key: cord-0312574-t93vc14m
authors: Garrido, Jose L.; Medina, Matias; Bravo, Felipe; McGee, Sarah; Fuentes, Francisco; Calvo, Mario; Bowman, James W.; Bahl, Christopher D.; Barría, Maria Inés; Brachman, Rebecca A.; Alvarez, Raymond A.
title: Humoral immune responses against seasonal coronaviruses predict efficiency of SARS-CoV-2 spike targeting, FcγR activation, and corresponding COVID-19 disease severity
date: 2021-09-16
journal: bioRxiv
DOI: 10.1101/2021.09.14.460338
sha: c06d958bb5c4eb67fca810c57470da1e5ee24442
doc_id: 312574
cord_uid: t93vc14m

Despite SARS-CoV-2 being a “novel” coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (FcγR2a & FcγR3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2’FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2’FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.

While SARS-CoV-2 has now evolved variant strains that could potentially act like heterologous 140 strains, these strains only arose several months after the initial outbreak and cannot account 141

for any ADE-like effects observed early on in severe COVID-19 patients. In contrast, the 142 seroprevalence of seasonal human coronaviruses (hCoVs) is fairly ubiquitous [53] , with some 143 seasonal hCoVs sharing regions of high sequence identity with SARS-CoV-2. In particular, the 144 spike protein of SARS-CoV-2, which is the predominant viral antigen targeted by neutralizing 145

Abs, contains regions of high sequence conservation, particularly in the S2 subunit. Notably, the 146 S2 subunit contains the structural domains required for viral fusion [54] . In contrast, the RBD-147

containing S1 subunit bears far less sequence similarity with seasonal hCoVs. Importantly, the 148 RBD region interacts with host ACE2 receptors, mediating viral attachment, fusion, and entry 149 into cells, and is therefore the predominant site against which neutralizing Abs have been found 150

to be directed in convalescent individuals [55] [56] [57] . Indeed, recent studies have found that SARS-151

CoV-2 naïve individuals possess IgG directed against the S2, but not the S1, subunit of the spike 152

protein [58] [59] [60] . Additionally, some pre-pandemic serum samples were also found to have 153 reactivity to the S2 subunit of the spike [58, 61] . Interestingly, pre-pandemic samples that 154 displayed high SARS-CoV-2 reactivity also displayed high reactivity to the spike protein of OC43, 155 a seasonal hCOV, and were found to be non-neutralizing against SARS-CoV-2 [58] . These 156 findings show that IgG targeting of the S2 region exists prior to SARS-CoV-2 infection in some 157 individuals, with the potential for these responses to become amplified upon SARS-CoV-2 158

infection. Indeed, some studies have already observed that cross-reactive Ab responses against 159

OC43 spike protein become elevated in SARS-CoV-2 convalescent individuals [10, 58, 62] . 160

However, the contribution of seasonal hCoV cross-reactive IgG to the humoral response against 161 SARS-CoV-2 remains unclear, with some studies showing no correlation with severity [63], while 162

others observed a correlation with severity [10, 62] .

Beyond the broad characterization of IgG S1 vs S2 binding, other studies have conducted 165 peptide walks to more precisely identify the linear epitope hotspots targeted by humoral 166 responses against the spike protein. These studies have identified several immunodominant 167 regions which overlap or flank several functional features in the SARS-CoV-2 spike protein, such 168 as the S1/S2 junction, S2' fusion peptide site, and the HR1 and HR2 sites [64] [65] [66] [67] . When these 169

immunodominant regions were examined in relation to COVID-19 severity, several epitopes 170

were highly correlated with severity [64, 66] . Interestingly, some epitopes were also highly 171

conserved amongst seasonal coronaviruses [62, 68] . These findings, coupled with IgG-172 recognition of the S2 region in naïve individuals and the elevated levels of OC43 cross-reactive 173

IgG in convalescent individuals, suggest that preexisting recall responses against seasonal 174 coronaviruses likely contribute to the humoral response against SARS-CoV-2. However, the 175 contribution of these cross-reactive Abs to the evolution of effective humoral immunity, 176 disease severity, and outcomes remains unclear; previous research has failed to establish a 177 conclusive, significant contribution-either protective or deleterious. However, these studies 178 either relied on pooled analyses, which masks complex signatures, or did not examine disease 179 severity. Therefore, there is an immediate need to characterize the FcγR activation and Ab 180 targeting profiles associated with mild versus severe SARS-CoV-2 infections. Additionally, there 181

is also a need to understand whether cross-reactive recall responses targeting non-neutralizing 182 regions could be inducing inefficient and/or ADE-like effects that are detrimental for outcomes. In this study, we investigate how anti-spike IgG responses correlate with disease 184 severity, FcγR activation, and epitope-targeting in a cohort of non-hospitalized SARS-CoV-2 185 convalescent donors, as well as dissect the contributions of divergent recall responses against 186 seasonal coronaviruses to the SARS-CoV-2 humoral response. 187

Non-hospitalized SARS-CoV-2 convalescent individuals displayed a spectrum of COVID- 19 191 symptom severity 192 193 In order to obtain a better understanding of the humoral responses generated by convalescent individuals, a total of 48 donors were recruited in the New York city area during 195 the first wave of the COVID-19 pandemic in the spring of 2020. The cohort was separated into 196 two groups based on the history of a positive or negative SARS-CoV-2 test (PCR or serology): 197 convalescent donors (positive test; CONVALESCENT) and negative controls (negative test; 198 NAÏVE). The negative control group was composed of age-and sex-matched SARS-CoV-2 naïve 199 donors (Table 1) . Importantly, in convalescent donors, the median time from the onset of 200 symptoms to the time of blood draw was 43 days, ensuring that anti-SARS-CoV-2 IgG levels 201

were sufficiently high for pathogen-specific IgG analysis in all convalescent donors.

