key: cord-0940218-i3q36klc
authors: Chan, Conrad En-Zuo; Ng, Ching-Ging; Lim, Angeline Pei-Chew; Seah, Shirley Lay-Kheng; Chye, De-Hoe; Wong, Steven Ka-Khuen; Lim, Jie-Hui; Lim, Vanessa Zi-Yun; Lai, Soak-Kuan; Wong, Pui-San; Leong, Kok-Mun; Liu, Yi-Chun; Sugrue, Richard J; Tan, Boon-Huan
title: The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies
date: 2022-04-18
journal: bioRxiv
DOI: 10.1101/2021.12.06.471528
sha: c2ff0406e2fcde6e21608a16f52ef9a1c6f1bf91
doc_id: 940218
cord_uid: i3q36klc

A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2- infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. Importance The SARS CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID19 patients to monitor the RBD in cells infected with SARS CoV-2 clinical isolates. These immunological reagents specifically recognise the correctly folded RBD and were used to monitor the appearance of the RBD in SARS CoV-2-infected cells and identified the site where the RDB first appears.

entry into host cells. The isolation of hMAbs from the B cells of convalescent serum can be 125 used in passive immunization for the timely treatment and prevention of SARS-CoV-2 126 infection, and the discovery of hMAbs from COVID19 convalescent patients have shown 127 therapeutic potential [16] [17] [18] [19] . A number of these have been granted emergency authorisation 128 and progressed through clinical trials for use as antibodies therapeutics [20, 21] . Although the 129 emphasis has been on the development of reagents that can block the RBD, the potential use in Singapore. These strains were first detected using PCR primers and thermocycling 155 conditions described in [24] . DSO's Institutional Ethics Review Board (IRB no 0008/2020) 156 approval was sought before using the de-identified nasopharyngeal samples for virus isolation.

The SARS-CoV-2 viruses were isolated in the African green monkey kidney epithelial (Vero 191 Discovery and isolation of human monoclonal antibodies. SC23 and SC29 were generated 192 by single B cell antibody interrogation from convalescent patient sample as described in [19] .

The human monoclonal antibodies, PD4, PD5 and PD7 were isolated from an immune phage 194 display library constructed from the convalescent patients' B cells according to the methods by 195 [27]. PD4, PD5 and PD7 were obtained by biopanning against recombinant expressed CoV-2 S protein using the method described in [19] . 197 Antibodies and specific reagents. The rabbit polyclonal antibody to S protein (polyS) (Sino 198 Biological, Singapore) and anti-N (Thermo Fisher Scientific) were purchased. 199 optimized full-length S gene (nucleotide residues 1 to 1273) was assembled as previously 238 described [19] and cloned into the recombinant pCAGGS vector to create pCAGGS/S. For S1 239 subunit and RBD expression, only residues 1-685 (S1) and 331-524 (RBD) were cloned 240 together with a c-flag and a c-myc tag respectively. The fragments were cloned into pCAGGS 241 to generate pCAGGS/S1 and pCAGGS/RBD. Bulk preparation of all plasmids was performed 242 using the plasmid midiprep kit (Qiagen). Cells (1x10 5 ) were transfected into HEK293 with 1µg In this study we used three SARS-CoV-2 viruses that were isolated during the early 267 phase of COVID19 pandemic in Singapore, which are referred to as SARS-CoV-2/1302, 268 SARS-CoV-2/0563 and SARS-CoV-2/0334. Since the characterization of the S protein hMAbs 269 was the major focus of this study, the complete S protein sequence for each isolate used in our 270 analysis was determined. Each virus isolate was passaged three times in Vero E6 cells and the 271 genetic material extracted from each passage was PCR-amplified with specific primers for the 272 S gene and sequenced using Sanger's sequencing. The sequences from overlapping amplicons 273 were assembled and analyzed. The data revealed that the nucleotide and amino acid sequences amino acid change R682W in the furin cleavage site. This created 682WRARS686 rather than 286 the complete furin consensus sequence 682RRARS686 that is found in the S protein sequence of 287 SARS-CoV-2/WIV04, SARS-CoV-2/0563 and SARS-CoV-2/0334. Virus variants, also 288 known as quasi-species, containing mutations in the region of the S1/S2 protein proteolytic 289 cleavage site have been reported to be present in SARS-CoV-2-infected patients [29] , and these 290 can be selected during passaging of the virus in tissue culture. The basis for this virus selection 291 and the selective advantage that these sequence changes impart to the virus isolate in tissue 292 culture is currently unclear. Apart from the sequence differences indicated above, the 293 remaining S protein sequence of each of the Singapore isolates was 100% identical to the S 294 protein sequence of SARS-CoV-2/WIV04. In particular, the sequence of the S protein RBD 295 was 100% identical in SARS-CoV-2/WIV04 and all three of the Singapore SARS-CoV-2 296 isolates. Since the S protein sequences of the SARS-CoV-2/0563 and SARS-CoV-2/0334 297 isolates were identical, all subsequent work on characterizing immune-reactivity of the hMAb 298 panel was performed mainly using the SARS-CoV-2/1302 and SARS-CoV-2/0334 isolates.

