Material and methods

Cell culture and transfections
Cell lines CHO-K1 and Vero E6 were purchased from American Type Cell Collection (Manassas, VA). Vero-E6 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% decomplemented fetal-calf serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Paisley, UK).
CHO cells are devoid of α1,2-fucosyltransferase activity and therefore of ABH antigens. To obtain the expression of the A antigen, they were transfected first with the rat Fut2 cDNA, and then with an A enzyme cDNA (Bureau et al. 2001; Cailleau-Thomas et al. 2002). The rat Fut2 cDNA was inserted into the pDR2 eukaryotic expression vector (Clontech, St Germain en Laye, France) deleted of the sequences lying between the EcoRV and ClaI sites. This vector possesses a hygromycin selection marker. The rat A enzyme cDNA was inserted into the pcDNA3.1 vector (Invitrogen, Paisley, UK) with a zeocin selection marker. Cells were transfected with the rat Fut2 cDNA using Lipofectamin 2000TM according to the manufacturer's instructions (Invitrogen). After selection in hygromycin containing medium (0.4 mg/mL), cells were cloned by limiting dilution, and a clone strongly expressing cell surface H antigen, as detected using the FITC-labeled UEA-I lectin, was selected. This clone was further transfected with the rat A enzyme cDNA. Transfectants were then selected in a zeocin-containing medium (0.6 mg/mL). After cloning by limiting dilutions, a clone strongly expressing the A histo-blood group antigen was selected. Control transfected cells were prepared by transfection with the empty vectors. These stable transfectants were cultured in RPMI 1640 supplemented with 10% decomplemented fetal-calf serum, 2 mM l-glutamine, 10 μg/mL free nucleotides, 100 U/mL penicillin, and 100 μg/mL streptomycin, 0.4 mg/mL hygromycin, and 0.6 mg/mL zeocin. To obtain the expression of the SARS-CoV S protein, control CHO cells, transfected CHO cells expressing the H antigen, and double transfected CHO cells expressing the A antigen were transfected with the previously described pEGFP-N1 vector containing an S protein–EGFP construction (CMV-SG) (Chou et al. 2005). Stable transfectants were obtained after selection with 1 mg/mL G418 (Gibco). Since the expression of the S protein was progressively lost, even when cells were continuously cultured in the presence of G418, part of the experiments were performed 48 h after transient transfection with the CMV-SG vector.
Cells were passaged at confluence after dispersal with 0.025% trypsin in 0.02% EDTA and routinely checked for mycoplasma contamination by Hoechst 33258 (Sigma, St Louis, MO) labeling.

Flow cytometry, immunofluorescence, and Western blot analyses
Cells at near confluence were detached by a brief 0.025% trypsin/0.02% EDTA treatment. Viable cells, 2 × 105 per well of 96 culture microtiter plates, were incubated with primary anti-H type 2 or broad reactive anti-A monoclonal antibodies 19-0LE and 3-3A respectively, in PBS containing 0.1% gelatin for 30 min at 4°C (Bara et al. 1988; Mollicone et al. 1996). After three washes with this same buffer, a 30-min incubation with the secondary anti-mouse IgG FITC-labeled antibody (Sigma) was performed at 4°C. After washing, fluorescence analysis was performed on a FACSCalibur (Becton-Dikinson, Heidelberg, Germany). The expression of the EGFP-S protein construct was detected by its autofluorescence on the FL1 channel.
CHO cells transfectants, cultivated on glass lamellae, were fixed by the addition of 2% formaldehyde in a culture medium for 10 min. After washing in PBS, the cell monolayer was incubated with the anti-A mAb 3-3A at 0.5 μg/mL in PBS for 1 h and washed thrice in PBS before incubation for 30 min with TRITC-labeled anti-mouse IgG (Sigma) diluted at 1/400. After three final washings in PBS, slides were mounted in Mowiol and observed under a Leica TCS SP (Heidelberg, Germany) confocal fluorescence microscope. Negative controls were incubated with the secondary antibody alone.
Confluent cells (CHO Fut2 simple transfectants, CHO Fut2/A double transfectants, and CHO Fut2/A/PS triple transfectants) were rinsed with ice-cold PBS, pH 7.2, and then recovered by scraping. After washing with ice-cold PBS, cells were solubilized in 50 mM potassium phosphate, pH 6.0, containing 2% (v/v) triton X-100 on ice for 30 min. Following a centrifugation at 13,000 × g for 10 min, the supernatant was collected and the protein concentration was determined using the BC assay protein quantification kit (Uptima, Montluçon, France). Thirty micrograms of total proteins of each extract were separated on 8% SDS–PAGE under reducing conditions and electrotransferred to immobilon P sheets (Millipore, Bedford, MA). Immunoblots were saturated for 1 h at room temperature with Western blocking reagent (Roche Diagnostics GmBH, Mannheim, Germany) and strips were cut and incubated overnight at 4°C with the anti-A 3-3A mAb at 2 μg/mL in antibody Western blocking reagent. Following three washes for 15 min with TBS containing 0.05% Tween 20, strips were incubated with horseradish peroxidase-labeled anti-mouse IgG (H + L) (Beckman Coulter, Fullerton, CA) for 1 h at room temperature. After three final washes, detection was performed with a chemiluminescence kit (Roche Diagnostics).

