbohydrates on the cell surface. After periodate treatment of human type O-positive RBCs, HA by rNV VLPs and by anti-H, a carbohydrate-dependent antibody (Immucor), was lost (Table 2). This same treatment did not reduce anti-D HA, which recognizes an Rh factor protein on RBCs. Furthermore, trypsin treatment of type O RBCs did not affect the rNV VLP HA titer (data not shown), indicating that rNV VLP HA was not dependent upon a trypsin-sensitive protein on the RBC surface.in;n, 百拇医药
fig.ommitteedin;n, 百拇医药
TABLE 2. Agglutination of periodate-treated human type O+ RBCs by rNV VLPsin;n, 百拇医药
Two cell surface carbohydrates to which viruses commonly bind are sialic acid and heparan sulfate. To test if the carbohydrate-dependent rNV VLP HA required sialic acid, pooled human RBC reagents were enzymatically treated with neuraminidase from A. ureafaciens, which removes both {alpha} -2,3 and {alpha} -2,6 linked sialic acid residues on the cell surface. This neuraminidase treatment of pooled human type O, A1, and A2 RBCs did not affect their rNV VLP HA titer (Table 1), whereas the sialic acid-dependent HA by rhesus rotavirus was lost. Thus, rNV VLP HA of human type O, A1, and A2 RBCs was sialic acid independent. Additionally, pretreating the rNV VLPs with heparan sulfate before performing the HA assay did not inhibit the VLPs' ability to hemagglutinate human type O RBCs (data not shown). Therefore, the rNV VLP binding was dependent upon a carbohydrate, and unlike many other viruses, this carbohydrate did not involve sialic acid or heparan sulfate.
An interesting observation was made when the pooled human type B RBCs were treated with neuraminidase and tested for rNV VLP HA (Table 1). As seen with the majority of individual type B RBC samples, the untreated pooled type B RBCs were not hemagglutinated by 400 ng of rNV VLPs per ml. However, after neuraminidase treatment, when the sialic acid-dependent rotavirus HA titer was abolished, the rNV VLPs were able to hemagglutinate the type B RBCs.-d#6};, http://www.100md.com
The rNV VLP HA is dependent upon H antigen. Individuals with the rare Bombay (Oh) blood type do not express the H histo-blood group antigen on their RBCs (35). Since the H antigen is the precursor substrate for A and B antigen expression, RBCs from Bombay-type individuals will appear to have an O phenotype regardless of their ABO genotype (35). Bombay-type individuals with an A or B and a secretor (Se+) genotype are A- or B-para-Bombay, respectively, where the A or B antigens may be expressed on additional carbohydrate substrates not normally expressed by RBCs (35). None of the five Bombay- or the two para-Bombay-type RBC samples assayed was hemagglutinated by 400 ng of rNV VLPs per ml (Fig. 3A). In fact, even 100 µg of rNV VLPs per ml did not hemagglutinate the seven Bombay-type RBC samples (data not shown). Furthermore, when monoclonal antibody BRIC 231 (Biogenesis, Poole, United Kingdom), which specifically recognizes the H type 2 antigen, was preincubated with human type O RBCs, the rNV VLPs did not hemagglutinate the RBCs (data not shown). These data strongly suggest that rNV VLP HA is dependent upon H antigen expression on human RBCs.
fig.ommitteed7?4vq, 百拇医药
FIG. 3. HA titers of rare-phenotype human RBC samples (A) and human umbilical cord blood samples (B) by rNV VLPs. Fifty microliters of rNV VLPs (400 ng/ml) or UEA-1 lectin (100 µg/ml) was serially diluted in PBS-H (pH 5.5) and combined with 50 µl of 0.5% unbranched (i/i), Bombay (Oh), para-Bombay (AOh and BOh), or normal adult type O RBC samples (A) and with 50 µl of 0.5% umbilical cord RBCs in (B).7?4vq, 百拇医药
The rNV VLP HA receptor may be a developmental antigen. Many RBC antigens, including the ABH antigens, are not expressed or are expressed at low levels on fetal and neonatal RBCs (37, 51). Four human umbilical cord RBC samples (neonatal RBCs) were obtained from full-term births and were tested for HA by rNV VLPs (Fig. 3B). One type A cord RBC sample showed an HA titer similar to that of some adult type A RBCs. The three additional cord blood samples (a second type A sample, a type O sample, and a type B sample) were not hemagglutinated by 400 ng of rNV VLPs per ml.
