key: cord-0276080-xj3sogrr
authors: Samuelsson, Ebba; Mirgorodskaya, Ekaterina; Nyström, Kristina; Bäckström, Malin; Liljeqvist, Jan-Åke; Nordén, Rickard
title: Sialic acid and fucose residues on the SARS-CoV-2 receptor binding domain modulate IgG reactivity
date: 2022-01-20
journal: bioRxiv
DOI: 10.1101/2022.01.20.477056
sha: d70e9118638b61978c95cb0a024e57a7867a51ca
doc_id: 276080
cord_uid: xj3sogrr

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of recombinant RBD produced in different mammalian cells. We found a higher degree of complex type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in Human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it might be possible to enhance the immunogenicity of recombinant viral proteins.

The adaptive immune response to SARS-COV-2 depends on T-cells that directs the immune 38 responses and contributes to killing of infected cells, and on antibody producing B-cells 39 (Rydyznski Moderbacher et al, 2020) . Seroconversion has been detected in 93-99 % of 40 patients with diagnosed SARS-CoV-2 infection, with disease severity correlating with 41 antibody titres (Kellam & Barclay, 2020; Lou et al, 2020; Zhao et al, 2020) . Neutralizing 42 antibodies (NAb) are a key component in the response towards viruses, and an important 43 aspect after immunization is whether the generated antibodies possess neutralizing 44 capabilities (Plotkin & Plotkin, 2008) . In the case of SARS-CoV-2 many neutralizing 45 glycosylation pattern of the S protein is not entirely established, although the presence of 71 several O-linked glycans have been identified within the RBD (Antonopoulos et al, 2021; 72 Bagdonaite et al, 2021; Sanda et al., 2021; Shajahan et al., 2020) . 73

The vaccines against SARS-CoV-2 induce antibodies that after immunization, target specific 75 domains of the S protein. The vector based DNA vaccines and mRNA vaccines utilize the 76 human glycosylation profile on the produced protein, while the glycosylation profile of 77 protein-based sub-unit vaccines is dependent on the cell type used for production (Croset et 78 al, 2012) . As the glycosylation profile of the S protein may affect the antibody epitopes, 79 leading to variability in the effectivity of the vaccine, the glycosylation of the target protein is 80 an important issue to study. 81

In this work we have characterized the glycan content of a recombinant RBD protein 83 expressed in three different mammalian cell lines and showed a diverse glycan composition 84 at each site. The N-and O-linked glycans were stepwise modulated using enzymatic 85 degradation. Serum samples from patients previously infected with SARS-CoV-2 were used 86 to assess the impact of glycan composition on antibody reactivity. A glycan hot spot within 87 the RBD was found to be essential for antibody reactivity. In addition, modulation of the 88 glycan content revealed specific monosaccharides that were able to enhance the antibody 89 reactivity. 90 91

Glycosylation pattern of the recombinant RBD produced in Recombinant RBD, produced in HEK293F-and CHO-S-cells respectively, was subjected to 94 nanoLC-MS/MS analysis. We defined the level of occupancy, composition, and structure of 95 121 In contrast to fucosylation, the sialylation level was lower for HEK293F compared to 1B, and Table 2 ). Also, the degree of sialylation (number of sialic acid residues per 123 glycan) differed between the cell lines (Table EV4) . CHO-S-cells produced multiple 124 sialylated forms in contrast to HEK293F, where mainly mono-sialylated structures were 125 observed. The lower sialylation level in glycans produced by HEK293F is likely a result of 126 the extensive fucosylation in this cell type. In both cell lines the major N-glycan type carrying 127 sialic acid was complex type glycans and a difference between the sites were noted, with N-128 linked glycans at position N331 displaying higher sialylation levels. 129

The O-linked glycans were similar for the two cell types (Fig. 1C , 1D, and Table EV5 ). The 131 O-linked glycan close to the N-terminal domain of the RBD could not be defined to a single 132 amino acid, due to the absence of fragment ions between the two adjacent potential sites, and 133 thus could be placed either at amino acid position T323 or S325. The site T323/S325 was 134 glycosylated to a high degree (97 % and 91 %, for CHO-S and HEK293F respectively), while 135 T523 was scarcely decorated and mainly remained non-glycosylated in both CHO-S-and 136 HEK293F-produced RBD (5 % and 1 %, respectively). Comparison of O-linked glycans at 137 the individual sites revealed more extensive processing and branching in the HEK293F 138 produced protein while the CHO-S produced O-linked glycans almost exclusively consisted 139 of core 1 structures ( Fig. 1C and 1D ). The degree of sialylated structures at site T323/S325 140 were similar between the cell lines (80 % and 82 % for CHO-S and HEK293F, respectively), 141 while the degree of monosialylated structures (66 % and 35 %, respectively) and disialylated 142 structures (15 % and 46 %, respectively) differed. Similarly, the frequency of sialylated 143 structures at site T523 was similar between CHO-S (80 %) and HEK293F (79 %) cells. The 144 degree of monosialylation was higher in the CHO-S produced RBD, as compared to the 145 HEK293F produced protein (38 % and 14 %, respectively), while a higher degree of 146 disialylation was seen on the HEK293F produced RBD (42 % and 62 %, respectively) (Fig. 147 1C and 1D) . 148