To assess the relative severity of disease, we surveyed the symptomological history of disease 204

for each convalescent donor, including the intensity and duration of each symptom. This 205

information was used to calculate both an average severity score for each symptom (sup Table  206 2), along with a composite symptom severity score for every convalescent donor. In doing so, 207

we observed that the convalescent group presented a wide range of symptoms and severities 208

ranging from mild to more severe COVID-19. The most commonly reported symptoms were 209 cough, headache, fatigue and myalgia, with greater than 80% of the cohort experiencing these 210

symptoms. The symptoms with the highest average scores were fatigue, myalgia, diarrhea, and 211

fever. The least frequently experienced symptoms were nausea and vomiting. In calculating a 212 composite symptom severity score for each convalescent individual, we observed that 213 convalescent donors clustered into two categories: those that experienced milder symptoms 214 (n=13), with composite severity scores below 45; and those with more severe disease (n=15), 215

with scores above 45. For simplicity, these two groups are henceforth referred to as MILD and 216 SEVERE, respectively, as they reflect these two ends of the CoV-2 convalescent  231  individuals  232  233  To elucidate the humoral immune responses associated with mild to severe COVID-19, we  234 began by quantifying the levels of IgG directed against the SARS-CoV-2 spike protein in 235 convalescent donors. To quantify the levels of anti-spike IgG in convalescent donors, we first 236 utilized a conventional ELISA-based assay [69] . Using this assay, we detected significantly higher 237 levels of anti-spike IgG titers in the convalescent versus SARS-CoV-2 naïve donors, with only a 238 few naïve donors possessing titers above background ( Figure 1A ). Amongst the convalescent 239 donors, we observed a range of anti-spike IgG levels, which differed approximately 30-fold 240 between lowest and highest samples ( Figure 1B) . Interestingly, using conventional ELISA, two 241

donors that tested PCR-positive for SARS-CoV-2 infection possessed no detectable anti-spike 242 titers and eight additional convalescent donors possessed titers that were less than 3-fold 243 above background, which was comparable to the anti-spike levels observed in some SARS-CoV-244 2 naïve donors. We next compared the anti-spike IgG levels against the composite severity 245 scores of each donor and observed a significant positive correlation between anti-spike titers 246

and higher severity scores ( Figure 1C ). Importantly, several studies have found similar 247

correlations between anti-spike IgG and COVID-19 severity in hospitalized patients [6-9, 11, 12] .

In addition to neutralization, IgG also mediates non-neutralizing effector responses against 250

virus-infected host cells, which involve the recognition of viral antigens expressed on the 251

surface of host cells [38] [39] [40] [41] . Therefore, we next quantified the levels of anti-spike IgG binding 252

to cell surface-expressed forms of the SARS-CoV-2 spike. To quantify cell surface IgG binding, 253

we employed a cell-based system using 293T endothelial cells transfected to express the SARS-254

CoV-2 spike. In this system, the levels of anti-spike IgG are quantified using a binding index that 255 accounts for both the percentage and Median Fluorescence Intensity (MFI) of IgG bound to 256 spike-expressing cells. We have previously used this method to discern viral antigen-specific IgG 257 levels in convalescent donors [70] [71] [72] . The benefit of this composite metric is that both 258 frequency and density of IgG-antigen binding are captured, as both parameters contribute to 259

the IgG-mediated activation of non-neutralizing effector functions. Additionally, more than in 260 ELISA, expressing spike protein in a human cell-based assay likely retains tertiary structure and 261 glycosylation consistent with natural infection, potentially capturing a greater range of 262 paratopes (i.e., higher sensitivity).

Using this cell-based assay, we detected an approximate 1.5-log difference in anti-spike IgG 265 titers between naïve and SARS-CoV-2 convalescent donors, and a further 2-log difference 266 amongst the convalescent donors ( Figure 1D , 1E). Interestingly, the two PCR-positive SARS-CoV-267 2 donors whose anti-spike IgG levels were undetectable by ELISA had detectable, albeit low, 268

anti-spike IgG levels in the cell-based assay. In the cell-based assay, these levels were 1 log 269

above the values obtained with IgG from naïve donors, demonstrating greater resolution as 270 compared to ELISA. Additionally, using the cell-based assay, anti-spike IgG levels were readily 271 detectable above negative control (naïve) IgG levels in the eight donors that possessed low 272

anti-spike IgG levels not clearly discernible above background using the ELISA assay, 273

demonstrating the high sensitivity and specificity of this cell-based assay (Supplementary Figure  274   1A ). Similar to the ELISA results, we observed a strong positive correlation between anti-spike 275

IgG levels and disease severity (r 2 = 0.27 p < 0.0001) ( Figure 1F ), with severe donors possessing 276 significantly higher anti-spike IgG levels (Supplementary Figure 1B) .

Higher levels of anti-spike IgG-mediated FcγR activation corelates with COVID-19 severity 280 281

Elevated levels of anti-spike IgG and proinflammatory cytokine levels are detected in severe 282

hospitalized COVID-19 patients, suggesting that IgG maybe exacerbating disease severity via 283

FcγR mediated ADE effects. Therefore, we next examined the levels of FcγR-signaling induced 284

by IgG from convalescent individuals. Specifically, we examined the levels of Fc-γ receptors 285

(FcγR), FcγR2a and FcγR3a, since these FcγR activate several important cellular effector 286 functions, like ADCC and ADCP [36, 37] . Importantly, both of these FcγR are capable of 287

activating an array of pro-inflammatory cytokines.

To quantify the ability of patient sera to activate FcγR, we transfected 293T cells to express the 290 SARS-CoV-2 spike. The spike-expressing 293T cells were co-cultured with FcγR2a or FcγR3a 291

reporter cell lines in the presence of various concentrations of purified donor IgG [70, 71] . Using 292 this system, we observed a wide range of FcγR activation in response to convalescent donor 293

IgG, but only background levels of activation in response to SARS-CoV-2-naïve donor IgG ( Figure  294 2A). In examining the relationship between disease severity and FcγR activation, we observed a 295 strong positive correlation between severity scores and FcγR2a and FcγR3a activation ( Figure  296 2B, 2D). This relationship was even more apparent in severe donors, as compared to mild 297 donors (Figure 2A and 2C). Interestingly, the majority of IgG from mild donors did not induce 298

greater FcγR2a-activation than SARS-CoV-2 naïve control IgG ( Figure 2A ). In contrast, the 299 majority of IgG from severe donors induced FcγR2a signaling that was 2-fold above naïve 300

control IgG. In regards to FcγR3a activation, both mild and severe convalescent donors induced 301

signaling that was at least 2-fold above naïve control IgG ( Figure 2C ). Similar to FcγR2a-302 activation, IgG from severe donors possessed significantly higher FcγR3a activity as compared 303

to IgG from mild donors. 304 305

Next, we evaluated the relationship between FcγR-activation and the levels of anti-spike IgG.

