key: cord-0874720-dpyz8uh4
authors: Dugan, Haley L.; Stamper, Christopher T.; Li, Lei; Changrob, Siriruk; Asby, Nicholas W.; Halfmann, Peter J.; Zheng, Nai-Ying; Huang, Min; Shaw, Dustin G.; Cobb, Mari S.; Erickson, Steven A.; Guthmiller, Jenna J.; Stovicek, Olivia; Wang, Jiaolong; Winkler, Emma S.; Madariaga, Maria Lucia; Shanmugarajah, Kumaran; Jansen, Maud O.; Amanat, Fatima; Stewart, Isabelle; Utset, Henry A.; Huang, Jun; Nelson, Christopher A.; Dai, Ya-Nan; Hall, Paige D.; Jedrzejczak, Robert P.; Joachimiak, Andrzej; Krammer, Florian; Diamond, Michael S.; Fremont, Daved H.; Kawaoka, Yoshihiro; Wilson, Patrick C.
title: Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets
date: 2021-05-06
journal: Immunity
DOI: 10.1016/j.immuni.2021.05.001
sha: 5f7b4db02f1b6a8dd644b7cf063bef1b4d11108a
doc_id: 874720
cord_uid: dpyz8uh4

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we utilized single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post-symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.

Finally, we did not identify substantial differences in serum titer to distinct 264 antigens across convalescent visit time points ( Figure S3H-S3J) . Similarly, reactivity 265 patterns in serological titer and probe hit to distinct antigens in individual subjects did 266 not appear to be correlated ( Figure S4A-S4E ). This may be related to differences in B 267 cell affinity to 3-dimensional probes in the bait sorting assay versus ELISA, or the fact 268 that the cellular response is sampled at one snapshot in time (over 1-month post-269 symptom onset), with serology reflective of antibody that has accumulated since initial The identification of B cells against distinct antigens is typically associated with 281 stereotypical VH and variable light chain kappa or lambda (VK; VL) gene usages. 282

Immunodominant and neutralizing spike and RBD epitopes are of particular interest, as 283 they represent key targets for vaccine-induced responses. To investigate whether 284 antigen-specific B cells displayed enriched variable gene usages, we analyzed VH and 285 VK/VL pairs for B cells targeting HCoV spike, non-RBD spike epitopes, and RBD-286 specific epitopes. A B cell was considered non-RBD spike-specific if it bound full-length 287 spike probe and not RBD probe, and a cell that bound both RBD and full-length spike 288 was considered to be RBD-specific. Using this approach, we found that B cells against 289

HCoV spike, non-SARS2 RBD spike epitopes, and the SARS2 RBD were enriched for 290 VH1-69 gene usage ( Figure 4A-4C ). VH1-69 is commonly utilized by broadly 291 neutralizing antibodies against the hemagglutinin stalk-domain of influenza viruses, as 292 well as the gp120 co-receptor binding site of HIV-1 due to its ability to bind conserved 293 hydrophobic regions of viral envelope glycoproteins (Chen et al., 2019). VH1-69 usage 294 by B cells that cross-react to SARS-CoV-2 and HCoV has also been indicated (Wec et 295 al., 2020) . However, VH1-69 usage for B cells targeting HCoV spike and SARS2 spike 296 non-RBD epitopes was predominantly enriched in convalescent visit 1 subjects and not 297 convalescent visit 2, suggesting that the repertoire may continue to evolve months after 298 infection ( Figure 4A and 4B; right). However, several VH gene usages were enriched in 299 both convalescent visits, regardless of antigen specificity. For SARS2 spike non-RBD-300 specific B cells, VH3-7 and VH1-24 were also commonly used, which we confirmed by 301 characterizing cloned mAbs from our cohort ( Figure 4B and Table S7 ). While NP- To better understand antigen-specific BCRs and how antigenic reactivity relates 310 to immune effectiveness, we next investigated the binding, neutralization potency, and 311 in vivo protective ability of mAbs cloned from select BCRs. To do so, we expressed 312 nearly 100 mAbs against the SARS2 spike, NP, and ORF8 from convalescent subjects, 313

representing a multitude of clusters (Table S7) . Cells from which to clone antibodies 314 were chosen at random, and were not chosen based on specific sequence features. 315