In summary, RBD produced in CHO-S cells carried O-linked glycans at two positions 150 although only position T323/S325 appeared to be glycosylated with high frequency. The 151 main type of O-linked glycan found at this position was a core 1 structure with a single sialic 152 acid at the distal galactose (Fig. 1E ). This RBD protein also carried two N-linked glycans at 153 position N331 and N343. The predominant type of N-linked glycan was the biantennary 154 complex type, although many variants of complex type glycans were found. RBD produced 155 in HEK293F cells predominantly carried core 1 O-linked glycans with two sialic acids, one 156 attached to the distal galactose and one to the innermost GalNAc residue. The N-linked 157 glycans on the HEK293F RBD were almost exclusively of complex type with a high degree 158 of fucosylation (Fig. 1F) . 159 160 Evaluation of convalescent sera from Covid-19 patients 161 Serum samples were collected from 24 individuals previously infected with SARS-CoV-2 as 162 determined by a PCR-positive nasopharyngeal sample. Blood samples were collected 25 -163 100 days following positive diagnosis. All sera were characterized with respect to anti-RBD 164

IgG levels and the capability to neutralize a DE-Gbg20 strain of SARS-CoV-2 grown in 165 VERO-cells. Based on the neutralization capability the sera were divided to three groups: 166 non-neutralizing (NT negative, n=7), weakly neutralizing (NT titre 3-6, n=7) and highly 167 neutralizing (NT titre 48-96, n=10) ( Fig. 2 and Table EV6 ). High neutralization capability 168 correlated well with high levels of IgG targeting the RBD, as all highly neutralizing sera also 169 were anti-RBD IgG positive while six of the seven serum samples in the weakly neutralizing 170 group were anti-RBD IgG negative. Interestingly, four out of seven serum samples in the NT 171 negative group were anti-RBD IgG positive. 172 173 Impact of glycan structures on antibody reactivity against RBD 174

To assess the impact of the different types of glycan structures found within the RBD we 175 removed the N-linked, the O-linked or a combination of both glycans using enzymatic 176 treatment. Removal of glycans was verified by a size shift on an SDS-page gel, visualized by 177 silver staining (Fig. EV1A ). The effect of glycan removal on the antibody reactivity against 178 the recombinant RBD was tested using the defined serum samples described above. RBD 179 produced in CHO-S-cells elicited a strong reactivity to the highly neutralizing sera, with 180 reduced reactivity following removal of N-linked glycans, O-linked glycans or a combination 181 of both (Fig. 3A) . The highly neutralizing sera showed reduced reactivity against the RBD 182 with removed N-linked glycans and the RBD lacking both N-and O-linked glycans produced 183 in HEK293F-cells, while no effect on reactivity against the RBD lacking only O-linked 184 glycans was observed (Fig. 3B ). Weakly neutralizing sera did not show any reactivity to the 185 CHO-S-or the HEK293F-produced RBD regardless of the glycosylation profile ( Fig. EV2A  186 and EV2C). The non-neutralizing serum samples displayed low reactivity against all 187 recombinant RBD with only a minor difference depending on glycosylation status (Fig. 188 EV2B and EV2D). The intensity of the reactivity of individual serum samples against the 189 recombinant RBD correlated well with the anti-RBD IgG levels detected in each serum (Fig. 190 EV3). 191 192 To further assess the impact of specific glycan residues on antibody reactivity, sialic acids 193 and fucose groups were enzymatically removed from the RBD. SDS-page gel electrophoresis and subsequent silver stain was used to confirm the 195 removal of sialic acids or fucose groups. Small, but distinct, size shifts were evident after the 196 enzymatic treatments, indicating successful removal of each glycan species (Fig. EV1B and 197 EV1C) . Removal of sialic acids from the CHO-S-produced RBD resulted in a significant 198 increase in the serum reactivity, as compared to fully glycosylated RBD. A similar effect was 199 seen following removal of sialic acids from the HEK293F-produced RBD, however this 200 difference was less prominent (Fig. 3C ). Removal of fucose groups also resulted in a 201 significant increase in serum reactivity for both the 202 with the HEK293F-produced construct showing a more prominent increase (Fig. 3D) . 203