One developmental change in human RBC antigens is the increase in carbohydrate branching. Fetal RBCs have no branched carbohydrates (phenotype i), and adult levels of branching are attained by 2 years of age (phenotype I) (20, 52). RBCs from adults lacking the {alpha} -1,6 galactosyltransferase carbohydrate branching enzyme resemble fetal RBCs in their lack of branched-carbohydrate expression (4). The rNV VLP HA did not depend on expression of branched carbohydrates (Fig. 3A). Two type O (i/i) RBC samples were hemagglutinated by 400 ng of rNV VLPs per ml to an HA titer of 128, similar to the HA titer of normal type O RBCs.28i5&, 百拇医药
rNV VLP HA of human type O RBCs is inhibited by mouse MAb 8812. The previously characterized rNV VLP-specific MAbs 8812, 3901, and 3912 were tested for their ability to inhibit rNV VLP HA (30). Also, IgG purified from mouse anti-rNV VLP hyperimmune serum was tested for rNV VLP HI. Only MAb 8812 inhibited rNV VLP HA of human type O RBCs (Table 3).28i5&, 百拇医药
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TABLE 3. HI titers of mouse anti-rNV VLP antibodies}ow%fut, 百拇医药
Convalescent-phase sera from NV-infected volunteers show an increase in rNV VLP HI titer. Prechallenge and convalescent-phase sera from seven NV-challenged volunteers were heat inactivated and kaolin treated to remove nonspecific hemagglutinating factors (9, 24). The treated sera were diluted (1:10) and had no nonspecific HA activity and lower HI titers than untreated sera. Increased HI titers were found in the convalescent-phase sera of NV-infected volunteers (n = 5; volunteers 1 to 5) compared to their prechallenge sera, while the HI titers did not increase in convalescent-phase sera from noninfected NV-challenged volunteers (n = 2; volunteers 6 and 7) (Table 4) (24).}ow%fut, 百拇医药
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TABLE 4. HI titers of preimmune and convalescent-phase sera from volunteers given NVa}ow%fut, 百拇医药
The rNV VLPs specifically bind to synthetic H and Lewis carbohydrate antigens. Based on the data that the H antigen may be the HA receptor for the rNV VLPs on human type O RBCs, the VLPs were tested for their ability to bind synthetic H and the structurally related Lewis carbohydrate reagents (Table 5). These synthetic H and Lewis antigens were chosen based on their relatedness to the H type 2 antigen found on human type O RBCs, their expression on gut mucosa, and their availability. When the rNV VLPs were incubated on carbohydrate-coated microtiter plates in the ELISA-based CMA in PBS (pH 6.0) at room temperature, the VLPs bound to the following carbohydrates, starting with the greatest rNV-specific detection: Lewis d (Led), Leb, H type 2, Ley, and H disaccharide (Fig. 4A). In HI assays, the same carbohydrates (CHO-PAA-biotin reagents) inhibited rNV VLP binding to human type O RBCs (Fig. 4B). The relative strength of detection of rNV VLPs bound to the immobilized carbohydrates in the ELISA-based CMA correlated with the relative magnitude of HI titers. Furthermore, when the free synthetic carbohydrates Led (H type 1), H type 2, and Lea were preincubated with the rNV VLPs before their addition to the ELISA-based CMA, Led and H type 2, but not Lea, inhibited rNV VLP binding to both the immobilized Led and H type 2 carbohydrates (Fig. 5).}ow%fut, 百拇医药
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TABLE 5. Structures of carbohydrates and rNV VLP binding}ow%fut, 百拇医药
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FIG. 4. rNV VLPs bind synthetic H and Lewis antigens. (A) Carbohydrate binding of rNV VLPs to synthetic H and Lewis antigens determined by ELISA-CMA. Multivalent CHO-PAA-biotin reagents were immobilized on microtiter plate wells and incubated with rNV VLPs in PBS (pH 7.0) at 25°C for 1 h. Bound rNV VLPs were detected with anti-rNV-specific rabbit serum, horseradish peroxidase-conjugated secondary antibody, TMB colorimetric development, and spectrophotometric absorption at 450 nm. Error bars indicate standard deviations. (B) HI titers of serially diluted multivalent CHO-PAA-biotin reagent (0.10 mg/ml) with 4 HAU of rNV VLPs and 50 µl of 0.5% human type O RBCs.(Anne M. Hutson Robert L. Atmar Donald M. Marcus and Mary K. Estes)
Received 5 July 2002/ Accepted 24 September 20028xm, http://www.100md.com
ABSTRACT8xm, http://www.100md.com
Noroviruses are a major cause of epidemic acute nonbacterial gastroenteritis worldwide. Here we report our discovery that recombinant Norwalk virus virus-like particles (rNV VLPs) agglutinate red blood cells (RBCs). Since histo-blood group antigens are expressed on gut mucosa as well as RBCs, we used rNV VLP hemagglutination (HA) as a model system for studying NV attachment to cells in order to help identify a potential NV receptor(s). rNV VLP HA is dependent on low temperature (4°C) and acidic pH. Of the 13 species of RBCs tested, rNV VLPs hemagglutinated only chimpanzee and human RBCs. The rNV VLPs hemagglutinated all human type O (11 of 11), A (9 of 9), and AB (4 of 4) RBCs; however, few human type B RBC samples (4 of 14) were hemagglutinated. HA with periodate- and neuraminidase-treated RBCs indicated that rNV VLP binding was carbohydrate dependent and did not require sialic acid. The rNV VLPs did not hemagglutinate Bombay RBCs (zero of seven) that lack H type 2 antigen, and an anti-H type 2 antibody inhibited rNV VLP HA of human type O RBCs. These data indicated that the H type 2 antigen functions as the rNV VLP HA receptor on human type O RBCs. The rNV VLP HA was also inhibited by rNV VLP-specific monoclonal antibody 8812, an antibody that inhibits VLP binding to Caco-2 cells. Convalescent-phase sera from NV-infected individuals showed increased rNV VLP HA inhibition titers compared to prechallenge sera. In carbohydrate binding assays, the rNV VLPs bound to synthetic Lewis d (Led), Leb, H type 2, and Ley antigens, and these antigens also inhibited rNV VLP HA of human type O RBCs. Overall, our results indicate that carbohydrate antigens in the gut are a previously unrecognized factor in NV pathogenesis.
