key: cord-0712728-6bx9b8io
authors: Fenwick, Craig; Turelli, Priscilla; Ni, Dongchun; Perez, Laurent; Lau, Kelvin; Lana, Erica; Pellaton, Céline; Raclot, Charlène; Esteves-Leuenberger, Line; Campos, Jérémy; Farina, Alex; Fiscalini, Flurin; Herate, Cécile; Marlin, Romain; Abdelnabi, Rana; Foo, Caroline S.; Neyts, Johan; Leyssen, Pieter; LeGrand, Roger; Lévy, Yves; Pojer, Florence; Stahlberg, Henning; Trono, Didier; Pantaleo, Giuseppe
title: SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties
date: 2022-03-18
journal: bioRxiv
DOI: 10.1101/2022.03.18.484873
sha: 21ac62b707bedc86f09eda517f7cf4e7765d2abd
doc_id: 712728
cord_uid: 6bx9b8io

The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one another’s escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.

Identification of P2G3, a highly potent SARS-CoV-2 neutralizing antibody 79 We screened for the presence of anti-Spike antibodies in serum samples from a cohort of >100 80 donors and focused on one post-infected donor that received two doses of the mRNA-1273 81 vaccine and displayed among the highest serum antibody levels with excellent breadth against 82 a panel of SARS-CoV-2 variants in a trimeric Spike-ACE2 surrogate neutralization assay 16 . 83 Screening of B cell clone supernatants for high affinity Spike binding led us to prioritize six 84 clones for mAb production via expression of paired heavy and light chains in ExpiCHO cells. 85 During initial profiling of these purified mAbs, P2G3 exhibited the strongest binding affinity Regeneron 17 , AZD8895 and AZD1061 from AstraZeneca 18 , ADG-2 from Adagio 19 , 91 S309/Sotrovimab from Vir/GSK 20 ) and mAbs previously described by our group 21 , 92 demonstrate that P2G3 binds an unique albeit overlapping epitope with those recognized by 93 both AZD1061 and S309/Sotrovimab, the latter of which acts by a mechanism distinct from 94 blocking the RBD/ACE2 interaction 20 (Extended Data Fig. 1b) . Importantly, our potent and 95 broadly active Class 1 mAb, P5C3, bound RBD non-competitively with P2G3, prompting us to 96 profile these mAbs both alone and in combination for subsequent studies. 97 Using a biochemical trimeric Spike-ACE2 surrogate neutralization assay 16 , we further 98 determined that P2G3 and P5C3 had the most potent and broad activity in blocking ACE2 99 binding to high quality structural grade Spike trimers from all past VOCs compared to our panel 100 of benchmark mAbs (Extended data Fig. 1 c-d) . P2G3 and P5C3 also display the most potent 101 activity in assays performed with the Spike protein from Omicron BA.1, BA.1.1 that includes 102 the R346K mutation and BA.2. (Fig. 1a and 1b) . P2G3 alone gave comparable activities to reached ~100% of inhibition in blocking ACE2 binding to Omicron Spike proteins at 1 µg/ml 108 total mAbs, P2G3 alone only blocked 50-72% of ACE2 binding at 20 µg/ml and P5C3 alone 109 reached a ~100% inhibition at 20 µg/ml. 110 We next compared P2G3 alone or in combination with P5C3 with our panel of current clinically 111 authorized and/or clinically advanced mAbs in pseudovirus neutralization assays. P2G3 had 112 potent neutralizing activity against lentiviruses pseudotyped with Spike from initial 2019-nCoV 113 (D614G), Alpha, Beta and Delta VOCs (IC80 value of 0.022, 0.051, 0.038 and 0.035 µg/ml, 114 respectively). Most importantly, P2G3 strongly neutralized the Omicron BA.1 Spike 115 pseudovirus with an IC80 value of 0.038 µg/ml (Fig. 2a) and thus showed no loss of activity as 116 compared to the other VOCs. In side-by-side comparisons, P2G3 was >42-fold more potent 117 than ADG-2, AZD1061, AZD8895, REGN10933 and REGN10987 mAbs and 19-fold more 118 potent than Sotrovimab at neutralizing Omicron BA.1 Spike pseudotyped lentiviral particles 119 (Fig. 2b) . Second most potent was P5C3, with an IC80 value of 0.223 µg/ml, and the 120 P2G3/P5C3 combination revealed a minor-enhanced activity over P2G3 alone in this assay, 121 with an IC80 value of 0.024 µg/ml for the total concentration of the two mAbs. Furthermore, 122 P2G3 and P5C3 maintained full neutralizing activity against the ancestral D614G and Omicron 123 BA.1.1 encoding the R346K Spike pseudovirus (Extended data Fig. 2a and b) , a mutation 124 present in ~10% of Omicron variant sequences in the GISAID 11 . 