Journal Pre-proof

Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75

Qian Wang, Sho Iketani, Zhiteng Li, Yicheng Guo, Andre Yanchen Yeh, Michael Liu,
Jian Yu, Zizhang Sheng, Yaoxing Huang, Lihong Liu, David D. Ho

PII: S1931-3128(22)00419-X
DOI: https://doi.org/10.1016/j.chom.2022.09.002
Reference: CHOM 2779

To appear in: Cell Host and Microbe

Received Date: 4 August 2022

Revised Date: 29 August 2022
Accepted Date: 1 September 2022

Please cite this article as: Wang, Q., Iketani, S., Li, Z., Guo, Y., Yeh, A.Y., Liu, M., Yu, J., Sheng, Z.,
Huang, Y., Liu, L., Ho, D.D., Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75,
Cell Host and Microbe (2022), doi: https://doi.org/10.1016/j.chom.2022.09.002.

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© 2022 The Author(s). Published by Elsevier Inc.

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 Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75

Qian Wang1,5, Sho Iketani1,5, Zhiteng Li1, Yicheng Guo1, Andre Yanchen Yeh2, Michael Liu1,
Jian Yu1, Zizhang Sheng1, Yaoxing Huang1, Lihong Liu1,*, and David D. Ho1,3,4,6,*

1
Aaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians

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and Surgeons, New York, NY 10032, USA.

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School of Medicine, National Taiwan University, Taipei 100233, Taiwan

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Department of Microbiology and Immunology, Columbia University Vagelos College of
Physicians and Surgeons, New York, NY 10032, USA.
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Division of Infectious Diseases, Department of Medicine, Columbia University Vagelos College

of Physicians and Surgeons, New York, NY 10032, USA.
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These authors contributed equally
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Lead contact
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*
Correspondence: ll3411@cumc.columbia.edu (L.L.), dh2994@cumc.columbia.edu (D.D.H.)
 Summary

1 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2.75
2 emerged recently and appears to be spreading. It has nine mutations in Spike compared to currently
3 circulating BA.2, raising concerns it may further evade vaccine-elicited and therapeutic antibodies.
4 We found BA.2.75 to be moderately more neutralization resistant to sera from vaccinated/boosted
5 individuals than BA.2 (1.8-fold), similar to BA.2.12.1 (1.1-fold), but more neutralization sensitive
6 than BA.4/5 (0.6-fold). Relative to BA.2, BA.2.75 showed heightened resistance to class 1 and
7 class 3 monoclonal antibodies targeting the Spike receptor-binding domain, while gaining
8 sensitivity to class 2 antibodies. Resistance was largely conferred by G446S and R460K mutations.

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9 BA.2.75 was slightly resistant (3.7-fold) to bebtelovimab, a therapeutic antibody with potent

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10 activity against all Omicron subvariants. BA.2.75 also exhibited higher binding affinity to host
11 receptor ACE2 than other Omicron subvariants. BA.2.75 provides further insight into SARS-CoV-
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2 evolution as it gains transmissibility while incrementally evading antibody neutralization.
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14 Keywords: SARS-CoV-2, Omicron, BA.2.75, COVID-19, vaccine, serum neutralization,

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15 monoclonal antibodies, antibody evasion, ACE2 affinity

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16 Main text
17 The coronavirus disease 2019 (COVID-19) pandemic is currently dominated by the SARS-CoV-
18 2 Omicron subvariant BA.5. Yet another subvariant known as BA.2.75 has recently emerged from
19 India, where it has spread rapidly, displacing the predominant BA.2 subvariant locally. Moreover,
20 BA.2.75 has now been identified in at least 27 countries worldwide (Shu and McCauley, 2017)
21 (Fig. S1A). Though a descendent from BA.2, it contains a distinct set of mutations in its spike
22 protein, including five substitutions in the N-terminal domain (NTD), K147E, W152R, F157L,
23 I210V, and G257S, and four substitutions in the receptor-binding domain (RBD), D339H, G446S,
24 N460K, and R493Q (Fig. S1B). Many of these mutations are located at sites targeted by

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25 neutralizing antibodies and may also affect the binding affinity of the spike to its receptor,
26 angiotensin-converting enzyme 2 (ACE2). In particular, two RBD mutations, D339H and N460K,

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27 are noteworthy because they have not been identified in previous SARS-CoV-2 variants and their
28
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impacts are not yet known. Newfound variants such as BA.2.75 that are increasing in frequency
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29 raise the concern that the viruses have developed additional mechanisms to escape from
30 neutralization by antibodies elicited by vaccination or previous infection, as well as by therapeutic
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31 monoclonal antibodies (mAbs) in clinical use. Therefore, we have evaluated the antibody evasion
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32 properties of BA.2.75, and our findings are reported here.

33
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34 We first set out to profile the antigenic differences of BA.2.75 from the wildtype SARS-CoV-2
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35 D614G and the other currently circulating Omicron subvariants BA.2, BA.2.12.1, and BA.4/5
36 (note that BA.4 and BA.5 share an identical spike). Vesicular stomatitis virus (VSV) - pseudotyped
37 viruses of each variant were produced and then assessed for their neutralization sensitivity to sera
38 from three different clinical cohorts: those who had received three doses of a COVID-19 mRNA
39 vaccine (boosted) and patients with either BA.1 or BA.2 breakthrough infection after vaccination.
40 We did not include sera from persons immunized with only two doses of COVID-19 mRNA
41 vaccines as we had previously observed that they lacked neutralization capacity against earlier
42 Omicron subvariants (Iketani et al., 2022; Liu et al., 2022b). The clinical information for our
43 cohorts is described in Table S1 and the serum neutralization profiles are shown in Fig. 1A.
44 Consistent with previous reports (Iketani et al., 2022; Liu et al., 2022b; Wang et al., 2022b),
45 neutralization ID50 (the 50% inhibitory dose) titers of the “boosted” sera were substantially lower
46 against BA.2, BA.2.12.1, and BA.4/5 (4.9-fold, 7.8-fold, and 14.8-fold, respectively), compared
47 with D614G. Neutralization titers against BA.2.75 were similar to those against BA.2.12.1, 8.7-
48 fold lower than D614G, 1.8-fold lower than BA.2, but 1.7-fold higher than BA.4/5. A similar trend
49 was observed in the “BA.1 and BA.2 breakthrough” cohorts.

