CORD-19-Sentences  

CORD-19:f20a4ca033637fcd68195ffdb405bcd7ff5c4ba3 JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-139 Sentence denotes Recurring and Adaptable Binding Motifs in Broadly Neutralizing Antibodies to Influenza Virus Are Encoded on the D3-9 Segment of the Ig Gene
TextSentencer_T2 141-149 Sentence denotes Abstract
TextSentencer_T3 150-697 Sentence denotes Highlights d Structure of bnAb S9-3-37 bound to influenza hemagglutinin stem was determined d D3-9-encoded region of S9-3-37 contributes majority of the interaction surface with HA d D3-9 gene segment of S9-3-37 can engage the HA stem in two different reading frames d D3-9 gene segment represents a recurring mechanism for antibody targeting of HA stem SUMMARY Discovery and characterization of broadly neutralizing antibodies (bnAbs) to the influenza hemagglutinin (HA) stem have provided insights for the development of a universal flu vaccine.
TextSentencer_T4 698-818 Sentence denotes Identification of signature features common to bnAbs from different individuals will be key to guiding immunogen design.
TextSentencer_T5 819-875 Sentence denotes S9-3-37 is a bnAb isolated from a healthy H5N1 vaccinee.
TextSentencer_T6 876-1045 Sentence denotes Here, structural characterization reveals that the D3-9 gene segment of S9-3-37 contributes most of the interaction surface with the highly conserved stem epitope on HA.
TextSentencer_T7 1046-1185 Sentence denotes Comparison with other influenza bnAb crystal structures indicates that the D3-9 segment provides a general mechanism for targeting HA stem.
TextSentencer_T8 1186-1283 Sentence denotes Interestingly, such bnAbs can approach the HA stem with vastly different angles and orientations.
TextSentencer_T9 1284-1405 Sentence denotes Moreover, D3-9 can be translated in different reading frames in different bnAbs yet still target the same HA stem pocket.
TextSentencer_T10 1406-1518 Sentence denotes Thus, the D3-9 gene segment in the human immune repertoire can provide a robust defense against influenza virus.
TextSentencer_T11 1520-1640 Sentence denotes Identifying signature features common to broadly neutralizing antibodies (bnAbs) is key to universal flu vaccine design.
TextSentencer_T12 1641-1864 Sentence denotes Wu et al. report that the D3-9 encoded segment of an influenza hemagglutinin stem-targeting bnAb contributes the majority of the interaction surface and is a recurring motif in antibodies that target the hemagglutinin stem.
TextSentencer_T13 1865-1957 Sentence denotes Development of a universal influenza vaccine has been of longterm interest for global heath.
TextSentencer_T14 1958-2251 Sentence denotes In the past decade, the discovery and characterization of broadly neutralizing antibodies (bnAbs) to the influenza hemagglutinin (HA) stem have provided invaluable insights for development of a universal influenza vaccine (Impagliazzo et al., 2015; Wu and Wilson, 2018; Yassine et al., 2015) .
TextSentencer_T15 2252-2396 Sentence denotes The HA stem domain, unlike the receptor-binding head domain, is highly conserved across influenza strains and subtypes (Nobusawa et al., 1991) .
TextSentencer_T16 2397-2642 Sentence denotes Therefore, antibodies that target the HA stem usually have much greater breadth than those that target the hypervariable head (Wu and Wilson, 2017) , which is the location of the major antigenic sites (Gerhard et al., 1981; Wiley et al., 1981) .
TextSentencer_T17 2643-2785 Sentence denotes Consequently, elicitation of stem-binding bnAbs has become a major goal for universal influenza vaccine development (Erbelding et al., 2018) .
TextSentencer_T18 2786-3007 Sentence denotes Understanding features that are common to bnAbs isolated from different individuals, whether through natural infection or vaccination, is important for immunogen design in the development of a universal influenza vaccine.
TextSentencer_T19 3008-3180 Sentence denotes As more and more influenza bnAbs are characterized, several multidonor classes of bnAbs have begun to emerge (Avnir et al., 2014; Joyce et al., 2016; Wu and Wilson, 2017) .
TextSentencer_T20 3181-3608 Sentence denotes Stem-binding bnAbs that utilize V H 1-69 germline genes are probably the most well-characterized multidonor class (Dreyfus et al., 2012; Ekiert et al., 2009; Kashyap et al., 2008; Lang et al., 2017; Sui et al., 2009; Throsby et al., 2008) , and the alleles used code for a signature motif consisting of Ile53 and Phe54 in complementarity-determining region (CDR) H2 and Tyr98 in CDR H3 (Avnir et al., 2014; Lang et al., 2017) .
TextSentencer_T21 3609-3967 Sentence denotes Recently, Joyce et al. reported three additional multidonor classes of bnAbs (Joyce et al., 2016) : (1) bnAbs that utilize V H 6-1 and D3-3 germline genes with a signature motif consisting of MIFGI in CDR H3; (2) bnAbs that utilize V H 1-18 and D3-9 with a signature motif of RXXILTG in CDR H3; and (3) bnAbs that utilize V H 1-18 and a QXXV motif in CDR H3.
TextSentencer_T22 3968-4045 Sentence denotes It is likely that more multidonor classes of bnAbs have yet to be discovered.
TextSentencer_T23 4046-4173 Sentence denotes We describe here the structure of bnAb S9-3-37, which targets the HA stem mainly using a relatively long CDR H3 of 21 residues.
