关于 IgM | 中科院微生物所等合作研发抗新冠病毒广谱IgM抗体

论文原文:

Highlights

• •

  Nanobodies R14 and S43 display not just pan-SARS-CoV-2 but varied pan-sarbecovirus activities

• •

  The multivalent nanobodies show enhanced neutralization, fitting the avidity effect

• •

  Intranasally administered MR14 potently prevents Omicron infections in mice

• •

  Structural studies reveal the neutralizing mechanisms of R14 and S43 as well as MR14

Summary

With the widespread vaccinations against coronavirus disease 2019 (COVID-19), we are witnessing gradually waning neutralizing antibodies and increasing cases of breakthrough infections, necessitating the development of drugs aside from vaccines, particularly ones that can be administered outside of hospitals. Here, we present two cross-reactive nanobodies (R14 and S43) and their multivalent derivatives, including decameric ones (fused to the immunoglobulin M [IgM] Fc) that maintain potent neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after aerosolization and display not only pan-SARS-CoV-2 but also varied pan-sarbecovirus activities. Through respiratory administration to mice, monovalent and decameric R14 significantly reduce the lung viral RNAs at low dose and display potent pre- and post-exposure protection. Furthermore, structural studies reveal the neutralizing mechanisms of R14 and S43 and the multiple inhibition effects that the multivalent derivatives exert. Our work demonstrates promising convenient drug candidates via respiratory administration against SARS-CoV-2 infection, which can contribute to containing the COVID-19 pandemic.

Graphical abstract

Keywords

• SARS-CoV-2
• Omicron
• nanobody
• IgM antibody
• inhalable administration
• cross-reactive antibody
• sarbecovirus

Introduction

In this study, we present two pan-SARS-CoV-2 and pan-sarbecovirus nanobodies, R14 and S43, that maintained neutralizing activity after being aerosolized. Both nanobodies significantly reduced lung viral RNAs via intranasal (i.n.) administration prior to SARS-CoV-2 infection. Specifically, aerosolized R14 also inhibited SARS-CoV-2 infection in mice in both pre- (PrEP) and post-exposure prophylactic (PEP) settings. To further increase their potencies and broaden their spectrum against Omicron VOC, we engineered the nanobodies into multiple constructs, including dimeric derivatives by tagging them to the Fc of the immunoglobulin G1 (IgG1) (DR14 and DS43), homo-trimeric ones by tandem-repeat constructions (TR14 and TS43), and decameric ones by fusing them to the Fc of IgM (MR14 and MS43). Notably, trimeric and decameric constructs maintained similar activities after aerosolization and exhibited higher antiviral activities against the Omicron sub-variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5. Compared with the parental nanobodies, the inhalable derivatives also displayed longer residence time in the lung, as well as longer half-life (t_1/2) in the blood when intraperitoneally (i.p.) administered. Additionally, using a mouse infection model, MR14 displayed great potency to prevent Omicron BA.2 infections in both PrEP and PEP settings via i.n. and i.p. administration routes. Further structural analysis revealed the neutralizing mechanisms of R14 and S43 and indicated the mechanisms by which the multivalent derivatives substantially increased their inhibition potencies. Thus, these data indicate that R14, S43, and their multivalent derivatives have great potential to be further developed as convenient inhalable therapeutics that are effective and broad spectrum for preventing infections of SARS-CoV-2 variants and potential emerging sarbecoviruses.

Results

Nanobodies prevented SARS-CoV-2 infection in vitro and in vivo

To obtain nanobodies, we immunized two alpacas with the SARS-CoV-2 spike (S) and RBD proteins, respectively. Nanobodies were displayed following the protocols previously described

²⁴

 (Figure S1A). Approximately 500 clones were identified by phage ELISA assays during panning, and the positive clones were sequenced. In our current study, we concentrated on two nanobodies, R14 and S43.Both R14 and S43 specifically recognized the SARS-CoV-2 RBD, as confirmed by phage ELISA (Figure S1). Moreover, S43 could also bind to SARS-CoV S, as shown by flow cytometry (Figure S2). Both R14 and S43 also blocked the binding between the SARS-CoV-2 RBD and human ACE2 (hACE2) (Figure S3). As determined by surface plasmon resonance (SPR) assays, S43 was bound to the SARS-CoV-2 RBD with high affinity, with an equilibrium dissociation constant (K_D) value of 0.18 nM, while R14 displayed no detectable dissociation from the SARS-CoV-2 RBD over 10 min (Figure 1A). These results indicate that R14 has a stronger binding affinity for SARS-CoV-2 than that of S43.

We next assessed the neutralizing capabilities of both nanobodies against pseudotyped and live SARS-CoV-2 in vitro and found that R14 and S43 have potent neutralizing activities against pseudotyped SARS-CoV-2, with half maximal inhibitory concentration (IC₅₀) values of 0.16 (0.0024 μg/mL) and 11 nM (0.16 μg/mL), respectively (Figure 1B). They also neutralized live SARS-CoV-2 with IC₅₀ values of 1.3 (0.02 μg/mL) and 40.7 nM (0.61 μg/mL), respectively (Figure 1C).

We further evaluated the in vivo prophylactic efficacy of the two nanobodies against SARS-CoV-2 in an Ad5-hACE2 mouse model. We found that 5 mg/kg R14 and S43 significantly reduced lung viral RNAs when administered via the i.n. route prior to SARS-CoV-2 infection (Figure 1D). In particular, R14 displayed an approximately 100-fold reduction of lung viral RNAs compared with the control mice. Lung pathology analyses revealed that SARS-CoV-2 can cause severe interstitial pneumonia, characterized by inflammatory cell infiltration and alveolar septal thickening in control mice, while only very mild lesions were observed when the mice received R14 or S43 by the i.n. route prior to challenge with SARS-CoV-2 (Figure 1E).

