key: cord-0975228-9bhi3rqu authors: Wang, Zijun; Schmidt, Fabian; Weisblum, Yiska; Muecksch, Frauke; Barnes, Christopher O.; Finkin, Shlomo; Schaefer-Babajew, Dennis; Cipolla, Melissa; Gaebler, Christian; Lieberman, Jenna A.; Oliveira, Thiago Y.; Yang, Zhi; Abernathy, Morgan E.; Huey-Tubman, Kathryn E.; Hurley, Arlene; Turroja, Martina; West, Kamille A.; Gordon, Kristie; Millard, Katrina G.; Ramos, Victor; Da Silva, Justin; Xu, Jianliang; Colbert, Robert A.; Patel, Roshni; Dizon, Juan; Unson-O’Brien, Cecille; Shimeliovich, Irina; Gazumyan, Anna; Caskey, Marina; Bjorkman, Pamela J.; Casellas, Rafael; Hatziioannou, Theodora; Bieniasz, Paul D.; Nussenzweig, Michel C. title: mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants date: 2021-01-30 journal: bioRxiv DOI: 10.1101/2021.01.15.426911 sha: 207b8ab66205ba19ade6c5f52153372dbcac056f doc_id: 975228 cord_uid: 9bhi3rqu To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines1,2. These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known3–6. Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers3,5,6. Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection7,8. However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy. against newly arising variants, and that mRNA vaccines may need to be updated 59 periodically to avoid potential loss of clinical efficacy. 60 obtained 1.3 and 6.2 months after infection was 0.5-to 29-and 0.5-to 20.2-fold less effective in 107 neutralizing the K417N:E484K:N501Y combination (p=0.001 and p<0.0001, respectively, Fig. 108 1j, Extended Data Table 2 ). We conclude that the plasma neutralizing activity elicited by either 109 mRNA vaccination or natural infection is variably but significantly less effective against 110 pseudoviruses that carry RBD mutations found in emerging SARS-CoV-2 variants. 111 Fig. 3a and b) . We focused on the RBD since it is the 117 target of the majority of the more potent SARS-CoV-2 neutralizing antibodies discovered to 118 date [21] [22] [23] [24] [25] [26] . Notably, the percentage of RBD-binding memory B cells in vaccinees was significantly 119 greater than in naturally infected individuals assayed after 1.3 months, but similar to the same 120 individuals assayed after 6.2 months (Fig. 2b) Table 3) . 128 Expanded clones of cells comprised 4-50% of the overall RBD binding memory B cell 129 compartment ( Fig. 2c and d, and Extended Data Fig. 3d ). Similar to natural infection, IGVH 3-130 53, and 3-30 and some IGVL genes were significantly over-represented in the RBD-binding 131 memory B cell compartment of vaccinated individuals (Fig. 2e, Extended Data Fig. 4a) . In 132 addition, antibodies that share the same combination of IGHV and IGLV genes in vaccinees 133 comprised 39% of all the clonal sequences (Extended Data Fig. 4b ) and 59% when combined 134 with naturally infected individuals 7,8 (Fig. 2f) , and some of these antibodies were nearly identical 135 (Extended Data Table 3 and 4). The number of V gene nucleotide mutations in vaccinees is 136 greater than in naturally infected individuals assayed after 1.3 months, but lower than that in the 137 same individuals assayed after 6.2 months ( Fig. 2g and Extended Data Fig. 5a ). The length of the 138 IgH CDR3 was similar in both natural infected individuals and vaccinees and hydrophobicity 139 was below average 27 ( Fig. 2h and Extended Data Fig. 5a and b) . Thus, the IgG memory response 140 is similar in individuals receiving the Pfizer-BioNTech and Moderna vaccines and both are rich 141 in recurrent and clonally expanded antibody sequences. 142 143 One hundred and twenty-seven representative antibodies from 8 individuals were expressed and 144 tested for reactivity to the RBD (Extended Data Table 5 ). The antibodies included: (1) 76 that 145 were randomly selected from those that appeared only once, and (2) 51 representatives of 146 expanded clones. Of the antibodies tested 98% (124 out of the 127) bound to RBD indicating 147 that single cell sorting by flow cytometry efficiently identified B cells producing anti-RBD 148 antibodies (Extended Data Fig. 6a -c and Table 5 ). In anti-RBD ELISAs the mean half-maximal 149 effective concentration (EC50) was higher than that observed in infected individuals after 6.2 150 months but not significantly different from antibodies obtained 1.3 months after infection 151 (Extended Data Fig. 6a , and Table 5 To examine the neutralizing breadth of the monoclonal antibodies and begin to map their target 164 epitopes we tested 17 of the most potent antibodies (Extended data antibodies were provisionally assigned to a defined antibody class or a combination (Fig. 3b) . As 172 seen in natural infection, a majority of the antibodies tested (9/17) were at least ten-fold less 173 effective against pseudotyped viruses carrying the E484K mutation 7,12,29 . In addition, 5 of the 174 antibodies were less potent against K417N and 4 against N501Y by ten-fold or more (Fig. 3b) . 