key: cord-0284019-g2y37i03
authors: Kaku, Chengzi I.; Bergeron, Alan J.; Ahlm, Clas; Normark, Johan; Sakharkar, Mrunal; Forsell, Mattias N. E.; Walker, Laura M.
title: Recall of pre-existing cross-reactive B cell memory following Omicron breakthrough infection
date: 2022-04-01
journal: bioRxiv
DOI: 10.1101/2022.04.01.486726
sha: ff8ed9c9c7309d7e6c64e602647e8e9c27fad232
doc_id: 284019
cord_uid: g2y37i03

Understanding immune responses following SARS-CoV-2 breakthrough infection will facilitate the development of next-generation vaccines. Here, we profiled spike (S)-specific B cell responses following Omicron/BA.1 infection in mRNA-vaccinated donors. The acute antibody response was characterized by high levels of somatic hypermutation (SHM) and a bias toward recognition of ancestral SARS-CoV-2 strains, suggesting the early activation of vaccine-induced memory B cells (MBCs). BA.1 breakthrough infection induced a shift in B cell immunodominance hierarchy from the S2 subunit toward the receptor binding domain (RBD). A large proportion of RBD-directed neutralizing antibodies isolated from BA.1 breakthrough infection donors displayed convergent sequence features and broadly recognized SARS-CoV-2 variants of concern (VOCs). Together, these findings provide fundamental insights into the role of pre-existing immunity in shaping the B cell response to heterologous SARS-CoV-2 variant exposure. One sentence summary BA.1 breakthrough infection activates pre-existing memory B cells with broad activity against SARS-CoV-2 variants.

variants of concern (VOCs). Together, these findings provide fundamental insights into the role 32 of pre-existing immunity in shaping the B cell response to heterologous SARS-CoV-2 variant 33 exposure. To investigate the impact of pre-existing vaccine-induced immunity on the B cell 115 response to BA.1 breakthrough infection, we enumerated B cells that displayed WT/BA.1 RBD 116 cross-reactivity in BA.1 breakthrough donors and uninfected/mRNA vaccinated individuals (Fig.  117 2C, Fig. S2A ). At one month post primary mRNA vaccination, only 48% of total RBD-directed 118 B cells displayed cross-reactivity with BA.1 (Fig. 2D) . The proportion of WT/BA.1 RBD cross-119 reactive B cells increased to 57% at 6-months post-primary vaccination and to 70% following labeled tetramers of full-length S, RBD, NTD, and prefusion-stabilized S2 (Fig. S2C ). In the 136 uninfected/vaccinated cohort, class-switched B cells targeting the NTD, RBD, and S2 137 subdomains comprised 18%, 25%, and 37% of the total S-directed response, respectively, and 138 these proportions remained largely unchanged at six months post-vaccination and after mRNA 139 booster immunization ( Fig. 2F-H) . In contrast, we observed significantly higher proportions of 140 RBD-directed B cells among donors with breakthrough infection, ranging from 35-63% 141 (median=46%) of the total activated (CD71 + ) B cell response to S ( Fig 2G) . Furthermore, S2-142 reactive B cells comprised a smaller fraction (median=16%) of the S-specific response in 143 breakthrough donors relative to uninfected/mRNA vaccinated individuals (Fig 2H) . This altered Sequence analysis revealed that the BA.1 RBD-reactive antibodies displayed a relatively 158 high level of clonal diversity, with 7-45% belonging to expanded clonal lineages (Fig. 3B) . We 159 observed a significant over-representation of heavy chain germline genes IGHV3-53, 3-66, 3-30, 160 1-69, 3-9, and 4-31 in BA.1 breakthrough infection repertoires relative to the baseline human 161 repertoire (17) (Fig. 3C ). While IGHV3-53, 3-66, and 3-30 germline gene families have also 162 been shown to be over-represented in the antibody response to ancestral SARS-CoV-2 strains, 163 IGHV1-69, 3-9, and 4-31 appear to be unique to the BA.1 breakthrough response (18) (Fig. 3C) . 164

The BA.1 RBD-reactive antibodies displayed a similar HCDR3 length distribution compared to 165 the baseline repertoire (Fig. 3D ). Ninety-five to 100% of antibodies derived from each donor 166 contained somatic mutations, with median SHM levels ranging from 8 to 11 nucleotide 167 substitutions in VH, supporting a memory B cell origin (Fig. 3E ). We conclude that the early B 168 cell response to breakthrough infection is dominated by highly mutated clones that cross-react 169 with both WT and BA.1 RBDs. 170

To further evaluate the binding properties of the BA.1 RBD-reactive antibodies, we 171 neutralizing antibodies utilized one of three VH germline genes (IGHV3-53/66, IGHV1-69, and 208 IGHV3-9) (Fig. 4F and Fig. S7 ). Similar to previously described IGHV3-53/66 antibodies 209 isolated from mRNA-vaccinated individuals, the BA.1 neutralizing IGHV3-53/66 antibodies 210 possessed short HCDR3s (11-12 residues) and displayed competitive binding with ACE2, the 211 class 1 mAb REGN10933, and the COVA1-16-like class 4 mAb ADI-62113 (21) (Fig. 4G and 212 (median IC50 = 0.016 and 0.051 µg/ml, respectively) (22, 23) ( Fig. 4E and Fig. S9 ). Thus, these 217 IGHV3-53/66-utilizing antibodies appear to recognize an antigenic site that is overlapping but 218 distinct from previously described IGHV3-53/66 antibodies induced by infection and 219

Neutralizing antibodies utilizing the IGHV1-69 and IGHV3-9 germline genes also 221 broadly recognized SARS-CoV-2 variants, including BA.2 (Fig. 4E) . In contrast to IGHV3-222 53/66, these germline genes have not been shown to be over-represented among RBD-directed 223 antibodies identified from mRNA-vaccinated donors (Fig. 3C) . Antibodies utilizing the IGHV1-224 69 germline gene segregated into two groups, one comprised of antibodies that targeted an 225 ACE2-and REGN10933-competitive region and the other containing antibodies that recognized 226 a non-ACE2 competitive site overlapping the COV2-2130 (class 3) epitope (Fig. 4G) . 227

Notably, >80% of non-ACE2 competitive clones utilized the light chain IGLV1-40 gene and 228 displayed highly similar LCDR3 sequences, suggesting a convergent mode of recognition (Fig.  229  S10) . Finally, 12/13 IGHV3-9 antibodies recognized an epitope outside of the ACE2 binding site 230 and competed with all three class 3 antibodies tested (S309, REGN10987, and COV2-2130), 231 suggesting a binding mode distinct from the IGHV1-69 antibodies (Fig. 4G) . Taken conserved site on SARS-CoV-2, variants of concern, and related viruses. 

SARS-CoV-2 Omicron triggers cross-reactive neutralization and Fc 288 effector functions in previously vaccinated, but not unvaccinated individuals. medRxiv

Vaccine Breakthrough Infection with the SARS-CoV-2 Delta or 291

BA.1) Variant Leads to Distinct Profiles of Neutralizing Antibody Responses. 292 medRxiv

Efficient recall of Omicron-reactive B cell memory after a third dose of 294 SARS-CoV-2 mRNA vaccine. bioRxiv

Increased Potency and Breadth of SARS-CoV-2 Neutralizing 296

Antibodies After a Third mRNA Vaccine Dose. bioRxiv

Commonality despite exceptional 298 diversity in the baseline human antibody repertoire

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. 300

SARS-CoV-2 neutralizing antibody structures inform therapeutic