In this this study we selected PD4, PD5, PD7, SC23 and SC29 from the original 300 antibody panel for further characterisation using a cellular virology approach. We failed to 301 detect binding of these antibodies to the S protein using Western blotting (SFig. 2A), but 302 recognition of S protein by each antibody in the panel was confirmed by using the purified 303 SARS-CoV-2 S protein ectodomain in ELISA (Fig. 1A) . In a similar analysis using a sequence 304 that corresponded to the S protein RBD, only PD5, PD7 and SC23 showed RBD binding (RBD-305 binders) (Fig. 1B(i) ). It is presumed that the epitopes recognized by the RBD-binders are 306 formed by different parts of the RBD once it had folded into the correct conformation. This 307 folding pattern is expected to be lost during the sample processing process in the Western blot 308 analysis. The inability of these antibodies to bind to the S protein in Western blot analysis 309 provided evidence that the RBD-binders bind to conformational-specific epitopes on the 310 correctly folded S protein. Although comparable binding affinities to the complete S protein 311 was noted for all hMAbs, the SC23 showed reduced binding-affinity for the RBD compared 312 with PD5 or PD7 (Figs. 1B(ii) and 1C). This indicate that SC23 may recognize a distinct 313 sequence in the RBD and that one or more sequences located outside of the RBD may facilitate 314 its binding to the RBD. No RBD-binding was detected by PD4 and SC29, suggesting that the 315 SC29 and PD4 epitope recognition sequences were outside the RBD region. Although we have 316 not mapped the binding domains of PD4 and SC29, we examined their binding activities using 317 an ELISA-based antibody-binding competition assay (SFig. 2B). This assay indicated that the 318 binding of PD4 and SC29 antibodies to the S protein was mutually exclusive, suggesting that 319 PD4 and SC29 bind to similar locations on the S protein. Prior binding of either the PD5, PD7 320 or SC23 antibodies to the S protein did not interfere with PD4 or SC29 binding, which was 321 consistent with PD4 and SC29 being non-RBD binders (SFig. 2B). Binding of the PD5, PD7 322 and SC23 antibodies to the S protein in the same antibody-binding competition assay were also 323 mutually exclusive indicating binding to similar locations on the S protein and consistent with 324 their RBD-binding properties. There was a general correlation between the RBD recognition 325 and the complementarity-determining regions (CDRs) sequences of the individual antibodies 326 in the hMAb panel. There was a high degree of similarity between the sequence of the CDRs 327 of PD5 and PD7 and to a lesser extent with SC23 (SFig. 2C), which correlated with the different 328 RBD binding activity of SC23. The PD4 and SC29 showed high levels of sequence similarity 329 in the CDR1 and CDR2 in the heavy chain and was distinct from the CDRs in the RBD-binders.

The differential recognition of the RBD was further supported by examining the ability 331 of the hMAb panel to block SARS-CoV-2 binding to Vero E6 cells using a virus neutralization 332 assay. Only PD5, PD7 and SC23 exhibited virus neutralizing activity, while PD4 and SC29 333 failed to inhibit infection (Fig. 1D ). These data indicated that inhibition of SARS-CoV-2 334 infection correlated with RBD recognition, and it is presumed that binding of the antibodies to 335 the RBD would create a steric hindrance that would interfere with the virus attachment to the 336 cell receptor.

