Tổng 2 số là 7/15

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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives và the global economy1,2. The discovery & development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 & admitted khổng lồ hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 1/2 virus-inhibitory concentrations of 0.7 to lớn 9 ng ml−1. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding tên miền (RBD) & those directed against the N-terminal domain name (NTD), indicating that both of these regions at the đứng đầu of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, ‘all RBD-down’ conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.

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The novel coronavirus SARS-CoV-21,2 has caused more than 14 million confirmed infections globally, & has caused more than 600,000 deaths. This pandemic has also put much of the world on pause, with unprecedented disruption of lives & unparalleled damage khổng lồ the economy. A return to lớn some semblance of normality will depend on the ability of science to deliver an effective solution, và the scientific community has responded admirably. Drug development is well underway, and vaccine candidates have entered clinical trials. Another promising approach is the isolation of SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) that could be used as therapeutic or prophylactic agents. The primary target for such antibodies is the viral spike, a trimeric protein3,4 that is responsible for binding of the virus to lớn the ACE2 receptor on the host cell1,3,5,6. The spike protein is comprised of two subunits. The S1 subunit has two major structural elements, RBD và NTD; the S2 subunit mediates virus–cell membrane fusion after the RBD has engaged ACE2. Reports of the discovery of neutralizing mAbs that target the RBD have been published recently7,8,9,10,11. We now describe our efforts in isolating & characterizing a collection of mAbs that not only target multiple epitopes on the viral spike but also show very high potency in neutralizing SARS-CoV-2.


Forty patients with PCR-confirmed SARS-CoV-2 infection were enrolled in a cohort study on virus-neutralizing antibodies. Plasma samples from all participants were first tested for neutralizing activity against SARS-CoV-2 pseudovirus (Wuhan-Hu-1 spike pseudotyped with vesicular stomatitis virus). Neutralizing titres varied widely, with half-maximal inhibitory concentrations (IC50s) ranging from a reciprocal plasma dilution of less than 100 to lớn roughly 13,000 (Fig. 1a). We selected five patients for isolation of mAbs because their plasma virus-neutralizing titres were among the highest. The clinical characteristics of these five patients are summarized in Extended Data Table 1. All were severely ill with acute respiratory distress syndrome requiring mechanical ventilation.


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a, Plasma neutralization profile of 40 patients against SARS-CoV-2 pseudovirus (highlighted are five top neutralizers chosen for further study). b, All 252 transfection supernatants were screened for binding lớn the S trimer và RBD, as well as for neutralization against SARS-CoV-2 pseudovirus & live virus. For pseudovirus neutralization, the 1/2 inhibitory dilutions (IC50) of each supernatant are plotted. For live virus, semiquantitative representation of the inhibition at a dilution of 1:50, with neutralization levels ranging from (−) for none khổng lồ (+++) for complete neutralization, is plotted. Potent antibodies later identified are marked by vertical lines & labelled at the bottom. The antibodies from each patient are coloured as in a.


Peripheral blood mononuclear cells from each patient were processed as shown in Extended Data Fig. 1a, starting with cell sorting by flow cytometry. The sorting strategy focused on live memory B lymphocytes that were CD3−, CD19+, và CD27+ (Extended Data Fig. 1b). The final step focused on those cells that bound the SARS-CoV-2 spike trimer (S trimer)4. A total of 602, 325, 14, 147, và 145 such B cells from patients 1, 2, 3, 4, and 5, respectively, were labelled with unique hashtags (Extended Data Fig. 1c). The cells were then placed into the 10X Chromium (10X Genomics) for single-cell 5′-mRNA & V(D)J sequencing khổng lồ obtain paired heavy (H) & light (L) chain sequences. A careful bioinformatic analysis was carried out on 1,145 paired sequences to downselect ‘high-confidence’ antigen-specific mAbs. We recovered 331 mAb sequences, representing 252 individual clones. Only six mAbs were from patient 3, whereas 44 to lớn 100 mAbs were identified from each of the other patients (Extended Data Fig. 2a). The VH and VL sequences of 252 antibodies (one per clone) were codon-optimized & synthesized, & each VH & VL gen was then cloned into an expression plasmid with corresponding constant regions of H chain and L chain of human IgG1. Monoclonal antibodies were then expressed by co-transfection of paired full-length H chain và L chain genes into Expi293 cells.