INTRODUCTIONjj3|k, 百拇医药
Norwalk virus (NV) was first isolated from an outbreak of winter vomiting disease, or acute gastroenteritis, at an elementary school in Norwalk, Ohio, in 1968 (1). Subsequent adult volunteer challenge studies with infectious stool filtrate from the Norwalk outbreak demonstrated that NV readily infects susceptible people, causing an acute illness associated with diarrhea, vomiting, myalgia, nausea, and fever within 15 to 24 h after exposure and lasting 24 to 72 h (16). Although these initial studies demonstrated that most people are highly susceptible to NV, presently no cell culture systems or animal models of infection exist, which limits the study of NV replication and pathogenesis.jj3|k, 百拇医药
NV is the prototype nonenveloped, positive-stranded RNA human virus in the genus Norovirus of the family Caliciviridae. Noroviruses are a major cause of acute gastroenteritis throughout the world and cause virtually all outbreaks of nonbacterial gastroenteritis in adults in the United States (18, 39). Furthermore, there are an increasing number of reports of gastroenteritis cases and outbreaks in children and the elderly caused by noroviruses, in part due to increased surveillance and better detection methods (15, 26, 39).
NV virions are detected in low numbers in infected stool. The virions are 27 to 38 nm in diameter, including 4.5-nm radial protrusions extending from the capsid shell that create the calix, or cup-like structures, apparent by electron microscopy (33, 44). The NV capsid proteins (open reading frames 2 and 3, which produce the structural viral proteins VP1 and VP2, respectively) spontaneously self-assemble into virus-like particles (VLPs) when synthesized in a recombinant baculovirus expression system. These recombinant NV VLPs (rNV VLPs) are structurally and antigenically similar to the capsids of native NV and are useful in modeling virus-cell interactions (27, 53).'f, 百拇医药
Hemagglutination (HA) is one method that has been helpful in identifying cell-binding receptors for many viruses, such as influenza A virus and parvovirus B19 (7, 45). The VLPs from human parvovirus B19, JC human polyomavirus, and SA11 simian rotavirus have HA properties that are similar to those of their virions (8, 14, 42).
This is the first report of HA by VLPs from a human calicivirus. Our data demonstrate that the H type 2 histo-blood group antigen is the rNV VLP HA receptor on human type O red blood cells (RBCs) and that the rNV VLPs also bind to synthetic H and structurally related Lewis carbohydrate antigens.kh9v, 百拇医药
MATERIALS AND METHODSkh9v, 百拇医药
rNV VLP purification. rNV VLPs were synthesized and purified by using methods described previously (53). Briefly, spinner flasks containing 3.5 x 106 Sf9 insect cells per 200 ml of Grace's insect cell medium were infected with pVL-NV ORF(2 + 3) recombinant baculovirus at a multiplicity of infection of 5. At 7 days postinfection the rNV VLPs were harvested from supernatants of spinner flask cultures. Cells were pelleted and discarded. The VLPs in the supernatant were pelleted by ultracentrifugation through a 30% sucrose cushion made with 0.2 M phosphate-buffered saline (PBS) for VLPs (PBS-V) (0.2 M sodium phosphate, 0.1 M NaCl, pH 6.0) in an SW28 rotor for 3 h at 120,000 x g and 4°C. The pellets were suspended in 0.39 g of CsCl (Invitrogen, Grand Island, N.Y.) per ml of PBS-V and subjected to isopycnic gradient centrifugation in an SW55 rotor for 18 to 24 h at 150,000 x g and 4°C. The visible band of rNV VLPs in the CsCl gradient was collected by micropipetting. CsCl was removed by diluting the VLPs in PBS-V and sedimenting by ultracentrifugation in an SW28 rotor for 3 h at 120,000 x g and 4°C. The VLP pellets were suspended in PBS-V containing protease inhibitors (aprotinin, leupeptin, and pepstatin [Sigma, St. Louis, Mo.] at 1 µg/ml each) and stored at 4°C. The rNV VLP protein concentrations were determined by the DC protein assay (Bio-Rad, Hercules, Calif.) microtiter plate method without detergent, and the rNV VLP structural integrity was ascertained by electron microscopic inspection of negatively stained (1.0% ammonium molybdate [Sigma], pH 5.0) VLPs on carbon-coated grids (Ted Pella, Reading, Calif.).