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51 We also investigated the impact of new point mutations found in BA.2.75 on serum antibody
52 evasion by conducting serum neutralization assays with pseudoviruses containing each point
53 mutation in the background of BA.2 (Fig. 1B). The mutations W152R, F157L, I210V, G257S,
54 D339H, and N446K each only slightly (0.8-fold to 1.3-fold) altered the neutralization titers of sera
55 from all three cohorts against BA.2. In contrast, K147E and N460K impaired the neutralization

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56 activity of sera significantly, by 1.6-fold to 1.8-fold and 1.5-fold to 2.4-fold, respectively, while

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57 the R493Q reversion mutation modestly enhanced the neutralization by 1.8-fold to 3.0-fold, as was

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58 observed previously (Wang et al., 2022b). re
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60 We next assessed the neutralization resistance of BA.2.75 to a panel of 23 mAbs directed to known
61 neutralizing epitopes on the viral spike. Among these, 21 target the four epitope classes in the RBD
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62 (Barnes et al., 2020), including REGN10987 (imdevimab) (Hansen et al., 2020), REGN10933
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63 (casirivimab) (Hansen et al., 2020), COV2-2196 (tixagevimab) (Zost et al., 2020), COV2-2130
64 (cilgavimab) (Zost et al., 2020), LY-CoV555 (bamlanivimab) (Jones et al., 2021), CB6
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65 (etesevimab) (Shi et al., 2020), Brii-196 (amubarvimab) (Ju et al., 2020), Brii-198 (romlusevimab)
66 (Ju et al., 2020), S309 (sotrovimab) (Pinto et al., 2020), LY-CoV1404 (bebtelovimab) (Westendorf
67 et al., 2022), CAB-A17 (Sheward et al., 2022), ZCB11 (Zhou et al., 2022), Omi-3 (Nutalai et al.,
68 2022), Omi-18 (Nutalai et al., 2022), XGv282 (Wang et al., 2022a), XGv347 (Wang et al., 2022a),
69 S2E12 (Starr et al., 2021), A19-46.1 (Wang et al., 2021), 35B5 (Wang et al., 2022c), and JMB2002
70 (Yin et al., 2022), as well as 10-40 (Liu et al., 2022a) from our group. The other two mAbs, C1717
71 (Wang et al., 2022d) and S3H3 (Hong et al., 2022), target NTD-SD2 and SD1, respectively. Many
72 of the recently isolated mAbs were chosen because they could neutralize earlier Omicron
73 subvariants (Hong et al., 2022; Nutalai et al., 2022; Sheward et al., 2022; Starr et al., 2021; Wang
74 et al., 2022a; Wang et al., 2021; Wang et al., 2022c; Wang et al., 2022d; Yin et al., 2022; Zhou et
75 al., 2022).
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77 The results are presented in Fig. 2A and Table S2. Unlike BA.2.12.1, which was largely similar
78 in its antigenic profile to BA.2, BA.2.75 differed from BA.2 across a wide range of the mAbs
79 tested. Antibodies across multiple classes were impaired against BA.2.75 compared to BA.2,
80 including some in class 1 (CAB-A17, Omi-3, and Omi-18) and class 3 (XGv282, LY-CoV1404,
81 JMB2002, COV2-2130, and REGN10987). Simultaneously, several antibodies showed
82 neutralization sensitivity against BA.2.75 relative to BA.2, all within class 2 (S2E12, COV2-2196,
83 and REGN10933). This also contrasts that of BA.4/5, which demonstrated heightened resistance
84 to class 2 and class 3 antibodies over BA.2. Moreover, even within the resistance to class 3

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85 antibodies, BA.2.75 and BA.4/5 only overlapped with additional resistance towards one antibody,

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86 JMB2002. The other impaired antibodies differed, with 35B5 and Brii-198 losing further activity
87 against BA.4/5 over BA.2. These data suggest that BA.2.75 has evolved to extend resistance
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towards some class 1- and class 3-directed antibodies over BA.2, yet has regained sensitivity for a
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89 subset of class 2 antibodies. Such differences may help to interpret the observations made on
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90 polyclonal serum neutralization (Fig. 1).

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92 Of particular note, BA.2.75 is the first SARS-CoV-2 variant that has demonstrated resistance to
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93 bebtelovimab (LY-CoV1404), albeit modestly at 3.7-fold loss in neutralization (Fig. 2A).

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94 Nevertheless, it remained the only clinical mAb that retained potent neutralizing activity against
95 all the Omicron subvariants with a IC50 (the 50% inhibitory concentration) below 0.01 µg/ml. All
96 other clinically authorized or approved antibodies or antibody combinations showed a substantial
97 loss of activity in vitro against BA.2.75.

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99 As we observed that BA.2.75 was resistant to mAbs in a unique way, we set out to identify the
100 mutations within BA.2.75 that conferred the observed antibody resistance profile. We generated
101 pseudoviruses carrying each of the point mutations in the background of D614G or BA.2 and tested
102 their neutralization sensitivity to the aforementioned panel of mAbs and combinations. These data
103 are shown in Fig. 2B and Table S2. G446S impaired or abolished the neutralizing activity of class
104 3 mAbs (XGv282, JMB2002, and REGN10987), as previously observed in our BA.1 studies (Liu
105 et al., 2022b). That this mutation did not result in a significant loss in polyclonal serum
106 neutralization (Fig. 1B) suggests that such antibodies may be rare in a polyclonal response. The
107 N460K substitution conferred resistance to all of the class 1 RBD mAbs tested, as well as one class
108 2 mAb (ZCB11). However, this resistance was only observed in the context of BA.2 for three of
109 the class 1 antibodies (CAB-A17, Omi-3, and Omi-18) and the class 2 antibody ZCB11, but not in
110 the context of D614G. In contrast, R493Q, also found in BA.4/5 (Wang et al., 2022b), sensitized
111 BA.2 to neutralization by several class 1 and 2 RBD mAbs, which is consistent with our previous
112 study (Wang et al., 2022b). We note that while the NTD mutation K147E had a significant impact
113 on polyclonal sera (Fig. 1B), we did not observe an effect by this mutation against the panel of

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114 mAbs tested here, suggesting that this mutation may be acting through non-RBD antibodies.