Human plasma preparation
Plasma samples from two blood group O healthy donors with anti-A titers 1/256 by classical hemagglutination were used. The presence of anti-A natural antibodies was confirmed by ELISA on synthetic A type 2 tetrasaccharide conjugated to polyacrylamide (Lectinity, Moscow, Russia). The polyacrylamide conjugate at 10 μg/mL in a carbonate–bicarbonate buffer, 100 mM, pH 9.6, was coated onto Maxisorp ELISA plates (NUNC, Roskilde, Denmark) by overnight incubation at 37°C. After three washes with PBS containing 0.05% Tween 20 (TPBS), wells were incubated with 5% defatted milk in PBS for 1 h at 37°C. PBS 2-fold serially diluted plasma samples were then incubated for 1 h at 37°C. After washing with TPBS, peroxidase-labeled anti-human IgG (H + L) (Uptima, Montluçon, France) diluted at 1/10,000 were incubated for 1 h at 37°C. After final washes with TPBS, reactivities were detected using TMB (5-tetramethylbenzidine) as a substrate (BD Bioscience, San Jose, CA) and read at A450 nm. To adsorb the anti-A natural antibodies, 1600 μL of plasma diluted 1/4 in PBS were incubated onto 120 mg of silica beads conjugated with either synthetic A type 2 tetrasaccharide or a methyl group (kind gift from the late Pr. R.U. Lemieux, Edmonton, Canada). The latter being used as mock adsorbed controls. After a 1-h incubation under gentle agitation and centrifugation at 13,000 × g for 10 min, the supernatant was collected and kept at 4°C until used.

Cell-based binding assay
Vero E6 cells were grown to a confluent layer in wells of 48-well plate (NUNC). The CHO transfected cells were labeled with Hoechst 33258 at 2 μM for 5 min, rinsed three times with PBS, and then suspended after incubation with 0.025% trypsin in 0.02% EDTA. 1 × 105 cells suspended in 500 μL were gently laid onto the Vero cell layer in at least triplicate wells. After 2-h incubation at 37°C, wells were gently rinsed with a culture medium three times, and cells were fixed by a 10-min incubation with 2% formaldehyde. Adherent CHO cells were counted under an epifluorescence microscope (Zeiss, Jena, Germany) with a 10× objective. Three to six fields/well were counted and results expressed as the mean number of cells per field. In the antibody blocking experiments, monoclonal antibodies or human plasma samples were added to the transfected CHO cells suspension before seeding on the Vero cell layer.

Modeling the effect of protection by natural anti-A or -B antibodies on the virus transmission in populations
A deterministic SIR (susceptible, infectious, recovered) model of the transmission dynamics of SARS that takes into account the effect of the protection by anti-histo-blood group natural antibodies was developed. In this model, the whole population was divided according to the different blood groups (A, B, O, or AB) into four interacting subpopulations NA, NB, NO and NAB. For each subpopulation N = S(t) + I(t) + R(t) where S(t), I(t), and R(t) represent, for each blood group, the number of individuals susceptible, infected, or recovered at time t, respectively. Four different populations reflecting the variability in blood groups distributions were studied (Table I): Chinese from Hong Kong, Aïnu from Japan, Amerindians and white Americans from the United States.
Table I  Distribution of ABO blood groups in four different populations used to model the impact of the ABO effect on SARS transmission.
[O]  [A]  [B]  [AB]
Aïnu (Japan)  0.17  0.32  0.337  0.173
Chinese (Hong Kong)  0.422  0.178  0.333  0.067
Caucasians (USA)  0.45  0.40  0.111  0.039
Amerindians (USA)  0.79  0.16  0.046  0.004
The frequencies of each phenotype were obtained from Mourant (1983). Mathematical details are given in the appendix available as supplementary data; the program was written in C++, a 1-day step was used for all the simulations. In each simulation, changes in the number of infected individuals (cases) over time were determined.