HA assay. The HA assay methods were based on those described by the Centers for Disease Control and Prevention (9). Whole blood was collected in 2 volumes of Alsever's solution (2.05% [wt/vol] glucose [Sigma], 0.8% sodium citrate [Fisher, Fair Lawn, N.J.], 0.055% citric acid [Fisher], 0.42% NaCl [Fisher], pH 6.1) and stored at 4°C. Individual human RBC samples were graciously provided by volunteer donors at the Baylor College of Medicine's Influenza Research Center and the Methodist Hospital Blood Donor Center (Houston, Tex.). Pooled human RBC reagents from at least five donors were purchased from Immucor (Atlanta, Ga.). Human cord RBC samples were kindly provided by the Children's Nutrition Research Center's Leukocyte Biology Laboratory (Houston, Tex.). Human RBC samples with rare phenotypes were generously donated by Immucor/Gamma Biologicals (Houston, Tex.). Chimpanzee, baboon, spider monkey, and rhesus RBC samples were graciously provided by the Southwest Foundation for Biomedical Research (San Antonio, Tex.). Chicken, guinea pig, canine, and feline blood samples were provided by the Center for Comparative Medicine at Baylor College of Medicine (Houston, Tex.). Murine RBC samples were obtained during routine blood draws from normal control BALB/c mice. Bovine, porcine, and lapine blood samples were purchased from Lampire (Pipersville, Pa.). The RBCs were packed by diluting the cells in 0.01 M PBS (without Ca2+ or Mg2+; pH 7.2) (Invitrogen, Carlsbad, Calif.) and centrifuging for 15 min at 500 x g. The HA activity of rNV VLPs was tested in untreated 96-well V-bottom plates (Nunc, Naperville, Ill.). Equal volumes (50 µl each) of 0.5% packed human RBCs in 0.85% saline (pH 6.2) and rNV VLPs serially diluted in 0.01 M PBS for HA (PBS-H) (0.01 M sodium phosphate [EM Science, Gibbstown, N.J.], 0.15 M NaCl, pH 5.5; filtered with a 0.2-µm-pore-size filter) were combined at 4°C. The plates were covered with adhesive film (Excel Scientific, Wrightwood, Calif.) and incubated for 75 to 120 min in a cold room (at 4 to 8°C), unless otherwise indicated. Incubations at other temperatures were performed at room temperature (22 to 25°C) or in a tissue culture incubator (36 to 37°C). The HA titer was the reciprocal of the greatest rNV VLP dilution that did not allow sedimentation of the RBCs compared to negative control wells that contained only buffer. The positive control for HA was an influenza A virus (H1N1, A/Texas/36/91), grown in eggs, for which the titer had been previously determined. For each animal RBC sample, the percentage of packed RBCs in saline used for each HA assay was determined by the minimum concentration of RBCs able to sediment in negative control wells (PBS-H) within 6 h of incubation at 4°C.
Periodate treatment of RBCs. Human type O RBCs were treated with freshly prepared 10.0 mM KIO4 (Sigma) for 20 min at room temperature and then rinsed and packed as described above. To test the RBCs' ability to be agglutinated, 40 µl of treated or control 2.0% RBCs was mixed with 10 µl of rNV VLPs (0.1 mg/ml), anti-D or anti-H monoclonal antibodies (MAbs) (Immucor) (0.1 mg/ml), or PBS-H on an ice-cold glass plate and incubated for 10 min at 4°C. The degree of HA was determined by visual comparison to controls and verified by an independent observer.|\1, http://www.100md.com
Enzyme treatment of RBCs. All enzyme reactions were performed with 50 µl of packed RBCs in a total enzyme reaction volume of 125 µl. Control reaction mixtures were incubated without enzyme. Neuraminidase from Arthrobacter ureafaciens (6 µU; Sigma) in Dulbecco's PBS (D-PBS) (Invitrogen) was combined with the RBCs and incubated for 60 min at 37°C. L-(Toslylamido-2-phenyl)ethyl chloromethyl ketone-treated trypsin (1.25 ng; Sigma) in D-PBS was added to the RBCs and incubated for 30 min at 37°C. Trypsin activity was stopped by adding 0.5 µg of soybean trypsin inhibitor (Sigma) to the reaction mixture. After enzyme treatment, the RBCs were rinsed three times with 6 ml of PBS and packed by centrifugation as described above. After removal of the PBS supernatant, the packed RBCs were diluted with cold 0.85% saline (pH 6.2) to make a 0.5% RBC suspension for HA assays.