To confirm the impact of sialic acids and fucose groups on the antibody reactivity, the RBD 205 was produced in Lec3.2.8.1-cells deficient in synthesis of complex type glycans. Highly 206 neutralizing serum samples showed a significantly higher reactivity against RBD produced in 207

Lec3.2.8.1-cells, as compared to the CHO-S-or HEK293F-produced RBD constructs (Fig. 208 4A). As expected, enzymatic removal of sialic acids and fucose from the Lec3.2.8.1 produced 209 RBD, did not confer any detectable size shift on an SDS-page gel (Fig. EV1D ) or a change in 210 antibody reactivity by highly neutralizing sera (Fig. 4B) . 211 212 Glycosylation of recombinant RBD produced in In order to verify that the recombinant RBD produced in Lec3.2.8.1-cells lacked complex 214 type glycans, it was subjected to nanoLC-MS/MS analysis. Both N-linked sites were found to 215 be glycosylated to a high degree (98 % and 88 %, respectively). The structural distribution 216 was similar between the sites, with high-mannose as the dominating glycan type (Table 1) . 217

No sialic acid or end-fucose were found, however 6 % of the structures at site N331 and 31 % 218 of the structures at site N343 carried core fucose (Table 2 ). Position T323/S325 was 219 frequently (98 %) decorated with an O-linked glycan, while site T523 were more sparsely 220 decorated (15 %). A single HexNAc was the most frequent structure at both O-linked sites 221 (Table EV5) (Brun et al, 2021; Nordén et al, 2015; Nordén et al., 2019) . This could imply that the 242 antibody response to a viral glycoprotein is more diverse than previously thought. Hence, 243 serum from infected individuals contain a polyclonal antibody pool which could recognize 244 multiple epitopes and various glycoforms can constitute parts of these epitopes. The S protein 245 of SARS-CoV-2 is highly glycosylated, with 17 to 22 previously identified sites carrying N-246 linked glycosylation that can shield B cell epitopes (Allen et al., 2021; Antonopoulos et al., 247 2021; Sanda et al., 2021) . Of these, 2 N-linked glycans are present in the RBD, and Yang et 248 al. identified as many as 10 O-linked glycans in this region, although most of them appeared 249 to be of low abundance and their biological significance is therefore uncertain (Yang et al, we found significant differences in the amount of sialic acid and fucose content when 270 comparing the RBD produced in CHO-S cells and HEK293F cells respectively. Interestingly, 271 the CHO-S-produced RBD also presented with a high degree of mannose-6-phospate (M-6-272 P). This structure has previously been observed in the SARS-CoV-2 spike protein when 273 expressed in cell lines but also when isolated from intact viral particles (Brun et al., 2021; 274 Gstöttner et al, 2021) . Mannose-6-phosphate is recognized by the M-6-P receptor present in 275 the trans-Golgi compartment and it directs tagged proteins to late endosomes/lysosomes. 276

Lysosomal egress dependent on M-6-P has been described for both HSV (26) and VZV (27). 277

Also, SARS-CoV-2 egress mediated by lysosomes has been proposed (Ghosh et al, 2020) . 278

However, we observed only a minor fraction of the peptides carrying M-6-P and to what 279 extent they potentially could contribute to viral particle egress remains to be clarified. 280 281 While each glycosite on the recombinant RBD was glycosylated at a similar frequency 282 independent of production cell line, RBD produced in HEK293F had a higher degree of 283 fucosylation compared to CHO-S. Selective removal of the fucose groups resulted in a 284 significantly increased antibody reactivity. While abundant fucosylation was a trait of the 285 HEK293F produced construct, RBD produced in CHO-S cells had a larger content of sialic 286 acid moieties. Selective removal of sialic acids enhanced antibody reactivity for both 287 constructs, but the effect was more prominent for the CHO-S construct. The use of the 288 Lec3.2.8.1-cell line resulted in RBD with a glycosylation profile completely deficient in 289 sialic acids and end-fucose. Consequently, the antibody reactivity towards the Lec3.2.8.1-290 produced RBD was enhanced and additional removal of the core-fucose did not result in any 291 change in the antibody reactivity. Altogether, these results points to an important function of 292 specific terminal-sugar residues in the antibody reactivity against glycosylated viral antigens 293 and suggests that that core-fucosylation is of minor importance, despite the report of an NAb 294 that specifically interacts with the core fucose of the N-linked glycan situated on position 295 N343 (Pinto et al., 2020) . 296