TextSentencer_T24 4174-4387 Sentence denotes Interestingly, CDR H3 of S9-3-37 has a very similar conformation as that of a pan-influenza A bnAb FI6v3 (Corti et al., 2011) , despite the huge disparity in their overall orientations with respect to the HA stem.
TextSentencer_T25 4388-4554 Sentence denotes Both S9-3-37 and FI6v3 utilize the D3-9 gene segment and encode an LXYFXWL motif in CDR H3 that is critical for targeting the most conserved part of the stem epitope.
TextSentencer_T26 4555-4693 Sentence denotes We further performed a structural comparison between S9-3-37 and 31.b.09 (Joyce et al., 2016) , which also utilizes the D3-9 gene segment.
TextSentencer_T27 4694-4809 Sentence denotes Our analysis reveals that the D3-9 gene segments of S9-3-37 and 31.b.09 are translated in different reading frames.
TextSentencer_T28 4810-4962 Sentence denotes Nonetheless, Phe in the D-3-9-encoded LXYFXWL motif of S9-3-37 and Leu in the D3-9-encoded ILTG motif of 31.b.09 targets the same pocket in the HA stem.
TextSentencer_T29 4963-5193 Sentence denotes Moreover, both reading frames of the D3-9 gene segment are utilized by influenza bnAbs from multiple vaccinated individuals (Joyce et al., 2016) and can be found in the B cell repertoires of healthy donors (DeKosky et al., 2015) .
TextSentencer_T30 5194-5407 Sentence denotes Overall, this study reveals that the D3-9 gene segment offers a recurrent and robust strategy for targeting the HA stem in which at least two different binding poses can be utilized depending on the reading frame.
TextSentencer_T31 5408-5604 Sentence denotes Structural Characterization of S9-3-37 S9-3-37 is a group 1-specific bnAb that was isolated from healthy volunteers who were vaccinated with the H5N1 prepandemic vaccine (Yamayoshi et al., 2018) .
TextSentencer_T32 5605-5808 Sentence denotes S9-3-37 uses the V H 1-18 and V k 2-24 heavy-and light-chain V genes, respectively ( Figure S1A -B), and binds to group 1 HAs with high affinity, but with no detectable binding to group 2 HAs (Table 1) .
TextSentencer_T33 5809-5981 Sentence denotes Here, we determined the crystal structure of S9-3-37 Fab in complex with the HA from an H5N1 strain A/Vietnam/1203/2004 (Viet04) to 2.9 Å resolution (Table S1; Figure S2 ).
TextSentencer_T34 5982-6157 Sentence denotes S9-3-7 binds to the HA stem region ( Figure 1A) , and one structural feature that immediately stands out is the extensive contact of CDR H3 with the stem region ( Figure 1B) .
TextSentencer_T35 6158-6299 Sentence denotes The overall conformation of CDR H3 of S9-3-37 at the HA-Fab interface is remarkably similar to that of CDR H3 of FI6v3 (Corti et al., 2011) .
TextSentencer_T36 6300-6419 Sentence denotes Moreover, the molecular interactions attributable to the D gene segment are almost identical between S9-3-37 and FI6v3.
TextSentencer_T37 6420-6576 Sentence denotes While S9-3-37 and FI6v3 utilize different V H and J gene segments, they both utilize the D3-9 gene segment and have a relatively long CDR H3 ( Figure S1C ).
TextSentencer_T38 6577-6666 Sentence denotes CDR H3 of FI6v3 is 20 residues, whereas that of S9-3-37 is 21 residues (Kabat numbering).
TextSentencer_T39 6667-6815 Sentence denotes Interestingly, despite the similarity in the conformation of CDR H3, the overall orientations of S9-3-37 and FI6v3 are very different ( Figure 1A ).
TextSentencer_T40 6816-7033 Sentence denotes When bound to HA, S9-3-37 adopts an $180 rotation relative to FI6v3, such that the positions of the heavy chain and light chain are swapped compared to FI6v3 with respect to their interaction with the HA stem epitope.
TextSentencer_T41 7034-7206 Sentence denotes To understand the difference in the HA binding orientation of S9-3-37 and FI6v3, structural alignments between their variable domains of heavy chains (V H ) were performed.
TextSentencer_T42 7207-7331 Sentence denotes When their D gene-encoded regions are superimposed, the base of their CDR H3s twist with respect to each other (Figure 1C) .
TextSentencer_T43 7332-7458 Sentence denotes When their V H framework regions are aligned, the CDR H3 loops of S9-3-37 and FI6v3 point in opposite directions (Figure 1D) .
TextSentencer_T44 7459-7782 Sentence denotes Together, our structural analysis reveals that the S9-3-37 and FI6v3 antibodies engage the HA stem surface in very different overall orientations and angles of approach, but nevertheless still have very similar CDR H3 conformations, especially for the residues that are important for engaging the conserved HA stem epitope.
TextSentencer_T45 7783-8039 Sentence denotes Paratope Analysis Reveals the Importance of D3-9 in Binding Next, we compared the paratopes of S9-3-37 and FI6v3 and computed the buried surface areas (BSAs) in the S9-3-37-HA complex and FI6v3-HA complex (PDB 3TZN) (Corti et al., 2011) (see STAR Methods).
TextSentencer_T46 8040-8264 Sentence denotes The paratope of S9-3-37 is dominated by CDR H3, similar to FI6v3 (Figures 2A and 2B ) (Corti et al., 2011) , and only one framework residue interacts with the HA (V L Asp1 hydrogen bonds with HA1 Asn289 glycan; Figure S3A ).