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116 We conducted structural modeling to further investigate the impact of the G446S and N460K
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117 mutations (Fig. 2C). Analysis on G446S revealed steric hindrance to binding by class 3 RBD
118 mAbs (XGv282 and JMB2002), as we reported previously (Liu et al., 2022b). In addition,
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119 structural modeling of N460K revealed that K460 abolished a common hydrogen bond between
120 RBD-N460 and S56 in CDRH2 of VH3-53 class antibodies (Barnes et al., 2020), such as CB6,
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121 Omi-3, and Omi-18, although it is not immediately clear why the loss in activity for Omi-3 and
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122 Omi-18 was only apparent in the background of BA.2 but not D614G.
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124 Finally, as receptor binding affinity may play a role in transmissibility, we investigated this
125 property for BA.2.75. The binding affinity of purified spike trimer proteins of D614G, BA.2,
126 BA.4/5, and BA.2.75 to dimeric human ACE2 (hACE2) was quantified using surface plasmon
127 resonance (SPR). We found BA.2.75 exhibited the highest receptor binding affinity, with a KD
128 value 7.0-fold and 2.4-fold lower than values for BA.2 and BA.4/5, respectively (Fig. 2D). To
129 validate these results, we tested pseudoviruses bearing these spikes for neutralization by dimeric
130 hACE2 (Fig. S2). A comparison of IC50 values suggested that BA.2.75 was slightly more sensitive
131 to hACE2 than the other pseudoviruses tested, in line with was observed in the SPR. To probe the
132 role of the mutations in BA.2.75 for ACE2 binding, we tested the neutralization by hACE2 of each
133 of the point mutants in the background of BA.2. R493Q was the most sensitive to hACE2
134 neutralization, and N460K was most resistant. These results parallel our previous studies, in which
135 we found R493Q could serve to restore the lost receptor binding affinity due to a resistance-
136 conferring mutation in BA.4/5 (Wang et al., 2022b). A similar mechanism, in which R493Q acts
137 to balance the compromised affinity caused by N460K, may be in action for BA.2.75.

138

139 In summary, we have systematically evaluated the antigenic properties of the new SARS-CoV-2
140 Omicron subvariant BA.2.75, which is spreading throughout the world. Our data suggest that
141 BA.2.75 exhibits a higher resistance to vaccine-induced and infection-induced serum neutralizing
142 activity than BA.2 (Fig. 1A). It is reassuring that BA.2.75 does not show greater immune evasion
143 from polyclonal sera than the BA.4/5 subvariant (Fig. 1A). The resistance profile of BA.2.75 to

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144 sera can be largely attributed to the K147E and N460K mutations (Fig. 1B). The latter mutation is

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145 consistent with findings from deep mutational scanning of the RBD (Greaney et al., 2021b). The

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146 impact of the former mutation is puzzling in that previous Omicron subvariants have already
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147 abolished the activity of the antibodies directed to the NTD antigenic supersite (Iketani et al., 2022;
148 Liu et al., 2022b; Wang et al., 2022b) and yet this new variant evolved to contain five additional
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149 NTD mutations. Why is SARS-CoV-2 doing so when NTD antibodies contribute only a small
150 portion of the serum virus-neutralizing activity (Garrett et al., 2021; Greaney et al., 2021a)?
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152 BA.2.75 exhibits a unique neutralizing profile for mAbs, with heightened resistance over BA.2 to
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153 class 1 and class 3 RBD antibodies, while gaining sensitivity towards class 2 RBD antibodies (Fig.
154 2A). Although the impairment is slight, BA.2.75 is the first SARS-CoV-2 variant to show
155 discernible resistance to betelovimab (LY-CoV1404). Profiling of the individual mutations within
156 BA.2.75 revealed that G446S and N460K could contribute to the resistance (Fig. 2B). These
157 mutations appear to be acting through steric hindrance or abrogation of a hydrogen bond (Fig. 2C).
158 More importantly, these findings on BA.2.75 demonstrate that our one remaining therapeutic
159 antibody with potent activity to treat COVID-19 is now under threat. Another mutation proximal
160 to residue 446 of the spike could knock out the current arsenal of therapeutic monoclonals.
161 Although numerous mAbs have been isolated and shown to neutralize the new Omicron
162 subvariants (Hong et al., 2022; Nutalai et al., 2022; Sheward et al., 2022; Starr et al., 2021; Wang
163 et al., 2022a; Wang et al., 2021; Wang et al., 2022c; Wang et al., 2022d; Yin et al., 2022; Zhou et
164 al., 2022), their development pathway to prove clinical efficacy has become rather daunting with
165 the availability of effective vaccines and antiviral drugs.

166

167 Finally, our data demonstrate that BA.2.75 has enhanced binding affinity to its receptor ACE2,
168 which may enhance its transmission (Fig. 2D). However, it is still unclear whether BA.2.75 could
169 out-compete BA.5, today’s dominant form globally. The features of BA.2.75 that diverged from
170 other Omicron subvariants serve to underscore the ability of SARS-CoV-2 to evolve,
171 incrementally gaining transmissibility and antibody evasion, and to reinforce the importance of
172 vaccination and booster campaigns as well as epidemiologic surveillance to detect the emergence

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173 of new SARS-CoV-2 variants.

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174 STAR METHODS

175 Detailed methods are provided in the online version of this paper and include the following:

176 • KEY RESOURCES TABLE

177 • RESOURCE AVAILABILITY
178 o Lead contact
179 o Materials availability
180 o Data and code availability
181 • EXPERIMENTAL MODEL AND SUBJECT DETAILS
o Patients and vaccinees

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183 o Cell lines

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184 • METHOD DETAILS
185 o Monoclonal antibodies
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186 o Construction of SARS-CoV-2 spike plasmids
187 o Expression and purification of SARS-CoV-2 stabilized spike trimers and human ACE2
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188 o Surface plasmon resonance (SPR)

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189 o Pseudovirus production

190 o Pseudovirus neutralization assay
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191 o Structural modeling of RBD mutations

