key: cord-0817521-tjfu02tx
authors: DiMuzio, Jillian M.; Heimbach, Baron C.; Howanski, Raymond J.; Dowling, John P.; Patel, Nirja B.; Henriquez, Noeleya; Nicolescu, Chris; Nath, Mitchell; Polley, Antonio; Bingaman, Jamie L.; Smith, Todd; Harman, Benjamin C.; Robinson, Matthew K.; Morin, Michael J.; Nikitin, Pavel A.
title: Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein
date: 2021-01-28
journal: bioRxiv
DOI: 10.1101/2021.01.27.428534
sha: d9275c71c5ffc70ef01b6bf6fb49bf5672a65c22
doc_id: 817521
cord_uid: tjfu02tx

Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

monoclonal antibodies that were selected on the basis of their binding to multiple SARS-92

CoV-2 proteins in both cell-based and target-based screens. Characterization of these 93 antibodies revealed broad responses to diverse viral antigens. Fewer than half of the 94 antibodies were directed at S protein, while the remainder were directed at other viral 95 proteins including N and ORF-encoded proteins. Even though the antibodies were 96 directed at highly diverse SARS-CoV-2 antigens, they were generally characterized as 97 having variable levels of somatic hypermutation (SHM) and a diversity of VL and VH gene 98 usage. Functional properties of anti-Spike antibodies were successfully confirmed in a 99 pseudovirus neutralization assay. These results indicate that an unbiased interrogation 100 of COVID-19 patient B cell repertoires is an effective approach to identifying specific anti-101 viral antibodies and antibody mixtures with the desired binding and functional properties. 102

Antibodies identified and characterized in this manner could be recombinantly produced 103 to yield therapeutic or prophylactic products to address the COVID-19 pandemic. The 104 rapidity with which antibodies from convalescent patients can be identified and 105

convalescent patients who demonstrated a high (2880) antibody titer to N and/or S 116 proteins. More than 17,000 hybridomas were generated from the memory B cells of six 117 patients (Table 1) , and the naturally occurring human antibodies (IgM, IgG, and IgA 118 isotypes) secreted by those hybridomas were screened for reactivity against a panel of 119 SARS-CoV-2 proteins. Antibody screening assays were developed for three structural 120 proteins (S, N, M) and a panel of accessory ORF proteins of SARS-CoV-2 ( Table 2) window. Viral protein expression in the cell-based assay was additionally confirmed by Consistent with previous reports [12, 14] , S protein was identified as the major antigen for 163 antibody responses in these patients. As shown in Figure 3A , S represented the single 164 most frequently identified target: 76 (44%) of the mono-specific antibodies bound 165 selectively to S protein. As expected, the epitopes for anti-Spike antibodies were 166 distributed across the RBD, non-RBD S1, and non-S1 regions of the Spike protein ( Figure  167 3B). However, consistent with the hypothesis that patients would be mounting antibody 168 responses to a broad range of SARS-CoV-2 proteins, more than half (56%) of identified 169 antibodies bound to non-Spike viral antigens (Figure 3 ). ORF8 -(28 antibodies, 17%) and 170 N -(23 antibodies, 14%) specific antibodies represented the second and third most-171 frequently identified targets, respectively. Of note, the antibodies identified in the hTRF 172 assay were biased toward the IgG class (>80%) ( Figure 4A ). This bias was likely due to 173 lack of sensitivity in the assay for either IgA or IgM, as demonstrated in the control antibody titrations shown in Figure 1 . While IgG was still the predominant isotype (~50%) 175 in the cell-based screening, IgA (~20%) and IgM (~30%) antibodies were also well-176 represented ( Figure 4B ). Further, these isotypes were also spread across the landscape 177 of antigens being evaluated ( Figure 4C) . among all "mutated" Igs that had more than 2% of their nucleotide sequence deviated 231 from the closest germline, there was an unusually high (26.4%) proportion of mutated 232 IgMs ( Figure 6B ), having a mean SHM rate of 5.73%. The functional basis of this 233 phenomenon is not known, but one could speculate that these IgMs came from non-234 switched memory B cells that had undergone affinity maturation. Second, a subset of 235 such somatically hypermutated IgMs recognized full-length Spike, but not the soluble 236 RBD or S1 subunit of Spike protein. And third, while the predominant isotype among 237 class-switched antibodies was, as expected, IgG ( Figure 6C ), we were able to capture a 238 panel of fairly mutated virus-specific IgAs. It is plausible that these antibodies play a major 239 role in mucosal neutralization of the incoming virus and may be of particular use for 240

prophylaxis of viral infection and for vaccine design ( Figure 6D ). 241 and Ab#26, potently neutralized pseudovirus infection with EC50 <500 ng/mL (Table 3) . Table 3 : Binding properties of anti-Spike antibodies 278