TextSentencer_T47 8265-8408 Sentence denotes The total BSA on S9-3-37 is 910 Å 2 (868 Å 2 from the heavy chain and 42 Å 2 from the light chain), with 83% (756 Å 2 ) attributable to CDR H3.
TextSentencer_T48 8409-8555 Sentence denotes In comparison, the total BSA on FI6v3 is 903 Å 2 (714 Å 2 from the heavy chain and 189 Å 2 from the light chain), with 71% (638 Å 2 ) from CDR H3.
TextSentencer_T49 8556-8668 Sentence denotes Similarly, the BSA on the HA is comparable in the S9-3-37-HA complex (832 Å 2 ) and FI6v3-HA complex (817 Å 2 ).
TextSentencer_T50 8669-8782 Sentence denotes Thus, binding of S9-3-37 to the HA is dominated by the relatively long CDR H3, which is similar to that of FI6v3.
TextSentencer_T51 8783-8886 Sentence denotes In CDR H3 of S9-3-37, the D3-9 gene encodes for seven residues 100b LGYFDWL 100h (Figures 2C and S1C) .
TextSentencer_T52 8887-9046 Sentence denotes With the exception of Gly100c and Asp100f, the other five hydrophobic residues (Leu100b, Tyr100d, Phe100e, Trp100g, and Leu100h) all interact with the HA stem.
TextSentencer_T53 9047-9177 Sentence denotes Of note, all of these five contacting residues are encoded by the germline D3-9, and therefore, none arise from somatic mutations.
TextSentencer_T54 9178-9307 Sentence denotes The buried surface area of this D3-9-encoded region is 495 Å 2 ( Figure 2D ), which accounts for 54% of the total BSA of S9-3-37.
TextSentencer_T55 9308-9410 Sentence denotes This observation further highlights the importance of the D3-9 gene segment in binding to the HA stem.
TextSentencer_T56 9411-9518 Sentence denotes Our structural analysis does, however, reveal a functional role for somatic mutations in CDR H2 of S9-3-37.
TextSentencer_T57 9519-9629 Sentence denotes Among the 17 residues in CDR H2, eight are somatically mutated from the inferred germline gene ( Figure S1A ).
TextSentencer_T58 9630-9749 Sentence denotes N54D and N56K in CDR H2 engage in electrostatic interactions with HA1 Lys32 and HA2 Asp57, respectively ( Figure S3B ).
TextSentencer_T59 9750-9829 Sentence denotes N56K, along with N58K, also hydrogen bonds with Gln99 in CDR H3 ( Figure S3C ).
TextSentencer_T60 9830-9974 Sentence denotes These observations suggest that somatic mutations in CDR H2 not only facilitate interactions with HA but also help stabilize the base of CDR H3.
TextSentencer_T61 9975-10087 Sentence denotes Therefore, somatic mutations in CDR H2 are likely to play a critical role during affinity maturation of S9-3-37.
TextSentencer_T62 10088-10386 Sentence denotes Epitope Analysis Explains the Group 1 Specificity of S9-3-37 Many HA residues are targeted by both S9-3-37 and FI6v3 (Figure 3A) , namely HA1 residue 289, and HA2 residues 18, 19, 20, 21, 38, 41, 42, 45, 46, 48, 49, 52, 53, 56 , and 57 (H3 numbering is used throughout, unless indicated otherwise).
TextSentencer_T63 10387-10593 Sentence denotes In contrast, some HA residues (HA1 18, 38, 39, 40, 291, 292, and 318; and (A) Comparison between structures of S9-3-37 in complex with H5 HA and FI6v3 in complex with H3 HA (PDB 3ZTJ) (Corti et al., 2011) .
TextSentencer_T64 10594-10654 Sentence denotes HA1 is in wheat, HA2 in white, V H in pink, and V L in cyan.
TextSentencer_T65 10655-10771 Sentence denotes For clarity, only one Fab is shown per trimer, and the two HA protomers without their corresponding Fab are in gray.
TextSentencer_T66 10772-10828 Sentence denotes (B) Comparison of the CDR H3 loops of S9-3-37 and FI6v3.
TextSentencer_T67 10829-10881 Sentence denotes The region corresponding to the D gene is in purple.
TextSentencer_T68 10882-11041 Sentence denotes Of note, despite occupying the same space, Leu100b in S9-3-37 is encoded by the D gene, whereas Leu100a in FI6v3 is encoded by N-region addition ( Figure S1 ).
TextSentencer_T69 11042-11155 Sentence denotes Similarly, Glu100i in S9-3-37 is encoded by N-region addition, whereas Ser100h in FI6v3 is encoded by the D gene.
TextSentencer_T70 11156-11279 Sentence denotes (C and D) Structural comparison between S9-3-37 and FI6v3 reveals a twist in the base of CDR H3 with respect to each other.
TextSentencer_T71 11280-11345 Sentence denotes Only heavy-chain variable domains of S9-3-37 and FI6v3 are shown.
TextSentencer_T72 11346-11418 Sentence denotes Heavy-chain constant domains and light chains are not shown for clarity.
TextSentencer_T73 11419-11467 Sentence denotes Regions corresponding to CDR H3 are highlighted.
TextSentencer_T74 11468-11564 Sentence denotes Alignments are based on (C) D gene encoded-region or (D) the entire heavy-chain variable domain.