Aerosolized R14 maintained neutralizing activity and prevented infection

We then tried to evaluate the effectiveness of the nanobody via inhalation (i.h.) delivery. Because R14 was superior to S43 in terms of neutralization activity in vitro and preventive efficacy in vivo via i.n. administration, we chose to assess the effects of R14 by aerosol delivery with a whole-body exposure platform (Figure 2A).

The whole-body exposure system designed in this study mainly contained an Aerogen Solo vibrating mesh nebulizer (Aerogen, Chicago, IL, USA) and an exposure chamber. Aerosol size distributions were measured at six monitoring sites (red triangles in Figure 2A) on the middle cross-section of the exposure chamber and showed no significant differences among the monitoring sites (Figure 2B), indicating that each mouse was exposed to similar conditions. To select the proper flowrate of the carrier air, the average mass concentration was monitored and suggested to be higher when the carrier air flowrate was maintained at 10 and 5 L per minute (LPM) compared with 20 LPM (Figure 2C). Finally, the flowrate of the carrier air was maintained at 10 LPM during exposure experiments due to remarkably deposited aerosol at the bottom of the chamber when the flowrate was 5 LPM.

Notably, the aerosolization process, a large environmental change, did not influence the neutralization activities of the two nanobodies against SARS-CoV-2 pseudotyped viruses (Figure 2D), providing the basis for further trials of i.h. administration. Thus, mice were administrated a single 0.25 mg/kg dose of R14 via the i.h. route prior to SARS-CoV-2 infection, while a single 5 mg/kg dose administrated via the i.n. route was used as a control. We found that the lung viral RNAs in the i.h. group decreased to a similar level to those mice in the i.n. group, though the administrated dose via the i.h. route was 20 times lower than that via the i.n. route (Figure 2E).

We further evaluated the therapeutic effect of R14 against SARS-CoV-2 via the i.h. route at different times. After SARS-CoV-2 infection, mice were treated with a single 0.75 mg/kg dose of R14 at 10 min, 1.5 h, or 7 h, while the control mice were administrated PBS (Figure 2F). We found that the viral RNAs in the lung were significantly reduced when the nanobody was administrated at 10 min or 1.5 h post-infection. Although no significant difference was observed in the 7 h group, the viral titer decreased compared with the control group, suggesting that the preventive effect is related to the time when the nanobody is delivered post-infection. Indeed, at 10 min, the viral copies decreased by three orders of magnitude, which is similar to the efficacy of R14 administered prior to infection by the i.h. route with a dose of 0.25 mg/kg (Figure 2E).

Both R14 and S43 displayed pan-SARS-CoV-2 and pan-sarbecovirus activities

The effectiveness of R14 and S43 against the prototype SARS-CoV-2 prompted us to further evaluate their efficacy against SARS-CoV-2 variants. We measured the binding affinities and neutralizing activities of R14 and S43 (Figures 3A and 3B ). Notably, both R14 and S43 could bind to all of the tested RBDs from Alpha, Beta, Gamma, Kappa, Delta, Delta plus, Lambda, and Mu (Figures 3A and 3C). Additionally, both R14 and S43 broadly neutralized multiple SARS-CoV-2 variants (Figures 3B and 3C). R14 displayed similar neutralizing ability against the SARS-CoV-2 prototype and its variants, with IC₅₀ values ranging from 0.16 to 1 nM (0.0024–0.015 μg/mL), except for the Lambda variant. The neutralizing activity of R14 against Lambda was 134 nM (2.01 μg/mL), which is ∼800-fold lower than that against the prototype strain. S43 displayed weaker neutralizing ability against these pseudotyped viruses than R14, with IC₅₀ values ranging from 2.75–13.05 nM (0.041–0.20 μg/mL). The results of the two nanobodies against the Omicron variant are covered in the next section.

To further explore the cross-reactivity of R14 and S43, we also tested the binding affinities of the two nanobodies to several sarbecoviruses, including SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX/P2V/2017, and GD/1/2019.11
 We found that R14 bound to the GD/1/2019 RBD with high affinity, but no binding was observed to the other sarbecoviruses (Figures 3D and 3E) at the highest tested concentration of 100 nM. Nevertheless, S43 bound to the RBDs from SARS-CoV, RaTG13, RshSTT182, RacC203, RsYN04, GX/P2V/2017, and GD/1/2019 (Figure 3D and 3E). These data indicate that S43 has broader cross-reactivity to sarbecoviruses than R14.

Multivalent derivatives presented improved efficacy against Omicron VOC

To increase their neutralization and broaden their activities against the Omicron VOC, we constructed multivalent forms of the two nanobodies, including dimeric derivatives by tagging them to the Fc of the IgG1 (DR14 and DS43), homo-trimeric ones by tandem-repeat constructions (TR14 and TS43), and decameric ones by fusing them to the Fc of IgM (MR14 and MS43) (Figure 4A), as it is known that multivalent mAbs can display improved efficacy.21^,25^,26
 Indeed, compared with the monomeric R14, both DR14 and TR14 greatly improved the neutralizing activities, with 27- to 500- and 280- to 4,000-fold enhancement, respectively, against pseudotyped Omicron sub-variants BA.1, BA.2, BA.2.12.1, BA.2.75, and BA.3. However, their activities against pseudotyped BA.4/5 were only slightly higher than that of R14. When R14 was constructed to the decameric MR14, the activity against BA.4/5 was substantially increased, with 350-fold change. Additionally, compared with DR14 and TR14, MR14 displayed further enhancement of the activities against sub-variants BA.1, BA.2, BA.2.12.1, BA.2.75, and BA.3, with 94,000-, 7,800-, 9,900-, 6,700-, and 2,600-fold changes, respectively, although, as with TR14, MR14 displayed similar binding affinities to the sub-variant RBDs compared with those of R14 (Figure S5).