The recognition of S protein by each antibody in hMAb panel was further confirmed 339 by examining their immuno-reactivity in SARS-CoV-2-infected Vero E6 cells ( Fig In non-infected Vero E6 cells expressing the recombinant S protein cell lysates were 364 prepared and examined by immunoblotting with polyS ( Fig. 2D (i)). S protein species 365 corresponding in size to the uncleaved S protein (S0) and to the S2 domain were detected, 366 which confirmed that the recombinant S protein was correctly processed in the transfected 367 cells. The recognition of the S protein in the immunoblotting assay by polyS also suggested 368 that recognition of the S protein by this antibody involved one or more of the liner epitopes 369 rather than conformational specific epitopes in the hMab panel. Cells expressing the 370 recombinant S protein were co-stained with polyS and either PD5, PD7, SC23 and SC29 and 371 examined using IF microscopy ( Fig. 2D (ii)-(v)) and staining of the transfected cells with either 372 antibody confirmed the S protein recognition. We also compared the PD5 staining in cells 373 expressing the full-length recombinant S protein with those expressing either only the S1 374 domain or the RBD (Fig. 2E) . A more widespread PD5 staining pattern was observed in cells 375 expressing the S1 domain and the RBD compared with the cell expressing the full-length S 376 protein sequence. This confirmed that the RBD can form into its distinct structure images were extracted at an optical plane from the Z-series that allowed the cytoplasmic 390 staining patterns of the different antibodies to be compared ( Fig 3A) . The polyS antibody 391 exhibited a diffuse cytoplasmic staining pattern, but a small degree of co-localization within 392 the distinct prominent punctate staining pattern exhibited by PD5, PD7 and SC23 was noted.

This punctate staining pattern was also exhibited in PD4-and SC29-stained virus-infected cells, 394 however these antibodies also exhibited an additional prominent diffuse SC29-and PD4-395 staining patterns. These different antibody staining patterns suggested that the PD5, PD7 and 396 SC23 only recognize a specific subpopulation of the total S protein in which the protein 397 conformation renders the RBD accessible to antibody binding. The recognition of the S protein 398 by PD4 and SC29 was not dependent on RBD recognition, and the additional diffuse antibody (Fig. 3D ). This revealed a high level of co-localization between the punctate PD5 staining 425 pattern and the Golgi complex in infected cells (Fig. 3D (ii) and (iii)) and SFig 3F) and 426 confirmed that the punctate PD5 staining pattern was mainly restricted to the Golgi complex.

The PD4 staining exhibited the diffuse antibody staining described above which only partially 428 localized with the anti-giantin staining at the Golgi complex ( Fig. 3D(ii) ). The infected cells 429 were also co-stained with either PD5 or PD4 and anti-NP and imaged using confocal 430 microscopy ( Fig 3E) . The anti-NP labelling gives rise to a prominent cytoplasmic staining 431 pattern that allowed delineation of the infected cells and the absence of nuclei staining with 432 this antibody enables the position of the nucleus to be visualized. This staining combination 433 therefore allows the respective PD5 and PD4 staining pattern to be visualized in the context of 434 the whole cell. 435 We also examined staining pattern of REGEN-10933 and REGEN-10987 whose 436 binding sites in the S protein RBD have been accurately defined using structural biology [31, Golgi-complex. Since the PD5 recognises conformational specific epitopes within the RBD of 466 the S protein, these data suggest that a conformational change in the S protein may occur that 467 leads to the correctly folded RBD at the Golgi complex that enabled PD5 recognition. (Fig. 4D ). As expected, the PD5-staining pattern observed on non-permeabilized 487 cells (Fig. 4D (i) ) was clearly distinct from the PD5 punctate cytoplasmic staining pattern 488 exhibited on permeabilized cells (Fig. 4D (ii) ). The non-permeabilized SARS-CoV-2/0334-489 infected cells were co-stained using PD5 and polyS and examined in greater detail using 490 confocal microscopy. A series of images was recorded in the Z-plane from individual 491 representative co-stained cells which allowed surface staining at the cell top ( Fig. 5A(i) ) and 492 at the cell periphery to be imaged (Fig. 5A(ii) ). Both antibodies exhibited a similar surface 493 staining pattern on the co-stained cells, which appeared as small spots and filament, and was 494 distinct from the cytoplasmic PD5 and polyS staining patterns in permeabilized cells described 495 above. The level of co-localization of these antibodies was examined using the Pearson's and 496 Mander's correlation coefficients (Fig. 5B ), which indicated a high level of co-localization 497 between the two antibodies. A high level of co-staining between both antibodies would be 498 expected since both antibodies would be expected to recognize the same population of the S 499 protein on the surface of infected cells. The surface-staining pattern exhibited by both 500 antibodies was more apparent in a 3-D reconstruction of the permeabilized co-stained infected 501 cell ( Fig. 5C (i) and (ii))), where the filamentous co-staining pattern was clearly distinguished 502 from the spotted antibody staining pattern.