TextSentencer_T75 11565-11721 Sentence denotes HA2 36, 50, and 54) are only targeted by S9-3-7, but not FI6v3, and vice versa (HA1 residues 8, 28, 29, 30, 287, 290, and 316; and HA2 residues 39 and 43) .
TextSentencer_T76 11722-11909 Sentence denotes Those shared epitope residues, which are targeted by CDR H3 ( Figure 3A ), are more conserved than those epitope residues that are unique to either S9-3-37 or FI6v3 ( Figures 3B and S4 ).
TextSentencer_T77 11910-12005 Sentence denotes In contrast to FI6v3, which bind to both groups 1 and 2 HAs, S9-3-37 only binds to group 1 HAs.
TextSentencer_T78 12006-12479 Sentence denotes Natural amino acid variants in HA1 residue 38 and HA2 residue (B) Sequence conservation of each epitope residue was quantified by sequence entropy, which was calculated based on the alignment of 20 representative strains from different types and subtypes shown in Figure S4 . ''Both,'' residues that are common between the epitopes of FI6v3 and S9-3-37. ''FI6v3,'' residues that are unique to the FI6v3 epitope. ''S9-3-37,'' residues that are unique to the S9-3-37 epitope.
TextSentencer_T79 12480-12603 Sentence denotes 50, both of which are epitope residues unique to S9-3-37, may explain the narrower breadth in S9-3-37 as compared to FI6v3.
TextSentencer_T80 12604-12736 Sentence denotes Most group 1 HAs carry an Asn at HA2 residue 50, whereas HA2 residue 50 is highly conserved as Gly among group 2 HAs ( Figure S3D ).
TextSentencer_T81 12737-12830 Sentence denotes In our crystal structure, HA2 Asn50 forms an H-bond with Arg97 of S9-3-37 V H ( Figure S3E ).
TextSentencer_T82 12831-12984 Sentence denotes When Gly is present at HA2 residue 50, this H-bond interaction would be abolished, which can partially explain lack of binding of S9-3-37 to group 2 HAs.
TextSentencer_T83 12985-13155 Sentence denotes In HA1 residue 38, an N-glycosylation site is present in most group 2 HAs, but not in group 1 HAs, and can provide steric hindrance to the HA stem epitope ( Figure S3F ).
TextSentencer_T84 13156-13318 Sentence denotes CDR L1 of S9-3-37 is spatially proximal to HA1 residue 38 ( Figure S3G ), and a glycan at HA1 residue 38 in group 2 HAs would likely clash with CDR L1 of S9-3-37.
TextSentencer_T85 13319-13498 Sentence denotes In comparison, although CDR H2 of FI6v3 occupies a similar space ( Figure S3G ), it is more distant from HA1 residue 38, which allows it to accommodate a glycan at HA1 residue 38.
TextSentencer_T86 13499-13664 Sentence denotes In fact, the N-glycosylation site at HA1 Asn38 has limited the ability of several stem-binding bnAbs to neutralize group 2 influenza subtypes (Wu and Wilson, 2017) .
TextSentencer_T87 13665-13934 Sentence denotes Subsequently, these analyses show that while both S9-3-37 and FI6v3 utilize a long CDR H3 to target the most conserved part of the epitope, other epitope residues that are unique to each antibody are less conserved and play a critical role in determining their breadth.
TextSentencer_T88 13935-14105 Sentence denotes The different angles of approach may also be critical for the difference in breadth between S9-3-37 and FI6v3, as shown for V H 1-69-encoded bnAbs (Lang et al., 2017) . .
TextSentencer_T89 14106-14265 Sentence denotes Their study discovered a number of bnAbs, including one called 31.b.09, that utilized V H 1-18 and D3-9 (Joyce et al., 2016) , which is the same as in S9-3-37.
TextSentencer_T90 14266-14439 Sentence denotes Nonetheless, when we compared the sequence of S9-3-37 to that of 31.b.09, we noticed that their D3-9 gene segments were translated in different reading frames ( Figure 4A ).
TextSentencer_T91 14440-14616 Sentence denotes In other words, despite the usage of the same D gene segment, the corresponding amino acid sequences in the D gene-encoded regions of S9-3-37 and 31.b.09 are totally different.
TextSentencer_T92 14617-14753 Sentence denotes The D3-9 gene segment in S9-3-37 encodes for amino acids LGYFDWL, whereas the D3-9 gene segment in 31.b.09 encodes for amino acids ILTG.
TextSentencer_T93 14754-15016 Sentence denotes When we compared the structure of S9-3-37-HA complex with that of 31.b.09-HA complex (PDB 5K9O) (Joyce et al., 2016), we found that the Phe within the LGYFDWL motif in S9-3-37 and Leu within the ILTG motif in 31.b.09 occupy the same pocket in the HA stem region.
TextSentencer_T94 15017-15131 Sentence denotes This observation demonstrates that the D3-9 gene segment can engage the HA stem with two different reading frames.
TextSentencer_T95 15132-15344 Sentence denotes Unlike the two reading frames outlined above, the third reading frame of D3-9 gene segment contains a stop codon in the middle of the translated protein sequence, making it unlikely to be utilized ( Figure S5A ).
TextSentencer_T96 15345-15517 Sentence denotes Five of the six subjects studied by Joyce et al. utilized the D3-9 gene segment in their cross-reactive B cells (Joyce et al., 2016) , with varying frequency ( Figure 4B ).