However, S43 displayed weak potency, with IC₅₀ >12 μg/mL against pseudotyped Omicron sub-variants, but enhanced neutralizing activity was also observed with DS43, TS43, and MS43. In addition, the multivalent forms of R14 and S43 demonstrated similar neutralizing activities as their respective monomeric forms against the SARS-CoV-2 prototype and Delta variants (Figures 4B and 4C).

We then chose the homo-trimeric and decameric derivatives for evaluation using live viruses since both strategies have been used to develop therapeutic antibodies that entered clinical trials as nasal administration drugs27^,28 (https://clinicaltrials.gov/). Both forms of constructs also greatly increased the neutralizing activities against sub-variants BA.1 and BA.2 compared with the monomeric nanobodies (Figure 4D).

MR14 displayed effective protection in both PrEP and PEP settings via i.n. and i.p. in vivo

As with the parental nanobodies, the aerosolization process did not influence the neutralization activities of both homo-trimeric and decameric versions of R14 and S43 (Figure 5A), which provides the basis for their applications as inhalable drugs. We further focused on R14, TR14, and MR14 to evaluate the decay rate, due to their superiorities in terms of neutralization activities and their broad potencies against Omicron sub-variants. We found that both aerosolized TR14 and MR14 displayed longer t_1/2 (7.2 and 28.1 h, respectively) in lungs compared with the aerosolized R14 (5.1 h) (Figure 5B), indicating an extended residence time and thereby higher protective efficacy by the two R14 multimers in the respiratory system when administered via the i.h. route. In addition, we also assessed the t_1/2 of TR14 and MR14 in blood after i.p. injection and found that they had substantially longer t_1/2 (12.9 and 58.1 h, respectively) compared with monovalent R14 (1.2 h) (Figure 5C).

We next evaluated the prophylactic and therapeutic efficacy of MR14 against Omicron sub-variant BA.2 using a K18-hACE2 mouse model. A single 5 mg/kg dose of MR14 was administered 6 h prior to BA.2 infection by the i.n. or i.p. route to evaluate prophylactic efficacy. Mice receiving PBS were used as a control. For therapeutic evaluation, MR14 was administered in three i.n. doses at 6, 30, and 54 h post-BA.2 infection, but only a single dose of MR14 was administered via the i.p. route 6 h post-infection. Subgenomic RNA (sgRNA) encoding for the SARS-CoV-2 E genes in lung tissues was analyzed 3 days post-infection. We found that MR14 demonstrated significant prophylactic efficacy when administered by the i.n. or i.p. route (Figure 5D). Notably, three mice (n = 5) in the i.n. group and four mice (n = 5) in the i.p. group showed undetectable sgRNA levels. MR14 also yielded decreased sgRNA loads in therapeutic groups, with two mice (n = 5) displaying undetectable sgRNA in the i.n. and i.p. groups. Pathological analyses of lung tissue revealed that MR14 alleviated lung injury, especially in prophylactic treatment via i.n. delivery (Figure 5E). These results suggest that MR14 has potent efficacy for the prevention and treatment of COVID-19.

Structural insight into the binding between SARS-CoV-2 RBD and R14 or S43

To illustrate the molecular basis of R14 and S43 neutralizing virus infection, we prepared SARS-CoV-2 prototype RBD/R14 complex and SARS-CoV-2 prototype RBD/S43 complex proteins (Figure S6) and solved their crystal structures at resolutions of 2.5 and 2.69 Å, respectively (Table S1). Overall, the two nanobodies recognized distinct conformational epitopes on the RBD.

R14 bound to the receptor-binding motif (RBM) with all three CDRs (CDR1-3) and one framework region (FR, FR2) involved in the interaction (Figure 6A), and it could span the epitopes of RBD-1, RBD-2, RBD-3, and RBD-4 antibodies due to its long CDR3 and large binding area (∼1,015 Å²).29
 We superimposed the prototype RBD/R14 complex onto the SARS-CoV-2 prototype S trimer (PDB: 6VYB), revealing that R14 preferentially bound to the RBD in the up conformation. However, when R14 interacted with the down RBD, it would sterically clash with the adjacent RBD in both up and down conformations (Figure 6B). Additionally, the R14 epitope would clash with the hACE2-binding site, as shown by superimposing the prototype RBD/R14 complex onto the prototype RBD/hACE2 complex (PDB: 6LZG) (Figure 6C). Therefore, R14 likely neutralizes SARS-CoV-2 by preventing viral attachment to host cells via competition with the ACE2 entry receptor.

Distinct from R14, S43 recognized a cryptic epitope on one side of the RBD (RBD-7)29 using only CDR3 (Figure 6D). When the prototype RBD/S43 complex was superimposed onto the SARS-CoV-2 prototype S trimer (PDB: 7LYK), S43 was observed to interact with the up RBD-containing S, which possesses at least two up RBDs (Figure 6E). Although the S43 epitope is away from the RBM, and no overlap with the ACE2-binding site was observed, we found that this nanobody displays steric hindrance with the ACE2 N322 glycan by superimposing the prototype RBD/S43 complex onto the prototype RBD/hACE2 complex (PDB: 6LZG) (Figure 6F). Therefore, S43 likely neutralizes SARS-CoV-2 infection by partially interfering with S binding to ACE2 caused by ACE2 glycosylation. This probable mechanism was also observed with SARS-CoV-2 neutralizing antibodies S30430 and EY6A,31 both of which also belong to RBD-7 and apply their constant regions to prevent the association between the receptor and the neighboring RBDs.