Enzyme-linked immunosorbent assay (ELISA)-based carbohydrate microtiter plate assay (CMA). Multivalent carbohydrate-biotin reagents conjugated to polyacrylamide (CHO-PAA-biotin; Glycotech, Rockville, Md.) were immobilized onto streptavidin-coated plates as suggested by the manufacturer, with all reagents added in volumes of 100 µl/well, unless otherwise specified. Briefly, PVC microtiter flat-bottom plates (Dynex Technologies, Chantilly, Va.) were coated with 10 µg of streptavidin (Sigma) per ml in PBS at 4°C overnight, blocked by addition of 1% fatty acid-free bovine serum albumin (faf-BSA) (Sigma) in PBS at room temperature for 4 h, and then rinsed three times with 200 µl of PBS per well. Next, 0.1 µg of biotin per ml or 10 µg of CHO-PAA-biotin conjugate per ml in PBS with 0.25% faf-BSA was incubated at room temperature for 1 h and rinsed as described above. The rNV VLP-carbohydrate binding was optimized at 10 µg of rNV VLPs per ml in PBS (pH 7.5) with 0.25% faf-BSA and incubation at room temperature for 1 h before rinsing as described above. rNV VLPs bound to carbohydrate were detected by incubation with rabbit anti-rNV VLP serum (1:5,000) in PBS with 0.25% faf-BSA for 1 h at room temperature; rinsing as described above; addition of goat anti-rabbit immunoglobulin A (IgA), IgM, and IgG conjugated to horseradish peroxidase (ICN Biomedicals, Philadelphia, Pa.) (1:50,000) in PBS with 0.25% faf-BSA; incubation for 1 h at room temperature; and rinsing. Plates were developed with the TMB microwell peroxidase endpoint assay (Kirkegaard & Perry Laboratories, Gaithersburg, Md.), and absorbance was read at 450 nm (Spectromax 190; Molecular Devices, Sunnyvale, Calif.). Carbohydrate competition assays for rNV VLP binding to the microtiter plate-immobilized CHO-PAA-biotin were performed by preincubating the rNV VLPs with CHO-PAA reagents (without biotin) in PBS (pH 7.2) for 30 min at 4°C before addition to the microtiter plates prepared as described above. The plates were incubated for 1 h at 4°C and then rinsed and detected as described above.
HI assay. Inhibition of rNV VLP HA was examined by using the multivalent CHO-PAA-biotin conjugates, human pre- and post-NV challenge sera (24), mouse hyperimmune serum IgG, and anti-rNV VLP MAbs (8812, 3901, and 3912) (30). The human sera were heat inactivated and kaolin treated for the HA inhibition (HI) assays as previously described (9). Mouse IgG from hyperimmune sera and monoclonal hybridoma ascites were purified by using UltraLink protein G columns (Pierce, Rockford, Ill.) according to the manufacturer's protocol and stored at 4°C in PBS with 0.02% NaN3. The HI activities of the carbohydrate conjugates, the treated human sera, and the mouse IgG were tested by serial dilutions of the inhibitors (starting concentrations of 100 µg/ml, 1:10, and 50 µg/ml, respectively) in a volume of 25 µl of PBS-H across 96-well V-bottom plates. An amount of rNV VLPs equal to 4 HA units (HAU) (1 HAU was defined as the amount of antigen present at the HA endpoint dilution) in a volume of 25 µl of PBS-H was added to each well, and the inhibitor was incubated with the rNV VLPs for 30 min at 4°C. (For the mouse IgG HI assays, the PBS-H contained 0.3% BSA fraction V [Calbiochem, San Diego, Calif.].) Next, 50 µl of 0.5% human type O RBCs in 0.85% saline was added to each well, and the plate was incubated as described above. Each inhibitor tested was negative for nonspecific HA of the RBCs at the highest concentration used in the assay. The HI titer was read as the last dilution of inhibitor that prevented HA of the RBCs by rNV VLPs.