All 252 transfection supernatants were screened for binding khổng lồ the S trimer and RBD by enzyme-linked immunosorbent assays (ELISAs), as well as for their ability lớn neutralize SARS-CoV-2 pseudovirus and live virus (Fig. 1b, Extended Data Fig. 2). A substantial percentage of the mAbs in the supernatants bound S trimer, and a subset of those bound RBD. Specifically, 121 supernatants were scored as positive for S trimer binding, yielding an overall hit rate of 48%. Of these, 38 were positive for RBD binding while the remaining 83 were negative. None of the 13 trimer-specific mAbs from patient 5 recognized RBD. In the pseudovirus neutralization screen, 61 supernatants were scored as positive, indicating that half of the trimer-specific mAbs were virus-neutralizing. In the screen for neutralization against SARS-CoV-2 (strain USA-WA1/2020), 41 supernatants were scored as positive. Overall, this screening strategy was quite effective in identifying neutralizing mAbs (vertical lines và labelled antibodies at the bottom of Fig. 1b) that were later identified as potent.


Of the 121 mAbs that bound the S trimer, 88% were IgG isotype, with IgG1 being predominant (Extended Data Fig. 3a). Comparison to lớn the IgG repertoires of three healthy human donors12 revealed a statistically significant over-representation of IGHV3-30, IGKV3-20, & IGHJ6 genes for this collection of SARS-CoV-2 mAbs (Extended Data Figs. 3b, c). In addition, the average CDRH3 length was also longer (Extended Data Fig. 3d). Notably, the average percentages of somatic hypermutation in VH and VL were 2.1 and 2.5, respectively, which were significantly lower than those found in healthy individuals (Extended Data Fig. 3e) and remarkably close lớn those of germline sequences.


Since the screening for pseudovirus neutralization was performed quantitatively with serial dilutions of the transfection supernatants, we plotted in Extended Data Fig. 2b the best-fit neutralization curves for 130 samples that were positive in at least one of the screens shown in Fig. 1b. Most were non-neutralizing or weakly neutralizing, but 18 showed better potency. One additional supernatant was initially missed in the pseudovirus screen (patient 1 in Extended Data Fig. 2b) but was later found lớn be a potent neutralizing mAb. Together, these 19 mAbs were purified from transfection supernatants & further characterized for their binding & neutralization properties. As shown in Fig. 2a, quantitative ELISA showed that all but one (2-43) of the mAbs bound the S trimer. Nine of the antibodies clearly bound RBD, with little or no binding lớn NTD. Eight antibodies bound NTD to varying degrees, with no binding lớn RBD. Two mAbs bound neither RBD nor NTD, và were therefore categorized as ‘other’.


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a, Binding profiles of 19 purified potent neutralizing mAbs against the S trimer (left), RBD (middle), and NTD (right) of SARS-CoV-2. Chú ý that mAb 2-30 bound multiple proteins at high concentrations. b, Neutralization profiles of the pseudovirus (top) và live vi khuẩn (bottom) for the 19 purified mAbs. Epitope classifications are listed above plots. A single replicate of the binding experiment và triplicates of neutralization are presented as mean ± s.e.m.


The pseudovirus neutralization profiles for these purified 19 mAbs are shown in Fig. 2b (top). The RBD-directed antibodies neutralized the pseudovirus with IC50 values of 0.005 khổng lồ 0.512 μg ml−1; the NTD-directed antibodies were slightly less potent, with IC50 values ranging from 0.013 lớn 0.767 μg ml−1. A common feature of the NTD mAbs was the plateauing of vi khuẩn neutralization at levels short of 100%. Two antibodies, categorized as ‘other’, neutralized with IC50 values of 0.071 và 0.652 μg ml−1. Antibody neutralization of the authentic or live SARS-CoV-2 (strain USA-WA1/2020) was carried out using Vero cells inoculated with a multiplicity of infection of 0.1. As shown in the bottom portion of Fig. 2b, the RBD-directed antibodies again neutralized the virus but with IC50 values of 0.0007 lớn 0.209 μg ml−1; the NTD-directed antibodies showed similar potency, with IC50 values ranging from 0.007 to lớn 0.109 μg ml−1. Here, the plateauing effect seen in the pseudovirus neutralization assay was less apparent. Antibodies 2-43 and 2-51 neutralized the live virut with IC50 values of 0.003 and 0.007 μg ml−1, respectively. Overall, nine mAbs exhibited high potency in neutralizing authentic SARS-CoV-2 in vitro with IC50 values of 0.009 μg ml−1 or less, including four against RBD (2-15, 2-7, 1-57, và 1-20), three against NTD (2-17, 5-24, and 4-8), & two against undetermined regions on the S trimer (2-43 và 2-51). Patient 2 alone contributed five of the đứng đầu nine SARS-CoV-2 neutralizing mAbs. A correlation of the results of the two virus-neutralizing assays is shown in Extended Data Fig. 4.