TextSentencer_T97 15518-15699 Sentence denotes Based on a motif searching approach (see STAR Methods), we classified the CDR H3 sequence of each B cell clone that utilized the D3-9 gene segment into S9-3-37-like or 31.b.09-like.
TextSentencer_T98 15700-15845 Sentence denotes Briefly, those D3-9-encoded CDR H3 sequences that contained an LXYFXWL motif, where X represents any amino acid, were classified as S9-3-37-like.
TextSentencer_T99 15846-16058 Sentence denotes Those D3-9-encoded CDR H3 sequences that contained an ILTG motif, with one mismatch allowed at position 1, 3, or 4 (i.e., no variation was allowed for Leu at the second position), were classified as 31.b.09-like.
TextSentencer_T100 16059-16324 Sentence denotes S9-3-37-like CDR H3 could be observed in four heterosubtypic cross-reactive B cells from subjects 31, 54, and 56, whereas 31.b.09-like CDR H3 could be observed in 78 heterosubtypic cross-reactive B cells from subjects 1, 16, and 31 ( Figures 4C and 4D ; Table S2 ).
TextSentencer_T101 16325-16591 Sentence denotes The four S9-3-37-like CDR H3 sequences identified in this analysis are relatively long (19-26 residues, Figure 4D ), as also seen in S9-3-37 (21 amino acid) and FI6v3 (20 amino acid), suggesting that they may bind to HA stem in a similar manner to S9-3-37 and FI6v3.
TextSentencer_T102 16592-16773 Sentence denotes Joyce et al. indeed showed that antibodies from three of those four B cell clones (31.d.01, 54.e.01, and 56.h.01) could neutralize multiple influenza subtypes (Joyce et al., 2016) .
TextSentencer_T103 16774-16905 Sentence denotes Of note, both 31.d.01 and 56.h.01 are group 1-specific bnAbs, whereas 54.e.01 can neutralize viruses from both group 1 and group 2.
TextSentencer_T104 16906-17036 Sentence denotes In addition, we analyzed four additional datasets to examine the frequency of D3-9 usage in heterosubtypic cross-reactive B cells.
TextSentencer_T105 17037-17324 Sentence denotes These include 306 H1/H3/H7 cross-reactive B cells (Andrews et al., 2017) , 55 H1/H3 cross-reactive B cells (McCarthy et al., 2018) , 42 HA stem-specific B cells (Andrews et al., 2015) , and 198 HA stem-specific B cells (Corti et al., 2011; Pappas et al., 2014) and Pappas et al. (2014) .
TextSentencer_T106 17325-17455 Sentence denotes Overall, 16% (198 out of 1,246) heterosubtypic cross-reactive B cells utilized the D3-9 gene segment across all analyzed datasets.
TextSentencer_T107 17456-17575 Sentence denotes These findings demonstrate preferences in the use of the D3-9 gene segment in HA heterosubtypic cross-reactive B cells.
TextSentencer_T108 17576-17761 Sentence denotes We further performed a structural comparison between S9-3-37 and a representative bnAb reported in Andrews et al. (2017) , namely 27-1C08 (V H 1-2+D3-9) that contains the LXYFXWL motif.
TextSentencer_T109 17762-17979 Sentence denotes Despite the vast difference in the overall conformations of the CDR H3 loops in 27-1C08 (PDB 5WCA) (Andrews et al., 2017) and S9-3-37, the conformations of their D3-9-encoded regions are highly similar ( Figure S6A ).
TextSentencer_T110 17980-18162 Sentence denotes While the structure of 27-1C08 in complex with HA is not available, our observations suggest that the D3-9-encoded regions from 27-1C08 would bind to the same stem pocket as S9-3-37.
TextSentencer_T111 18163-18280 Sentence denotes These results also support the notion that the D3-9 gene segment provides a recurring strategy to target the HA stem.
TextSentencer_T112 18281-18378 Sentence denotes We further aimed to analyze the D3-9-encoded CDR H3s in the B cell repertoires of healthy donors.
TextSentencer_T113 18379-18586 Sentence denotes We utilized a dataset from DeKosky et al., which contained a total of 134,345 unique CDR H3 sequences from three healthy donors that were derived from next-generation sequencing data (DeKosky et al., 2015) .
TextSentencer_T114 18587-18800 Sentence denotes Both 31.b.09-like and S9-3-37-like CDR H3 sequences could be found ( Figure 5A ), although the frequency of 31.b.09-like CDR H3 sequences ($1%) was much higher than S9-3-37-like CDR H3 sequences ($0.01% to $0.1%).
TextSentencer_T115 18801-18923 Sentence denotes S9-3-37-like CDR H3s with at least 20 amino acids long could be observed in two of the three healthy donors ( Figure 5B ).
TextSentencer_T116 18924-19101 Sentence denotes This analysis shows that B cells that utilize S9-3-37-like CDR H3, despite being less common than 31.b.09-like CDR H3, are prevalent in the B cell repertoires of healthy donors.
TextSentencer_T117 19102-19271 Sentence denotes To assess the relative prevalence of 31.b.09-like (ILTG motif) and S9-3-37-like (LXYFXWL motif) CDR H3s in targeting non-HA antigens, we analyzed two published datasets.
TextSentencer_T118 19272-19421 Sentence denotes The first dataset describes the memory B cell repertoires from four donors after administration of two meningococcal vaccines (Galson et al., 2015) .
TextSentencer_T119 19422-19577 Sentence denotes The second dataset describes the memory B cell repertoire from three time points that are 1-2 years apart in an HIV-infected patient (Huang et al., 2016) .