We observed a significant positive correlation with both FcγR2a and FcγR3a signaling and the 307 levels of anti-spike IgG in all convalescent donors, as quantified by both ELISA and cell-based 308

anti-spike IgG binding assays ( Figure 2E -H). However, anti-spike IgG levels as quantified by the 309 cell-based assay were much more highly correlated with the levels of FcγR3a signaling (R 2 = 310 0.66)( Figure 2F ), as compared to anti-spike titers obtained by ELISA (R 2 = 0.48)( Figure 2H ). In 311 addition, FcγR3a signaling in general was much more directly correlated with anti-spike IgG 312 levels (R 2 = 0.66; R 2 = 0.48)( Figure 2F , 2H), as compared to FcγR2a signaling (R 2 = 0.43; R 2 = 313 0.29)( Figure 2E , 2G). Altogether, these data show a strong positive correlation between anti-314

spike IgG titers, the levels of FcγR activation, and COVID-19 severity. 315 316 317 COVID-19 disease severity is correlated with higher anti-spike IgG cross-reactivity against 318 betacoronaviruses 319 320

Normally, IgG responses against novel antigens arise days to weeks after the establishment of 321 infection, since naïve B cells require 7-14 days to become activated, class-switch, and begin 322

producing IgG [73] . However, in the case of COVID-19, elevated IgG has been reported within 4 323 days of infection [7, 9, 11] . The seroprevalence of seasonal human alpha-and 324 betacoronaviruses are ubiquitous [53] , with 4-27% of the population testing positive for any 325

given hCoV at any given time [61] . The spike proteins of these seasonal hCoVs share 326 approximately 25% and 30% sequence identity with the spike protein of SARS-CoV-2, 327 respectively (Table 3 ). The high seroprevalence of IgG against common coronaviruses, coupled 328

with the high degree of identity between common seasonal hCoVs and SARS-CoV-2 spike, lead 329

us to hypothesize that humoral responses against common coronaviruses may contribute to 330 SARS-CoV-2 anti-spike IgG responses.

To examine this hypothesis, we measured the levels of anti-spike IgG binding against common 333 hCoV spike proteins using IgG from SARS-CoV-2 convalescent and naïve donors. Specifically, we 334 measured the amount of IgG binding to the spike of a seasonal betacoronavirus, OC43, as well 335

as two alphacoronaviruses, NL63 and 229E. Additionally, we examined the level of cross-336 reactivity of SARS-CoV-2 convalescent IgG against the spike protein of SARS-CoV-1 (SARS1). 337

Since we observed more sensitive detection of anti-spike IgG binding using the cell-based assay 338

versus ELISA, we quantified the levels of seasonal hCoV anti-spike IgG binding using cell-based 339

assays. Similar to SARS-CoV-2, cell-based IgG binding assays showed that naïve donor IgG had 340 no recognition of SARS1 spike protein ( Figure 3A ). The majority of SARS-CoV-2 convalescent 341 donors, however, did possess IgG which recognized the SARS1 spike protein. IgG-recognition of 342 the OC43 spike was significantly elevated in SARS-CoV-2 convalescent donors as compared to 343 SARS-CoV-2 naïve donors ( Figure 3B ). In contrast, IgG binding to the alphacoronavirus spike 344 proteins NL63 and 229E was not significantly different amongst SARS-CoV-2 convalescent and 345 naïve donors ( Figure 3C , 3D). This finding suggests that IgG responses against SARS-CoV-2 did 346 not boost IgG responses against alphacoronaviruses. When further explored in relation to 347 disease severity, IgG from severe donors possessed higher cross-reactivity to the spike protein 348 of SARS1 and OC43 as compared to mild donors ( Figure 3E , 3F). These findings show that 349

infection with SARS-CoV-2 induces IgG that are cross-reactive with other betacoronaviruses, 350

and higher levels of cross-reactive IgG correlate with more severe disease.

Immunodominant regions with high OC43 sequence identity differentially correlate with 354 COVID-19 severity 355 356

Recently, several groups have conducted SARS-CoV-2 peptide walk experiments using Abs from 357

hospitalized COVID-19 patients to identify immunodominant epitopes that correlate with 358

severe . Notably, some of the immunodominant epitopes that have been 359

identified overlap with functional regions within the SARS-CoV-2 spike protein, which include 360 the S1/S2 furin cleavage site (S1/S2) and S2' cleavage fusion protein region (S2'FP), as well as 361 regions in and around heptad repeat 1 and 2 (HR1 & HR2). Interestingly, several of these 362 regions share high sequence identity with seasonal coronaviruses [62, 68] , suggesting that IgG-363 recognition of these immunodominant epitopes could be due to recall responses to seasonal 364 coronaviruses such as OC43.

To explore how epitope-targeting relates to infection severity in non-hospitalized individuals, 367

we screened our cohort against a panel of immunodominant SARS-CoV-2 peptides that 368 possessed high identity with seasonal hCoVs (Table 3 ), in particular betacoronavirus OC43 369

( Figure 4 ). To contrast these conserved regions, we also screened the levels of IgG targeting 370 immunodominant regions that possessed little identity with seasonal CoVs. Broadly, this set of 371

peptides represent most of the functional regions within the spike protein, including S1/S2 furin 372 cleavage site, S2' cleavage site, and the HR2 region ( Table 3 ). In addition, we quantified the 373 levels of IgG targeting the RBD region, since several studies have identified that Abs targeting 374 the RBD region mediate potent neutralization in vitro and control of SARS-CoV-2 infection in 375

vivo [55] [56] [57] 74] .

To quantify the levels of IgG targeting these regions, we developed a luciferase-based ELISA to 378 allow for the sensitive detection of IgG binding. Using this assay, we first examined the levels of 379

IgG targeting the RBD region. We observed a significant inverse correlation between the levels 380 of anti-RBD IgG and overall severity scores among convalescent donors (P=<0.0001)( Figure 5A ). 381

In comparing donors with milder versus severe disease, we observed significantly higher levels 382

of anti-RBD IgG-targeting in donors with mild disease (sup Figure 3A) . Notably, no significant 383 correlation was observed between anti-RBD IgG and total anti-spike IgG levels ( Figure 5B ).