The surface topology of Vero E6 cells infected with SARS-CoV-2 isolates was further 504 examined using scanning electron microscopy (SEM) to better understand the relevance of the 505 different surface-staining patterns detected in the confocal microscope analysis described 506 above. Cells were either mock-infected or infected with SARS-CoV-2/1302 and SARS-CoV-507 2/0334 isolates and at 18 hpi imaged using SEM (Fig. 5D ). Numerous surface projections were 508 observed on the surface of the mock-infected cells that was consistent with the presence of 509 microvilli ( Fig. 5D (i)), and these structures were also present on the surface of cells infected 510 with SARS-CoV-2/1302 ( Fig. 5D(ii) ) and SARS-CoV-2/0334 ( Fig. 5D(iii) ). The SARS-CoV- compared with SARS-CoV-2/0334 virus-infected Vero E6 cells. Since the cells were exposed 545 to similar levels of each virus isolate (moi of 0.1) and examined at the same time of infection, 546 these differences suggested a slower rate of appearance of virus particles (i.e. virus particle 547 assembly) in SARS-CoV-2/1302-infected Vero E6 cells and this was examined further. Vero 548 E6 cell monolayers were infected with SARS-CoV-2/1302 or SARS-CoV-2/0334 using a moi 549 of 0.001 (Fig.6A) , 0.01 (Fig. 6B ) and 0.1 (Fig. 6C) , and at 18 hpi the cell monolayers were co- Since all antibodies showed similar clustered staining patterns, the difference in rate of cell-to-591 cell spread between the two virus isolates was not antibody-specific e.g. due to differences in 592 RBD recognition by PD5.

Our data is consistent with localized cell-to-cell spread in the cell monolayer that 594 correlated with high levels of cell-associated virus. However the reason for the high level of 595 cell-associated virus at this time of infection is currently uncertain and will require further 596 investigation. In this context, high resolution structures of the S protein on isolated SARS-

CoV-2 particles have demonstrated that low levels of the S protein trimer disassociate leaving 598 the S2 domain radiating from the virus envelope in an extended post-fusion conformation [39] . 599 It is possible that the freely exposed fusion peptide at the N-terminus of the S2 protein may isolate in these cells. This suggests that the differences in virus spread may be due to differences 606 between the isolates at the later stages of the virus replication, however the mechanism behind 607 this phenomenon will require further investigation.

Furin is enriched at the Golgi complex [41] , and post-translational cleavage of the S0 611 protein into the S1 and S2 domains at the S1/S2 site would be expected to occur as it is 612 trafficked through the Golgi complex. Furthermore, processing of the N-linked glycan from 613 high-mannose cores to terminally differentiated complex glycans also occurs in the Golgi 614 complex. The S protein contains several potential N-linked glycosylation sites, including two 615 sites within the RBD. We therefore examined if maturation of the associated N-linked glycans 616 and furin cleavage of the S protein was required for the recognition of the RBD by PD5.

Inhibition of the processing of S protein N-linked glycans into complex glycans was 618 performed using the Golgi mannosidase-1 inhibitor deoxymannojirmycin (DMJ) (SFig. 8). We The effect of RVKR-treatment on PD5 recognition in Vero E6 cells infected with 670 SARS-CoV-2/1302 ( Fig 8C) and SARS-CoV-2/0334 (Fig 8D) was also examined. Virus-671 infected Vero E6 cells were either non-treated or treated with 40 µM RVKR at 4 hpi, and at 18 672 hpi the cells were co-stained with polyS and PD5 (Fig. 8C (i) and D(i)) and with anti-NP ( 