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192 QUANTIFICATION AND STATISTICAL ANAYLYSIS

193

194 ACKNOWLEDGEMENTS
195 This study was supported by funding from the Gates Foundation, JPB Foundation, Andrew and
196 Peggy Cherng, Samuel Yin, Carol Ludwig, David and Roger Wu, Regeneron Pharmaceuticals,
197 and the NIH SARS-CoV-2 Assessment of Viral Evolution (SAVE) Program. We are grateful to
198 Michael T. Yin, Magdalena E. Sobieszczyk, Jennifer Y. Chang, Jayesh G. Shah, and David
199 S. Perlin for providing serum samples from COVID-19 patients. We thank all who have
200 contributed their data to GISAID.
201
202 AUTHOR CONTRIBUTIONS
203 D.D.H. and L.L. conceived this project. Q.W., A.Y.Y., and L.L. constructed the spike expression
204 plasmids and produced pseudoviruses. Q.W., S.I., and L.L. conducted pseudovirus neutralization
205 experiments. Q.W., Z.L., A.Y.Y., and L.L. purified SARS-CoV-2 spike and ACE2 proteins and
206 performed SPR. Y.G. and Z.S. conducted bioinformatic analyses. Q.W. managed the project. J.Y.
207 and M.L. produced antibodies. D.D.H. and L.L. directed and supervised the project. Q.W., S.I.,
208 Y.G., L.L., and D.D.H. analyzed the results and wrote the manuscript.
209

210 DECLARATION OF INTERESTS

211 S.I, J.Y., Y.H., L.L., and D.D.H. are inventors on patent applications (WO2021236998) or

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212 provisional patent applications (63/271,627) filed by Columbia University for a number of SARS-

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213 CoV-2 neutralizing antibodies described in this manuscript. Both sets of applications are under

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214 review. D.D.H. is a co-founder of TaiMed Biologics and RenBio, consultant to WuXi Biologics
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215 and Brii Biosciences, and board director for Vicarious Surgical.
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272 Wang, X., Chen, X., Tan, J., Yue, S., Zhou, R., Xu, Y., Lin, Y., Yang, Y., Zhou, Y., Deng, K., et al. (2022c). 35B5
273 antibody potently neutralizes SARS-CoV-2 Omicron by disrupting the N-glycan switch via a conserved spike
274 epitope. Cell Host Microbe 30, 887-895 e884.
275 Wang, Z., Muecksch, F., Cho, A., Gaebler, C., Hoffmann, H.H., Ramos, V., Zong, S., Cipolla, M., Johnson, B.,
276 Schmidt, F., et al. (2022d). Analysis of memory B cells identifies conserved neutralizing epitopes on the N-terminal
277 domain of variant SARS-Cov-2 spike proteins. Immunity 55, 998-1012 e1018.
278 Westendorf, K., Zentelis, S., Wang, L., Foster, D., Vaillancourt, P., Wiggin, M., Lovett, E., van der Lee, R., Hendle,
279 J., Pustilnik, A., et al. (2022). LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants. Cell Rep
280 39, 110812.
281 Wrapp, D., Wang, N., Corbett, K.S., Goldsmith, J.A., Hsieh, C.L., Abiona, O., Graham, B.S., and McLellan, J.S.
282 (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263.
283 Yin, W., Xu, Y., Xu, P., Cao, X., Wu, C., Gu, C., He, X., Wang, X., Huang, S., Yuan, Q., et al. (2022). Structures of
284 the Omicron spike trimer with ACE2 and an anti-Omicron antibody. Science 375, 1048-1053.
285 Zhou, B., Zhou, R., Tang, B., Chan, J.F., Luo, M., Peng, Q., Yuan, S., Liu, H., Mok, B.W., Chen, B., et al. (2022).
286

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A broadly neutralizing antibody protects Syrian hamsters against SARS-CoV-2 Omicron challenge. Nat Commun
287 13, 3589.
288 Zost, S.J., Gilchuk, P., Case, J.B., Binshtein, E., Chen, R.E., Nkolola, J.P., Schafer, A., Reidy, J.X., Trivette, A.,

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289 Nargi, R.S., et al. (2020). Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature 584,
290 443-449.

291
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292 Figure legends

293 Fig. 1 | Serum neutralization profile of BA.2.75. A, Neutralization of pseudotyped D614G and
294 Omicron subvariants by sera from three different clinical cohorts. Boosted refers to individuals
295 who received three doses of a COVID-19 mRNA vaccine, and breakthrough refers to individuals
296 who were infected and received COVID-19 vaccines. B, Serum neutralization of pseudotyped
297 BA.2 or BA.2 with point mutations from BA.2.75. For both panels, values above the symbols
298 denote the geometric mean ID50 values and values on the lower left indicate the sample size (n)
299 for each group. The limit of detection (LOD) is 100 (dotted line), and values below the LOD are
300 arbitrarily plotted to allow for visualization of each sample. P values were determined by using

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301 two-tailed Wilcoxon matched-pairs signed-rank tests. In B, comparisons were made against BA.2.

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302 Significance is denoted with asterisks and the fold-change is also denoted. ns, not significant; *, P
303 < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

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304 See also Figure S1 and Table S1.
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305

306 Fig. 2 | Neutralization of BA.2.75 by monoclonal antibodies and receptor binding affinity. A,
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307 Neutralization of pseudotyped D614G and Omicron subvariants by NTD-SD2-, SD1-, and RBD-
directed mAbs. Values above the LOD of 10 μg/mL (dotted line) are arbitrarily plotted to allow
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308
309 for visualization of each sample. B, Fold change in IC50 values for the neutralization of
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310 pseudotyped point mutants, relative to D614G or BA.2, with resistance colored red and
311 sensitization colored green. C, Modeling of the impact of G446S and N460K on antibody
312 neutralization. Clashes are shown as red discs and hydrogen bonds are shown as dashed lines. D,
313 Binding affinity of D614G, BA.2, BA.2.75, and BA.4/5 stabilized spike trimers to dimeric human
314 ACE2. NA, not applicable.

315 See also Figure S1, S2 and Table S2.

316 RESOURCE AVAILABILITY

317 Lead contact

318 Further information and requests for resources and reagents should be directed to and will be
319 fulfilled by the Lead Contact Author David D. Ho (dh2994@cumc.columbia.edu).

320 Materials availability

321 All reagents generated in this study are available from the Lead Contact with a completed Materials
322 Transfer Agreement.

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323 Data and code availability

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324 Data reported in this paper will be shared by the lead contact upon request.

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325 This paper does not report original code. re
326 Any additional information required to reanalyze the data reported in this paper is available from
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327 the lead contact upon request.