RESULTScatx!r', http://www.100md.com
HA by rNV VLPs is temperature and pH dependent. The rNV VLP HA of RBCs was tested at 4, 25, and 37°C in PBS-H (pH 7.2). The rNV VLPs were able to hemagglutinate human type O-positive RBCs at 4°C but not at 25 or 37°C (Fig. 1A). Microscopic inspection of the human type O-positive RBCs and HA of the RBCs by influenza A virus at all three temperatures demonstrated that the cells were intact at these temperatures. The HA of human type O-positive RBCs by rNV VLPs at 4°C was lost within 30 min when plates were warmed to room temperature. The loss of HA at room temperature was reversible. The HA titer was restored to a level equal to or up to fourfold lower than the initially observed titer when the plates were first cooled to 4°C, shaken to suspend the RBCs, and then incubated a second time at 4°C (data not shown). Because several enteric viruses have HA titers that are optimal at pHs of below 7.2 (9), we evaluated the effect of pH on rNV VLP HA of type O-positive RBCs. With pH increments of 0.5, the rNV VLPs hemagglutinated human type O-positive RBCs when diluted in PBS-H at pH 5.0 to 7.0 but not at pH 7.5 and 8.0 (Fig. 1B). HA by influenza A virus was pH independent between pH 5.0 and 8.0. Upon further examination of HA at pHs of between 7.0 and 7.5, rNV VLP HA was observed at pH 7.0 to 7.2 but was lost at pH 7.3 to 7.5 (data not shown). Therefore, for rNV VLP HA of human type O-positive RBCs, an incubation temperature of 4 to 8°C and a pH of between 5.0 and 7.2 were required. At the chosen conditions, i.e., incubation at 4°C and pH 5.5, an HA endpoint of 0.625 ng of rNV VLPs (3.6 x 107 particles) was regularly able to hemagglutinate approximately 3 x 106 human type O-positive RBCs. Thus, to further characterize the HA properties of rNV VLPs, we made our standard HA assay with 400 ng of rNV VLPs per ml as the initial VLP concentration that was serially diluted in PBS-H at pH 5.5 and incubated for 75 to 90 min at 4°C, unless otherwise noted.
fig.ommitteedps:pu-, 百拇医药
FIG. 1. HA titers of human type O RBCs by rNV VLPs. Fifty microliters of rNV VLPs (400 ng/ml) influenza A/TX/36/91 virus grown in eggs , or PBS-H was serially diluted in PBS-H and mixed with 50 µl of 0.5% RBCs. (A) The PBS-H was at pH 7.2, and the HA assay incubation times, as determined by sedimentation of RBCs in negative control wells, were 30 min at 37°C, 40 min at 25°C, and 90 min at 4°C. (B) The pH of the PBS-H ranged from 5.0 to 8.0 as indicated, and the HA assay incubation time was 90 min at 4°C.ps:pu-, 百拇医药
rNV VLPs hemagglutinate chimpanzee RBCs. The ability of a virus to agglutinate RBCs depends on the expression of the HA receptor on the cells, and this expression may be limited to a subset of animal species RBCs. Therefore, we tested RBCs from different species to gain insight into the nature and expression pattern of the rNV VLP HA receptor. Additionally, if the rNV VLP HA was a biologically relevant interaction, we hypothesized that the narrow host restriction of NV infection, i.e., to most humans and some chimpanzees (24, 43, 54), would be reflected in the ability of the VLPs to agglutinate RBCs from susceptible species. A panel of RBC samples from 12 nonhuman animal species was chosen based on their susceptibility to other caliciviruses, evolutionary relatedness to humans, availability, and use in other viral HA assays. Of these RBC samples, only the chimpanzee RBCs (0.5%) were hemagglutinated by the rNV VLPs (Fig. 2A). This sample of chimpanzee RBCs also reacted with an anti-A histo-blood group typing reagent (Immucor), indicating the presence of A or A-like histo-blood group antigens on the chimpanzee RBCs (data not shown) (3). The HA titer for the chimpanzee RBCs was 256, similar to the high HA titers exhibited by several human type A RBC samples (Fig. 2B). The baboon (0.5%), spider monkey (0.5%), rhesus macaque (1.5%), chicken (0.5%), guinea pig (1.0%), murine (1.0%), canine (1.0%), bovine (1.0%), porcine (1.0%), feline (1.5%), and lapine (2.0%) RBC samples were not hemagglutinated by 400 ng of rNV VLPs per ml.
fig.ommitteedn7#42, 百拇医药
FIG. 2. HA titers of individual RBC samples by rNV VLPs. Fifty microliters of rNV VLPs (400 ng/ml) serially diluted in PBS-H (pH 5.5) were combined with 50 µl of RBC samples (0.5 to 2.0%) from the indicated 12 animal species (A) and adult human RBC samples (0.5%) (separated by their ABO phenotypes) (B). The RBC concentrations used in HA assays were determined by sedimentation of RBCs in PBS-H (pH 5.5) negative control wells.n7#42, 百拇医药
rNV VLPs hemagglutinate adult human type O, A (A1 and A2), and AB RBCs and a subset of adult human type B RBCs. Many individual RBC samples and pooled human RBC reagent samples were tested for rNV VLP HA to determine the frequency of expression of the rNV VLP HA receptor on human RBCs. For convenience, the human RBC samples were grouped by their ABO and Rh phenotypes. The rNV VLPs hemagglutinated all adult human type O, A, and AB individual RBC samples tested (11, 9, and 4 individual samples, respectively) (Fig. 2B). However, only 4 of the 14 type B individual human RBC samples tested were hemagglutinated by 400 ng of rNV VLPs per ml (Fig. 2B). In HA assays starting at a much higher concentration of rNV VLPs (100 µg/ml), an additional six of the individual human type B RBC samples were hemagglutinated by the VLPs (data not shown). For all human RBC samples tested, the Rh factor did not affect rNV VLP HA of human RBCs. Additional rNV VLP HA assays were performed on pooled human A1, A2, B, and O RBC reagents. In agreement with our individual RBC sample data, 400 ng of rNV VLPs per ml hemagglutinated the pooled type A1, A2, and O RBCs but not the pooled type B RBCs (Table 1).