In a final analysis we examined if furin cleavage of the S protein was required for the 687 recognition of the RBD by PD5 on the cell surface of Vero E6 cells infected with SARS-CoV-688 2/1302 ( Fig. 9A(i) ) and SARS-CoV-2/0334 ( Fig. 9A(ii) ). While PD5 staining on non-treated 689 cells was detected, a reduced level of PD5 surface staining on drug treated cells infected with 690 either virus isolate was noted. Quantification of the PD5 staining intensity on SARS-CoV-691 2/0334-infected cells indicated an approximate 80% reduction in PD5 staining in RVKR-692 treated cells (Fig. 9A (iii) and (iv)). Individual representative non-treated and RVKR-treated 693 non-permeabilized and permeabilized virus-infected cells that were co-stained with PD5 and 694 polyS was also examined using confocal microscopy and a series of images were recorded in 695 the Z-plane (SFig. 9). An individual representative image slice was extracted from the image 696 series to highlight the differences in the surface-staining intensity (Fig.9B(i) SC29 do not bind to the RBD, an additional punctate staining pattern that resembles that in the 740 RBD-binders was also apparent, suggesting that they also recognise the form of the S protein 741 that is recognised by the RBD-binders. We do not know which domain PD4 and SC29 bind in 742 the S protein trimer, but our data indicates that they are able to recognise multiple different 743 forms of the S protein in a manner similar to polyS antibody. There are currently several high-744 resolution structures that have been described that have used the soluble trimeric S protein 745 ectodomain of both the uncleaved (S0) and the furin cleaved (S1/S2) forms of the S protein 746 [22, 23] . In these structures it has been reported that one of the RBD in the S protein trimer can forms that are recognized by the PD5, PD7 and SC23 is related to the orientation of the RBD.

It is possible that these antibodies recognise an alternative conformational change in the S 760 protein during virus particle assembly that is not related to the elevation of the RBD. We can 761 speculate that since the RBD-binders recognise conformational specific epitopes within the 762 RBD these antibodies are able to detect formation of the correctly folded RBD prior to or during 763 virus particle assembly. Future work will focus on identifying the precise binding sites of these 764 antibodies in the S protein trimer to better understand the conformational changes in the S 765 protein that is recognised by the RBD-binders in virus-infected cells.

Limitations of this study. 767 The work described in this study primarily relies on the use of Vero E6 cells, which is 768 an established and accepted highly permissive cell system used to propagate SARS-CoV-2.

These cells lead to the production of easily definable SARS-CoV2 particles and are useful to representative of the human airway (e.g., nasal epithelial cells), to obtain a complete and more 777 physiological picture of SARS-CoV-2 S protein antibody recognition. 778 We have used low passaged clinical virus isolates that were isolated using tissue culture 779 during the early stages of the pandemic in Singapore. The focus of this study was on the S 780 protein, and in this context the S protein sequence of the individual virus isolates used in this 781 study were genetically characterised. Although we did observe some differences in the S 782 protein sequences that were restricted to the vicinity of the furin cleavage site, the virus isolates 783 showed high levels of sequence identity with the corresponding S protein sequence of SARS 784 CoV-2/WIV04, including the sequence of the RBD. Understanding the complex biology of 785 these virus isolates was not within the scope of the current study, but the differences that we 786 observed in the localised virus transmission of these virus isolates in tissue culture suggest 787 subtle differences in their replication characteristics. While it is unclear how genetic variation 788 in other virus genes would directly influence the recognition of the S protein by the antibodies 789 used in this study, future work on the complete genetic characterisation of these viruses may 790 help to explain these differences in their biological properties that we observe. 

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In each case the associated inset is an enlargement (white bar =500nm). The 1026 individual virus particles on the cell surface (short arrow) and clusters of virus particles 1027 associated with cell microvilli (long arrows) are highlighted. The microvilli on the surface of 1028 mock

Comparison of Vero E6 cells infected with SARS-CoV-2/1302 and

/0334 at different multiplicity of infections. Vero E6 cells were infected with (i) SARS-1031

CoV-2/1302 and (ii) SARS-CoV-2/0334 using a multiplicity of infection (moi) of (A) 0

01 and (C) 0.1, and at 18 hrs post-infection (hpi) the cells were co-stained using PD5 and 1033 polyS, and imaged using immunofluorescence microscopy (objective x20 magnification)