175 Similar results were obtained with antibodies being developed for clinical use (REGN10987, 176 REGN10933, COV2-2196, COV2-2130, C135 and C144 (Extended data Fig. 7) ). However, 177 antibody combinations remained effective against all of the variants tested confirming the 178 importance of using antibody combinations in the clinic (Extended data Fig. 7) . Whether less 179 potent antibodies show similar degrees of sensitivity to these mutations remains to be 180 determined. 181 To determine whether antibody-imposed selection pressure could also drive the emergence of 183 resistance mutations in vitro, we cultured an rVSV/SARS-CoV-2 recombinant virus in the 184 presence of each of 18 neutralizing monoclonal antibodies. All of the tested antibodies selected 185 for RBD mutations. Moreover, in all cases the selected mutations corresponded to residues in the 186 binding sites of their presumptive antibody class ( Fig. 3b and c) . For example, antibody C627, 187 which was assigned to class 2 based on sensitivity to the E484K mutation, selected for the 188 emergence of the E484K mutation in vitro (Fig 3c) To further characterize antibody epitopes and mechanisms of neutralization, we characterized 196 seven complexes between mAb Fab fragments and the prefusion, stabilized ectodomain trimer of 197 SARS-CoV-2 S glycoprotein 37 using single-particle cryo-EM (Fig 4 and Extended Data Table 7) . 198 Overall resolutions ranged from 5-8 Å (Extended Data Fig. 8 ) and coordinates from S trimer and 199 representative Fab crystal structures were fit by rigid body docking into the cryo-EM density 200 maps to provide a general assessment of antibody footprints/RBD epitopes. Fab-S complexes 201 exhibited multiple RBD-binding orientations recognizing either 'up'/'down' (Fig 4a-j) or solely 202 'up' (Fig 4k-n) RBD conformations, consistent with structurally defined antibody classes from 203 natural infection (Fig 4o) 29 . The majority of mAbs characterized (6 of 7) recognized epitopes that 204 included RBD residues involved in ACE2 recognition, suggesting a neutralization mechanism 205 that directly blocks ACE2-RBD interactions. Additionally, structurally defined antibody epitopes 206 were consistent with RBD positions that were selected in rVSV/SARS-CoV-2 recombinant virus 207 outgrowth experiments, including residues K417, N439/N440, E484, and N501 (Fig 3c and The comparatively modest effects of the mutations on viral sensitivity to plasma reflects the 244 polyclonal nature of the neutralizing antibodies in vaccinee plasma. Nevertheless, emergence of 245 these particular variants is consistent with the dominance of the class 1 and 2 antibody response 246 in infected or vaccinated individuals. We speculate that these mutations emerged in response to 247 immune selection in individuals with non-sterilizing immunity. What the long-term effect of 248 accumulation of mutations on the SARS-CoV-2 pandemic will be is not known, but the common 249 cold coronavirus HCoV-229E evolves antigenic variants that are comparatively resistant to the 250 older sera but remain sensitive to contemporaneous sera 50 . Thus, it is possible that these 251 mutations and others that emerge in individuals with suboptimal or waning immunity will erode 252 the effectiveness of natural and vaccine elicited immunity. The data suggests that SARS-CoV-2 253 vaccines and antibody therapies may need to be updated and immunity monitored in order to 254 compensate for viral evolution. Sequence Read Archive accession SRP010970 (orange), and vaccinees (blue). A two-sided 300 binomial test was used to compare the frequency distributions, significant differences are 301 denoted with stars (* p < 0.05, ** p < 0.01, *** p < 0.001, **** = p < 0.0001). f, Clonal 302 relationships between sequences from 14 vaccinated individuals (Moderna in black, Pfizer-303 BioNTech in red Extended Data Table 3 ) and naturally infected individuals (in green, from 7,8 ). 304 Interconnecting lines indicate the relationship between antibodies that share V and J gene 305 segment sequences at both IGH and IGL. Purple, green and grey lines connect related clones, 306 clones and singles, and singles to each other, respectively. g, Number of somatic nucleotide 307 mutations in the IGVH (top) and IGVL (bottom) in vaccinee antibodies (Extended Data Table 3 ) 308 compared to natural infection obtained 1.3 or 6.2 months after infection 7,8 . Statistical 309 significance was determined using the two-tailed Mann-Whitney U-tests and red horizontal bars 310 indicate mean values. h, as in g, but for CDR3 length. Polymerase (Millipore Sigma) was used for amplification of cDNA using primers flanking the S-433 encoding sequence. The PCR products were purified and sequenced as previously described 7,12 . 