We next quantified the levels of IgG-targeting of three representative immunodominant 386

peptides with high sequence identity to seasonal hCoVs. One selected peptide contains the 387 immunodominant region of S2'FP and possesses the highest sequence identity with OC43 388 among the screened immunodominant regions. The fusion protein (FP) region becomes 389 exposed after cleavage of the spike protein, which induces conformational rearrangement in 390 the spike allowing for insertion of the FP into the host cell membrane, thereby facilitating viral 391 fusion [75, 76] . We observed a significant positive correlation between the levels of IgG binding 392 the S2'FP region and severity ( Figure 5C ). We also observed that IgG-targeting of this region 393

increased with anti-spike IgG titers ( Figure 5D ). Next, we examined the levels of IgG binding to 394

the Heptad Repeat 2 (HR2) domain. HR2 functions to mediate viral fusion and entry into host 395 cells through the formation of a six-helix bundle in conjunction with the HR1 domain [77] . In 396

contrast to IgG targeting the S2'FP region, we observed a significant inverse correlation with 397 the levels of IgG targeting the HR2 region and severity ( Figure 5E ); on average, mild donors 398 possessed higher levels of IgG targeting the HR2 region, as compared to severe donors (sup 399 Figure 3E ). We also observed that IgG-targeting of the HR2 region was inversely correlated with 400 overall anti-spike IgG titers ( Figure 5F ). Lastly, we examined IgG-targeting of an 401

immunodominant and hCoV conserved region located just upstream of the HR2 domain, aka 402 the 5' flank HR2 (5'fHR2). The levels of IgG binding in this region was also inversely correlated 403

with severity ( Figure 5G ), similar to direct targeting of the HR2 region; however, IgG levels were 404 not inversely correlated with overall anti-spike IgG titers ( Figure 5H ). 405

To contrast and compare the conserved region targeting data, we next examined the levels of 407

IgG targeting immunodominant regions that were not highly conserved with OC43 or any other 408 seasonal hCoV ( Figure 6 ). One region we examined contained the novel furin cleavage site at 409 the S1/S2 junction, while the other two regions were located within the C-terminal domain 410

(CTD) just downstream of the RBD region ( Figure 4 ). In screening these regions, we observed a 411 significant positive correlation with the levels of CTD1 targeting and severity ( Figure 6A ). 412

However, we detected no correlation between IgG-targeting of the CTD2 or S1/S2 regions and 413 severity ( Figure 6C, 6E) . In comparing the overall anti-spike levels with IgG-targeting of these 414

regions, we detected a significant correlation between IgG-targeting of CTD2 and higher anti-415

spike IgG levels ( Figure 6D ), but did not observe any other significant correlation with the other 416 regions ( Figure 6B , 6F). However, targeting of CTD1 trended towards a significant positive 417 correlation with overall anti-spike IgG levels ( Figure 6B ). 418 419 420

Multivariable analysis Identifies that severe non-hospitalized COVID-19 correlates with high 421

FcγR activation and IgG targeting of spike protein's S2' fusion protein site.

To gain insights into the associations between all of the Ab features tested and disease severity, 424

we performed a Spearman's chart correlation on all of the variables analyzed ( Figure 7A ). Our 425 analysis recapitulated the significance of all of the previous findings in this study, such as the 426 correlations between overall anti-spike levels, betacoronavirus cross-reactivity, and elevated 427 levels of FcγR signaling. Additionally, this analysis allowed us to compare these variables with 428

IgG-targeting of various immunodominant/functional regions.

In examining all convalescent donors, we observed that individuals with more severe COVID-19 431

were characterized by higher anti-SARS CoV-2 spike IgG (R=0.66), higher IgG cross-reactivity to 432 betacoronaviruses (SARS1 R=0.54; OC43 R=0.36), and higher proinflammatory FcγR activation 433 (R=0.62), along with higher levels of IgG targeting the CTD1 (R=0.26) and S2'FP region (R=0.35), 434

as compared to mild donors ( Figure 7A ). Interestingly, higher severity scores were inversely 435 correlated with the levels of IgG targeting the RBD and HR2 regions, suggesting that in severe 436

individuals there was a relatively lack of IgG targeting neutralizing regions as a proportion of 437 total anti-spike levels. Of note, the levels of betacoronavirus (OC43; SARS1) cross-reactivity 438

were more closely associated with IgG targeting of SARS2 immunodominant regions as 439 compared to the levels of alphacoronavirus (NL63; 229E) targeting ( Figure 7A ). Interestingly, 440

while we observed that both FcγR2a and FcγR3a activation were positively correlated with 441 betacoronavirus cross-reactivity, the levels of FcγR2a signaling were more highly correlated 442

with IgG targeting the CTD1 (R2=0.42) and S2'FP (R2=0.55) regions, as compared to FcγR3a 443 signaling (CTD1 R=0.14; S2'FP R=0.35). These findings suggest that seasonal hCoV IgG recall 444 responses and epitope targeting contribute to the efficiency of FcγR signaling against the SARS-445

CoV-2 spike, especially in the case of FcγR2a activation.

To further explore the contribution of different IgG features, we performed an unsupervised 448 analysis of principal components (PCA) using all 28 convalescent donors ( Figure 7B , 7C variance), which was primarily represented by IgG-targeting of the RBD, S1/S2 furin site, HR2 459 region, and 5f'HR2 regions. This analysis suggests a differential course of disease when the IgG 460 response is directed against different regions of the SARS-CoV-2 spike. Interestingly, regarding RBD-targeting in mild donors, we did not observe a relationship 477 between anti-RBD levels and HR2 region targeting (R=-0.077), possibly suggesting two divergent 478 protective signatures, one recall and one de novo, respectively. In severe profiles, we observed 479 that total anti-spike IgG levels were inversely correlated with IgG targeting the S1/S2 (R=-0.39) 480

and HR2 region (R=-0.42) and positively correlated with S2'FP (R=0.37) region IgG-targeting 481

(Supplementary figure 4D) . Interestingly, anti-RBD IgG levels in severe donors were not strongly 482 correlated with the levels of IgG-targeting of any immunodominant regions.

