Saliva glycoproteins bind to Spike protein of SARS-CoV-2

We analyzed the affinity-proteomics data of saliva absorbed to plate-bound Spike protein of SARS-CoV-2, and identified major virus-binding proteins as MUC7, MUC5B, DMBT1, and neutrophil defensins. Furthermore, we found that saliva from healthy donors inhibited the binding of Spike-protein-specific polyclonal antibodies to Spike antigen. These data suggest that the Spike protein’s glycoprotein-binding domains (GBD) may be targeted to block virus adherence or entry of SARS-CoV-2.


Spike protein of SARS-CoV-2 binds to multiple saliva proteins
A total of 138 proteins with ion abundance higher than 10 7 were absorbed by plate-bound Spikeprotein from 3 volunteers (Table 1 and supplemental Tables 1-3

Saliva proteins inhibit the binding of polyclonal antibodies to Spike protein
To test whether saliva proteins inhibit the binding of polyclonal antibodies specific to Spike protein of SARS-CoV-2, we collected saliva of healthy donors of different gender and age groups (study protocol: 2020tjdx055, Tongji University). The inhibition rate was defined as the percentage of maximum inhibition in serial diluted antibody sample pooled from Spike proteinvaccinated mice. We found above 50% inhibition rate in 13 of the 22 donors ( Figure 1). No significant difference were found among different age or gender groups. The maximum inhibition was observed at the sera dilution fold of 1:6075 to 1:54675 (Table 2 and Supplemental Saliva proteins are known to contain virus-binding proteins and be involved in viral pathogenesis.

Sugar binding domain of PEDV-CoV and MERS-CoV Spike protein is considered as important
for the critical entry functions [13,18]. Four monoclonal antibodies (MAbs 56, 60, 63, and 72) targeting the sialic acid-binding domain could neutralize GDU strain of PEDV-CoV virus at 0.015 to 0.039 µg/ml of concentration [13]. Passive immunization of mice with a mAb 1.10f3 which target sialic acid-binding domain of MERS-CoV resulted in 40% protection from mortality following MERS-CoV infection [18]. In our study, we observed the maximum inhibition rate of saliva protein in competing with the antibody binding to Spike protein at the titer of 1:6075 to 1:54675. The exact nature of the domains that bind to saliva glycoproteins and whether they are targets are neutralizing antibodies remain to be studied. Ongoing studies are being focused to isolate potential neutralizing antibodies in SARS-CoV-2 infected or vaccinated individuals that may block the Spike protein's glycoprotein-binding domains (GBD).
In summary, our study clearly identified saliva glycoproteins as binders of Spike protein of SARS-CoV-2. Glycoprotein-binding domains of SARS-CoV-2 virus are potential targets for the development of antibody therapeutics and vaccines.

Affinity proteomics by recombinant Spike protein
Recombinant insect cell-derived Spike proteins for SARS-CoV-2 (GenBank Accession Number: MN908947) was prepared as described [19]. A plate-bound assay was used to purify saliva proteins samples were reduced and digested by trypsin (Promega) as described (19). The trypsindigested peptides were desalted with mono-Spin C18 column (GL Sciences). The desalted peptide mixture were analyzed by LC-MS-MS as described using an Easy-nLC 1000 system (Thermo Scientific, San Jose, CA) and orbitrap analyzer (Q Exactive mass spectrometer, Thermo Scientific, San Jose, CA).

Preparation of anti-sera to Spike protein
All animal studies were approved by the animal care and use committee of Tongji University.
All experiments were carried in SPF housing facilities. C57/BL6 strain of mice (male, 8 weeks old) were immunized by recombinant S protein with polyI:C as adjuvant [20]. 5 µg of S protein mixed with 50 µg of polyI:C adjuvant (Yisheng Biopharma, Beijing) were injected per mouse intramuscularly [20]. Two vaccination schedule were tested. In Schedule A, mice were immunized at days 0, 7 and 16. In Schedule B, mice were immunized at days 0, 3 and 7. Sera from immunized mice were collected by tail vein bleeding, and anti-S protein antibody titer was measured by ELISA. 50 µl of 1 µg/ml S protein in PBS was bound to 96-well Clear Flat Bottom Polystyrene High Bind Microplate (Corning) at 4ºC for overnight, and washed five times with 0.05% Tween in PBS (every time for 2 minutes using a mini shaker). The S proteincoated plates were blocked with 100 µl 1% bovine serum albumin at 37ºC for one hour, and washed five times with 0.05% Tween in PBS, followed by incubation with 50 µl serially diluted sera for one hour. The plates were washed for five times, and the anti-S protein mouse IgG was visualized by a secondary antibody (goat anti-mouse IgG, Southern Biotech) followed by colorimetric detection. One percent bovine serum albumin in PBS was used as blank to determine the cutoff value.

Measurement of inhibition rate of saliva on antibodies generated against S protein
Saliva of healthy donors of different gender and age groups were collected to test their inhibition rate on pooled mouse sera containing polyclonal anti-Spike protein antibodies (study protocol: 37ºC for one hour, and washed five times with 0.05% Tween in PBS. The plates were incubated with 50 µl saliva per well at 37 ºC for one hour, and washed for five times. Pooled sera from vaccinated mice (with anti-S antibody titer of 1:54675) were serially diluted and added, and anti-S protein mouse IgG was visualized by a secondary antibody (goat anti-mouse IgG, Southern Biotech) followed by colorimetric detection. The inhibition rate was determined as the percentage inhibition of absorbance at OD450, at the antibody dilution that showed maximum rate of inhibition.
Dapeng Zhou designed this study. Dapeng Zhou and Chenghao Wu contributed to the collection, analysis and interpretation of data. Dapeng Zhou wrote the manuscript. All authors read and approved the final manuscript. for one hour before ELISA experiment. The inhibition rate was determined as the percentage inhibition of absorbance at OD450, at the antibody dilution that showed maximum rate of inhibition ( Table 2, Supplemental Figure 2).

Supplemental Online Materials
Supplemental