The detailed interactions, containing van der Waals (vdw), hydrogen bond (H-bond), and salt bridge interactions, between R14 and RBD are displayed (Table S2). However, we found 10 mutations (K417N, G446S, L452R/Q/M, T478K, F486V, Q493R, G496S, Q498R, N501Y, and Y505H) in the Omicron RBD that belong to R14-binding sites (Figure S7A; Table S2). They break partial H-bond (G446S, Q493R, Q498R, N501Y, and Y505H) and hydrophobic (G446S, L452R, F486V, and G496S) interactions, and some showed potential clashes with R14 due to their large side chain (such as Q498R) (Figures S7B and S7D). On the contrary, T478K added a salt bridge interaction with D62, and N501Y added a π-π interaction with Y119 and a hydrophobic interaction with F27, L29, and P100. Nevertheless, R14 ultimately displayed decreased binding and neutralizing activities to Omicron sub-variants due to the combination of several mutations. Notably, because the BA.2 RBD has no G446S and G496S mutations, R14 demonstrated higher binding and neutralizing abilities to BA.2 than to BA.1.S43 recognized a cryptic epitope, defined by previous studies,³²

 and its footprint did not overlap with the ACE2-binding site (Figure 6F). The detailed interactions between S43 and the RBD are shown in Table S3. Notably, all of the interactions were contributed by CDR3, which adopted a pan-like conformation parallel to the RBD. Notably, only the S375F mutation in the BA.1 RBD is included in the S43-binding site, which leads to the loss of two H-bonds formed by S375 with G110 and T112 in S43 (Figure S7E). This may reveal why S43 showed slightly reduced binding to the BA.1 RBD compared with the prototype RBD. However, due to the preferential conformation of BA.1 S in one-RBD-up,33 S43 displayed significantly reduced neutralization against BA.1. Aside from the S375F mutation, the T376A, D405N, and R408S mutations in the BA.2, BA.2.12.1, and BA.4/5 RBDs also belong to the S43-binding site, which further impaired the interaction with S43 (Figure S7F). Therefore, S43 displayed poor neutralization against other Omicron sub-variants besides BA.1.

Both TR14 and MR14 bind to the BA.1 RBD at similar levels to R14 but display extremely higher neutralizing activity against pseudotyped and authentic BA.1 than R14. To explain this phenomenon, we negatively stained samples of S protein incubated with antibodies and imaged them with transmission electron microscopy (TEM) (Figure 6G), which suggested that particles are formed between BA.1 S protein with TR14 or MR14 but not with R14. Indeed, large particles without free S protein were observed in samples of BA.1 S protein with the MR14 antibody, explaining its high neutralizing activity. The clustering effect of TR14 or MR14 to SARS-CoV-2 prototype S protein was not obvious, consistent with their similar potencies against SARS-CoV-2 prototype infections compared with the parental R14.

Discussion

Thus, we focused on both of the above strategies to engineer R14 and S43 in this study. The homo-trimer of R14 or S43 was linked by a (G₄S)₅ linker, and IgM derivatives were constructed by fusing the nanobodies with the Fc of IgM. Especially, as the single variable domain of the nanobody without the light chain, the complexities for the assembly as well as the manufacturing of these IgM derivatives decreased from the traditional 21 chains (10 heavy chains, 10 light chain, and 1 J chain) to the current 11 chains (10 heavy chains and 1 J chain). Since the manufacturing is a challenge for the development of IgM antibodies, the nanobody-based IgM derivatives reported here provide a potential design that would solve this problem and advance their further applications.

The data suggest that these multivalences, including the dimer, homo-trimer, and decamer, enable them to have pan-SARS-CoV-2 neutralizing activities, especially against the latest Omicron VOC. As expected, dimeric (DR14 and DS43), homo-trimeric (TR14 and TS43), and decameric derivatives (MR14 and MS43) displayed significant improvement against Omicron sub-variants compared with monomer. Particularly, MR14 exhibited more than 300-fold enhancement and also had potent in vivo prophylactic and therapeutic efficacy against the BA.2 sub-variant when delivered by i.n. and i.p. administration in mice.

As indicated by the complex structures, R14 has large binding area on the RBD and could overlap with RBD-1, RBD-2, RBD-3, and RBD-4 antibodies. It binds to the RBD in the up conformation and blocks the interaction between the virus and the receptor. S43 belongs to the RBD-7 and needs to function when at least two RBDs are in the up conformations. Thus, R14 would be greatly influenced by the residue mutations on the RBM region, such as L452Q/R/M, F486V, and Q493R. Although S43 targets the relatively conserved region and shows broader spectrum against sarbecoviruses than R14, the conformational shifts of the S protein would influence its activity. When constructed to the multivalent derivatives, their remarkably increased efficacy for preventing viral infection was observed in Omicron sub-variants but not with the prototype or Delta. Negatively stained TEM images showed that multivalent R14, especially the decameric MR14, engages more S proteins and causes the aggregation of the BA.1 S proteins. It is reported that, compared with the prototype S protein, the S trimer of BA.1 predominantly has one RBD in the up position, allowing the stable binding of the R14 protomer in the multimers. This also provides a reasonable explanation for why the activity of MS43 did not increase a lot.