All 19 potent neutralizing mAbs (Fig. 2) were further studied in antibody competition experiments lớn gain insight into their epitopes. We also chose 12 mAbs that bound the S trimer strongly during the initial supernatant screen, including 5 that bound RBD (1-97, 2-26, 4-13, 4-24, and 4-29) and 7 that did not bind RBD (1-21, 2-29, 4-15, 4-32, 4-33, 4-41, và 5-45). Four of these mAbs were weak in neutralizing SARS-CoV-2 pseudovirus, and the remaining eight were non-neutralizing (Extended Data Fig. 5). We used ELISA to lớn evaluate 16 non-RBD mAbs for competition in binding khổng lồ the S trimer in a ‘checkerboard’ experiment. The extent of the antibody competition is reflected by the intensity of the heatmap in Fig. 3a. There is one large cluster (A) of mAbs that competed with one another, which partially overlaps with a small cluster (B). A third cluster (C) does not overlap at all. Chú ý that all but one of the antibodies in cluster A recognized NTD. Antibody 2-51 is clearly directed against the NTD region even though it could not bind NTD. Moreover, one mAb from each of clusters B and C also recognized NTD, thereby indicating that all three clusters are within or near the NTD. One mAb, 1-21, appears to lớn have a chất lượng non-overlapping epitope (epitope region D).


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a, Competition results of non-RBD binders (left) & RBD binders (right) in blocking binding of ACE2 or biotinylated mAb to lớn the S trimer. In addition, the ability of each mAb lớn bind NTD và RBDmut is shown. The numbers in each box show the area under each competition curve (AUC) as tested by ELISA. Plus và minus signs indicate binding và no binding, respectively, of the mAb lớn the protein. The letters A to lớn H at the bottom denote clusters of antibody epitopes defined by the competition experiments. b, Venn diagram interpretation of results from a and Extended Data Fig. 6b.

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We carried out a similar ‘checkerboard’ competition experiment by ELISA for 14 of our RBD-directed mAbs plus CR302213,14. Here, the heatmap shows four epitope clusters that are serially overlapping (Fig. 3a). There is one large cluster (E) that contains mAbs that can largely block ACE2 binding. Furthermore, four antibodies in this cluster lost the ability khổng lồ bind to lớn a mutant RBD (L455R, A475R, G502R) that could no longer bind ACE2 (unpublished data). Together, these results suggest that most of the mAbs in cluster E are directed against the ACE2-binding interface of RBD. The next cluster (F) connects khổng lồ both cluster E và cluster G, the location of which is defined by its thành viên CR302215. Last, cluster G overlaps another cluster (H), which includes 1-97, which strongly inhibited the binding of 2-30 lớn the S trimer. This finding suggests that cluster H may be proximal to lớn one edge of cluster E.

One potent neutralizing mAb, 2-43, did not bind the S trimer in ELISA (Fig. 2a) và thus could not be tested in the above competition experiments. However, 2-43 did strongly bind the S trimer when expressed on the cell surface, as determined by flow cytometry (Extended Data Fig. 6a), and this binding was competed out by itself but not by RBD, NTD, ACE2, or the soluble S trimer4 (Extended Data Fig. 6b). NTD-directed mAbs had only a modest effect on its binding lớn cell-surface S trimer, but numerous RBD-directed mAbs in cluster E potently blocked the binding of 2-43, demonstrating that this antibody is likely lớn target a quaternary epitope on the đứng đầu of RBD.

These mapping results could be represented by two sets of Venn diagrams shown in Fig. 3b. In the non-RBD region, the potent neutralizing mAbs reside exclusively in cluster A and bind a patch on the NTD. Weaker neutralizing mAbs recognize a region at the interface between clusters A và B. In the RBD region, the most potent neutralizing mAbs also group together within one cluster (E). Given that all block ACE2 binding, it is likely they recognize the đứng đầu of RBD & neutralize the vi khuẩn by competitive inhibition of receptor binding. Cluster G contains CR3022, a mAb known to be directed against an epitope on a cryptic site on the side of RBD when it is in the ‘up’ position15. Cluster F is therefore likely situated between the top & this ‘cryptic’ site. The Venn diagram also suggests that cluster H may occupy a different side surface of RBD, perhaps in the region recognized by S309, a mAb isolated from a patient with SARS-CoV-18.


We produced cryo-electron microscopy (cryo-EM) reconstructions of antigen-binding fragments (Fabs) from three mAbs in complex with the S trimer4. First, single-particle analysis of the complex with the Fab of mAb 2-4 (RBD-directed) yielded maps of high chất lượng (Fig. 4a; Extended Data Table 2; Extended Data Fig. 7a–d), with the most abundant particle class representing a 3-Fab-per-trimer complex, refined khổng lồ an overall resolution of 3.2 Å. While density for the constant portion of the Fabs was visible, it was blurred as a result of molecular motion, & thus only the variable domains were included in the molecular model. Fab 2-4 bound the spike protein near the apex, with all RBDs in the ‘down’ orientation, and the structure of the antibody-bound spike protein was highly similar to lớn previously published unliganded spike structures in the ‘all-down’ conformation3,4. Detailed interactions between mAb 2-4 & RBD are shown in Extended Data Fig. 7e–i. Overall, the structure of the 2-4 Fab–spike complex shows that neutralization of SARS-CoV-2 by this mAb is likely to lớn result from locking the RBD in the down conformation while also occluding access to ACE2.


