1. Introduction

Figure 1. Chemical structures of heparin and marine sulfated glycans.

Figure 1. Chemical structures of heparin and marine sulfated glycans.

2. Results

2.1. SARS-CoV-2 Variants and XBB S-protein RBD Mutations

Throughout the Covid-19 pandemic numerous SARS-CoV-2 variants have emerged and posed weighty challenges to both human health and global healthcare systems. Several of these variants are of particular concern due to their increased transmissibility, reduced vaccine and antibody effectiveness and increased virulence. Five variants of concern (VOC) have been declared by the WHO - Alpha (V1, B.1.1.7), Beta (V2, B.1.351), Gamma (V3, P.1), Delta (B.1.617.2) and Omicron (B.1.1.529). The latter was identified in November 2021 both in South Africa and Botswana and named as Omicron [2]. Studies showed that this variant has many mutations leading to an increased risk of reinfection and transmissibility [21,22]. Omicron rapidly spread worldwide and became the main variant. As the pandemic evolved, a number of new Omicron subvariants emerged, including BA.1, BA.2, BA.2.75, BA.2.12.1, BA.4, BA.5 and XBB (Figure 2A). XBB emerged and became predominant in India and Singapore in September 2022, and soon thereafter this variant became the dominant variant in several countries [23]. By the end of 2022, XBB’s sublineage XBB.1.5 outcompeted other VOCs, and became the most dominant variant in the USA. Sequence comparison between WT and XBB.1.5 showed that 21 amino acid mutations emerged in the S-protein RBD (Arg319-Phe541, Figure 2B). Among these amino acid mutations, XBB.1.5 harbors an F486P substitution, which enables the XBB.1.5 subvariant outcompete other Omicron variants.

Figure 2. Omicron phylogenetic tree and S-protein RBD amino acid multiple sequence alignment. (A) Omicron phylogenetic tree, adapted from Nextstrain and CoVariants. (B) Mutation profile of S-protein RBD of WT and XBB.1.5 strains. Multiple sequence alignment was carried out by Clustal Omega (1.2.4). Asterisks (*) indicate positions with a single, fully conserved residue.

Figure 2. Omicron phylogenetic tree and S-protein RBD amino acid multiple sequence alignment. (A) Omicron phylogenetic tree, adapted from Nextstrain and CoVariants. (B) Mutation profile of S-protein RBD of WT and XBB.1.5 strains. Multiple sequence alignment was carried out by Clustal Omega (1.2.4). Asterisks (*) indicate positions with a single, fully conserved residue.

2.2. Binding Affinity and Kinetics Measurement of Heparin-SARS-CoV-2 S-proteins Interactions

Heparin/HS is a group of highly sulfated, polydisperse anionic linear polysaccharides which consist of variably repeating disaccharide building blocks, D-glucuronic acid (GlcA) or L-iduronic acid linked to N-acetylated or N-sulfated glucosamine [25]. Heparin/HS is widely expressed in the extracellular matrix and on the surface of mammalian cells. Through binding and regulating a wide range of proteins, heparin/HS regulates various biological processes such as blood coagulation, tumor metastasis and pathogen invasion [26]. Heparan sulfates are covalently attached to various core proteins in the extracellular matrix and at the cell surfaces forming HS proteoglycans (HSPGs), which play critical roles in pathogen infection, especially in cellular attachment. Many studies suggest that the highly negatively charged and ubiquitously expressed HSPGs provide an ideal adhesive primary attachment point for viruses [27,28,29]. Heparin, the highest sulfated GAG, is well studied as an anticoagulant, and heparin and its analogs are inhibitors to different viruses through blocking viral-HSPGs interactions. Our previous studies showed that full-length heparin and its oligomers can interact with some viral proteins, such as monkeypox A35/A29 proteins, SARS-CoV-2 S-proteins and respiratory syncytial virus glycoproteins [19,30,31]. In this study, a heparin SPR chip was prepared to measure the binding kinetics of heparin and S-protein interactions using S-protein RBD from WT and XBB.1.5 variants. Sensorgrams for interactions of heparin with these two S-protein RBDs are shown in Figure 3.

Figure 3. SPR sensorgrams of S-protein RBD of WT and XBB.1.5 binding to heparin. SPR sensorgrams of S-protein RBD binding with heparin; (A) WT and (B) XBB.1.5. Concentrations of S-protein RBD (from top to bottom) are 1000, 500, 250, 125, and 63 nM, respectively.