STAR★Methods

Data and code availability

The atomic coordinates for the complex of the SARS-CoV-2 RBD with R14 or S43 have been deposited in the PDB (www.rcsb.org). The PDB IDs are 7WD1 and 7WD2. This paper does not report the original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

We thank Dr. Linhui Wang for generous support of pMES4, phage VSCM13, and E. coli TG1. We are grateful for the help from the staff of BSL-3 at the Institute of Microbiology, Chinese Academy of Sciences. We thank Dr. Xin Zhou from Yangzhou University for supporting our project. We thank Kun Xu (Beijing Institutes of Life Science, Chinese Academy of Sciences) for generous support of Ad5-hACE2. We thank Zheng Fan (Institutional Center for Shared Technologies and Facilities, Institute of Microbiology, Chinese Academy of Sciences) and Yuanyuan Chen (Institute of Biophysics, Chinese Academy of Sciences) for technical help with Biacore experiments. We thank Jingnan Liang (Institute of Microbiology, Chinese Academy of Sciences) for technical help with negative-stain experiments by TEM. We also thank Qingwen He, Xinxin Yan, Xiaoyun Wang, Anqi Zheng, Chunli Wu, Shihua Li, and Yunfei Jia (Institute of Microbiology, Chinese Academy of Sciences) for supporting our project. This work was supported by CAS Project for Young Scientists in Basic Research (YSBR-010); National Natural Science Foundation of China (81922044, 52091541, 82041047, and 82225021); Ministry of Science and Technology of the People’s Republic of China (2021YFC0863300 and 2022YFC2303403); Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29040203); and Youth Innovation Promotion Association CAS (2018119).

Author contributions

Q.W., G.F.G., and H.L. designed this study. S.Q. and R.F. conducted the alpaca immunization. H.L. selected the two nanobodies R14 and S43 by phage display technology. H.L., B.L., and X.R. performed protein expression. H.L. and B.L. performed flow cytometry, SPR, and crystallization assays. H.L. and X.R. performed neutralization assay of pseudotyped virus. K.X. and W.L. performed neutralization assay of live virus in BSL-3 facility. J.J., J.D., D.W., and L. Wang designed the whole-body exposure system and detected aerosol distribution and mass concentration. H.L. and B.L. performed the bronchoalveolar lavage of murine lungs. With the help of W.J.L. and G.W., W.L. and K.X. performed animal experiments using live virus in the BSL-3 Laboratory of the Chinese Center for Disease Control and Prevention. With the help of Y.B., L. Wu and P.D. performed animal experiments using live virus in the BSL-3 Laboratory of the Institute of Microbiology, Chinese Academy of Sciences. J.Q. and C.S. collected the diffraction data and built structural model. Q.W. and L. Wu analyzed the complex structure. H.C. and X.Z. analyzed the mutations of multiple variants. H.L., L. Wu, and P.D. wrote the manuscript. Q.W., G.F.G., J. J., and Q.C. revised the manuscript with inputs from other co-authors.

Declaration of interests

Q.W., G.F.G., H.L., and L. Wu are listed as inventors on patent applications for R14 and S43 based antiviral treatment. Q.W., G.F.G., H.L., B.L., and L. Wu are listed as inventors on patent applications for MR14 and MS43 based antiviral treatment.

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References

1. • Gao G.F.

   From "A"IV to "Z"IKV: attacks from emerging and re-emerging pathogens.

   Cell. 2018; 172: 1157-1159View in Article
   • Scopus (191)
   • PubMed
   • Abstract
   • Full Text
   • Full Text PDF
   • Google Scholar
2. • Katoh K.
   • Standley D.M.

   MAFFT multiple sequence alignment software version 7: improvements in performance and usability.

   Mol. Biol. Evol. 2013; 30: 772-780View in Article
   • Scopus (25214)
   • PubMed
   • Crossref
   • Google Scholar
3. • Zhu N.
   • Zhang D.
   • Wang W.
   • Li X.
   • Yang B.
   • Song J.
   • Zhao X.
   • Huang B.
   • Shi W.
   • Lu R.
   • et al.

   A novel coronavirus from patients with pneumonia in China, 2019.

   N. Engl. J. Med. 2020; 382: 727-733View in Article
   • Scopus (17972)
   • PubMed
   • Crossref
   • Google Scholar
4. • Xu Z.
   • Liu K.
   • Gao G.F.

   Omicron variant of SARS-CoV-2 imposes a new challenge for the global public health.

   Biosaf. Health. 2022; 4: 147-149View in Article
   • Scopus (22)
   • PubMed
   • Crossref
   • Google Scholar
5. • Du P.
   • Gao G.F.
   • Wang Q.

   The mysterious origins of the Omicron variant of SARS-CoV-2.

   Innovation. 2022; 3: 100206View in Article
   • Google Scholar
6. • Huang M.
   • Wu L.
   • Zheng A.
   • Xie Y.
   • He Q.
   • Rong X.
   • Han P.
   • Du P.
   • Han P.
   • Zhang Z.
   • et al.

   Atlas of currently available human neutralizing antibodies against SARS-CoV-2 and escape by Omicron sub-variants BA.1/BA.1.1/BA.2/BA.3.

   Immunity. 2022; 55: 1501-1514.e3View in Article
   • Scopus (39)
   • PubMed
   • Abstract
   • Full Text
   • Full Text PDF
   • Google Scholar
7. • Cao Y.
   • Yisimayi A.
   • Jian F.
   • Song W.
   • Xiao T.
   • Wang L.
   • Du S.
   • Wang J.
   • Li Q.
   • Chen X.
   • et al.

   BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection.

   Nature. 2022; 608: 593-602View in Article
   • Scopus (605)
   • PubMed
   • Crossref
   • Google Scholar
8. • Niu S.
   • Wang J.
   • Bai B.
   • Wu L.
   • Zheng A.
   • Chen Q.
   • Du P.
   • Han P.
   • Zhang Y.
   • Jia Y.
   • et al.

   Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin.

   EMBO J. 2021; 40: e107786View in Article
   • Scopus (36)
   • PubMed
   • Crossref
   • Google Scholar
9. • Liu K.
   • Pan X.
   • Li L.
   • Yu F.
   • Zheng A.
   • Du P.
   • Han P.
   • Meng Y.
   • Zhang Y.
   • Wu L.
   • et al.

   Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

   Cell. 2021; 184: 3438-3451.e10View in Article
   • Scopus (82)
   • PubMed
   • Abstract
   • Full Text
   • Full Text PDF
   • Google Scholar
10. • Xiao K.
    • Zhai J.
    • Feng Y.
    • Zhou N.
    • Zhang X.
    • Zou J.J.
    • Li N.
    • Guo Y.
    • Li X.
    • Shen X.
    • et al.

    Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

    Nature. 2020; 583: 286-289View in Article
    • Scopus (437)
    • PubMed
    • Crossref
    • Google Scholar
11. • Wang Q.
    • Chen H.
    • Shi Y.
    • Hughes A.C.
    • Liu W.J.
    • Jiang J.
    • Gao G.F.
    • Xue Y.
    • Tong Y.

    Tracing the origins of SARS-CoV-2: lessons learned from the past.

    Cell Res. 2021; 31: 1139-1141View in Article
    • Scopus (19)
    • PubMed
    • Crossref
    • Google Scholar
12. • Lam T.T.Y.
    • Jia N.
    • Zhang Y.W.
    • Shum M.H.H.
    • Jiang J.F.
    • Zhu H.C.
    • Tong Y.G.
    • Shi Y.X.
    • Ni X.B.
    • Liao Y.S.
    • et al.

    Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

    Nature. 2020; 583: 282-285View in Article
    • Scopus (1106)
    • PubMed
    • Crossref
    • Google Scholar
13. • Wang Q.
    • Zhang Y.
    • Wu L.
    • Niu S.
    • Song C.
    • Zhang Z.
    • Lu G.
    • Qiao C.
    • Hu Y.
    • Yuen K.Y.
    • et al.

    Structural and functional basis of SARS-CoV-2 entry by using human ACE2.

    Cell. 2020; 181: 894-904.e9View in Article
    • Scopus (1947)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar
14. • Shi R.
    • Shan C.
    • Duan X.
    • Chen Z.
    • Liu P.
    • Song J.
    • Song T.
    • Bi X.
    • Han C.
    • Wu L.
    • et al.

    A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.

    Nature. 2020; 584: 120-124View in Article
    • Scopus (875)
    • PubMed
    • Crossref
    • Google Scholar
15. • Wu Y.
    • Wang F.
    • Shen C.
    • Peng W.
    • Li D.
    • Zhao C.
    • et al.

    A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2.

    Science. 2020; 368: 1274-1278View in Article
    • Scopus (698)
    • PubMed
    • Crossref
    • Google Scholar
16. • Earle K.A.
    • Ambrosino D.M.
    • Fiore-Gartland A.
    • Goldblatt D.
    • Gilbert P.B.
    • Siber G.R.
    • Dull P.
    • Plotkin S.A.

    Evidence for antibody as a protective correlate for COVID-19 vaccines.

    Vaccine. 2021; 39: 4423-4428View in Article
    • Scopus (520)
    • PubMed
    • Crossref
    • Google Scholar
17. • Forchette L.
    • Sebastian W.
    • Liu T.

    A comprehensive review of COVID-19 virology, vaccines, variants, and therapeutics.

    Curr. Med. Sci. 2021; 41: 1037-1051View in Article
    • Scopus (107)
    • PubMed
    • Crossref
    • Google Scholar
18. • Guilleminault L.
    • Azzopardi N.
    • Arnoult C.
    • Sobilo J.
    • Hervé V.
    • Montharu J.
    • Guillon A.
    • Andres C.
    • Herault O.
    • Le Pape A.
    • et al.

    Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system.

    J. Control. Release. 2014; 196: 344-354View in Article
    • Scopus (84)
    • PubMed
    • Crossref
    • Google Scholar
19. • Respaud R.
    • Vecellio L.
    • Diot P.
    • Heuzé-Vourc'h N.

    Nebulization as a delivery method for mAbs in respiratory diseases.

    Expert Opin. Drug Deliv. 2015; 12: 1027-1039View in Article
    • Scopus (90)
    • PubMed
    • Crossref
    • Google Scholar
20. • Jovčevska I.
    • Muyldermans S.

    The therapeutic potential of nanobodies.

    BioDrugs. 2020; 34: 11-26View in Article
    • Scopus (352)
    • PubMed
    • Crossref
    • Google Scholar
21. • Xiang Y.
    • Nambulli S.
    • Xiao Z.
    • Liu H.
    • Sang Z.
    • Duprex W.P.
    • Schneidman-Duhovny D.
    • Zhang C.
    • Shi Y.

    Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2.

    Science. 2020; 370: 1479-1484View in Article
    • Scopus (223)
    • PubMed
    • Crossref
    • Google Scholar
22. • Wu X.
    • Cheng L.
    • Fu M.
    • Huang B.
    • Zhu L.
    • Xu S.
    • Shi H.
    • Zhang D.
    • Yuan H.
    • Nawaz W.
    • et al.

    A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration.

    Cell Rep. 2021; 37: 109869View in Article
    • Scopus (35)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar
23. • Li T.
    • Cai H.
    • Yao H.
    • Zhou B.
    • Zhang N.
    • van Vlissingen M.F.
    • Kuiken T.
    • Han W.
    • GeurtsvanKessel C.H.
    • Gong Y.
    • et al.