To further elucidate the overall profiles between mild and severe individuals, the mean of Z-485 score values for each antibody feature was represented on a polar plot graph ( Figure 7D, 7E) . 486

Notably, for mild individuals, the IgG recognition of betacoronaviruses was lower than in severe 487 individuals ( Figure 7D, 7E) . However, in comparing the relative targeting of betacoronaviruses 488

vs alphacoronaviruses, we observed that mild individuals expressed higher Z scores for IgG 489 targeting alphacoronaviruses, with 229E being more highly targeted as compared to NL63 490 ( Figure 7D ). In examining only severe profiles, the inverse pattern was observed, where more 491 severe individuals possessed higher Z scores for betacoronavirus-targeting when compared to 492 alphacoronaviruses ( Figure 7E ). In support of the PCA analysis relating disease severity to FcγR 493 function, the polar plot shows that individuals with mild disease had lower FcγR2a and FcγR3a 494 enrichment scores ( Figure 7D ). Crucially, despite severe donors possessing higher overall levels 495

of anti-spike IgG, the enrichment scores for RBD IgG-targeting was higher in mild donors ( Figure  496 7D). This observation may imply that the elevated levels of anti-spike Abs observed in more 497

severe individuals could be attributable to inefficient non-neutralizing responses against SARS-498

CoV-2, since milder profiles are seen to be enriched for a higher proportion of IgG targeting the 499 RBD, HR2, and HR2 flanking regions, which have been previously shown to neutralize both 500 SARS1 and SARS2 [55] [56] [57] [78] [79] [80] [81] [82] [83] . Additionally, Z-scores in mild individuals indicate a relative 501 lower enrichment of IgG targeting the CTD1 and S2'FP regions, both of which were significantly 502 correlated with elevated FcγR activity in severe individuals ( Figure 7D ). In severe individuals, the 503 inverse was observed: IgG targeting CTD1 and S2'FP regions was enriched, and HR2 region and 504 5'fHR2 targeting was decreased, relative to total anti-spike IgG ( Figure 7E ). Taken together, 505

these data suggest that efficacy of IgG responses-and corresponding disease severity-is 506 highly dependent on the specific epitopes targeted in the SARS-CoV-2 spike protein, which in 507 turn may be influenced by prior hCoV exposure. 508

COVID-19 is expected to remain an ongoing global threat, driven in part by emerging variants 511 and challenges to vaccine distribution worldwide. Unlike many other pathogens that induce 512 long-lived immunity, seasonal coronaviruses have been observed to induce transient immunity, 513

which rapidly wanes following natural infection. Vulnerability to seasonal coronavirus 514 reinfection is the norm starting 6 months to 3 years after each seasonal hCoV infection [84] . 515

Evidence has emerged that SARS-CoV-2 reinfection is possible after 3-6 months, particularly 516

with emerging variants of concern [85-90]. Moreover, vaccine-induced immunity wanes 517 significantly after 6-8 months [91, 92] . This uncertainty surrounding the duration of immunity 518

(whether natural or vaccine-induced), combined with the continuing evolution of novel SARS-519

CoV-2 mutants, reinforces the need to continue to identify the immune correlates of protection 520

in SARS-CoV-2 infections in humans. Identifying the protective correlates of Ab-targeting is 521

particularly important, as they may provide new insights into mechanisms of protection that 522

can be leveraged for more effective or broadly-acting therapeutics and vaccines. 523 524

Early in the pandemic, researchers noted an unusual aspect of COVID-19 disease course; 525

namely, some patients were showing IgG within days of contracting SARS-CoV-2. [7, 9, 11] 526

Furthermore, early anti-spike IgG was associated with disease severity-not protection. Higher 527

anti-spike IgG titers, along with elevated levels of proinflammatory cytokines, correlated with 528 more severe disease in hospitalized individuals [9, 12] . In this study, we observed that 529

increasing anti-spike IgG titers were correlated with higher symptom severity scores, 530

substantiating and extending this phenotype to non-hospitalized individuals. We detected 531

significantly higher levels of anti-spike IgG using both ELISA and cell-based assays. Of note, the 532 cell-based assay detected anti-spike IgG with greater sensitivity than ELISA. Other studies have 533 also observed higher sensitivity with cell-based detection assays [93, 94] . This may be 534

attributable to the display of conformational epitopes that are dependent on tertiary or 535 quaternary structure which occur in natively folded trimeric spike protein. Moreover, 536

commercial recombinant antigens produced in bacteria or insect cells may not accurately 537 reflect the glycosylation pattern produced in human cells [95, 96] . This difference in sensitivity 538

should be considered when selecting IgG assays for varying research and clinical uses. 539 540

In this study, we observed a significant positive correlation between severity, anti-SARS-CoV-2 541

spike IgG levels, FcγR signaling, and IgG cross-reactivity with other betacoronaviruses; with the 542 highest levels of FcγR-signaling and IgG cross-reactivity in the donors with the highest symptom 543 severity scores. These findings suggest that anti-spike IgG may be contributing to COVID-19 544 severity through the amplification of inefficient and cross-reactive IgG responses against 545 seasonal hCoVs, potentially elevating proinflammatory cytokine levels through IgG-induced 546

FcγR activation. Early in the pandemic, some researchers had optimistically posited that Abs 547 from previous hCoVs might be protective against SARS-CoV-2. However, subsequent studies 548

identified that pre-pandemic and SARS-CoV-2 naïve samples did not mediate effective virus 549 neutralization, and predominantly targeted the S2 subunit of the SARS-CoV-2 spike protein [58-550 60]. Importantly, the RBD region, located in the S1 subunit of the spike protein, interacts with 551 the human ACE2 receptor to mediate viral attachment and entry into host cells [74] . In studies 552 characterizing neutralizing Abs from convalescent donors, Abs targeting the SARS-CoV-2 RBD 553 region were shown to potently inhibit virus replication and control infection in vivo [55] [56] [57] . In 554

contrast to the S1 subunit that is largely non-homologous with seasonal hCoVs, the S2 subunit 555 contains the highest sequence and structural similarity with seasonal coronaviruses [54] . 556

Therefore, the seroreactivity observed in some pre-pandemic and naïve donors is likely a result 557

of SARS-CoV-2 cross-reactive humoral memory responses against seasonal hCoVs. In light of 558 this possibility, we initially hypothesized that any seasonal hCoV recall responses amplified 559 during acute infections would favor S2 subunit targeting of the spike protein. Moreover, if 560 these recall Abs dominated the humoral response within an individual, we further hypothesized 561 this would lead to an inefficient non-neutralizing response (ala Original Antigenic Sin), 562

correlating with worse outcomes. In line with this hypothesis, the early and elevated levels of 563

anti-spike IgG observed in severe COVID-19 patients could be a consequence of amplifying 564

cross-reactive recall responses against S2 targeting non-neutralizing epitopes. In this manner, 565

anti-spike Ab titers may increase due to inefficient Ab-targeting and uncontrolled virus 566

replication, leading to an elevated inflammatory environment and worse outcomes. Whereas 567 de novo responses (i.e., S1/RBD/novel sequence)-which are broader and inherently SARS-CoV-568

2 sequence-specific-would be protective.