Figure 3. SPR sensorgrams of S-protein RBD of WT and XBB.1.5 binding to heparin. SPR sensorgrams of S-protein RBD binding with heparin; (A) WT and (B) XBB.1.5. Concentrations of S-protein RBD (from top to bottom) are 1000, 500, 250, 125, and 63 nM, respectively.

The binding kinetics and affinity (k_a, association rate constant; k_d, dissociation rate constant; and K_D=k_a/k_d, binding equilibrium dissociation constant) between SARS-CoV-2 S-protein RBD (both WT and XBB.1.5) and heparin were obtained by globally fitting the association and dissociation phases using a 1:1 Langmuir binding model. The kinetic parameters of the interaction between SARS-CoV-2 S-protein RBD (WT and XBB.1.5) with heparin are shown in Table 1. The binding affinities of S-protein RBD with heparin are nanomolar: XBB.1.5 (K_D =160 nM) is slightly stronger than WT (K_D =350 nM). From Figure 2 we can find that among these 20 mutations in the S-protein RBD region, half of the mutations result in reduced amino acid hydrophilicity, while others enhanced the hydrophilicity. Among amino acids from N440 to T500, there are nine mutations, eight of which enhance hydrophilicity. Notably, F486P is known to aid the virus escape the immune system’s detection. These relatively concentrated mutations that enhance hydrophilicity could be responsible for XBB.1.5’s slightly stronger binding affinities than WT. In addition, interactions between protein and heparin are mainly based on the electrostatic attraction, and therefore, negatively charged GAGs are expected to interact with positively charged amino acids including lysine (K), arginine (R), and histidine (H). Comparing the sequences of S-protein of XBB with WT (Figure 2), an additional six positively charged amino acid residues are found on the XBB RBD, which can enhance heparin binding affinity.

Table 1. Kinetic data of interactions of S-protein RBD of WT and XBB.1.5 with heparin*.

Table 1. Kinetic data of interactions of S-protein RBD of WT and XBB.1.5 with heparin*.

* The data with (±) in parentheses are the standard deviations (SD) from global fitting of five injections.

2.3. SPR Solution Competition between Surface-Immobilized Heparin and Isostichopusbadionotus-sourced Sulfated Glycans IbSF, IbSFucCS, desIbSF and desIbFucCS

Two sulfated glycans, sulfated fucan (IbSF) and fucosylated chondroitin sulfate (IbSFucCS), were originally isolated and characterized from Isostichopus badionotus (a species of sea cucumber in the family Stichopodidae) by Chen et.al [32,33]. The structure of IbSF is [→3)-α-Fuc2,4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→3)-α-Fuc-(1→]_n and IbFucCS’s structure is of [→3)-β-GalNAc4,6S-(1→4)β-GlcA[(3→1)Y]-(1→]_n, where Y = α-Fuc2,4S (96%) or α-Fuc4S (4%) (Figure 1). Both IbSF and IbFucCS showed good anticoagulant and antithrombotic activities. Our previous study demonstrated that these two marine sulfated glycans also were promising inhibitors towards monkeypox virus (MPXV). Their fully chemical desulfated derivatives, desulfated IbSF (desIbSF) and IbFucCS (desIbFucCS) were prepared as described previously [34], and showed weak competitive inhibition activity between heparin and MPXV A29 and A35 proteins [19]. Pomin’s group indicated that IbSF and IbFucCS showed excellent anti-SARS-CoV-2 activity on both WT and Delta variants, by disrupting the entry of virus into the host cells [17].

SPR was used to perform solution/surface competition experiments for studying the ability of Isostichopus badionotus-sourced glycans (IbSF, IbFucCS, desIbSF and IbFucCS) to inhibit the interactions between SARS-CoV-2 S-proteins (WT and XBB.1.5) and immobilized heparin (Figure 4A,C). The same concentration of Ib glycans (5 µg/mL) was mixed with 1 µM S-proteins (WT and XBB.1.5 individually). Both Ib-sourced sulfated glycans, IbSF and IbFucCS, significantly inhibited the binding of surface-immobilized heparin to the S-proteins (WT and XBB.1.5). Soluble heparin inhibited the binding of WT and XBB.1.5 SARS-CoV-2 S-protein to surface-immobilized heparin by 53.4% and 54.6%, respectively (Figure 4B,D). IbSF and IbFucCS had a better result on the inhibition of immobilized heparin binding to WT S-protein, with 94.8% and 99.5%, respectively. At the same time, IbSF and IbFucCS also showed very strong inhibition activity, with normalized XBB. 1.5 ratio of 92.5% and 91.3%, respectively. The fully chemical desulfated derivatives, desIbSF and desIbFucCS, showed significantly lower competitive inhibition of heparin binding to both WT and XBB.1.5 S-proteins. Our results indicate that sulfation of these marine-sourced glycans play a critical role in the interaction of S-proteins and their anti-SARS-CoV-2 activity.