However, we note that the results described herein may be affected by sampling bias, 316

as only a small subset of antigen-specific mAbs were cloned. We confirmed that cells 317 designated as specific bound with moderate to high affinity to their corresponding 318 antigens ( Figure 5A ), and cells identified as multi-reactive exhibited features of 319 polyreactivity or bound to PE ( Figure S4F ). We next tested the antibodies for viral 320 neutralization by SARS-CoV-2/UW-001/Human/2020/Wisconsin virus plaque assays, 321

where lower plaque forming units (PFU)/ml equates to increased neutralization. 322

Whereas 82% percent of mAbs to the RBD were neutralizing including 42% exhibiting 323 complete inhibition, only 23% of mAbs to spike regions outside of the RBD were 324 neutralizing, and these showed relatively low potency ( Figure 5B ). NP-and ORF8-325 specific mAbs were entirely non-neutralizing ( Figure 5B ). Using animal models of 326 SARS-CoV-2 infection, we confirmed that anti-RBD antibodies were therapeutically 327 protective in vivo, preventing weight loss and reducing lung viral titers relative to PBS 328 control and an irrelevant Ebola anti-GP133 mAb ( Figure 5C and 5D) . 329

While mAbs to NP and ORF8 were non-neutralizing in vitro, they might still 330 provide protection in vivo, potentially through Fc-mediated pathways if the proteins were 331 exposed on the virus or cell surface at appreciable levels. However, neither ORF8-332 reactive mAbs nor NP-reactive mAbs conferred protection from weight loss or viral 333 infection in the lung in vivo ( Figure 5E-5H (Table S1) , as defined previously (Guthmiller et al., 2021) . 346

We found that reactivity of total B cells toward different antigens varied widely by 347 subject, likely reflecting host-intrinsic differences ( Figure 6A ). With age, we identified a 348 decrease in the generation of spike-specific B cells, and an increase in ORF8 and NP-349 specific B cells ( Figure 6B) . Similarly, the percentage of total spike-specific B cells was 350 reduced in subjects with more severe disease, whereas ORF8-specific B cells were 351 increased ( Figure 6C ). Lastly, we identified females had increased percentages of 352 ORF8-reactive cells, whereas males showed slightly greater percentages of NP-reactive 353 cells ( Figure 6D ). To address whether differences in B cell reactivity with age and 354 severity were associated with naïve-like or MBC subsets, we analyzed reactivity by 355 subset. We observed a substantial decrease in spike-specific MBCs and an increase in 356 NP-and ORF8-reactive MBCs with age, while naïve-like B cell subsets were more 357 evenly distributed in reactivity across age groups ( Figure 6E , Figure S5A ). We identified 358 a significant correlation with age and the percentage of ORF8-reactive MBCs in 359 females, but not in males ( Figure S5B and S5C). By contrast, the generation of specific 360

MBCs was not different between mild and severe cases, though naïve-like subsets 361 targeting ORF8 were increased across mild, moderate, and severe disease ( Figure 6F , 362 Figure S5D ). 363

While B cell memory to the spike was decreased in older patients, the overall 364 median number of VH SHMs for antigen-specific MBCs was increased relative to 365 younger patients ( Figure 6G ). However, whereas the majority of MBCs harboring the 366 most mutations targeted the SARS2 spike in younger age groups ( Figure 6H and 6I), 367 mutated MBCs against NP and ORF8 were proportionately increased relative to the 368 spike in older patients ( Figure 6J ). Finally, we observed variability in the percentages of 369

MBCs and naïve-like B cells across subjects ( Figure 6K In summary, our study highlights the diversity of B cell subsets expanded upon 379 novel infection with SARS-CoV-2. Using this approach, we identified that B cells against 380 the spike, ORF8, and NP differ in their ability to neutralize and derive from functionally 381 distinct and differentially adapted B cell subsets; that memory B cell output overtime 382 shifts from the spike to intracellular antigens; and that targeting of these antigens is 383 impacted by age, sex, and disease severity. The COVID-19 pandemic continues to pose one of the greatest public health and 387 policy challenges in modern history, and robust data on long-term immunity is critically 388 needed to evaluate future decisions regarding COVID-19 responses. Our approach 389 combined three powerful aspects of B cell biology to address human immunity to SARS-390