fig.ommitteedin;n, 百拇医药
TABLE 1. HA titers of untreated and neuraminidase-treated pooled human RBCs by rNV VLPsin;n, 百拇医药
rNV VLP binding to RBCs is carbohydrate dependent. Because the B antigen is a carbohydrate moiety, we tested the carbohydrate dependence of rNV VLP-RBC binding by periodate treatment, which oxidizes all carbohydrates on the cell surface. After periodate treatment of human type O-positive RBCs, HA by rNV VLPs and by anti-H, a carbohydrate-dependent antibody (Immucor), was lost (Table 2). This same treatment did not reduce anti-D HA, which recognizes an Rh factor protein on RBCs. Furthermore, trypsin treatment of type O RBCs did not affect the rNV VLP HA titer (data not shown), indicating that rNV VLP HA was not dependent upon a trypsin-sensitive protein on the RBC surface.in;n, 百拇医药
fig.ommitteedin;n, 百拇医药
TABLE 2. Agglutination of periodate-treated human type O+ RBCs by rNV VLPsin;n, 百拇医药
Two cell surface carbohydrates to which viruses commonly bind are sialic acid and heparan sulfate. To test if the carbohydrate-dependent rNV VLP HA required sialic acid, pooled human RBC reagents were enzymatically treated with neuraminidase from A. ureafaciens, which removes both {alpha} -2,3 and {alpha} -2,6 linked sialic acid residues on the cell surface. This neuraminidase treatment of pooled human type O, A1, and A2 RBCs did not affect their rNV VLP HA titer (Table 1), whereas the sialic acid-dependent HA by rhesus rotavirus was lost. Thus, rNV VLP HA of human type O, A1, and A2 RBCs was sialic acid independent. Additionally, pretreating the rNV VLPs with heparan sulfate before performing the HA assay did not inhibit the VLPs' ability to hemagglutinate human type O RBCs (data not shown). Therefore, the rNV VLP binding was dependent upon a carbohydrate, and unlike many other viruses, this carbohydrate did not involve sialic acid or heparan sulfate.
An interesting observation was made when the pooled human type B RBCs were treated with neuraminidase and tested for rNV VLP HA (Table 1). As seen with the majority of individual type B RBC samples, the untreated pooled type B RBCs were not hemagglutinated by 400 ng of rNV VLPs per ml. However, after neuraminidase treatment, when the sialic acid-dependent rotavirus HA titer was abolished, the rNV VLPs were able to hemagglutinate the type B RBCs.-d#6};, http://www.100md.com
The rNV VLP HA is dependent upon H antigen. Individuals with the rare Bombay (Oh) blood type do not express the H histo-blood group antigen on their RBCs (35). Since the H antigen is the precursor substrate for A and B antigen expression, RBCs from Bombay-type individuals will appear to have an O phenotype regardless of their ABO genotype (35). Bombay-type individuals with an A or B and a secretor (Se+) genotype are A- or B-para-Bombay, respectively, where the A or B antigens may be expressed on additional carbohydrate substrates not normally expressed by RBCs (35). None of the five Bombay- or the two para-Bombay-type RBC samples assayed was hemagglutinated by 400 ng of rNV VLPs per ml (Fig. 3A). In fact, even 100 µg of rNV VLPs per ml did not hemagglutinate the seven Bombay-type RBC samples (data not shown). Furthermore, when monoclonal antibody BRIC 231 (Biogenesis, Poole, United Kingdom), which specifically recognizes the H type 2 antigen, was preincubated with human type O RBCs, the rNV VLPs did not hemagglutinate the RBCs (data not shown). These data strongly suggest that rNV VLP HA is dependent upon H antigen expression on human RBCs.
fig.ommitteed7?4vq, 百拇医药
FIG. 3. HA titers of rare-phenotype human RBC samples (A) and human umbilical cord blood samples (B) by rNV VLPs. Fifty microliters of rNV VLPs (400 ng/ml) or UEA-1 lectin (100 µg/ml) was serially diluted in PBS-H (pH 5.5) and combined with 50 µl of 0.5% unbranched (i/i), Bombay (Oh), para-Bombay (AOh and BOh), or normal adult type O RBC samples (A) and with 50 µl of 0.5% umbilical cord RBCs in (B).7?4vq, 百拇医药
The rNV VLP HA receptor may be a developmental antigen. Many RBC antigens, including the ABH antigens, are not expressed or are expressed at low levels on fetal and neonatal RBCs (37, 51). Four human umbilical cord RBC samples (neonatal RBCs) were obtained from full-term births and were tested for HA by rNV VLPs (Fig. 3B). One type A cord RBC sample showed an HA titer similar to that of some adult type A RBCs. The three additional cord blood samples (a second type A sample, a type O sample, and a type B sample) were not hemagglutinated by 400 ng of rNV VLPs per ml.