Figure 4. Solution competition between heparin and Ib glycans. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with different Ib glycans. The concentration of the WT SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Ib glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Ib glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different Ib glycans. The concentration of the the XBB.1.5 SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Ib glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Ib glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control, **: p ≤ 0.01 compared with the control).

Figure 4. Solution competition between heparin and Ib glycans. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with different Ib glycans. The concentration of the WT SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Ib glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Ib glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different Ib glycans. The concentration of the the XBB.1.5 SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Ib glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Ib glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control, **: p ≤ 0.01 compared with the control).

2.4. SPR Solution Competition between Surface-Immobilized Heparin and Holothuria floridana-Sourced Glycans HfSF and HfFucCS

Two marine-sulfated glycans, HfSF and HfFucCS, from the sea cucumber Holothuriafloridana (Hf) were firstly reported by Shi et al. 2019. HfSF is a sulfated fucan whose structure is [→3)-α-Fuc2,4S-(1→3)-α-Fuc-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→]_n, while HfFucCS is a fucosylated chondroitin sulfate structure with the following structure [→3)-β-GalNAc4,6S-(1→4)-β-GlcA-[(3→1)Y]-(1→]_n, where Y = αFuc2,4S (45%), α-Fuc3,4S (35%), or α-Fuc4S (20%) [35] (Figure 1). These two Hf glycans were reported to have good inhibition activities towards some SARS-CoV-2 variants and MPXV [17,19].

Again, we used solution/surface competition SPR experiments for studying the ability of HfSF and HfFucCS to inhibit the interactions between SARS-CoV-2 S-proteins (WT and XBB.1.5) and immobilized heparin. The same concentration of Hf glycans (5 µg/mL) was mixed with 1 µM S-proteins (WT and XBB.1.5 individually). Solution competition SPR results are shown in Figure 5A,C. Heparin inhibited the binding of WT and XBB.1.5 S-proteins binding to surface-immobilized heparin by 53.4% and 54.5%, respectively. HfSF and HfFucCS showed excellent activity for the inhibition of WT S-protein binding to surface-immobilized heparin, with 88.9% and 93.1%, respectively. Meanwhile, HfSF and HfFucCS also showed good results for the inhibitions of XBB.1.5 S-protein binding to surface-immobilized heparin, with 84.0% and 83.8%, respectively (Figure 5B,D).

Figure 5. Solution competition between heparin and Hf glycans. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with different Hf glycans. The concentration of the WT SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Hf glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Hf glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different Hf glycans. The concentration of the XBB.1.5 SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Hf glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Hf glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control).

Figure 5. Solution competition between heparin and Hf glycans. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with different Hf glycans. The concentration of the WT SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Hf glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Hf glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different Hf glycans. The concentration of the XBB.1.5 SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different Hf glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different Hf glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control).

2.5. SPR Solution Competition between Surface-Immobilized Heparin and two Marine-Soured Sulfated Glycans LvSF and PpFucCS

Sulfated fucan LvSF is a polysaccharide isolated from the sea urchin Lytechinus variegatus with the structure of [→3)-α-Fuc2,4S-(1→3)-αFuc2S-(1→3)-α-Fuc2S-(1→3)-α-Fuc4S-(1→]_n [36], while the fucosylated chondroitin sulfate PpFucCS is isolated from the sea cucumber Pentacta pygmaea with a structure of [→3)-β-GalNAcX(1→4)-β-GlcA-[(3→1)Y]-(1→]_n, where X = 4S (80%), 6S (10%), or non-sulfated (10%), and Y = α-Fuc2,4S (40%), αFuc2,4S(1→4)-α-Fuc (30%), or α-Fuc4S (30%) (Figure 1) [37].