Despite these reasonable assumptions, in screening the levels of IgG targeting highly conserved 571 immunodominant regions, we detected no uniform association between high sequence identity 572 and severity. We instead observed that IgG-targeting of two highly conserved regions (HR2 573 region, 5'HR2 flanking region) was significantly correlated with milder infections, while 574 targeting of another conserved region (S2'FP site) was correlated with more severe infections. with IgG-targeting of the HR2 region is consistent with our data showing that IgG-targeting of 586 the HR2 region is correlated with milder infections, IgG-targeting of the S2'FP region correlated 587 with more severe infections. Importantly, studies identifying the HR2 and S2'FP regions as 588 immunodominant did not examine the relationship between immunodominance and infection 589 severity [68] . Therefore, despite the potential for Ab-targeting of the S2'FP region to mediate 590 some level of neutralization against SARS-CoV-2, whether or not targeting this region correlates 591

with milder outcomes and presumably more effective control of infection in vivo-remains 592

undefined.

While we did not examine IgG neutralization activity in this study, we did examine the capacity 595

of IgG to induce FcγR activation, a prerequisite for inducing non-neutralizing effector functions 596 like proinflammatory cytokine release. In this analysis, we identify that increased FcγR-signaling 597 is highly correlated with higher IgG targeting of the S2'FP region and more severe This finding provides a rationale as to why targeting this epitope may correlate with severity, 599

since FcγR activation in the context of ADE can induce proinflammatory cytokines, which are 600

highly associated with severe hospitalized cases of COVID-19. Notably, anti-SARS-CoV-2 Ab-601

targeting of the S2'FP region was shown to be one of the most immunodominant regions 602 targeted within a severe COVID-19 patient after prolonged hospitalization and intensive care 603

[61]. Altogether, our data shows clear correlations between all three of the immunodominant 604 conserved regions and severity, whether inverse or positive. In contrast, we only noted a 605 significant correlation between IgG-targeting and severity with one of three non-homologous 606

regions. 607 608

Importantly, these data demonstrate that the contribution of recall antibodies to COVID-19 609 disease severity is nuanced; IgG-targeting of some conserved neutralizing epitopes may be 610

protective, while targeting of other conserved epitopes may activate high levels of FcγR-611 mediated proinflammatory signaling, which in some instances may be detrimental. Importantly, 612

while cross-reactive responses, like those against the S2'FP region, may contribute to the 613 severity of infection, cross-reactivity in and of itself may not be the sole factor in governing 614 inefficient humoral responses during SARS-CoV-2 infections. These findings highlight that the 615 relationship between seasonal hCoV humoral cross-reactivity and COVID-19 disease outcomes 616 may be highly dependent on an individual's pre-existing hCoV IgG repertoire prior to becoming 617

infected with SARS-CoV-2. Therefore, further examination of how specific IgG-targeting and 618

pre-existing memory responses influence and contribute to pathogenesis in vivo will be key to 619 fully understand how cross-reactivity contributes to effective humoral responses. 620 621

Beyond defining the spike protein immunodominant regions based on their similarity to 622 betacoronaviruses (OC43), these regions also represent known functional regions within the 623 spike protein. Examining the profiles of IgG-targeting among convalescent individuals in the 624 context of functional regions, we observed that IgG-targeting of RBD, HR2, and HR flanking 625 regions significantly correlated with lower severity scores and-in the case of HR2 region-626

targeting-also correlated with lower levels of anti-spike IgG. This latter finding suggests that 627 humoral responses with a higher proportion of IgG-targeting against the HR2 region may be 628

highly efficient at controlling infection, since lower anti-spike titers were independently 629 associated with milder infections in this study and others [7] [8] [9] 12] . In previous studies 630

examining Ab-targeting of the RBD and HR2 regions, targeting both of these regions have been 631

shown to mediate virus neutralization [55] [56] [57] 83] . In experiments conducted with SARS1, 632

targeting of the HR2 flanking region also neutralizes virus replication [79, 82] . Interestingly, 633

when we examined the levels of anti-RBD IgG targeting in mild donors, we observed a positive 634 correlation with S1/S2 furin site and HR2 flanking region IgG-targeting, but not with HR2 region 635

targeting. Therefore, it is possible that targeting the HR2 flanking region sufficiently disrupts the 636 interaction between HR2 and HR1, thereby preventing viral fusion. Moreover, when we 637 compared the levels of RBD vs immunodominant epitope targeting in mild donors, we observed 638 no correlation between RBD and HR2 region IgG-targeting, despite both being correlated with 639 mild disease. This raises the possibility that one profile of neutralization predominantly arises in 640 some individuals and not in others, possibly influenced by whether or not humoral responses 641 are predominately recall or de novo. A closer examination of the relationship between RBD and 642

HR2 region neutralization will be the subject of future studies.

In regard to which IgG-targeting profiles correlated with overall severity amongst all 645 convalescent donors, we detected a correlation with IgG targeting the non-conserved CTD2 646 region and the conserved S2'FP region. In further examining the profiles of only severe donors, 647

we resolved that these profiles were characterized by higher levels of IgG targeting S2'FP 648 region, with an absence of IgG targeting the S1/S2 furin site and HR2 region (Supplementary 649 Figure 4C ). Notably, milder IgG profiles were not only characterized by higher levels of RBD and 650

HR2 region IgG-targeting, but also lower proportions of CTD1 and S2'FP region targeting in 651 relation to overall anti-spike titers (Figure supplementary 4B) . These findings strongly suggest 652 that targeting of the S2'FP region-coupled with the absence of anti-RBD and/or anti-HR2 653

region IgG-targeting-is linked with severity; further implying that these IgG profiles represent 654 inefficient responses against the SARS-CoV-2 spike.