328
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329 EXPERIMENTAL MODEL AND SUBJECT DETAILS

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330 Patients and vaccinees

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331 Sera from individuals who received three doses of the mRNA-1273 or BNT162b2 vaccines were
332 collected at Columbia University Irving Medical Center (referred to as “boosted” in the text). Sera
333 from individuals who were infected by an Omicron subvariant (BA.1 or BA.2) following
334 vaccinations were collected from December 2021 to May 2022 at Columbia University Irving
335 Medical Center (referred to as “BA.1 or BA.2 breakthrough” in the text). All samples were
336 confirmed for prior SARS-CoV-2 infection status by anti-nucleoprotein (NP) enzyme-linked
337 immunosorbent assay (ELISA) , and the variant involved in breakthrough cases were determined
338 by sequencing. All collections were conducted under protocols reviewed and approved by the
339 Institutional Review Board of Columbia University. All participants provided written informed
340 consent. Clinical information on the different cohorts of study subjects is provided in Table S1.
341
342 Cell lines
343 Expi293 cells were obtained from Thermo Fisher Scientific (A14527); Vero-E6 cells were
344 obtained from the ATCC (CRL-1586); HEK293T cells were obtained from the ATCC (CRL-3216).
345 Expi293 cells were maintained in Expi293TM Expression Medium, supplemented with 0.5%
346 penicillin-streptomycin (Thermo Fisher Scientific, Cambridge, MA), and were incubated at 37°C,
347 8% CO2, 125 rpm. Vero-E6 and HEK293T cells were cultured in Dulbecco modified Eagle
348 medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-
349 streptomycin at 37°C, 5% CO2. HEK293T cells and Expi293 cells are of female origin. Vero-E6
350 cells are from African green monkey kidneys. Cells were purchased from authenticated vendors
351 and morphology was confirmed visually before use. All cell lines tested mycoplasma negative.

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353 METHOD DETAILS

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354 Monoclonal antibodies re
355 Antibodies were expressed in-house as previously described (Liu et al., 2020). For each of the
356 antibodies in this study, heavy chain variable (VH) and light chain variable (VL) genes were
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357 synthesized (GenScript), cloned into an expression vector, transfected into Expi293 cells (Thermo
358 Fisher Scientific), and purified from the cellular supernatant by affinity purification using rProtein
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359 A sepharose (GE). REGN10987, REGN10933, COV2-2196, and COV2-2130 were provided by
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360 Regeneron Pharmaceuticals; Brii-196 and Brii-198 were provided by Brii Biosciences; CB6 was
361 provided by B. Zhang and P. Kwong (NIH); and ZCB11 was provided by Z. Chen (HKU).
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362
363 Construction of SARS-CoV-2 spike plasmids
364 Spike expression constructs for D614G, BA.2, BA.2.12.1, and BA.4/5 were previously generated
365 (Iketani et al., 2022; Liu et al., 2022b; Wang et al., 2022b). Expression constructs encoding the
366 BA.2.75 spike, as well as the individual mutations found in BA.2.75, were generated using the
367 QuikChange II XL site-directed mutagenesis kit according to the manufacturer’s instructions
368 (Agilent). For expression of stabilized soluble S2P spike trimer proteins, 2P substitutions (K986P
369 and V987P in WA1) and a “GSAS” substitution of the furin cleavage site (682-685aa in WA1)
370 were introduced into the spike-expressing plasmids for stabilization as previously described
371 (Wrapp et al., 2020), and then the ectodomain (1-1208aa in WA1) of the spike was fused with a
372 C-terminal 8x His-tag and cloned into the paH vector. All constructs were confirmed by Sanger
373 sequencing prior to use.
374
375 Expression and purification of SARS-CoV-2 stabilized spike trimers and human ACE2
376 Stabilized SARS-CoV-2 spike trimer proteins of D614G and the Omicron subvariants were
377 generated by transfecting Expi293 cells with each of the stabilized spike trimer expression
378 constructs using 1 mg mL-1 polyethylenimine (PEI), and then purifying the spike trimer from the
379 supernatants five days post-transfection using nickel-nitrilotriacetic acid (Ni-NTA) resin
380 (Invitrogen) according to the manufacturer’s instructions (Liu et al., 2020). Dimeric human ACE2-

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381 IgG1 (hACE2) was generated by transfecting Expi293 cells with pcDNA3-sACE2-WT(732)-IgG1
382 (Chan et al., 2020) (Addgene plasmid #154104, gift from Erik Procko) using 1 mg mL-1 PEI and

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383 then purifying from the supernatant five days post-transfection using rProtein A sepharose (GE)
384
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according to the manufacturer’s instructions. All proteins were confirmed for purity and size by
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385 sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) prior to use.
386
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387 Surface plasmon resonance (SPR)

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388 Surface plasmon resonance binding assays for hACE2 binding to SARS-CoV-2 stabilized spike
389 trimers were performed using a Biacore T200 biosensor equipped with a Series S CM5 chip
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390 (Cytiva), in a running buffer of 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

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391 (HEPES) pH 7.4, 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA) , 0.05% P-20
392 (Cytiva) at 25 °C. Spike proteins were captured through their C-terminal His-tag over an anti-His
393 antibody surface. These surfaces were generated using the His-capture kit (Cytiva) according to
394 the manufacturer’s instructions, resulting in approximately 10,000 resonance units (RU) of anti-
395 His antibody over each surface. An anti-His antibody surface without antigen was used as a
396 reference flow cell to remove bulk shift changes from the binding signal. For each spike, binding
397 to hACE2 was tested using a three-fold dilution series with concentrations ranging from 2.46 nM
398 to 200 nM. The association and dissociation rates were each monitored for 60 s and 600 s
399 respectively, at 30 µL/min. The bound spike/hACE2 complex was regenerated from the anti-His
400 antibody surface using 10 mM glycine pH 1.5. Blank buffer cycles were performed by injecting
401 running buffer instead of hACE2 remove systematic noise from the binding signal. The resulting
402 data was processed and fit to a 1:1 binding model using Biacore Evaluation Software.
403
404 Pseudovirus production
405 Pseudotyped SARS-CoV-2 (pseudoviruses) were produced in the vesicular stomatitis virus (VSV)
406 background, in which the native VSV glycoprotein was replaced by that of SARS-CoV-2 and its
407 variants, as previously described (Liu et al., 2020). HEK293T cells were transfected with the
408 appropriate spike expression construct using 1 mg mL-1 PEI and cultured overnight at 37 °C under
409 5% CO2, and then infected with VSV-G pseudotyped ΔG-luciferase (G*ΔG-luciferase, Kerafast)
410 24 h post-transfection. After 2 h of infection, cells were washed three times, changed to fresh
411 medium, and then cultured for approximately another 24 h before the supernatants were collected,