125 We next profiled P2G3 using the initial D614G strain and all current VOCs in a live virus BA.2 SARS-CoV-2 variants ( Fig. 2c-2f) . All tested mAbs displayed good activity against the 132 Delta variant, although Sotrovimab was ~6 times less potent than ADG-2 and >10 times less 133 potent than P2G3, P5C3 and both the AZD and REGN cocktails against this virus. However 134 and most notably, P2G3 was by far the most active mAb against Omicron BA.1 with an IC80 135 value of 0.035 µg/ml, that is ~23-fold, 60-fold and 88-fold more potent than those of 136 AZD1061/AZD8895, Sotrovimab and ADG-2, respectively, the REGN combination being 137 completely ineffective against this variant ( Fig. 2d and 2f) . Of note, while P5C3 alone 138 displayed an IC80 against Omicron in the range of that of the AZD cocktail, the P2G3/P5C3 139 combination showed high neutralization activity comparable to P2G3 alone with an IC80 of 140 0.039 µg/ml total mAb. P2G3 was again the most potent mAb in neutralizing the Omicron BA.2 141 variant with an IC80 values of 0.014 µg/ml, that is 4.9-fold, 14-fold, 907-fold and 607-fold more 142 potent than AZD1061/AZD8895, REGN10933/REGN10987, ADG-2 and Sotrovimab, 143 respectively ( Fig. 2e-2f) . Importantly, although the Spike-ACE2 surrogate neutralization assay 144 correlates well with cell-based neutralization assays 16 , P2G3 only blocked ~50-72% of ACE2 145 binding to Omicron Spike proteins (Fig. 1a) despite P2G3 reaching 100% of the maximum 146 signal in a direct binding assay (Extended data Fig. 1e ). This suggests that at least with the 147 Omicron variant, virus inhibition by P2G3 may not be solely mediated through the Spike-ACE2 148 interaction, but rather a mechanism analogous with that of S309/Sotrovimab, reported to be Fig. 4 b) . Complete prophylactic protection was observed with 185 P2G3 mAb plasma levels >6.2 µg/ml at the time of viral inoculation and P2G3 treatment groups 186 showed a significant ~4-log reduction of genomic viral RNA levels (Extended data Fig. 4 c) . 187 We next evaluated P2G3 mediate protection from SARS-CoV-2 Omicron BA.1 infection in a 188 cynomolgus macaques pre-exposure challenge study. Monkeys were administered 10 mg/kg of 189 P2G3 LS intravenously and challenged 72 hrs later via combined intranasal and intratracheal 190 routes with 1x10 5 TCID50 of SARS-CoVo-2 B.1.1.519 Omicron BA.1 virus (Fig. 3a) . 191 Following viral challenge, control animals showed similar genomic (g)RNA levels and kinetics 192 with median peak viral loads (VL) of 6.9-and 6.6-log10 copies/ml gRNA at 2-3 days post 193 challenge in tracheal swabs and bronchoalveolar lavage (BAL) samples, respectively (Fig. 3b) . 194 Nasopharyngeal swabs showed a higher-level variability in VL between control animals but 195 still showed median peaks of 6.9-log10 copies/ml for gRNA. In comparison, the two P2G3 LS 196 treated monkey had a strong median peak VL reduction of 3.8-, 2.5-and 3.9-log10 copies/ml 197 gRNA for tracheal, nasopharyngeal and BAL samples, respectively. (<2.9-log10) to 5.4-log10 copies/ml (Fig. 3c) . P2G3 LS treated monkeys had sgRNA levels 202 that were at or below the limit of detection, exhibiting 1.9-, 2.2-and 1.6-log10 reduced levels 203 in tracheal, nasopharyngeal and BAL samples, respectively. Consistent with viral protection 204 resulting in reduced detection of gRNA and sgRNA, P2G3 treated monkeys exhibited stable 205 lymphocyte levels throughout the study, whereas strong lymphopenia, determined by 206 lymphocyte levels below 2.1x 10 3 cells/µl, was observed in all control animals challenged with 207 the Omicron variant of SARS-COV-2 (Fig. 3d) . 208 Structural analysis of P2G3 and P5C3 Fab bound to Omicron Spike trimer. 209 To decipher the molecular features underlying P2G3 and P5C3 potent neutralization of 210 Omicron Spike, we performed single particle cryo-EM reconstruction of the Omicron Spike 211 trimeric ectodomain 12,28,29 bound to both Fabs, at a 3.04 Å resolution (Fig. 4a , Extended Data . CDRH2 extends the epitope by interacting with R346 that is engaged by residue W53 by a 232 potential cation-pi interaction ( Fig. 4d and Extended Data Fig. 8e) , an interaction that is likely 233 conserved with the R346K Spike substitution (Extended data Fig. 2a-b) . The only potential 234 contact from the light chain derives from the CDRL1 Y32 forming a hydrophobic interaction 235 with V445 of the RBD (Extended Data Fig. 8c) . Moreover, P2G3 is only observed to contact 236 RBD amino acid residues and the distance to the nearest atom of the glycan branch is ~10Å 237 from P2G3. Importantly, the epitope defined by our structural studies rationalizes the potent 238 neutralizing activity of P2G3 against the Omicron variant relative to other Class 3 mAbs.