    A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.

    Nat. Commun. 2021; 12: 4635View in Article
    • Scopus (53)
    • PubMed
    • Crossref
    • Google Scholar
24. • Wrapp D.
    • De Vlieger D.
    • Corbett K.S.
    • Torres G.M.
    • Wang N.
    • Van Breedam W.
    • Roose K.
    • van Schie L.
    • Hoffmann M.
    • et al.
    • VIB-CMB COVID-19 Response Team

    Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies.

    Cell. 2020; 181: 1436-1441View in Article
    • Scopus (61)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar
25. • Schoof M.
    • Faust B.
    • Saunders R.A.
    • Sangwan S.
    • Rezelj V.
    • Hoppe N.
    • Boone M.
    • Billesbølle C.B.
    • Puchades C.
    • Azumaya C.M.
    • et al.

    An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

    Science. 2020; 370: 1473-1479View in Article
    • Scopus (247)
    • PubMed
    • Crossref
    • Google Scholar
26. • Yang Z.
    • Wang Y.
    • Jin Y.
    • Zhu Y.
    • Wu Y.
    • Li C.
    • Kong Y.
    • Song W.
    • Tian X.
    • Zhan W.
    • et al.

    A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants.

    Signal Transduct. Target. Ther. 2021; 6: 378View in Article
    • Scopus (21)
    • PubMed
    • Crossref
    • Google Scholar
27. • Ku Z.
    • Xie X.
    • Hinton P.R.
    • Liu X.
    • Ye X.
    • Muruato A.E.
    • Ng D.C.
    • Biswas S.
    • Zou J.
    • Liu Y.
    • et al.

    Nasal delivery of an IgM offers broad protection from SARS-CoV-2 variants.

    Nature. 2021; 595: 718-723View in Article
    • Scopus (95)
    • PubMed
    • Crossref
    • Google Scholar
28. • Piedra P.A.
    • Martinon-Torres F.
    • Szymanski H.
    • Brackeva B.
    • Dombrecht E.
    • Detalle L.
    • Fleurinck C.
    • Cunningham S.
    • Piedra P.A.
    • et al.
    • RESPIRE study group

    Nebulised ALX-0171 for respiratory syncytial virus lower respiratory tract infection in hospitalised children: a double-blind, randomised, placebo-controlled, phase 2b trial.

    Lancet Respir. Med. 2021; 9: 21-32View in Article
    • Scopus (54)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar
29. • Hastie K.M.
    • Li H.
    • Bedinger D.
    • Schendel S.L.
    • Dennison S.M.
    • Li K.
    • Rayaprolu V.
    • Yu X.
    • Mann C.
    • Zandonatti M.
    • et al.

    Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: a global consortium study.

    Science. 2021; 374: 472-478View in Article
    • Scopus (156)
    • PubMed
    • Crossref
    • Google Scholar
30. • Piccoli L.
    • Park Y.J.
    • Tortorici M.A.
    • Czudnochowski N.
    • Walls A.C.
    • Beltramello M.
    • Silacci-Fregni C.
    • Pinto D.
    • Rosen L.E.
    • Bowen J.E.
    • et al.

    Mapping neutralizing and immunodominant sites on the SARS-CoV-2 spike receptor-binding domain by structure-guided high-resolution serology.

    Cell. 2020; 183: 1024-1042.e21View in Article
    • Scopus (814)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar
31. • Zhou D.
    • Duyvesteyn H.M.E.
    • Chen C.P.
    • Huang C.G.
    • Chen T.H.
    • Shih S.R.
    • Lin Y.C.
    • Cheng C.Y.
    • Cheng S.H.
    • Huang Y.C.
    • et al.

    Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient.

    Nat. Struct. Mol. Biol. 2020; 27: 950-958View in Article
    • Scopus (201)
    • PubMed
    • Crossref
    • Google Scholar
32. • Yuan M.
    • Wu N.C.
    • Zhu X.
    • Lee C.-C.D.
    • So R.T.Y.
    • Lv H.
    • Mok C.K.P.
    • Wilson I.A.

    A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV.

    Science. 2020; 368: 630-633View in Article
    • Scopus (975)
    • PubMed
    • Crossref
    • Google Scholar
33. • Cui Z.
    • Liu P.
    • Wang N.
    • Wang L.
    • Fan K.
    • Zhu Q.
    • Wang K.
    • Chen R.
    • Feng R.
    • Jia Z.
    • et al.

    Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron.

    Cell. 2022; 185: 860-871.e13View in Article
    • Scopus (210)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar
34. • Ren S.Y.
    • Wang W.B.
    • Gao R.D.
    • Zhou A.M.

    Omicron variant (B.1.1.529) of SARS-CoV-2: mutation, infectivity, transmission, and vaccine resistance.

    World J. Clin. Cases. 2022; 10: 1-11View in Article
    • PubMed
    • Crossref
    • Google Scholar
35. • Zhao X.
    • Li D.
    • Ruan W.
    • Chen Z.
    • Zhang R.
    • Zheng A.
    • Qiao S.
    • Zheng X.
    • Zhao Y.
    • Dai L.
    • et al.

    Effects of a prolonged booster interval on neutralization of Omicron variant.

    N. Engl. J. Med. 2022; 386: 894-896View in Article
    • Scopus (68)
    • PubMed
    • Crossref
    • Google Scholar
36. • Ai J.
    • Zhang H.
    • Zhang Y.
    • Lin K.
    • Zhang Y.
    • Wu J.
    • Wan Y.
    • Huang Y.
    • Song J.
    • Fu Z.
    • et al.