TextSentencer_T120 19578-19741 Sentence denotes As compared to the 1,246 HA cross-reactive antibodies that were analyzed above ( Figures 4B, 4C , and S5B-S5E), the S9-3-37-like CDR H3 is rare in the Figure 6 ).
TextSentencer_T121 19742-19913 Sentence denotes Among HA cross-reactive antibodies, the frequency of S9-3-37-like CDR H3 (2.2%, 27 out of 1,246) is $3-fold lower than that of 31.b.09-like CDR H3 (6.7%, 84 out of 1,246).
TextSentencer_T122 19914-20133 Sentence denotes In contrast, in the memory B cell repertoires of meningococcal-vaccinated individuals and the HIV-infected patient, an S9-3-37-like CDR H3 is either undetectable or >100-fold less prevalent than the 31.b.09-like CDR H3.
TextSentencer_T123 20134-20340 Sentence denotes This analysis suggests that, while 31.b.09-like CDR H3 may be involved at very low frequencies in targeting other non-HA antigens, S9-3-37-like CDR H3 is much more specific for targeting the HA stem region.
TextSentencer_T124 20341-20535 Sentence denotes We also analyzed the germline usage of 53 HIV bnAbs from different clonotypes (Eroshkin et al., 2014) , in which two of them (PGT136 and VRC-PG04b) utilized the D3-9 gene segment ( Figure S7A ).
TextSentencer_T125 20536-20660 Sentence denotes Nonetheless, in these two HIV bnAbs, the D3-9 gene segment only encodes for two to three amino acids (Figures S7B and S7C) .
TextSentencer_T126 20661-20768 Sentence denotes Therefore, D3-9 gene segment in these two HIV bnAbs is unlikely to be as critical for engaging the epitope.
TextSentencer_T127 20769-20999 Sentence denotes Consistent with the analysis of memory B cell repertoires (Figure 6 ), the result here implies that the D3-9 gene segment has a specific role in bnAbs that target influenza HA, but not in other microbial antigens analyzed to date.
TextSentencer_T128 21000-21148 Sentence denotes From the X-ray structure of group 1 influenza bnAb S9-3-37, we uncovered an important role of the D3-9 gene segment in targeting the HA stem region.
TextSentencer_T129 21149-21340 Sentence denotes In particular, the binding of the D3-9-encoded CDR H3 region to the HA stem is resilient to different reading (translation) frames and has minimum dependency on the V H and J H gene segments.
TextSentencer_T130 21341-21452 Sentence denotes Importantly, the D3-9 gene drews et al., 2015 Corti et al., 2011; McCarthy et al., 2018; Pappas et al., 2014) .
TextSentencer_T131 21453-21541 Sentence denotes Four subjects were analyzed in the Meningococcal vaccine dataset (Galson et al., 2015) .
TextSentencer_T132 21542-21641 Sentence denotes Three time points that were 1-2 years apart were analyzed in the HIV dataset (Huang et al., 2016) .
TextSentencer_T133 21642-21720 Sentence denotes The number of CDR H3 sequences being analyzed (n) in each sample is indicated.
TextSentencer_T134 21721-21955 Sentence denotes Data points are plotted on the x axis if their occurrence frequency equals zero. segment is utilized by HA stem-binding bnAbs from multiple donors (Andrews et al., 2017; Corti et al., 2011; Joyce et al., 2016; Wyrzucki et al., 2014 ).
TextSentencer_T135 21956-22167 Sentence denotes An ''SOS component'' of the antibody repertoire has been used to describe a germline response that is immediately available upon microbial infection with minimum somatic mutations (Lerner, 2011 (Lerner, , 2016 .
TextSentencer_T136 22168-22407 Sentence denotes V H 1-69 is perhaps the most well-known ''SOS component'' of the antibody repertoire (Dreyfus et al., 2012; Ekiert et al., 2009; Kashyap et al., 2008; Lang et al., 2017; Lerner, 2011 Lerner, , 2016 Sui et al., 2009; Throsby et al., 2008) .
TextSentencer_T137 22408-22511 Sentence denotes Our results suggest that the D3-9 gene segment is another ''SOS component'' of the antibody repertoire.
TextSentencer_T138 22512-22596 Sentence denotes However, it seems the response from D3-9 gene segment is not as general as V H 1-69.
TextSentencer_T139 22597-22863 Sentence denotes V H 1-69 is frequently observed in immune response against pathogens other than influenza virus, such as hepatitis C virus (Marasca et al., 2001) , HIV (Gorny et al., 2012; Luftig et al., 2006) , and middle east respiratory syndrome coronavirus (Ying et al., 2015) .
TextSentencer_T140 22864-23062 Sentence denotes In contrast, our analysis indicates that D3-9 gene segment, especially the reading frame that encodes the motif LXYFXWL (S9-3-37-like CDR H3), is more specific to influenza virus (Figures 6 and S7).
TextSentencer_T141 23063-23150 Sentence denotes To date, most large-scale analyses on germline usage have focused on V H gene segments.
TextSentencer_T142 23151-23306 Sentence denotes Future analysis should investigate whether other D gene segments and even J H gene segments contribute to the ''SOS component'' of the antibody repertoire.
TextSentencer_T143 23307-23474 Sentence denotes Structural characterization of multidonor class bnAbs has been highly valuable for development of the universal influenza vaccine using a reverse-engineering approach.