SPR was applied to perform solution/ surface competition experiments for studying the ability of the marine-sourced glycans LvSF and PpFucCS to inhibit the interactions between SARS-CoV-2 S-proteins (WT and XBB.1.5) and immobilized heparin. The same concentration of Hf glycans (5 µg/mL) was mixed with 1 µM S-proteins (WT and XBB.1.5 individually). Solution competition results between these marine-sourced glycans and heparin are indicated in Figure 6A,C. Heparin inhibited the binding of WT and XBB.1.5 S-proteins binding to surface-immobilized heparin by 53.4% and 54.5%, respectively. PpFucCS and LvSF showed excellent results for the inhibitions of WT S-protein binding to surface-immobilized heparin, with 97.9% and 86.0%, respectively. Meanwhile, PpFucCS and LvSF also showed good results for the inhibitions of XBB.1.5 S-protein binding to surface-immobilized heparin, with 91/5% and 88.6%, respectively (Figure 6B,D).

Figure 6. Solution competition between heparin and PpFucCS and LvSF. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with different PpFucCS and LvSF. The concentration of the WT SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different PpFucCS and LvSF glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different PpFucCS and LvSF glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different PpFucCS and LvSF glycans. The concentration of the the XBB.1.5 SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different PpFucCS and LvSF glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different PpFucCS and LvSF glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control).

Figure 6. Solution competition between heparin and PpFucCS and LvSF. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with different PpFucCS and LvSF. The concentration of the WT SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different PpFucCS and LvSF glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different PpFucCS and LvSF glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different PpFucCS and LvSF glycans. The concentration of the the XBB.1.5 SARS-CoV-2 S-protein was 1 µM mixed with 5 µg/mL of different PpFucCS and LvSF glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different PpFucCS and LvSF glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control).

2.6. SPR Solution Competition between Surface-Immobilized Heparin and two Marine-Soured Sulfated Glycans RPI-27 and RPI-28

RPI-27 and RPI-28 are a family of sulfated heteropolysaccharides derived from the brown seaweed Saccharina japonica consisting of two types of polysaccharide backbones: (1) a sulfated glucuronomannan and a glucuronomannan backbone with repeating 4-linked GlcA and 2-linked mannose (Man), and a Man residue with the first C-6 sulfated mannopyranose residue from the nonreducing terminus (2) a glucuronan with a backbone of 3-linked GlcA. There are some other branched chains including GlcA-(1→3)-Man-(1→4)-GlcA, Man-(1→3)-GlcA-(1→4)-GlcA, Fuc-(1→4)-GlcA and Fuc-(1→3)-Fuc. (Figure 1) [38]. RPI-27 and RPI-28 share the same structure but have a different average molecular weight with 100 kDa and 12 kDa respectively.

In this competition SPR analysis, the same concentration of RPI-27 and RPI-28 glycans (50nM) was mixed with 1 µM S-proteins (WT and XBB.1.5 individually). Solution competition results between these glycans and heparin are shown in Figure 7A,C. Heparin inhibited the binding of WT and XBB.1.5 S-proteins binding to surface-immobilized heparin by 53.4% and 54.6%, respectively. RPI-27 and RPI-28 showed better results for inhibition of WT S-protein binding to surface-immobilized heparin, with 83.7% and 75.4%, respectively. Meanwhile, RPI-27 and RPI-28 also showed good results for the inhibitions of XBB.1.5 S-protein binding to surface-immobilized heparin, with 68.3% and 74.4%, respectively (Figure 7B,D).

Figure 7. Solution competition between heparin and RPI-27/ RPI-28. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with RPI-27/ RPI-28. The concentration of the WT SARS-CoV-2 S-protein was 1µM mixed with 5 µg/mL of different RPI-27/ RPI-28 glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different RPI-27/ RPI-28 glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different RPI-27/ RPI-28 glycans. The concentration of the the XBB.1.5 SARS-CoV-2 S-protein was 1µM mixed with 5 µg/mL of different RPI-27/ RPI-28 glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different RPI-27/ RPI-28 glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control).