Omicron mutations S371L, N440K, G446S and the minor R346K sub-variant are all situated 240 outside of, adjacent to or have little effect on recognition of the P2G3-binding epitope, whereas 241 two or more of these mutations directly impinge on epitopes recognized by REGN10987, 242 AZD1061 and S309/Sotrovimab (Fig. 4e) . Furthermore, P2G3 displays a unique binding 243 orientation on the RBD with its Fab angling away from most of these Omicron mutations (Fig.   244 4f). In modelling the observed angles of attack of various Class 3 mAbs, it is likely that 245 REGN10987 only binds to the up-RBD form while AZD1061 binds one up-and one down-246 RBD form, with steric hindrance blocking the third RBD site on the Omicron Spike trimer 247 (Extended data Fig. 10) . In contrast, P2G3 and S309/Sotrovimab likely binds both the up and 248 down forms of Omicron RBD without clashes (Fig. 4g, Extended data Fig. 10c) , a 249 characteristic that may contribute to the largely conserved and high potency of P2G3 across animal models compared to other VOCs 31,32 .

Despite the exceptional neutralization profile of P2G3 against currently circulating variants, 305 development of resistance is almost inevitable when a virus is under selective pressure. We 306 reveal here that P5C3, a previously described potent and broadly active neutralizing mAb that 307 acts by blocking the Spike-ACE2 interaction, not only targets a highly conserved region of the 308 RBD but also can bind Omicron Spike concomitantly with P2G3. We further identify mutants 309 capable of escaping neutralization by either one of these mAbs, but demonstrate that i) they are 310 lowly infectious, ii) they are extremely rare in the wild, suggesting poor fitness, and iii) P5C3 of CDRH2 residue W53 that forms a potential cation-pi interaction with RBD residue R346.

The mesh represents the Cryo-EM density. Table   635 3) designed on consensus sequence from available Omicron sequences, and introduced by In- purification. The trimeric Spike variants were produced and purified as previously described 16 .

The purity of Omicron Spike trimers used for cryo-EM was determined to be >99% pure by Anti-Spike B cell sorting, immortalization and cloning 671 The blood from a ImmunoVax study donors were collected in EDTA tubes and the isolation of Table 3 . Delta B1.617.2 clone was generated by gene synthesis with a 717 codon-optimized Spike ORF (GenScript). The Omicron ORF was amplified from an RNA as 718 described for protein production with primers listed in Table 3 Cryo-electron microscopy 811 Cryo-EM grids were prepared with a Vitrobot Mark IV (Thermofisher Scientific (TFS)). 

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