434 Briefly, tagmentation reactions were performed using 1ul diluted cDNA, 0.25 µl Nextera TDE1 435 Tagment DNA enzyme (catalog no. 15027865), and 1.25 µl TD Tagment DNA buffer (catalog 436 no. 15027866; Illumina). Next, the DNA was ligated to unique i5/i7 barcoded primer 437 combinations using the Illumina Nextera XT Index Kit v2 and KAPA HiFi HotStart ReadyMix 438 (2X; KAPA Biosystems) and purified using AmPure Beads XP (Agencourt), after which the 439 samples were pooled into one library and subjected to paired-end sequencing using Illumina 440 MiSeq Nano 300 V2 cycle kits (Illumina) at a concentration of 12pM. 441 For analysis of the sequencing data, the raw paired-end reads were pre-processed to remove trim 442 adapter sequences and to remove low-quality reads (Phred quality score < 20) using BBDuk. Antibodies were identified and sequenced as described previously 8 . In brief, RNA from single 507 cells was reverse-transcribed (SuperScript III Reverse Transcriptase, Invitrogen, 18080-044) and 508 the cDNA stored at −20 °C or used for subsequent amplification of the variable IGH, IGL and 509 IGK genes by nested PCR and Sanger sequencing. Sequence analysis was performed using 510 MacVector. Amplicons from the first PCR reaction were used as templates for sequence-and 511 ligation-independent cloning into antibody expression vectors. Recombinant monoclonal 512 antibodies and Fabs were produced and purified as previously described 8 . 513 514 Expression and purification of SARS-CoV-2 6P stabilized S trimers 37 was conducted as 516 previously described 53 . Purified Fab and S 6P trimer were incubated at a 1.1:1 molar ratio per 517 protomer on ice for 30 minutes prior to deposition on a freshly glow-discharged 300 mesh, 518 1.2/1.3 Quantifoil copper grid. Immediately before 3 µl of complex was applied to the grid, 519 fluorinated octyl-malotiside was added to the Fab-S complex to a final detergent concentration of 520 0.02% w/v, resulting in a final complex concentration of 3 mg/ml. Samples were vitrified in 521 100% liquid ethane using a Mark IV Vitrobot after blotting for 3 s with Whatman No. 1 filter 522 paper at 22˚C and 100% humidity. 523 524 Cryo-EM data collection and processing 525 Data collection and processing followed a similar workflow to what has been previously 526 described in detail 29 . Briefly, micrographs were collected on a Talos Arctica transmission 527 electron microscope (Thermo Fisher) operating at 200 kV for all Fab-S complexes. Data were 528 collected using SerialEM automated data collection software 54 and movies were recorded with a 529 K3 camera (Gatan). For all datasets, cryo-EM movies were patch motion corrected for beam-530 induced motion including dose-weighting within cryoSPARC v2.15 55 after binning super 531 resolution movies. The non-dose-weighted images were used to estimate CTF parameters using 532 cryoSPARC implementation of the Patch CTF job. Particles were picked using Blob picker and 533 extracted 4x binned and 2D classified. Class averages corresponding to distinct views with 534 secondary structure features were chosen and ab initio models were generated. 3D classes that 535 showed features of a Fab-S complex were re-extracted, unbinned (0.869 Å/pixel) and 536 homogenously refined with C1 symmetry. Overall resolutions were reported based on gold 537 standard FSC calculations. 538 539 Coordinates for initial complexes were generated by docking individual chains from reference 541 structures into cryo-EM density using UCSF Chimera 56 (S trimer: PDB 6KXL, Fab: PDB 6XCA 542 or 7K8P after trimming CDR3 loops and converting to a polyalanine model). Models were then 543 refined into cryo-EM maps by rigid body and real space refinement in Phenix 57 . If the resolution 544 allowed, partial CDR3 loops were built manually in Coot 58 and then refined using real-space 545 refinement in Phenix. 546 547 Antibody sequences were trimmed based on quality and annotated using Igblastn v.1.14. with 549 IMGT domain delineation system. Annotation was performed systematically using Change-O 550 toolkit v.0.4.540 59 . Heavy and light chains derived from the same cell were paired, and 551 clonotypes were assigned based on their V and J genes using in-house R and Perl scripts (Fig. 2c 552 and f, Extended data Fig. 3d, Extended data Fig. 4b) . All scripts and the data used to process 553 antibody sequences are publicly available on GitHub (https://github.com/stratust/igpipeline). 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