The SARS-CoV-2 pandemic has stimulated extraordinary efforts to study anti-viral 282 response and to develop means for treatment and prophylaxis, in both the academic and 283 the biopharmaceutical research communities. By the end of October 2020, a mere 11 284 months after the virus was first identified, the Clinicaltrials.gov database listed more than 285 3500 distinct clinical trial activities directed at patients infected with SARS-CoV-2. There 286 is significant diversity among these efforts, from the assessment of existing drugs, to the 287 use of convalescent plasma from recovered patients, to the use of specific vaccines and 288 antibodies directed at the viral S protein. It is not yet apparent that there will be a single 289 The majority of anti-S antibodies, as expected, recognized a full-length S protein (Table  322 3). Further, all but one of the six COVID-19 convalescent patients produced antibodies to 323 a soluble receptor-binding domain of S protein (RBD). Of note, while most of the RBD-324 and S1-specific antibodies were less mutated than those specific to the full-length S 325 protein, we identified several highly mutated RBD-specific outliers. Lower rate of SHM in 326 the S-specific antibody group may connote limited rounds of affinity-maturation for a high 327 Immunome. The samples were deidentified and the B cells were isolated from those 376 deidentified blood samples. Immunome did not seek IRB approval of a "research project" 377 because analysis of peripheral blood samples that are obtained with consent, 378 deidentified, coded or anonymized are not believed to be subject to human tissue 379 research regulations. Immunome used a commercial vendor to obtain additional blood 380 samples, one with a standing IRB approval in place for their donor collection efforts.

Hybridomas were generated following protocols for isolating and expanding primary B- Nano Kit V2 following bead-based cleanup of RNA. Immunoglobulin sequences 464 containing CDR1, 2 and 3 and framework regions were amplified using IgG and IgA-465 specific mixes for IgH, and kappa and lambda-specific primers for IgL. IgM-expressing 466 hybridoma samples, from which IgG or IgA heavy chains were not amplified using this 467 approach, were sequenced using the iRepertoire iPair system. Final sequences were 468 exported using iPair software. 469

The analysis of primary NGS data was performed by iRepertoire. Immunoglobulin 470 sequences were analyzed for predicted CDR sequences, % identity to appropriate 471 germlines, isotype of the constant regions and read counts. Sequence pairing was performed based on the read count information. In the event more than one LC:HC pair 473 was discovered in a single well, each LC:HC combination was analyzed as a separate 474 antibody. In wells exhibiting 5' truncation in the V region, the germline sequence was used 475 to create an expression construct. Final sequences were translated and analyzed for 476 potential stop codons and frame shifts. PureFection reagent (System Biosciences; Cat # LV750A-1) was added to each 1mL 505 tube, vortexed for 10 seconds, and incubated at room temperature for 15 minutes. The 506 plasmid and PureFection mixture was added to a T150 flask containing 293TN cells and 507 placed in a 37°C incubator containing 5% CO2 for 48 hours. Pseudovirus-containing 508 supernatants were harvested at 48 hours and passed through a 0.45-micron PVDF filter 509 to remove cellular debris. 5x PEG-it Virus Precipitation Solution (System Biosciences, 510

Cat # LV810A-1) was added to supernatants and incubated 4°C overnight. 511

were then spun at 1500 x g for 30 minutes. Pseudovirus-containing pellets were 513 resuspended in plain DMEM to achieve at 10x concentration and frozen at -80°C in 514 single use aliquots. 515

Pseudovirus infection and neutralization assays were performed by adapting 517 established protocols [27, 28] . In summary, 10 4 ACE2-293T cells were plated in the 518 inner 60 wells of a 96 well flat bottom plate in 100uL of ACE2-293T media overnight in a 519 37°C incubator containing 5% CO2. To determine infectivity of each lot of pseudovirus, 520 pseudovirus-containing supernatants were thawed from -80°C and two-fold dilutions 521 were performed. 100 mL of pseudovirus at various dilutions was added to ACE2-293T 522 cells. To test neutralization activity of antibodies, indicated antibody concentrations 523

were pre-incubated with pseudovirus for 1 hour in a 37°C incubator containing 5% CO2. 

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