TextSentencer_T144 23475-23743 Sentence denotes For example, two different headless HA immunogens (Impagliazzo et al., 2015; Yassine et al., 2015) were designed based on the binding modes of CR9114 (Dreyfus et al., 2012) and CR6261 (Ekiert et al., 2009; Throsby et al., 2008) , which are both V H 1-69-encoded bnAbs.
TextSentencer_T145 23744-23859 Sentence denotes Structural characterization of such bnAbs also provides templates for antiviral development (Wu and Wilson, 2018) .
TextSentencer_T146 23860-24077 Sentence denotes These HA stem-binding bnAbs have also inspired the design of small proteins and peptides that display antiviral activities (Chevalier et al., 2017; Fleishman et al., 2011; Kadam et al., 2017; Whitehead et al., 2012) .
TextSentencer_T147 24078-24282 Sentence denotes In fact, a small neutralizing peptide that binds to the HA stem was designed primarily based on CDR H3 of bnAb FI6v3 containing the D3-9-encoded LXYFXWL motif (Kadam et al., 2017) (PDB 5W6T; Figure S6B ).
TextSentencer_T148 24283-24457 Sentence denotes We anticipate that future discovery and characterization of multidonor class bnAbs will continue to provide insight into antiviral and vaccine design against influenza virus.
TextSentencer_T149 24458-24674 Sentence denotes As several HA stem-binding bnAbs are currently being tested in clinical trials for both prophylactic and therapeutic usage (Sparrow et al., 2016) , it is equally important to understand the potential for bnAb escape.
TextSentencer_T150 24675-25160 Sentence denotes Although it is generally more difficult for influenza virus to escape from stem antibodies compared to head antibodies (Anderson et al., 2017; Chai et al., 2016; Doud et al., 2018; Ekiert et al., 2009; Sui et al., 2009; Throsby et al., 2008; Yamayoshi et al., 2018) , strong escape mutations have been identified for several HA stem-binding bnAbs (Anderson et al., 2017; Chai et al., 2016; Ekiert et al., 2011; Friesen et al., 2014; Henry Dunand et al., 2015; Yamayoshi et al., 2018) .
TextSentencer_T151 25161-25242 Sentence denotes Most of the escape mutations dramatically decrease the binding affinity to bnAbs.
TextSentencer_T152 25243-25372 Sentence denotes Nonetheless, increasing the membrane fusion pH of the HA can also contribute to HA stem-binding bnAb escape (Chai et al., 2016) .
TextSentencer_T153 25373-25663 Sentence denotes A previous study by some of the authors here has shown that HA1 N21S/Y (residue 11 in H1 numbering), which eliminates the highly conserved N-glycosylation site at HA1 Asn21, abolished the neutralization activity of several HA stem-binding bnAbs, including S9-3-37 (Yamayoshi et al., 2018) .
TextSentencer_T154 25664-25876 Sentence denotes Interestingly, Asn21 is not part of the S9-3-37 epitope ( Figures S6C and S6D) , suggesting that the escape mechanism of N21S/Y is not due to a loss of a direct interaction that could decrease binding to S9-3-37.
TextSentencer_T155 25877-26147 Sentence denotes Rather, such S9-3-37 escape could be due to an increase in the membrane fusion pH, since removal of the N-glycosylation site at Asn21 has been suggested to decrease the pH stability of the virus and, hence, make it more fusion sensitive at higher pH (Yin et al., 2017) .
TextSentencer_T156 26148-26341 Sentence denotes While the HA stem is a promising target for antiviral and vaccine design, future studies of potential escape mechanisms will be critical for the development of a robust anti-influenza strategy.
TextSentencer_T157 26342-26720 Sentence denotes Detailed methods are provided in the online version of this paper and include the following: and R.U. discovered and prepared the S9-3-37 antibody; N.C.W. performed the binding kinetic assays, X-ray data collection, structure determination, refinement, structural analysis, and sequence analysis; N.C.W. and I.A.W. wrote the paper, and all authors reviewed and edited the paper.
TextSentencer_T158 26721-26943 Sentence denotes Y.K. has received speaker's honoraria from Toyama Chemical and grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Tsumura and Co, and Denka Seiken Co., Ltd.
TextSentencer_T159 26944-26975 Sentence denotes Y.K. is a co-founder of FluGen.
TextSentencer_T160 26976-27120 Sentence denotes Corti, D., Voss, J., Gamblin, S.J., Codoni, G., Macagno, A., Jarrossay, D., Vachieri, S.G., Pinna, D., Minola, A., Vanzetta, F., et al. (2011) .
TextSentencer_T161 27121-27233 Sentence denotes A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins.
TextSentencer_T162 27234-27255 Sentence denotes Science 333, 850-856.
TextSentencer_T163 27257-27388 Sentence denotes Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ian A.
TextSentencer_T164 27389-27417 Sentence denotes Wilson (wilson@scripps.edu).
TextSentencer_T165 27418-27520 Sentence denotes ExpiCHO cells were maintained according to the manufacturer's instructions (Thermo Fisher Scientific).
TextSentencer_T166 27521-27621 Sentence denotes Sf9 cells and High Five cells were maintained in HyClone insect cell culture medium (GE Healthcare).
TextSentencer_T167 27622-27752 Sentence denotes The heavy and light chains of S9-3-37 Fab were cloned into phCMV3 vector with an N-terminal secretion signal METDTLLLWVLLLWVPGSTG.