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412 clarified by centrifugation, and aliquoted and stored at -80 °C until further use.
413

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414 Pseudovirus neutralization assay
415
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Prior to use in the neutralization assay, all pseudoviruses were first titrated to equilibrate the viral
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416 input between assays. Five-fold serial dilutions of heat-inactivated sera, antibodies, or hACE2
417 were prepared in media in 96-well plates in triplicate, starting at 1:100 dilution for sera and
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418 10 µg mL−1 for antibodies and hACE2. Pseudoviruses were then added to wells and the virus–
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419 sample mixture was incubated at 37 °C for 1 h, except for hACE2, where no incubation was
420 conducted. Control wells with the virus only were included on all plates. Vero-E6 cells were then
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421 added at a density of 3 × 104 cells per well and the plates were incubated at 37 °C for approximately
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422 10 h. Cells were then lysed and luciferase activity was quantified using the Luciferase Assay
423 System (Promega) according to the manufacturer’s instructions with SoftMax Pro v.7.0.2
424 (Molecular Devices). Neutralization curves and IC50 values were derived by fitting a nonlinear
425 five-parameter dose-response curve to the data in GraphPad Prism v.9.2.
426
427 Structural modeling of RBD mutations
428 The structures of antibody-spike complexes for modeling were obtained from PDB (7WLC
429 (XGv282), 7XOD (JMB2002), 7C01 (CB6), 7ZF3 (Omi-3), and 7ZFB (Omi-18)). PyMOL v.2.3.2
430 was used to perform mutagenesis, to identify steric clashes and hydrogen bonds between RBD and
431 antibodies, and to generate structural plots (Schrödinger, LLC).
432
433 QUANTIFICATION AND STATISTICAL ANAYLYSIS
434 Serum neutralization ID50 values and antibody and hACE2 neutralization IC50 values were
435 calculated using a five-parameter dose-response curve in GraphPad Prism v.9.2. Evaluations of
436 statistical significance were performed employing two-tailed Wilcoxon matched-pairs signed-rank
437 tests using GraphPad Prism v.9.2 software. Levels of significance are indicated as follows: ns, not
438 significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. The SPR data was
439 processed and fit to a 1:1 binding model using Biacore Evaluation Software.

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 Boosted BA.1 breakthrough BA.2 breakthrough
A 10 6 5,332 1,083 684 614 360 10,290 3,126 1,622 1,587 824 10,425 3,696 3,135 2,707 1,653
*** (12.5X) *** (6.3X)
**** (14.8X)
*** (6.5X) *** (3.9X)
**** (8.7X)
*** (6.3X)
*** (3.3X)
10 5 **** (7.8X) *** (3.8X)
*** (3.3X) *** (2.0X) *** (2.2X)
*** (2.8X)
**** (4.9X) ********
(3.0X)
** (2.0X) * (1.4X)
(1.8X)
*** (1.9X) ns (1.0X) *** (1.9X)
* (1.2X) ns (1.2X)
*** (1.9X)
** (1.6X) ns (1.1X) ** (1.9X) ** (1.6X)
ID50

10 4
** (1.7X)

10 3

n = 16 n = 13 n = 11
10 2

f
4G

4G

4G

5
.2

.2

.2

5
.

/
7

7
oo
12

.4

12

.4

12

.4
BA

BA

BA
2.

2.

2.
61

61

61
BA

BA

BA
2.

2.

2.
.

.
BA

BA

BA
D

D
.

.
BA

BA

BA
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B Boosted BA.1 breakthrough BA.2 breakthrough

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10 5 606 1,045 1,100 1,241 3,205 1,967 2,739 2,792 2,500 5,799 2,029 3,194 4,375 4,161 6,731
1,083 867 1,434 1,099 734 3,126 2,484 3,098 2,342 1,299 0.5X 3,696 2,762 4,509 3,509 2,016 0.5X

lP
0.3X
1.0X 1.1X **** 0.8X
0.8X
ns 0.9X ****
**** 1.3X 1.1X ns ns 1.3X 1.3X 1.2X
*
1.1X ns
1.6X
ns ns * ns 1.8X
*** **
1.3X
0.8X 1.0X 1.0X 0.9X 1.5X ** 2.4X
1.8X ***
1.2X
ns ns ns ns *** *** ***
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10 4 1.8X ** 1.0X
ns ***
****
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ID50

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10 3

10 2
n = 16 n = 13 n = 11
BA W1 E

BA -D3 S
BA -G4 H
BA -N4 S

BA W1 E

BA -D3 S
BA -G4 H
BA -N4 S

BA W1 E

BA -D3 S
BA -G4 H
BA -N4 S
. 2 52R

. 2 52R

. 2 52R
-R K

-R K

-R K
. 2 57L

BA -G2 V

BA 2-I2 L
BA -G2 V

. 2 57L

BA -G2 V
3Q

3Q

3Q
BA 2-K .2

BA 2-K .2

BA 2-K .2
. 57
.2 147

.2 57

.2 46

.2 147

.2 57

.2 46

.2 147

.2 57

.2 46
.2 60

.2 60

.2 60
.2 10

.2 10

.2 10
.2 39

.2 39

.2 39
A

A
49

49

49
BA B

BA B

BA B
BA F1

BA F1

BA F1
BA -I2

BA -I2
-

-
-

-
.

.
A Non-RBD mAbs RBD mAbs Combinations D
200 D614G + hACE2 BA.2 + hACE2
Class 1 Class 2 Class 3 Class 4 KD = 2.52 nM KD = 1.19 nM
10 -4 C1717 CAB-A17 XGv347 A19-46.1 XGv282 35B5 10-40 REGN10987 + REGN10933
S3H3 CB6 ZCB11 COV2-2196 LY-CoV1404 COV2-2130 COV2-2196 + COV2-2130 150
Brii-196 S2E12 LY-CoV555 S309 REGN10987 LY-CoV555 + CB6
Omi-3 REGN10933 JMB2002 Brii-198 Brii-196 + Brii-198
Omi-18 100
10 -3

Ka = 1.82 x 105 M-1s-1

50
Ka = 1.94 x 105 M-1s-1

Response (AU)
10 -2 Kd = 4.89 x 10-4 s-1 Kd = 2.16 x 10-4 s-1
IC50 (μg/mL)

