Low Levels of Natural Anti-α- N-Acetylgalactosamine (Tn) Antibodies Are Associated With COVID-19

Abstract

Human serum contains large amounts of anti-carbohydrate antibodies, some of which may recognize epitopes on viral glycans. Here, we tested the hypothesis that such antibodies may confer protection against COVID-19 so that patients would be preferentially found among people with low amounts of specific anti-carbohydrate antibodies since individual repertoires vary considerably. After selecting glycan epitopes commonly represented in the human anti-carbohydrate antibody repertoire that may also be expressed on viral glycans, plasma levels of the corresponding antibodies were determined by ELISA in 88 SARS-CoV-2 infected individuals, including 13 asymptomatic, and in 82 non-infected controls. We observed that anti-Tn antibodies levels were significantly lower in patients as compared to non-infected individuals. This was not observed for any of the other tested carbohydrate epitopes, including anti-αGal antibodies used as a negative control since the epitope cannot be synthesized by humans. Owing to structural homologies with blood groups A and B antigens, we also observed that anti-Tn and anti-αGal antibodies levels were lower in blood group A and B, respectively. Analyses of correlations between anti-Tn and the other anti-carbohydrates tested revealed divergent patterns of correlations between patients and controls, suggesting qualitative differences in addition to the quantitative difference. Furthermore, anti-Tn levels correlated with anti-S protein levels in the patients' group, suggesting that anti-Tn might contribute to the development of the specific antiviral response. Overall, this first analysis allows to hypothesize that natural anti-Tn antibodies might be protective against COVID-19.

Keywords: COVID-19; O-glycans; Tn antigen; histo-blood group antigens; natural antibodies.

FIGURE 1

Structures of the selected glycan motifs. The Tn, T or core 1 and core 3 motifs correspond to short O-glycans in alpha linkage to either serine or threonine of the peptide chain. The Le^c (Lewis c), GlcNAcLac and αGal epitopes can be present either on N– and O–linked glycans of glycoproteins, or on glycolipids, whilst the Gb3 trisaccharide (globotrihexosyl), also called Pk antigen, is only known on glycosphingolipids. The GlcNAcLac, Gb3 and αGal motifs contain either a lactose or an N-acetyllactosamine inner core (Lac and LacNAc, respectively, in gray). Yellow squares = GalNAc (N-acetylgalactosamine), yellow circles = Gal (galactose), blue squares = GlcNAc (N-acetylglucosamine), blue circles = Glc (glucose). Linkages are indicated as α or β anomers on either position 3 or 4 of the subjacent monosaccharide unit.

FIGURE 2

Relationships between the anti-Tn and anti-αGal levels, ABO phenotypes and infection status. Antibodies levels are shown as OD450 nm values at 1: 30 and 1: 50 plasma dilutions, respectively. Infected patients are defined as SARS-CoV-2 +, regardless of their clinical status or subdivided as asymptomatic and symptomatic COVID-19 +. Controls presented no sign of disease, were RT-PCR negative and anti-S negative. Violin plots show median values and quartiles (horizontal bars) for each group. P-values from Mann-Whitney between-groups comparisons are indicated: *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 3

Relationships between natural anti-carbohydrate antibodies, ABO phenotypes and infection status. Analyzed carbohydrate antigens are indicated on each panel. Antibodies levels are shown as OD450 nm values at 1: 30 plasma dilutions. Infected patients are defined as SARS-CoV-2 +, regardless of their clinical status or subdivided as asymptomatic and symptomatic COVID-19 +. Controls presented no sign of disease, were RT-PCR negative and anti-S negative. Plots show median values and quartiles (horizontal bars). P-values from Mann-Whitney between-groups comparisons are indicated: *p < 0.05.

FIGURE 4

Correlations between levels of anti-Tn natural antibodies and the other assayed natural anti-carbohydrate antibodies. Antibodies levels are shown as OD450 nm values at 1: 30 or 1: 50 (αGal) plasma dilutions. Correlations were assessed using Spearman r and two-tailed p-values after Holm correction for multiple testing are shown. Data sets with p-values <0.05 are boxed.

FIGURE 5

Relationships between the anti-Tn and anti-αGal levels, ABO phenotypes and the levels of anti-S protein. Anti-S protein antibodies of COVID-19 patients were detected and quantified by the S-Flow assay at a plasma dilution of 1: 300. Data are shown as percentage of positive cells. Only positive plasma samples were considered. (A) Correlation between anti-S and anti-Tn, Spearman r p value is shown; (B) correlation between anti-S and anti-αGal, Spearman r P-value is shown; (C) relationship between ABO phenotypes and anti-S. Only anti-S positive individuals (with cut-off values >30%) were considered. P-values from Mann-Whitney comparisons are indicated: *p < 0.05, ***p < 0.001.

FIGURE 6

Expression of the Tn epitope in lung and trachea. (A) Binding of the anti-Tn monoclonal antibody NaM217-2A9 to immobilized polyacrylamide-conjugated carbohydrate epitopes (Tn, A blood group trisaccharide, A type 2 and A type 3), human salivary mucins from A, B or O secretor individuals and desialylated bovine submaxillary mucin (asialoBSM) were assayed by ELISA. Strong reactivity was observed with the Tn-PAA conjugate, and asialoBSM that contains large amounts of Tn epitope. (B) Reactivity of the anti-Tn on cell lines detected by flow cytometry. Jurkat cells known to express large amounts of Tn epitope were used as a positive control. Negative controls are shown as red plots the blue plots showing data in the presence of the anti-Tn NaM217-2A9. (C) Staining of tracheal (middle panel) and bronchial epithelial cells (lower panel) by the anti-Tn NaM217-2A9 shown by the brown-red color (stars) in a blood group B and a blood group O donor, respectively (type B, type O). The upper panel (negative control) shows the lack of staining in absence of the primary anti-Tn antibody. Black bar = 100 μm.