The average numbers of infected cells per infected cell cluster in cells infected with SARS-1036

CoV-2/1302 (1302) virus and SARS-CoV-2/0334 (0334) using a moi of 0.01 is shown

The relative levels of cell-associated (CA) and cell-free (CF) virus infectivity recovered from 1038 cells infected with SARS-CoV-2/0334 (0334) and SARS-CoV-2/1302 (1302) using a moi of 1039 0.01

Temporal appearance of PD5 and anti-polyS staining on Vero E6 cells infected 1042 with the SARS-CoV-2/1302 and SARS-CoV-2/0334

SARS-CoV-2/0334 using a multiplicity of infection of 0.01 1044 and at (i) 6 hrs post-infection (hpi), (ii) 18 hpi, (iii) 24 hpi and (iv) 30 hpi the cells were co-1045 stained with PD5 and polyS. The stained cells were imaged using immunofluorescence 1046 microscopy (objective x20 magnification). A sporadic infected cell at 6 hpi is highlighted 1047 (white arrow), and in both (A) and (B) clusters of

RVKR-treated cells expressing the recombinant S protein were stained with PD5 and polyS, 1055 and imaged using immunofluorescence (IF) microscopy (objective x20 magnification). (ii)

Imaging of polyS-stained NT and RVKR-treated cells expressing the recombinant S protein at 1057 16 hpt (objective x40 magnification)

At 18 hpi, the cells were co-stained using (i) PD5 and polyS, and (ii) anti-NP 1060 and PD5, and imaged using IF microscopy (objective x20 magnification). The reduced size of 1061 the infected cell clusters in RVKR

Furin cleavage of the S protein is required for surface display of the PD5 epitope 1063 in virus-infected Vero E6 cells. (A) Vero E6 cells were infected with (i) SARS-CoV-2/1302 1064 and (ii) SARS-CoV-2/0334 and either non-treated (NT) or treated with

At 18 hrs post-infection (hpi), the cells were non-permeabilized and 1066 stained with PD5 and imaged by immunofluorescence (IF) microscopy (objective x20 1067 magnification). The reduced staining in RVKR-treated cells is highlighted (*). (iii) Non-treated 1068 (NT) and RVKR-treated cells infected with SARS-CoV-2/0334 were non-permeabilized and 1069 stained with PD5 and imaged by IF microscopy

The average image intensity of non-treated (-) and RVKR-treated (+) SARS-CoV-2/0334-1071 infected cells stained with PD5 is shown. n=50 in each case. (B) The non-treated (NT)

SARS-CoV-2/0334-infected cells were (i) non-permeabilized and 1073 (ii) permeabilized, and co-stained with PD5 and polyS. An individual image taken from a Z-1074 stack series of images of representative cells is shown, and the individual channel and merged 1075 images are presented. The punctate cytoplasmic PD5 staining pattern (*) is highlighted

Cells were (i) mock-transfected or transfected with pCAGGS/S and either (ii) non-treated (NT)

or (iii) RVKR-treated (RVKR) or (iii). The non-permeabilized cells were co-stained with PD5 1078 and polyS, and imaged by IF microscopy

SARS-CoV-2/0334, SARS-CoV-2/0563, and SARS-CoV-2/1302. The receptor 1083 binding domain (RBD) at amino acid positions 319-540 (green highlight), deleted sequence at 1084 amino acid position 675-679

The lane representing blank refers to a blank lane and the lanes 1092 representing Anti-Strep refers to incubation with Anti-Strep conjugated to HRP as is the 1093 loading control (positive S protein control). The protein species corresponding in size to the 1094 uncleaved S0 and S2 are highlighted. The Ladder represents protein standards from 37 to 250 1095 kDa

Different hMAbs (hMab2) were then added to the bound S protein and 1098 binding assessed by ELISA. Red = binding of the specific hMab1 inhibits the specific hMab2 1099 antibody binding; while green = binding of the specific hMab1 has no effect on the specific 1100 hMab2 antibody binding. (C) The sequences of the complementarity-determining regions 1101 (CDRs) for member of the antibody panel. The CDR1-3 sequences are shown for the heavy 1102

The reconstructed Z-stack images obtained by confocal microscopy from virus-1104 infected Vero E6 cells stained with the anti-SARS-CoV-2 human monoclonal antibodies