10 -1 200 BA.2.75 + hACE2 BA.4/5 + hACE2

KD = 0.17 nM KD = 0.40 nM
150
10 0
100

50 Ka = 2.28 x 105 M-1s-1

10 1 Ka = 1.94 x 105 M-1s-1
Kd = 3.77 x 10-5 s-1 Kd = 7.72 x 10-5 s-1
0
0 300 600 900 1200 0 300 600 900 1200
.2 .2

.2 .2

.2 .2

.2 .2

.2 .2

.2 .2
/5

/5

/5

/5

/5

/5
BA 5

BA 5

BA 5

BA 5

BA 5

BA 5
4G

4G

4G

4G

4G

4G
BA 2.1

BA 2.1

BA 2.1

BA 2.1

BA 2.1

f BA 2.1
.7

.7

.7

.7

.7

.7
BA BA

BA BA

BA BA

BA BA

BA BA

BA BA
.4

.4

.4

.4

.4

.4
61

61

61

61

61

61
Time (s)
.2

.2

.2

.2

.2

.2
.1

.1

.1

.1

.1

.1
oo
D

D
B

r
NTD- RBD mAbs

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SD1 Class 4 Combinations
Fold change SD2 Class 1 Class 2 Class 3
in IC50 CAB- Brii-196 Omi-18 XGv347 ZCB11 S2E12
A19- COV2- LY-CoV REGN
XGv282
LY-CoV JMB COV2- REGN
Brii-198 REGN10987 + COV2-2196 + LY-CoV555 Brii-196 +
C1717 S3H3 CB6 Omi-3 46.1 2196 555 10933 1404 S309 35B5 2002 2130 10987 10-40 REGN10933 COV2-2130 + CB6 Brii-198

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A17
D614G-K147E -1.0 1.2 1.3 1.0 1.1 1.1 1.1 -1.0 1.4 1.5 1.2 1.4 1.7 1.2 1.1 1.2 -1.1 1.0 -1.3 1.5 1.4 -1.7 -1.4 -1.1 -1.5 1.1 -1.0
D614G-W152R 1.0 1.1 1.2 -1.0 -1.5 -1.4 -1.3 1.2 -1.0 1.3 1.1 1.1 1.3 -1.0 -1.3 -1.3 -1.2 1.4 -1.5 1.1 1.1 1.0 -1.0 -1.2 -1.4 -1.1 -1.0
Compared with

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D614G-F157L 1.1 1.5 1.3 1.3 1.2 1.5 2.4 1.5 2.0 2.2 2.8 1.2 2.0 1.7 1.0 1.3 -1.2 1.3 -1.1 1.8 -1.1 -1.4 -1.2 1.1 -1.2 1.5 1.5
D614G

D614G-I210V 1.1 1.3 1.1 1.1 1.1 -1.2 1.5 1.0 1.1 1.6 1.5 1.0 1.5 1.1 -1.1 1.1 1.1 1.4 -1.3 1.3 -1.0 -1.8 -1.2 1.1 -1.5 -1.2 1.2
D614G-G257S -1.2 1.8 1.2 1.1 1.3 -1.2 1.2 1.4 1.2 1.1 1.6 1.6 1.4 1.1 -1.1 -1.1 -1.1 1.3 -1.4 -1.0 -1.3 -1.2 -1.4 -1.1 -1.3 -1.1 1.3
na
D614G-G339H 1.1 1.6 1.1 1.1 -1.3 -1.6 1.2 -1.2 -1.2 -1.0 1.4 1.0 1.3 1.0 -1.4 1.1 -1.1 1.1 -1.5 1.3 -1.4 -1.3 -1.5 -1.6 -1.5 -1.3 -1.0
D614G-G446S 1.1 2.0 1.2 1.2 1.1 1.1 1.3 1.0 1.1 1.3 -1.5 1.1 1.2 1.2 -3.5 -1.9 1.3 -1.0 -27 -3.1 -2462 -1.1 1.2 -4.0 -2.5 -1.1 1.4
D614G-N460K 1.0 1.9 -1.2 -172 -8.6 -1.9 -1.2 -1.1 -1.0 -1.0 -1.5 -1.1 1.3 -1.4 -1.3 1.0 -1.1 -1.1 -1.6 1.2 -1.2 -2.4 -1.7 -1.3 -1.7 -1.7 -4.7
ur

BA.2-K147E 1.1 -1.3 -2.4 NA 1.3 -2.2 -1.1 -1.4 -2.7 -1.4 1.1 -1.2 NA NA -1.6 1.0 1.0 1.2 1.2 1.2 -1.5 1.0 -1.0 1.4 -1.2 NA -1.3
BA.2-W152R 1.5 1.4 -1.4 NA 1.8 -1.7 1.2 1.3 -1.7 1.5 2.0 1.1 NA NA -1.1 1.4 1.5 3.2 1.1 1.1 1.4 1.4 1.6 1.5 1.1 NA -1.2
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Compared with

BA.2-F157L -1.0 1.1 -1.8 NA 1.9 -2.0 -1.3 -1.1 -2.4 -1.2 1.0 -1.7 NA NA 1.0 1.6 -1.0 -1.0 1.4 1.2 1.8 1.4 3.8 1.0 -1.2 NA -1.5
BA.2-I210V 1.6 -1.0 -1.6 NA 1.5 -1.9 -1.2 -1.1 -1.6 1.0 1.3 2.1 NA NA -1.3 1.1 1.1 1.1 1.3 1.1 -1.3 1.5 1.5 1.5 1.3 NA 1.3
BA.2

BA.2-G257S 1.2 1.2 -1.8 NA -2.1 -2.4 -1.3 1.1 -2.1 1.0 1.3 1.3 NA NA 1.6 1.2 1.5 1.2 1.3 1.3 1.0 1.3 2.0 2.6 1.6 NA -1.0
BA.2-D339H 1.1 -1.0 -1.6 NA 1.5 -1.6 1.2 1.4 -2.3 1.1 1.3 -1.2 NA NA -1.0 1.3 2.4 1.5 1.5 1.2 1.0 1.9 2.1 1.1 1.1 NA -1.0
BA.2-G446S -1.0 1.7 1.0 NA 1.8 -1.3 -1.1 1.0 -2.2 -1.0 -1.0 1.6 NA NA -14 1.1 1.2 1.4 -4.1 -1.2 <-9.4 1.1 1.0 <-3.7 -1.2 NA 1.5
BA.2-N460K -2.1 1.2 <-558 NA <-3.9 -75 -4.3 -1.2 -35 -2.8 -1.5 -1.3 NA NA 1.1 1.2 -1.3 -2.5 -1.3 -1.2 -2.8 -1.8 <-2.3 -1.8 1.0 NA <-8.6
BA.2-R493Q 2.2 -1.2 3.2 NA 150 2.4 -1.1 2.9 3.6 29 3.0 76 NA >12 2.4 1.2 1.3 2.9 1.7 1.7 1.5 1.5 3.0 3.8 1.8 NA 23.4

