key: cord-0941469-apt3sr91
authors: Li, Min; Liu, Jiaojiao; Lu, Renfei; Zhang, Yuchao; Du, Meng; Xing, Man; Wu, Zhenchuan; Kong, Xiangyin; Zhu, Yufei; Zhou, Xianchao; Hu, Landian; Zhang, Chiyu; Zhou, Dongming; Jin, Xia
title: Longitudinal immune profiling reveals dominant epitopes mediating long-term humoral immunity in COVID-19 convalescent individuals
date: 2022-01-21
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2022.01.005
sha: 338959e86c9b9fd137a4ab45fc3696f4c1677608
doc_id: 941469
cord_uid: apt3sr91

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic and contagious coronavirus that caused a global pandemic with 5.2 million fatalities. Questions concerning serological features of long-term immunity, especially dominant epitopes mediating durable antibody responses after SARS-CoV-2 infection, still remain to be elucidated. Objective We aimed to dissect the kinetics and longevity of immune responses in COVID-19 patients, as well as epitopes responsible for sustained long-term humoral immunity against SARS-CoV-2. Methods We assessed SARS-CoV-2 immune dynamics up to 180-220 days after disease onset in 31 individuals who predominantly experienced moderate symptoms of COVID-19 and performed a proteome-wide profiling of dominant epitopes responsible for persistent humoral immune responses. Results Longitudinal analysis revealed sustained SARS-CoV-2 spike protein-specific antibodies and neutralizing antibodies in COVID-19 individuals, along with activation of cytokine production at early stages after SARS-CoV-2 infection. Highly reactive epitopes that were capable of mediating long-term antibody responses were shown to be located at the spike and ORF1ab proteins. Key epitopes of the SARS-CoV-2 spike protein were mapped to the N-terminal domain of the S1 subunit and the S2 subunit, with varying degrees of sequence homology among endemic human coronaviruses and high sequence identity between the early SARS-CoV-2 (Wuhan-Hu-1) and current circulating variants. Conclusion SARS-CoV-2 infection induces persistent humoral immunity in COVID-19 convalescent individuals, by targeting dominant epitopes located at the spike and ORF1ab proteins that mediate long-term immune responses. Our findings provide a path to aid rational vaccine design and diagnostic development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen 121 of the ongoing coronavirus disease 2019 pandemic, belongs to the beta-122 coronavirus genus, which includes other known highly pathogenic human coronaviruses, 123 severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory 124 syndrome coronavirus (MERS-CoV) 1 . Since the onset of the first recorded case in late 125 responses necessitate a systematic characterization of SARS-CoV-2 epitopes, as well as 166 properties of long-lasting antibodies targeting neutralizing or non-neutralizing epitopes. 167 Despite recent progress on kinetics and duration of antibody responses following phase (days 122 to 214 after disease onset). Accordingly, sera (n=20) from age-and sex-210 matched healthy donors were also included as the control group. All study participants did 211 not have a documented prior SARS or MERS infection. The study was approved by the 212 Ethical Committee of Nantong Third Hospital Affiliated to Nantong University (approval 213 number: EL2020006). Written informed consent was obtained from each of the study 214 participants. Serum was separated from peripheral blood in serum-gel tubes via 215 dilution at which the adjusted OD value (OD450 -OD630) = 0 was calculated to give an 241 endpoint titer. 242 Antigen-specific IgG antibodies in peptide-immunized mice were assessed by peptide-243 based ELISA. Briefly, 96-well ELISA plates (Thermo Fisher Scientific) were coated with 244 1μg/well of 4 mixed peptides (peptides No. 318, No.356, No.510 and No.530) in carbonate 245 buffer (pH 9.6) overnight at 4℃. After blocked with PBS-T containing 5% nonfat milk, 246 wells were incubated with serum samples diluted at 1:40 for 1 h at 37℃. Subsequently, 247 plates were washed and incubated with HRP-conjugated anti-mouse IgG antibody (Abcam).