At 18 hrs post-infection (hpi) SARS-CoV-2/0334-infected Vero E6 cells were co-stained with 1106 anti-polyS and either (A) SC23, (B) SC29, (C) PD5, (D) PD7 and (E) PD4, and imaged using 1107 confocal microscopy. A series of images from the same cell were obtained in the Z-projection 1108 and the individual images reconstructed into 3-dimentional images of the co-stained cells

At 18 hpi SARS-CoV-2/0334-infected cells were co-stained with PD5 and anti-giantin, and 1110 imaged using confocal microscopy. A series of images from the same cell were

Z-projection and the individual images reconstructed into 3-dimentional images of the co-1112 stained cells

SARS-CoV-2/0334-infected Vero E6 1115 cells were co-stained with either (A) REGEN-10987 (Reg10987) or (B and C) REGEN-10933 1116 (Reg10933) and anti-NP (recognizes the SARS-CoV-2 N protein). In all cases images were 1117 recorded using immunofluorescence microscopy. Plates (A) and (B)

Direct evidence for cell-to-cell transmission of SARS-CoV-2 particles via cell 1120 surface projections in virus-infected Vero E6 cells. (A) Mock-infected Vero E6 cells and 1121 cells infected with (B) SARS-CoV-2/0334

infected (I) and non-infected (N) cells, the borders between different cells 1124 (broken yellow line), individual virus particles (white arrow head), virus particles on microvilli 1125 (white arrows), virus particles on microvilli spanning different cells (yellow arrow) and 1126 microvilli spanning different cells in mock

B(ii) and C(ii) at 80,000x magnification, C(i) at 1128 30,000x magnification, C(ii) at 60,000x magnification, and D(i) at 16,000x magnification

D(ii) Plates a and b are enlarged images from the area demarcated by the open white boxes in 1130

D(i). The white bars in D(ii) represent 1µm

Vero E6 cells infected with SARS-CoV-2 at low multiplicity of infection and 1132 stained with PD7, SC23, SC29 and polyS. (A) Cells were infected with SARS-CoV-2/0334

SARS-CoV-2/0563 and SARS-CoV-2/1302 as indicated using a multiplicity of infection (moi)

Cells were 1136 infected with (B) SARS-CoV-2/1302 and (C) SARS-CoV-2/0334 using a multiplicity of 1137 infection (moi) of 0.01 and at 18 hpi the cells were stained with PD7

Temporal appearance of PD7 and anti-N protein staining on Vero E6 cells 1140 infected with SARS-CoV-2/1302 and SARS-CoV-2/0334. Cells were infected with

SARS-CoV-2/0334 using a multiplicity of infection of 0.01 and at 1142 (i) 6 hrs post-infection (hpi), (ii) 18 hpi, (iii) 24 hpi and (iv) 30 hpi the cells were co-stained 1143 with PD7 and anti-N protein (NP). The stained cells were imaged using immunofluorescence 1144 microscopy (objective x20). In both (A) and (B), clusters of

A) (i) Cells were mock-transfected and transfected with pCAGGS/S in the 1148 absence (NT) and presence of 1mM deoxymannojirmycin (DMJ) and the cell lysates 1149 immunoblotted with polyS

DMJ-treated cells were either mock-digested (Mock) or treated with EndoH and PNGase 1153 F and the samples immunoblotted with polyS. The migration of the EndoH resistant

In all cases 1155 immunoblotting with anti-tubulin(tub) is the loading control. (B) Non-treated (NT) and DMJ-1156 treated cells expressing the recombinant S protein were co-stained with polyS and PD5 and 1157 imaged by IF microscopy (objective x40 magnification)

The reconstructed Z-stack images obtained by confocal microscopy from non-1162 treated and decanoyl-RVKR-cmk-treated virus-infected Vero E6 cells stained with PD5

At 18 hrs post-infection, (i) non-treated and (ii) decanoyl-RVKR-cmk-treated SARS-CoV-1164 2/0334-infected Vero E6 cells were (A and B) non-permeabilized and

A 1166 series of images from the same cell were obtained in the Z-projection and the individual images 1167 reconstructed into 3-dimentional images of the co-stained cells

The work was supported by DSO National Laboratories, and Nanyang Technological Lay-Tin Aw) for their assistance.

The authors declared no conflict of interest.