CoV-2: B cell transcriptome, Ig sequencing, and recombinant mAb characterization. We 391

show that antibodies targeting key protective spike epitopes are enriched within MBC 392

populations, but over time the MBC pool continues to adapt toward non-protective 393 intracellular antigens, which could be a molecular hallmark of waning protection. This is 394 further evidence that widespread vaccination, which only elicits a response to the spike, 395 may be critical to end the pandemic. 396

Through this study, we revealed that the landscape of antigen targeting and B 397 cell subsets varied widely across severe acute subjects and convalescent subjects 398 antigen-specific B cell subsets directly impacts susceptibility and disease severity, and 435 conversely, whether age or disease severity shape memory formation. Addressing 436 these questions will be critical for understanding the disease course, determining 437 correlates of protection, and developing vaccines capable of protecting against SARS-438

CoV-2 and emerging variants. 439 440

A primary limitation to this study is the assumption that the total number and overall 442 distribution of antigen-specific B cells is accurately captured by the bait-sorting method 443 described herein. These parameters could be altered by a number of factors, including 444 probe preparation, staining dilution, flow cytometry gating, and bioinformatics analysis. 445

However, this approach is inherently more sensitive and high-throughput compared to 446 serology and lower-throughput methods for single B cell cloning. The frequencies of 447 memory B cells examined will also be dependent on the numbers that could be isolated 448 J o u r n a l P r e -p r o o f 15 by bait-sorting starting from purified B cells, rather than examining reactivity within a 449 controlled number of memory B cells per subject. An additional limitation is that we were 450 unable to analyze acute subjects with a range of disease severity. Finally, we were 451 unable to obtain longitudinal samples from the same individuals across acute, early and 452 late convalescent time points, and future longitudinal studies assessing the evolution of 453 memory B cells to SARS-CoV-2 will be important. Table S5 and Table S6 . Table S7 . 

To assess the quality of B cell subsets identified in this study we used ROGUE scoring, 786 an entropy-based metric for assessing the purity of single cell populations, adapted from 787 a previous study . The expression entropy for each gene was 788 calculated using "SE_fun" from the "ROGUE" package (version 1.0). Based on the 789 expression entropy, the ROGUE score for each cluster was calculated using the "rogue" 790 function from the same package with parameters "platform" set to "UMI" and "span" set 791 to 0.6. 792 793 Antigen probe reactivity assignment 794

Antigen probe signals were normalized by a centered log-ratio transformation 795 individually for each subject. All B cells were subsequently clustered into multiple probe-796 specific groups according to their normalized probe signals. By investigating all 797 normalized antigen-probe binding signals, we arbitrarily set a threshold equal to 1 for all 798 normalized probe signals to distinguish probe binding cells as "positive" or "negative". 799

Cells that were negative to all probes were clustered into the "negative" group; those 800 positive to only one probe were clustered into corresponding probe-specific groups; and 801 those that were positive to multiple probes were further investigated. Only cells whose 802 top hit probe value was at least two-fold greater than their second hit probe value were 803 clustered into the top hit probe-specific group; others were clustered into the "multi-804 reactive" group that indicates non-specific cells. To account for the inclusion of endemic 805

HCoV spike protein reactivity in some samples, cells positive to both SARS2 spike and 806 endemic spike were further clustered into a group we assigned as "spike cross-reactive" 807 in the code. For samples in which we included separate SARS2 spike and RBD oligo 808 tags, we placed cells positive to both SARS2 spike and SARS2 RBD into the "spike" 809 group. 810 811

Scores for B-cell-genotype-related gene modules (e.g. MBC score, naïve score, ASC 813 score, and GC emigrant score) were calculated using the "AddModuleScore" function 814 to intranasal inoculation with 10 3 PFU of SARS-CoV-2 (n-CoV/USA_WA1/2020 strain). 911

Weight change was monitored daily and lungs were harvested at 7 days post-infection. 912

Viral RNA levels in lung homogenates were determined by qRT-PCR quantifying N 913 gene copy number and compared to a standard curve as described previously (Winkler 914 et al., 2020) . NP:

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