>10 >3 <-3 <-10 <-100 <-1000

C
XGv282 JMB2002 CB6 CDRH2 Omi-3 CDRH2 Omi-18 CDRH2
CDRL3 CDRL3
G55HC
L94LC
S56HC
S56HC G54HC
S56HC
G446S
W92LC
G446S N460K N460K N460K

RBD RBD RBD RBD RBD

In brief

Wang et al. have systematically evaluated the antigenic properties of the new SARS-CoV-2 Omicron
subvariant BA.2.75. They found that BA.2.75 exhibits greater neutralization resistance to monoclonal
antibodies and vaccine- and infection-induced sera than previous Omicron subvariant BA.2, while
showing higher binding affinity for human ACE2.

Highlights

• BA.2.75 is more resistant to neutralization by polyclonal sera than BA.2

• BA.2.75 shows heightened resistance to class 1 and class 3 RBD-directed antibodies
• BA.2.75 is the first variant to show discernible resistance to bebtelovimab
• BA.2.75 exhibits higher human ACE2-binding affinity than other Omicron subvariants

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KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
C1717 Wang et al., 2022d N/A
S3H3 Hong et al., 2022 N/A
CAB-A17 Sheward et al., 2022 N/A
CB6 Shi et al., 2020 N/A
Provided by P. Kwong (NIH)
Brii-196 Ju et al., 2020 N/A
Omi-3 Nutalai et al., 2022 N/A
Omi-18 Nutalai et al., 2022 N/A
XGv347 Wang et al., 2022a N/A
ZCB11 Zhou et al., 2022 N/A

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Provided by Z. Chen (HKU)
S2E12 Starr et al., 2021 N/A

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A19-46.1 Wang et al., 2021 N/A
COV2-2196 Zost et al., 2020 N/A

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Provided by Regeneron
Pharmaceuticals
LY-CoV555 Jones et al., 2021 N/A
re
REGN10933 Hansen et al., 2020 N/A
Provided by Regeneron
lP

Pharmaceuticals
XGv282 Wang et al., 2022a N/A
LY-CoV1404 Westendorf et al., 2022 N/A
na

S309 Pinto et al., 2020 N/A

35B5 Wang et al., 2022c N/A
ur

JMB2002 Yin et al., 2022 N/A

COV2-2130 Zost et al., 2020 N/A
Jo

Provided by Regeneron
Pharmaceuticals
REGN10987 Hansen et al., 2020 N/A
Provided by Regeneron
Pharmaceuticals
Brii-198 Ju et al., 2020 N/A
10-40 Liu et al., 2022a N/A
Bacterial and virus strains
VSV-G pseudotyped ΔG-luciferase Kerafast Cat#EH1020-PM
Biological samples
Boosted sera Wang et al., 2022 N/A
BA.1 breakthrough sera Wang et al., 2022 N/A
BA.2 breakthrough sera Wang et al., 2022 N/A
Chemicals, peptides, and recombinant proteins
Polyethylenimine (PEI) Polysciences Inc. Cat#23966-100
hACE2 This paper N/A
SARS-CoV-2 D614G S2P Wang et al., 2022 N/A
SARS-CoV-2 BA.2 S2P Wang et al., 2022 N/A
SARS-CoV-2 BA.4/5 S2P Wang et al., 2022 N/A
SARS-CoV-2 BA.2.75 S2P This paper N/A
Critical commercial assays
Luciferase Assay System Promega Cat#E4550
QuikChange Lightning Site-Directed Agilent Cat#210518
Mutagenesis Kit
Series S sensor chip CM5 Cytiva Cat#BR100530
His-capture kit Cytiva Cat#28995056
Deposited data

Experimental models: cell lines

HEK293T ATCC Cat#CRL-3216;
RRID: CVCL_0063
Vero-E6 ATCC Cat#CRL-1586;

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RRID: CVCL_0574
Expi293 cells Thermo Fisher Scientific Cat#A14527

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Experimental models: organisms/strains
Oligonucleotides

-p
re
Recombinant DNA
pCMV3-D614G Wang et al., 2022 N/A
lP

pCMV3-BA.2 Wang et al., 2022 N/A

pCMV3-BA.2.12.1 Wang et al., 2022 N/A
pCMV3-BA.4/5 Wang et al., 2022 N/A
na

pCMV3-BA.2.75 This paper N/A

pCMV3-D614G-K147E This paper N/A
ur

pCMV3-D614G-W152R This paper N/A

pCMV3-D614G-F157L This paper N/A
This paper N/A
Jo

pCMV3-D614G-I210V
pCMV3-D614G-G257S This paper N/A
pCMV3-D614G-G339H This paper N/A
pCMV3-D614G-G446S This paper N/A
pCMV3-D614G-N460K This paper N/A
pCMV3-BA.2-K147E This paper N/A
pCMV3-BA.2-W152R This paper N/A
pCMV3-BA.2-F157L This paper N/A
pCMV3-BA.2-I210V This paper N/A
pCMV3-BA.2-G257S This paper N/A
pCMV3-BA.2-D339H This paper N/A
pCMV3-BA.2-G446S This paper N/A
pCMV3-BA.2-N460K This paper N/A
pCMV3-BA.2-R493Q This paper N/A
paH-D614G S2P Wang et al., 2022 N/A
paH-BA.2 S2P Wang et al., 2022 N/A
paH-BA.4/5 S2P Wang et al., 2022 N/A
paH-BA.2.75 S2P This paper N/A
pcDNA3-sACE2-WT(732)-IgG1 Chan et al., 2020 RRID: Addgene_154104
Software and algorithms
GraphPad Prism 9 GraphPad Software Inc https://www.graphpad.co
m/scientific-
software/prism/
PyMOL v.2.3.2 Schrödinger, LLC https://pymol.org/2/#pag
e-top
Biacore T200 Evaluation Software Cytiva NA
(Version 1.0)
Other

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lP
na
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