Reactions were visualized with TMB substrate (Life Technologies) and the absorbance at 249 450 nm was measured after stopping reactions with 2M H2SO4. according to the manufacture's instruction. Briefly, Sulfo-SMCC was added at 30-fold 297 molar excess to BSA, followed by dialysis to PBS. Subsequently, the peptide containing 298 cysteine was then added in a ratio of 1:1 (w/w) and incubated for 2 h, and further dialyzed 299 with PBS to eliminate free peptides. To prepare the peptide microarray, peptides of SARS-CoV-2, as well as the negative control 303 (BSA) and positive controls (anti-human IgG and anti-human IgM antibodies, Sigma-304 Aldrich) were immobilized on PATH substrate slides (Grace Bio-Labs) in triplicate using 305 a Super Marathon printer (Arrayjet). Then peptide microarrays were preserved at −80 °C 306 for further use.

Microarray-based serum analysis was performed as Li, Y. et al. 36 with minor modifications. 310 In order to create individual chambers for the identical subarrays, a 14-chamber rubber 311 gasket was mounted onto each peptide microarray slides. The slide arrays were warmed to 312 room temperature before used, and then blocked with 3% BSA in PBS-T for 3 h. The serum The IgG and IgM data were analyzed respectively. The signal intensity of each spot was 325 defined as the foreground minus the background, and averaged the triple spots for each 326 peptide. The cut-off value for positive peptide response of COVID-19 samples was set as 327 twice the signal intensity of the healthy donor control. Peptides with positive rates above 328 80% among samples tested at all three sampling timepoints (days 10-60, 100-150, 180-220 after disease onset) were defined as "dominant and persistent" peptides, and peptides with 330 a positive response frequency above 60% at all three timepoints were considered as 331 "subdominant and persistent". The average signal intensity of each peptide in three 332 sampling timepoint groups was calculated respectively, and peptides showing high signal 333 intensity (above mean plus standard deviation of signal intensities of all tested samples) 334 were also selected. The data processing and analysis were done in R (version 3.6.3), and 335 significant signal intensity changes among different sampling timepoint groups was 336 evaluated based on Limma. Differences with p values less than 0.05 were considered 337 statistically significant. 

For immunization, groups of mice (n=5) were injected intramuscularly with 25 345 μg/dose of each peptide (peptides No. 318, No.356, No.510 and No.530 respectively) 346 mixed with 50 μg alum (InvivoGen) and 10 μg CpG adjuvants, and boosted twice with 50 347 μg/dose of peptides in the presence of adjuvants at two-week intervals. Vaccination with 348 adjuvants alone served as the negative control. Sera were collected from immunized mice 349 at day 10 or day 14 after the 2 nd and 3 rd immunization.

All graphs were plotted using GraphPad Prism software (version 8.0). Statistical 353 comparisons between groups in Fig 1, Fig 2 and (Fig 1, A) . Study participants included 26 patients with moderate 370 one with asymptomatic presentation, two with mild illness and two with severe symptoms 371 ( Table E1) . Patients enrolled in this study ranged from 17 to 66 years of age (median age: 372 45 years), with an approximately equal distribution of males (51.6%) and females (48.4%).