One developmental change in human RBC antigens is the increase in carbohydrate branching. Fetal RBCs have no branched carbohydrates (phenotype i), and adult levels of branching are attained by 2 years of age (phenotype I) (20, 52). RBCs from adults lacking the {alpha} -1,6 galactosyltransferase carbohydrate branching enzyme resemble fetal RBCs in their lack of branched-carbohydrate expression (4). The rNV VLP HA did not depend on expression of branched carbohydrates (Fig. 3A). Two type O (i/i) RBC samples were hemagglutinated by 400 ng of rNV VLPs per ml to an HA titer of 128, similar to the HA titer of normal type O RBCs.28i5&, 百拇医药
rNV VLP HA of human type O RBCs is inhibited by mouse MAb 8812. The previously characterized rNV VLP-specific MAbs 8812, 3901, and 3912 were tested for their ability to inhibit rNV VLP HA (30). Also, IgG purified from mouse anti-rNV VLP hyperimmune serum was tested for rNV VLP HI. Only MAb 8812 inhibited rNV VLP HA of human type O RBCs (Table 3).28i5&, 百拇医药
fig.ommitteed}ow%fut, 百拇医药
TABLE 3. HI titers of mouse anti-rNV VLP antibodies}ow%fut, 百拇医药
Convalescent-phase sera from NV-infected volunteers show an increase in rNV VLP HI titer. Prechallenge and convalescent-phase sera from seven NV-challenged volunteers were heat inactivated and kaolin treated to remove nonspecific hemagglutinating factors (9, 24). The treated sera were diluted (1:10) and had no nonspecific HA activity and lower HI titers than untreated sera. Increased HI titers were found in the convalescent-phase sera of NV-infected volunteers (n = 5; volunteers 1 to 5) compared to their prechallenge sera, while the HI titers did not increase in convalescent-phase sera from noninfected NV-challenged volunteers (n = 2; volunteers 6 and 7) (Table 4) (24).}ow%fut, 百拇医药
fig.ommitteed}ow%fut, 百拇医药
TABLE 4. HI titers of preimmune and convalescent-phase sera from volunteers given NVa}ow%fut, 百拇医药
The rNV VLPs specifically bind to synthetic H and Lewis carbohydrate antigens. Based on the data that the H antigen may be the HA receptor for the rNV VLPs on human type O RBCs, the VLPs were tested for their ability to bind synthetic H and the structurally related Lewis carbohydrate reagents (Table 5). These synthetic H and Lewis antigens were chosen based on their relatedness to the H type 2 antigen found on human type O RBCs, their expression on gut mucosa, and their availability. When the rNV VLPs were incubated on carbohydrate-coated microtiter plates in the ELISA-based CMA in PBS (pH 6.0) at room temperature, the VLPs bound to the following carbohydrates, starting with the greatest rNV-specific detection: Lewis d (Led), Leb, H type 2, Ley, and H disaccharide (Fig. 4A). In HI assays, the same carbohydrates (CHO-PAA-biotin reagents) inhibited rNV VLP binding to human type O RBCs (Fig. 4B). The relative strength of detection of rNV VLPs bound to the immobilized carbohydrates in the ELISA-based CMA correlated with the relative magnitude of HI titers. Furthermore, when the free synthetic carbohydrates Led (H type 1), H type 2, and Lea were preincubated with the rNV VLPs before their addition to the ELISA-based CMA, Led and H type 2, but not Lea, inhibited rNV VLP binding to both the immobilized Led and H type 2 carbohydrates (Fig. 5).}ow%fut, 百拇医药
fig.ommitteed}ow%fut, 百拇医药
TABLE 5. Structures of carbohydrates and rNV VLP binding}ow%fut, 百拇医药
fig.ommitteed}ow%fut, 百拇医药
FIG. 4. rNV VLPs bind synthetic H and Lewis antigens. (A) Carbohydrate binding of rNV VLPs to synthetic H and Lewis antigens determined by ELISA-CMA. Multivalent CHO-PAA-biotin reagents were immobilized on microtiter plate wells and incubated with rNV VLPs in PBS (pH 7.0) at 25°C for 1 h. Bound rNV VLPs were detected with anti-rNV-specific rabbit serum, horseradish peroxidase-conjugated secondary antibody, TMB colorimetric development, and spectrophotometric absorption at 450 nm. Error bars indicate standard deviations. (B) HI titers of serially diluted multivalent CHO-PAA-biotin reagent (0.10 mg/ml) with 4 HAU of rNV VLPs and 50 µl of 0.5% human type O RBCs.(Anne M. Hutson Robert L. Atmar Donald M. Marcus and Mary K. Estes)