Altogether, these findings suggest that humoral memory responses contribute to COVID-19 657 disease severity, conferring either protection or risk depending on epitope targeting. This may 658 explain the atypical bimodality of COVID-19 disease severity-an observation which was 659

previously obscured by aggregate data/epitope analysis. The correlation we observed with IgG-660

targeting of the spike protein highlights an important point since to date it's been unclear why 661 some people respond so severely to infection while others have a mild infection. Thus far, it has 662 been challenging to enumerate the underlying factors that accurately predict disease outcome, 663 even among individuals who are at high risk of severe symptoms. The findings reported here 664

have the potential to help improve prediction accuracy and explain the underlying mechanisms 665

of COVID-19 pathogenesis, as pre-existing cross-reactive immunity with seasonal coronaviruses 666 is correlated with, and likely contributes to, disease outcome. This epitope-based Original 667

Antigenic Sin immunosurveillance, or OASiS, could be readily adapted to a clinical prognostic, 668 offering a novel approach for the prediction of disease severity risk, as well as vaccine efficacy, 669 on a patient-by-patient basis. Dissecting the interplay between these epitopes and the 670 efficiency of IgG-mediated immunity will be the focus of future work.

Beyond defining how IgG-targeting of immunodominant regions correlates with COVID-19 673 outcomes, we also show that higher levels of FcγR2a and FcγR3a activation correlate with more 674

severe SARS-CoV-2 infections. Interestingly, both FcγR2a and FcγR3a are correlated with more 675 severe dengue pathogenesis in humans and animal models, and have been shown to promote 676 ADE effects in vitro [33] [34] [35] 99] . In SARS1 infections, in vitro experiments using IgG from severe 677 SARS1 patients have been shown to induce the FcγR-mediated activation of proinflammatory 678 cytokines from macrophages [13] . In studies examining the role IgG in severe COVID-19, higher 679 levels of the afucosylated glycoform of IgG-which inherently possess a higher capacity to 680

activate FcγRs-have been correlated with more severe 24, 100] , and have also 681 been shown to activate cytokine expression from macrophages [24, 100] . Notably, the levels of 682 afucosylated anti-spike IgG were correlated with higher C-reactive protein and IL-6 levels in 683 severe hospitalized COVID-19 patients, as compared to convalescent non-hospitalized 684 individuals with milder symptoms [22] . Importantly, afucosylated anti-spike IgG were also 685

shown to possess higher affinity for FcγR3a and induce high levels of IL-6 and TNF-α release 686 from monocytes and macrophages in vitro [24, 100] . These findings suggest that FcγR-activation 687

contributes to the proinflammatory profile in COVID-19 patients. However, these results do not 688 define the contribution of FcγR effector functions in the control of SARS-CoV-2 infections in 689

vivo. Whether or not this increased Fc-activation is a compensatory mechanism for controlling 690 virus replication in the face of inefficient IgG neutralization, or the driver of severe 691 pathogenesis though increasing inflammation via the induction of proinflammatory cytokines,

remains an open question.

Interestingly, in murine models of SARS-CoV-2 infection, the passive infusion of neutralizing IgG 695 directed against the RBD region was shown to effectively protect animals when administered 696

prophylactically In this study, we observed that more severe SARS-CoV-2 infections were characterized by lower 711 levels of IgG targeting the RBD and HR regions. This suggests that high FcγR activation in the 712 absence of high titers of neutralizing IgG may be deleterious. One caveat to the previous studies 713 that found FcγR activity to be critical for effective humoral immunity is that all of those studies 714

were conducted with neutralizing IgG. Therefore, we cannot rule out that FcγR activation in the 715 absence of a neutralizing response does not contribute to severe COVID-19 pathogenesis, as 716 may be the case with individuals with anti-S2'FP dominant IgG profiles. Therefore, it will be 717 important to dissect how IgG-targeting of neutralizing and non-neutralizing epitopes influences 718 the relationship between FcγR activation and disease outcomes. Additionally, it will be 719 important to examine the contribution of FcγR activation to systemic inflammation and control 720 of virus replication in animal models that faithfully mimic human FcγR-signaling effects. 721 722

In conclusion, the data presented here demonstrate that immunological history-particularly 723 antibody repertoire from seasonal betacoronaviruses-predicts and potentially determines 724 COVID-19 disease severity. Specifically, an anti-HR2-dominant Ab profile represents an efficient 725 protective recall response, an anti-S2'FP dominant Ab profile suggests a deleterious inefficient 726 recall response, and a predominantly anti-RBD profile likely reflects a protective de novo 727 response. These data become particularly important in assessing epitope-targeting early in 728 disease, which could allow earlier interventions with treatments like monoclonal Ab therapies, 729

preventing progression to ARDS. Additionally, assessing the humoral profiles induced by 730 vaccination will be important. Vaccination will boost SARS-CoV-2-specific IgG, both novel and 731 recall responses, initially giving some level of protection, but could lead to higher frequencies of 732 breakthrough infections over time if inefficient IgG-targeting remains the predominant 733 response. Fortunately, vaccine studies have shown that current mRNA vaccines appear to 734

induce high titers of RBD-specific antibodies; however, anti-RBD-targeting Abs are 735

predominantly induced only after the second dose [103, 104] . This finding suggests potentially 736 meaningful variation in IgG profiles induced by different vaccines, particularly multi-vs single-737

dose regimens, such as J&J. Therefore, it will be important to assess the IgG-targeting profiles 738

induced by all available vaccines, as this type of analysis may help to explain why some vaccines 739

induce effective anti-SARS-CoV-2 Abs while others do not produce lasting or robust protection, 740

such as SinoVac. Additionally, it will be important to examine the ratios of RBD to other 741 immunodominant epitope IgG targeting and assess the longevity of immunity and the 742 frequencies of breakthrough infections over time. Altogether, epitope-profiling may be 743 essential to ensure long-term vaccine-induced protection in individuals, as individuals with 744

inefficient IgG profiles (i.e., anti-S2'FP-dominant), even after vaccination, may require an RBD-745 specific or HR2-specific vaccine to change their targeting profiles and induce long-lived 746 protective humoral responses. Promisingly, HR2 offers a good target for universal hCoV 747 treatment and vaccine design. 748 Fc-gamma receptor signaling assay 818 819

FcR2a and FcR3a signaling was assessed using a reporter cell co-culture system that we have 820 previous used to assess FcγR signaling in response to viral antigens ([70, 71] ). For this assay, 821