Figure 7. Solution competition between heparin and RPI-27/ RPI-28. (A) SPR sensorgrams of the WT SARS-CoV-2 S-protein–heparin interaction competing with RPI-27/ RPI-28. The concentration of the WT SARS-CoV-2 S-protein was 1µM mixed with 5 µg/mL of different RPI-27/ RPI-28 glycans. (B) Bar graphs (based on triplicate experiments with standard deviation) of normalized WT SARS-CoV-2 S-protein binding preference to surface heparin by competing with different RPI-27/ RPI-28 glycans. (C) SPR sensorgrams of the XBB.1.5 SARS-CoV-2 S-protein–heparin interaction competing with different RPI-27/ RPI-28 glycans. The concentration of the the XBB.1.5 SARS-CoV-2 S-protein was 1µM mixed with 5 µg/mL of different RPI-27/ RPI-28 glycans. (D) Bar graphs (based on triplicate experiments with standard deviations) of the normalized XBB.1.5 SARS-CoV-2 S-protein binding preference to surface heparin by competing with different RPI-27/ RPI-28 glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control).

All eight naturally occurring marine-sourced sulfated glycans (IbSF, IbFucCS, HfSF, HfFucCS, PpFucCS, LvSF, RPI-27, PRI-28) showed the ability to inhibit the interactions between SARS-CoV-2 S-proteins (both WT and XBB.1.5) and surface-immobilized heparin. However, the chemically desulfated glycans, both desIbSF and desIbFucCS, showed significantly reduced binding activity of both S-proteins to surface-immobilized heparin (Table 2). Our study shows that sulfation plays critical roles in the inhibitory activity of marine sulfated glycans. All eight naturally occurring marine-sourced sulfated glycans exhibited outstanding inhibition activity against surface-immobilized heparin binding with WT and XBB. 1.5 SARS-CoV-2 S-proteins. Among the three different marine-sourced fucosylated chondroitin sulfates, IbFucCS had the highest sulfation level (96% branching disulfated fucoses) and exhibited the best inhibitory activity towards WT S-protein. HfFucCS (80% branching disulfated fucoses) shows slightly better inhibitory activity than PpFucCS (70% branching disulfated fucoses) against both WT and XBB.1.5. Clearly, sulfation levels are an important factor for the inhibitory activities of marine-sourced FucCS glycans. Despite sharing the same fucan tetrasaccharide repeating unit, IbSF, LvSF and HfSF differ in their sulfation patterns. The higher sulfated LvSF has pentasulfated tetrasaccharide building blocks, while IbSF and HfSF have tetrasulfated tetrasaccharide building blocks. Among them, IbSF showed the best inhibitory activity, while LvSF and HfSF showed very similar inhibitory activities. This suggests that sulfation pattern has a more pronounced influence on the interactions with SARS-CoV-2 S-proteins than the degree of sulfation. RPI-27 and RPI-28 showed similar inhibitory activity, indicating that no obvious correlations between this seaweed-derived fucoidan molecular weight and binding properties. Despite the strong inhibitory activity of all tested sulfated glycans against both viral proteins in the SPR-based binding to the surface-immobilized heparin, there are no clear correlations between the structural features of these glycans and their binding properties. Similar results were found in our previous study on the inhibitory activity of these sulfated glycans against other evolving SARS-CoV-2 strains and Monkeypox virus [17,19].

Table 2. Summary of solution competition between heparin and eight marine-derived glycans binding to S-proteins ^a.

Table 2. Summary of solution competition between heparin and eight marine-derived glycans binding to S-proteins ^a.

Normalized S-protein binding	Controlb	Heparin	IbSF	desIbSF	IbFucCS	desIbFucCS
WT (%)	100	46.6*	5.2**	77.7**	0.5*	89.8*
XBB.1.5 (%)	100	45.4*	7.5**	58.4*	8.7*	72.9**
Normalized S-protein binding	HfSF	HfFucCS	PpFucCS	LvSF	RPI-27	RPI-28
WT (%)	11.1*	6.9*	2.1*	14.0*	16.3*	24.6*
XBB.1.5 (%)	16.0*	16.2*	8.5*	11.4*	31.7*	25.5*

^aSPR sensorgrams of WT/XXB.1.5 S-protein–heparin interaction competing with 10 different marine-derived glycans. The concentration of proteins was 1 µM mixed with 5 µg/mL of different glycans. ^bThe control had the same concentration of WT/XXB.1.5 S-protein (1 µM) without any mixed glycans. Statistical analysis was performed using an unpaired two-tailed t-test (*: p ≤ 0.05 compared with the control, **: p ≤ 0.01 compared with the control).

3. Materials and Methods

4. Conclusions

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