Fig. 4: Cryo-EM reconstructions of Fab–spike complexes và visualization of neutralizing epitopes on the spike surface.

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a, Cryo-EM reconstruction of 2-4 Fab in complex with the S trimer at 3.2 Å overall resolution. Density is coloured with RBD in green, NTD in orange, and other regions in grey. b, Cryo-EM reconstruction of 4-8 Fab in complex with the S trimer (ribbon diagram, coloured as in a) at 3.9 Å overall resolution, with RBDs in the ‘all-down’ configuration. c, Cryo-EM reconstruction of the 2-43 Fab in complex with the S trimer at 5.8 Å resolution reveals a quaternary epitope involving RBD from one subunit & another RBD from the next. d, Mapping of the Venn diagrams from Fig. 3b onto the surface of the viral spike.


We also produced 3d cryo-EM reconstructions of 4-8 Fab (NTD-directed) in complex with the S trimer (Extended Data Table 2, Extended Data Fig. 8a–f). Two main particle classes were observed—one for a 3-Fab-bound complex with all RBDs ‘down’ at 3.9 Å resolution (Fig. 4b), và another a 3-Fab-bound complex with one RBD ‘up’ at 4.0 Å resolution (Extended Data Fig. 8g). However, molecular motion prevented visualization of the interaction at high resolution. Nevertheless, the mật độ trùng lặp từ khóa in the 4-8 bản đồ reveals the overall positions of the antibody chains that target the NTD. It is unclear how binding to lớn the tip of the NTD results in neutralization of SARS-CoV-2.

Third, a 5.8 Å resolution reconstruction of 2-43 Fab in complex with the S trimer (Extended Data Table 2, Extended Data Fig. 8h–k) revealed three bound Fabs, each targeting a quaternary epitope on the đứng đầu of the spike that included elements of the RBDs from two adjacent S1 protomers (Fig. 4c), consistent with the epitope mapping results (Fig. 3b, Extended Data Fig. 6b), including the lack of binding khổng lồ isolated RBD (Fig. 2a). Given these findings, the inability of 2-43 khổng lồ bind the S trimer in ELISA studies is likely lớn be an artefact of the assay format, as this mAb did bind the spike expressed on the cell surface and in the cryo-EM study.

Armed with these three cryo-EM reconstructions, we used the Venn diagrams from Fig. 3b to bản đồ the epitopes of many of our SARS-CoV-2-neutralizing mAbs onto the surface of the spike (Fig. 4d). This is obviously a rough approximation because antibody footprints are much larger than the area occupied by the mAb number. However, the spatial relationship of the antibody epitopes should be reasonably represented by Fig. 4d.


To assess the in vivo potency of mAb 2-15, we performed a protection experiment in a golden Syrian hamster mã sản phẩm of SARS-CoV-2 infection. The hamsters were first given an intraperitoneal injection of the antibody at a dose of 1.5 mg kg−1 or 0.3 mg kg−1, or PBS alone. Intranasal inoculations of 105 plaque-forming units (PFU) of the HKU-001a strain of SARS-CoV-2 were carried out 24 h later. Four days after vi khuẩn challenge, lung tissues were removed to lớn quantify the viral load. As shown in Fig. 5, both viral RNA copy numbers và infectious virut titres were reduced by 4 logs or more in hamsters given 1.5 mg kg−1 of mAb 2-15. The protection at 0.3 mg kg−1 was borderline, as we had estimated. This pilot animal study demonstrates that the potency of mAb 2-15 in vitro is reflected in vivo, with complete elimination of infectious SARS-CoV-2 at a relatively modest antibody dose.


Fig. 5: Efficacy of mAb 2-15 in protecting against SARS-CoV-2 infection in lung tissues of hamsters.

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One day before intranasal challenge with SARS-CoV-2, each group of hamsters was given a single intraperitoneal dose of 1.5 mg kg−1 of mAb 2-15 (n = 4), 0.3 mg kg−1 of mAb 2-15 (n = 4), or saline as control (n = 4). The viral loads in the lung tissues on day 4 after viral challenge were determined by quantitative PCR with reverse transcription (qRT–PCR; red), as well as by an assay to lớn quantify PFUs of infectious SARS-CoV-2 (blue). All data points are shown, along with the mean ± s.d. The differences between the 1.5 mg kg−1 group và the control group are statistically significant at phường Full size image

Methods


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