293T cells are transfected with SARS-CoV-2 spike expression vector and co-cultured with either 822 a FcγR2a, or FcγR3a, CD4 + Jurkat reporter cell line, which expresses firefly luciferase upon FcR 823 activation. For this assay, 1x10 5 SARS-CoV-2 spike-expressing 293T cells were plated in each 824

well of a 96-well round bottom plate. The cells were then preincubated with a 5-fold dilution 825 series of convalescent donor-derived IgG starting at a maximum concentration of 25g/ml. IgG 826 opsonized 293T cells were then co-cultured with FcR2a or FcR3a reporter cells at a 2:1 827

reporter-to-target cell ratio for 24hrs at 37C. After 24 hours, all cells were lysed with cell lysis 828 buffer (Promega; E1531), and the levels of firefly luciferase activity determined using a 829 luciferase assay kit according to manufacturer's instructions (Promega; E1500). To quantify 830 background (i.e., IgG activation-independent) luciferase production, reporter cells were co-831 cultured with the spike-expressing 293T cells in the absence of any IgG. Background levels were 832

subsequently subtracted from the signal to yield IgG-specific activation in relative light units 

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Anti-spike IgG levels in convalescent donors correlates with COVID-19 severity. 1260 (A) Anti-spike IgG titers in SARS-CoV-2 naïve (CoV-2-) and SARS-CoV-2 positive convalescent 1261 (CoV-2+) donors, as quantified by recombinant spike protein ELISA. (B) Variations in anti-spike 1262 titers among convalescent (CoV-2+) donors as quantified by ELISA. Red line represents 3-fold 1263 above the mean anti-spike levels of naïve (CoV-2-) donors, as quantified by ELISA

Comparison of anti-spike IgG titers, as quantified by ELISA, vs. composite severity scores 1265 amongst convalescent donors. (D-F) Levels of anti-spike IgG in naïve (CoV-2-) and convalescent 1266 (CoV-2+) donors as quantified using a cell-based assay. (D) Anti-spike IgG titers in naïve (CoV-2-) 1267 and convalescent (CoV-2+) donors as quantified by cell-based assay. (E) Variations in anti-spike 1268 titers amongst convalescent (CoV-2+) donors as quantified by cell-based IgG binding assay

The SEM of N=3 experiments are shown

A-H) Graphs compare the level of IgG binding to spike proteins of (A,E) SARS1, (B,F) OC43, 1355 (C,G) NL63 and (D,H) 229E coronavirus as assessed by cell-based assay and detected by flow 1356 cytometry. Graphs A-D compare the level anti-spike IgG in SARS-CoV-2 naïve versus 1357 convalescent donors. Graphs E-H compare the levels of anti-spike IgG among donor groups 1358 separated by SARS-CoV-2 status and COVID-19 severity scores. For all cross-reactive Ab binding 1359 results, the SEM of N=3 experiments are shown

 (RLUs) . Luminescence was measured on a Cytation 3 image reader using Gen5 software. 834

For this assay, N-terminus biotinylated peptides were synthesized by Genscript. N-terminal 837 GSGS linker sequence was added to all peptide sequences. The RBD peptide contained a C-838terminal avitag (GLNDIFEAQKIEWHE), for biotinylation via BirA enzyme; a Protein C tag 839 (EDQVDPRLIDGK), and a polyhisidine tag (HHHHHHHHHH), to enable immobilized metal affinity 840 chromatography purification. Lyophilized peptides and RBD were initially resuspended in DMSO 841and then used to make 5g/ml working dilutions in TBST ELISA wash buffer. Pierce TM white 842 streptavidin-coated high binding capacity 96-well binding plates (ThermoFisher Scientific; 843 15502) were washed twice with ELISA wash buffer and coated with 5g/ml of biotiylated 844peptides at room temp. Plates were coated for 2hrs while shaking at 500rpm on a Benchmark 845Orbi-Shaker. respectively). In each protomer of the spike, the protein mainchain is shown as a cartoon 1408representation and colored white, except for the RBD, which is colored red. The atoms in the 1409six peptide epitopes that were tested are shown as space-filling models, colored according to 1410peptide number. There are regions of missing density in the models, presumably due to 1411 conformational flexibility, and these regions are omitted here; CTD1 (magenta) and S2'FP 1412(green) are fully resolved, CTD2 (cyan), S1/S2 (orange), and 5'fHR2 (yellow) are partially 1413 resolved, and HR2 is completely absent in the structure. 1415  1416  1417  1418  1419  1420  1421  1422  1423  1424  1425  1426  1427  1428  1429  1430  1431  1432  1433  1434  1435  1436  1437  1438  1439  1440  1441  1442  1443  1444  1445  1446  1447  1448  1449  1450  1451  1452  1453  1454  1455  1456  1457  1458   Table 3 

The level of sequence identity between spike proteins were assessed using PRALINE software (IBIVU), by comparing SARS-CoV-2 spike protein sequence (Genbank YP_009724390.1) to spike protein sequences of SARS1 (Genbank AAP13567.1), OC43 (Genbank AVR40344.1), HKU1 (YP_173238.1), NL63 (APF29071.1), or 229E (APT69883.1). Percent sequence identity was measured by the level of exact AA conservation in reference to the SARS-CoV-2 spike sequence. Gaps in hCoV sequences were treated as no conservation .   1459  1460  1461  1462  1463  1464  1465  1466  1467  1468  1469  1470  1471  1472  1473  1474  1475  1476  1477  1478  1479  1480  1481  1482  1483  1484  1485  1486  1487  1488  1489  1490  1491  1492 1547  1548  1549  1550  1551  1552  1553  1554  1555  1556  1557  1558  1559  1560  1561  1562  1563  1564  1565  1566  1567  1568  1569  1570  1571  1572  1573  1574  1575  1576  1577 1578 Figure 7 . Multivariable analysis identifies that mild and more severe COVID19 is 1579 differentiated by distinct humoral immune profiles.

(A) Scatter matrix chart summarizes the Spearman's correlation (upper) and the scatter plots 1581(lower) between all analyzed variables using the entire cohort (n=28). The Spearman's r values 1582 are shown inside the colored squares and the scale of blue-to-red color indicates a negative-to-1583 positive correlation. The small bar graphs (diagonal) represent the distribution of data for each 1584variable (B) Biplot shows the principal component analysis (PCA) depicting the mild-scored 1585(n=13) and more severe-scored (n=15) COVID-19 patients, according to their severity scores (C) 1586The contribution of each variable to PCA for dimension 1 and 2 is represented by bars, and its 1587 threshold is indicated as a red dotted line. (D,E) Polar plots show the different profiles of 1588 humoral response for mild and more severe groups. Each bar in the plot represents the mean of 1589 z-scores for each variable. 1590