The most common symptoms among study participants included fever (83.9%), cough Table E2 ). Sampling from the other 11 participants was 378 performed at a single time point during late convalescence (days 122 to 214 post-symptom 379 onset). Additionally, samples from 20 healthy donors with matching age and gender 380 distribution were included as a control group (Table E1) . 381 Antigen-specific IgG antibodies in serum samples were quantified by ELISA pre- 180-220 days after disease onset, in comparison to the healthy donor group (Fig 1, B and 391 C). Correlation analysis revealed a significant correlation between log10 anti-N IgG titers 392 and anti-S IgG titers (r = 0.50 and p < 0.001 ; Fig 1, F) . 393 In addition to quantifying binding antibodies, dynamics of functional neutralizing 394 antibodies in COVID-19 individuals was further determined using pseudotyped SARS-395 CoV-2. Over 95% patient serum samples (35/37) collected between 4 and 30 days post-396 symptom onset presented robust neutralizing activities against SARS-CoV-2, and a large 397 proportion of samples exhibiting moderate (NT50 80-320) to strong neutralizing activity 398 (NT50 > 320) (Fig 1, D and E) . Of note, two samples with no or low neutralizing titers month of illness, including IL-1α (pro-inflammatory), IL-6 (pro-inflammatory) and IL-10 442 (anti-inflammatory) that have been linked to cytokine release syndrome in severe COVID-443 19 cases 38, 39 , as well as IFN-γ (Th1-type) and IL-4 (Th2-type) (Fig 2, A and Fig E8) . 444 Specifically, serum levels of IL-6, IL-10 and IFN-γ increased within 15 days after 445 symptoms onset in patients, and decreased in later phases; whereas the release of IL-1α 446 and IL-4 was shown to be remarkably increased during 16-30 days from onset of disease, 447 and rapidly dropped to normal afterward (Fig 2, A) . Correlation analysis indicated a weak 448 or no linear association between antigen-specific antibody responses and cytokine 449 production following SARS-CoV-2 infection, although a statistical significance was 450 observed between levels of IL-10 and SARS-CoV-2 N protein binding antibody (r = 0.321 451 and p < 0.05), between IL-1β production and S protein binding antibody (r = -0.335 and p 452 < 0.05), and between TNF-α and S protein binding antibody levels (r = -0.335 and p < 0.05, 453 Fig E9) . 454 To allow a direct visualization and comparison among patient samples across multiple cytokine responses over time, we constructed a heatmap showing fold changes of cytokine 456 release, relative to the healthy donor control group (Fig 2, B) . Consistent with our findings 457 presented above, elevated serum cytokine levels after SARS-CoV-2 infection were 458 predominantly observed during the acute phase and an early period of convalescence 459 (within 30 days after disease onset) (Fig 2, A and B) . Among cytokines tested, pro-460 inflammatory IL-6 exhibited the most robust response with a 4.9-fold increase and a 2.9-461 fold increase on average for samples collected during 1-15 days and 16-30 post-symptoms 462 onset, respectively (Fig 2, B) . Of note, one serum sample (sample ID: Pt-S22, day 18 after 463 disease onset) obtained from a COVID-19 individual with moderate disease, presented a 464 marked increase in the production of multiple pro-inflammatory cytokines, including IL-465 1α, IL-1β, IL-6 and TNF-α (Fig 2, B) . Additionally, hyper-production of cytokines 466 including IL-1α, IL-4, IL-6, IL-10, IL-13 and IFN-γ was also detected in one sample 467 (sample ID: Pt-S11, day 15 after disease onset) collected from a severe case of 468 presumably being associated with disease severity and outcome (Fig 2, B) . These results To better characterize the distinguishing features of humoral immunity to SARS-CoV-2 475 over time, we applied a proteome-wide epitope mapping using peptide-based microarrays.

A peptide library covering the SARS-CoV-2 proteome was generated and immobilized 477 onto slides, in which each peptide was 15 amino acids in length with 11 amino acids overlap.

after SARS-CoV-2 infection (days 10-60 post-disease onset, Fig E10) . The number of 502 positive-binding peptides for IgM was shown to be associated with serum levels of IL-6 503 and IL-10; likewise, the average IgM signal intensity of total reactive peptides and that of 504 ORF1ab binding peptides presented positive associations with IL-6 and IFN-γ production 505 in serum samples. These data suggest that changes in cytokine levels following SARS-

CoV-2 infection may influence the magnitude and breaths of epitopes recognized by 507 antigen-specific humoral responses.

On the basis of identified positive epitopes, we further selected the most common 509 epitopes that consistently remain reactive in more than 80% of the COVID-19 samples 510 among each of the three sampling groups (termed dominant and persistent epitopes).