In line with our findings that removal of sialic acids leads to increased antibody reactivity, the 298 non-sialylated glycan structures of yeast-cell produced proteins could possibly be part of the 299 explanation of the highly efficient yeast-produced vaccines against HBV (Doering et al, 300 2015; Ho et al, 2020) . This suggests that it is possible to optimize recombinantly expressed 301 RBD or S proteins in order to generate effective vaccine candidates. However, important to 302 note is the possibility that immunization with a recombinant expressed subunit vaccine 303 directs the humoral immune response towards B-cell epitopes with species-specific 304 glycosylation profiles. This can possibly result in skewed immunodominance, directing the 305 antibody response towards epitopes that are not exposed after a natural infection with the 306 virus, resulting in disturbed efficiency of the vaccine (Abbott & Crotty, 2020). The data 307 presented in this work confirms the necessity of correct glycosylation, and shows that also 308 small differences in the glycosylation profile of a viral antigen can have a large impact on the 309 reactivity by antibodies generated after a natural infection with SARS-CoV-2. A conscious 310 decision regarding the glycosylation traits of the production cell line could hence affect the 311 antibody response triggered by a recombinant protein. We suggest the glycosylation 312 characteristics should be considered during the production of recombinant vaccines towards 313 SARS-CoV-2 but also other enveloped viruses which carry glycoproteins. 314 315

The receptor-binding domain of the SARS-CoV-2 spike protein (amino acids 319-541) was 318 produced in three cell lines using an expression vector obtained through BEI Resources, 319 NIAID, NIH, which is vector pCAGGS containing the SARS-CoV-2, Wuhan-Hu-1 spike 320 glycoprotein gene RBD with C-terminal Hexa-Histidine tag (NR-52309) (Table EV1) The RBD proteolytic preparations were analyzed on a QExactive HF mass spectrometer 370 interfaced with Easy-nLC1200 liquid chromatography system (Thermo Fisher Scientific). 371

Peptides were trapped on an Acclaim Pepmap 100 C18 trap column (100 μm x 2 cm, particle 372 size 5 μm, Thermo Fischer Scientific), and separated on an in-house packed analytical 373 column (75 μm × 300 mm, particle size 3 μm, Reprosil-Pur C18, Dr. Maisch) using a 374 gradient from 7 % to 50 % B over 75 min, followed by an increase to 100 % B for 5 min at a 375 flow of 300 nL/min, where Solvent A was 0. 

The antibody reactivity towards glycosidase treated proteins were assessed using an enzyme-464 linked immunosorbent assay (ELISA). Briefly, Nunc Maxisorp™ 96-well plates (Thermo 465

Fischer Scientific) were coated with 0.1 µg glycosidase treated peptides or heat-treated 466 controls diluted in carbonate buffer (pH 9.6). Coating was performed over night at 4 °C 467 followed by washing three times with 0.05 % tween20 in phosphate buffered saline (PBS). 468

The plates were blocked in 2 % milk for 30 minutes at room temperature prior to addition of 469 sera (diluted 1:100 in 1 % milk in PBS with 0.05 % tween20) and 1.5 hours incubation at 37 470 °C. The plates were washed three times before addition of alkaline phosphatase-conjugated 471 (1)Hex (1) HexNAc (2)Hex (1) HexNAc (1)Hex (1)NeuAc (1) HexNAc (2)Hex (2) HexNAc (3)Hex (1) HexNAc (2)Hex (1)NeuAc (1) HexNAc (1)Hex (1)NeuAc (2) HexNAc (2)Hex (2)NeuAc (1) HexNAc (3)Hex (1)NeuAc (1) HexNAc (2)Hex (2)NeuAc (2) 

RBD following removal of N-linked, O-linked or both N-linked and O-linked glycans

Antibody reactivity of weakly neutralizing sera (NT nitre 3-6, n=7) against RBD produced in

Antibody reactivity of non-neutralizing sera (NT negative, n=7) against

Antibody reactivity of weakly neutralizing sera (NT nitre 745 3-6. n=7) against RBD produced in HEK293F-cells. D. Antibody reactivity of non-746 neutralizing sera (NT negative, n=7) against RBD produced in HEK293F-cells