    Omicron variant showed lower neutralizing sensitivity than other SARS-CoV-2 variants to immune sera elicited by vaccines after boost.

    Emerg. Microbes Infect. 2022; 11: 337-343View in Article
    • Scopus (237)
    • PubMed
    • Crossref
    • Google Scholar
37. • Xu K.
    • Gao P.
    • Liu S.
    • Lu S.
    • Lei W.
    • Zheng T.
    • Liu X.
    • Xie Y.
    • Zhao Z.
    • Guo S.
    • et al.

    Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2.

    Cell. 2022; 185: 2265-2278.e14View in Article
    • Scopus (51)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar
38. • Detalle L.
    • Stohr T.
    • Palomo C.
    • Piedra P.A.
    • Gilbert B.E.
    • Mas V.
    • Millar A.
    • Power U.F.
    • Stortelers C.
    • Allosery K.
    • et al.

    Generation and characterization of ALX-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection.

    Antimicrob. Agents Chemother. 2016; 60: 6-13View in Article
    • Scopus (186)
    • PubMed
    • Crossref
    • Google Scholar
39. • Vollmers H.P.
    • Brändlein S.

    Nature’s best weapons to fight cancer. Revival of human monoclonal IgM antibodies.

    Hum. Antibodies. 2002; 11: 131-142View in Article
    • PubMed
    • Crossref
    • Google Scholar
40. • Willyard C.

    How antiviral pill molnupiravir shot ahead in the COVID drug hunt.

    Nature. 2021;View in Article
    • Crossref
    • Google Scholar
41. • Zhang Y.
    • Huo M.
    • Zhou J.
    • Xie S.

    PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.

    Comput. Methods Programs Biomed. 2010; 99: 306-314View in Article
    • Scopus (1464)
    • PubMed
    • Crossref
    • Google Scholar
42. • Otwinowski Z.
    • Minor W.

    Processing of X-ray diffraction data collected in oscillation mode.

    Methods Enzymol. 1997; 276: 307-326View in Article
    • Scopus (38806)
    • PubMed
    • Crossref
    • Google Scholar
43. • Read R.J.

    Pushing the boundaries of molecular replacement with maximum likelihood.

    Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1373-1382View in Article
    • Scopus (799)
    • PubMed
    • Crossref
    • Google Scholar
44. • Emsley P.
    • Cowtan K.

    Coot: model-building tools for molecular graphics.

    Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132View in Article
    • Scopus (24411)
    • PubMed
    • Crossref
    • Google Scholar
45. • Adams P.D.
    • Afonine P.V.
    • Bunkóczi G.
    • Chen V.B.
    • Davis I.W.
    • Echols N.
    • Headd J.J.
    • Hung L.W.
    • Kapral G.J.
    • Grosse-Kunstleve R.W.
    • et al.

    PHENIX: a comprehensive Python-based system for macromolecular structure solution.

    Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221View in Article
    • Scopus (17554)
    • PubMed
    • Crossref
    • Google Scholar
46. • Williams C.J.
    • Headd J.J.
    • Moriarty N.W.
    • Prisant M.G.
    • Videau L.L.
    • Deis L.N.
    • Verma V.
    • Keedy D.A.
    • Hintze B.J.
    • Chen V.B.
    • et al.

    MolProbity: more and better reference data for improved all-atom structure validation.

    Protein Sci. 2018; 27: 293-315View in Article
    • Scopus (1882)
    • PubMed
    • Crossref
    • Google Scholar
47. • Pardon E.
    • Laeremans T.
    • Triest S.
    • Rasmussen S.G.F.
    • Wohlkönig A.
    • Ruf A.
    • Muyldermans S.
    • Hol W.G.J.
    • Kobilka B.K.
    • Steyaert J.

    A general protocol for the generation of nanobodies for structural biology.

    Nat. Protoc. 2014; 9: 674-693View in Article
    • Scopus (449)
    • PubMed
    • Crossref
    • Google Scholar
48. • Nie J.
    • Li Q.
    • Wu J.
    • Zhao C.
    • Hao H.
    • Liu H.
    • Zhang L.
    • Nie L.
    • Qin H.
    • Wang M.
    • et al.

    Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.

    Nat. Protoc. 2020; 15: 3699-3715View in Article
    • Scopus (209)
    • PubMed
    • Crossref
    • Google Scholar
49. • Sun J.
    • Zhuang Z.
    • Zheng J.
    • Li K.
    • Wong R.L.Y.
    • Liu D.
    • Huang J.
    • He J.
    • Zhu A.
    • Zhao J.
    • et al.

    Generation of a broadly useful model for COVID-19 pathogenesis, vaccination, and treatment.

    Cell. 2020; 182: 734-743.e5View in Article
    • Scopus (296)
    • PubMed
    • Abstract
    • Full Text
    • Full Text PDF
    • Google Scholar

Article info

Figures

• 

  Graphical Abstract

• 

  Figure 1Anti-SARS-CoV-2 activity of R14 and S43 in vitro and in vivo

• 

  Figure 2Aerosolization and the prophylactic and therapeutic efficacy of R14 against SARS-CoV-2

• 

  Figure 3The cross-binding and cross-neutralization of R14 and S43 to SARS-CoV-2 and its variants

• 

  Figure 4Enhanced neutralization by dimer, homo-trimer, and IgM versions of R14 or S43

• 

  Figure 5The prophylactic and therapeutic efficacy of MR14 against Omicron BA.2 in vivo

• 

  Figure 6The complex structure of R14 or S43 bound to the SARS-CoV-2 RBD

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