Results revealed that these highly dominant epitopes capable of mediating long-term 512 humoral immune responses were located at the SARS-CoV-2 ORF1ab polyprotein and S 513 protein, with more epitopes recognized by IgM antibodies (n=33) than those recognized by 514 IgG antibodies (n=10) (Fig 3, B and Table 2 ; Fig E11 and Fig E12) . The ORF1ab 515 polyprotein possessed maximal number of dominant epitopes mediating long-term 516 responses, and epitopes were broadly distributed on the regions of non-structural proteins 517 (nsp) 2-5, nsp 8-10, nsp 12-14 and nsp 16 ( Table 2) . Notably, we identified one 518 immunodominant epitope No.2073 (ORF1ab, aa 5801-5815) that could be recognized by 519 IgG and IgM antibodies from 100% COVID-19 individuals, regardless of serum sampling 520 time points (Fig 3, B and Table 2 ; Fig E13 and Fig E14) . This highly reactive peptide is 521 located within the helicase (nsp13) region of the ORF1ab polyprotein, which is essential on the nsp 13 presented the most robust binding intensities (Fig 3, B) . Considering the 528 variation in baseline signals (pooled sera from healthy donors) for different peptides (Fig   529   3, B) , we further calculated fold changes of signal intensities regarding each key peptide, connecting region between FP and the first heptad repeat (HR1) of S2 subunit (Fig 3, B   541 and Table 2 ). Among these key peptides of S protein selected, peptide No.318 located at 542 the NTD of S protein possessed the most robust binding intensity (fold change relative to 543 the control; Fig E13 and Fig E14) . Structural analyses revealed that these epitopes are 544 fully exposed on the surface of monomeric S protein, however, some residues of epitopes 545 for peptides No.318, No.356 and No.530 are concealed under the surface of the trimeric S 546 protein (Fig 4, A and B) , suggesting that both S monomer and trimer structures are recognized efficiently by host immune system under certain circumstances. To be specific, mostly cryptic, with only a small fraction of the loop (aa 893-895) exposed on the trimeric 554 S protein (Fig 4, C) . 555 Sequence homology analysis among seven common human coronaviruses revealed 556 that two epitopes located at the S2 subunit (No. 510 and No. 530) share high sequence 557 identity with other coronaviruses, suggesting serological cross-reactivity targeting these 558 epitopes among human coronaviruses (Fig 4, D) . The sequence of No.318 exhibited high 559 similarity with SARS-CoV, while a low level of sequence homology for No.356 was shown 560 among coronaviruses, suggesting SARS-CoV-2 specific antibody responses targeting this 561 region (Fig 4, D) . 562 Considering new emerging and circulating SARS-CoV-2 variants globally, we further 563 performed sequence alignment with regard to key epitopes of the S protein between the 564 early SARS-CoV-2 strain (Wuhan-Hu-1) and five variants of concern (Alpha, Beta, (Fig 4, E) . These data indicate that antibodies generated by early SARS-CoV-2 strains may consistently recognize current 571 circulating variants, and that these dominant epitopes may be capable of mediating 572 sustained long-term antibody responses upon infection of SARS-CoV-2 variants.

To further determine the immunological characteristics of identified epitopes on the S 574 protein, and to further investigate the potential value of these S-protein epitopes as peptide 575 vaccine candidates, we performed a mouse immunization study using selected peptides.

BALB/c mice were inoculated three times with each peptide in the presence of alum and 577 CpG adjuvants (Fig 4, F) . Among the four peptides selected, vaccination with peptide 578

No.356 elicited antigen-specific antibodies following the second and third doses (Fig 4, G) . 579 The results of the serum neutralization assay revealed that immunization with linear 580 peptides did not generate significant levels of neutralizing antibodies against SARS-CoV-581 2 (Fig 4, H) , suggesting these linear peptides perform poorly in inducing robust 582 neutralizing antibody responses. (Fig 5, A-B and Table E4 (Fig 6, B) . Notably, despite largely overlapped with each other, two residues that are fully exposed on the surface of trimeric S protein (Fig 6, C) . These results 635 revealed new features of epitopes that may ultimately contribute to the long-lasting and 636 stronger humoral immunity against SARS-CoV-2. In this study, we identified 4 dominant epitopes (No.318, No.356, No.510 and No.530) 652 within the SARS-CoV-2 S protein that were capable of persistently reactive with more than 653 80% COVID-19 patient samples tested, up to 180-220 days post-symptom onset. Peptide against SARS-CoV-2 26, 33 , in spite of highly exposed on the surface of the S protein (Fig 4,   658 A-C). In the case of peptide No.530 (S, aa 893-907) that is positioned between the FP and 659 HR1 of the S2 subunit, residues are generally buried inside the trimeric structure of the S 660 protein and makes it hard to be accessible by robust neutralizing antibodies against SARS-CoV-2 (Fig 4, A-C) . Additionally, two S1-NTD-directed peptides that could mediate long-662 term antibody responses of SARS-CoV-2 were also selected. Analyses regarding the 663 peptide sequence and location indicated that residues of these two peptides No. 318 (S, aa 664 45-59) and No.356 (S, are in close proximity to the reported epitopes 665 recognized by infection-enhancing antibodies 23, 25 , but apart from the key sites of highly 666 potent neutralizing antibodies targeting NTD of the S1 subunit 16, 19, 49 , suggesting the 667 possibility of epitope recognition by non-neutralizing antibodies towards these two 668 identified peptides. Besides the details mentioned above, mouse immunization with 669 selected peptides further indicated the low efficacy of these non-RBD linear peptides in 670 inducing robust neutralizing antibody responses (Fig 4, G) . Collectively, these data suggest J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

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