TextSentencer_T168 27753-27973 Sentence denotes The phCMV3-S9-3-37 heavy chain and phCMV3-S9-3-37 light chain were produced in the ExpiCHO expression system (Thermo Fisher Scientific) according to the Max Titer Protocol as described in the manufacturer's instructions.
TextSentencer_T169 27974-28094 Sentence denotes S9-3-37 Fab was purified by the 5 mL HiTrap Protein G HP antibody purification columns and buffer exchanged into 1x PBS.
TextSentencer_T170 28095-28204 Sentence denotes Influenza hemagglutinin (HA) was prepared for binding studies as previously described (Ekiert et al., 2011) .
TextSentencer_T171 28205-28545 Sentence denotes Briefly, the HA ectodomain, which corresponds to 11-329 (HA1) and 1-176 (HA2) based on H3 numbering, was fused with an N-terminal gp67 signal peptide and a C-terminal BirA biotinylation site, thrombin cleavage site, trimerization domain, and a His 6 tag, and then cloned into a customized baculovirus transfer vector (Ekiert et al., 2011) .
TextSentencer_T172 28546-28631 Sentence denotes Recombinant bacmid DNA was generated using the Bac-to-Bac system (Life Technologies).
TextSentencer_T173 28632-28891 Sentence denotes Baculovirus was generated by transfecting purified bacmid DNA into Sf9 cells using FuGene HD (Promega the C-terminal tag (BirA biotinylation site, thrombin cleavage site, trimerization domain, and the His 6 tag) and to produce the cleaved mature HA (HA1/HA2).
TextSentencer_T174 28892-29080 Sentence denotes The trypsin-digested Viet04 HA was then purified by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN 3 .
TextSentencer_T175 29081-29203 Sentence denotes The binding assay was performed by biolayer interferometry (BLI) using an Octet Red instrument (ForteBio, Menlo Park, CA).
TextSentencer_T176 29204-29443 Sentence denotes Biotinylated HA0 at $10-50 mg mL -1 in 1x kinetics buffer (1x PBS with 0.01% BSA and 0.002% Tween 20) was loaded onto streptavidin biosensors and incubated with supernatant from transfected cells or with the indicated concentration of Fab.
TextSentencer_T177 29444-29562 Sentence denotes Streptavidin biosensors that were not loaded were used as a reference for subtracting background binding from signals.
TextSentencer_T178 29563-29606 Sentence denotes Briefly, the assay consisted of five steps:
TextSentencer_T179 29607-29619 Sentence denotes 1) baseline:
TextSentencer_T180 29620-29661 Sentence denotes 60 s with 1x kinetics buffer; 2) loading:
TextSentencer_T181 29662-29703 Sentence denotes 120 s with biotinylated HA0; 3) baseline:
TextSentencer_T182 29704-29749 Sentence denotes 60 s with 1x kinetics buffer; 4) association:
TextSentencer_T183 29750-29843 Sentence denotes 120 s with samples (supernatant from transfected cells or purified Fab); and 5) dissociation:
TextSentencer_T184 29844-29874 Sentence denotes 120 s with 1x kinetics buffer.
TextSentencer_T185 29875-29929 Sentence denotes For estimating the K d , a 1:1 binding model was used.
TextSentencer_T186 29930-30079 Sentence denotes Crystallization and structural determination S9-3-37 Fab was incubated with the purified Viet04 HA trimer in a molar ratio of 4.5:1 overnight at 4 C.
TextSentencer_T187 30080-30347 Sentence denotes The S9-3-37 Fab-Viet04 HA complex was purified by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN 3 and concentrated to 9 mg mL -1 in 10 mM Tris pH 8.0, 50 mM NaCl, and 0.02% NaN 3 .
TextSentencer_T188 30348-30455 Sentence denotes Crystal screening was carried out using our high-throughput, robotic CrystalMation system (Rigaku) at TSRI.
TextSentencer_T189 30456-30626 Sentence denotes The initial crystal screening was based on sitting drop vapor diffusion method with 35 mL reservoir solution and each drop consisting 0.1 mL protein + 0.1 mL precipitant.
TextSentencer_T190 30627-30748 Sentence denotes Diffraction-quality crystals were obtained with a reservoir solution containing 20% PEG 6000, 0.1 M HEPES pH 7.0 at 20 C.
TextSentencer_T191 30749-30841 Sentence denotes Diffraction data were collected at Stanford Synchrotron Radiation Lightsource beamline 12-2.
TextSentencer_T192 30842-30946 Sentence denotes The data were indexed, integrated and scaled using HKL2000 (HKL Research) (Otwinowski and Minor, 1997) .
TextSentencer_T193 30947-31128 Sentence denotes The structure was solved by molecular replacement using Phaser (McCoy et al., 2007) , modeled using Coot (Emsley et al., 2010) , and refined using Refmac5 (Murshudov et al., 2011) .
TextSentencer_T194 31129-31310 Sentence denotes For molecular replacement, PDB 2FK0 (Stevens et al., 2006) was used as the model for Viet04 HA, and a homology model generated by PIGSPro (Lepore et al., 2017) was used for S9-3-37.
TextSentencer_T195 31311-31389 Sentence denotes Ramachandran statistics were calculated using MolProbity (Chen et al., 2010) .
TextSentencer_T196 31390-31465 Sentence denotes IgBLAST (Ye et al., 2013) was employed to identify the CDRs on S9-3-37 Fab.
TextSentencer_T197 31466-31502 Sentence denotes The Kabat numbering scheme was used.
TextSentencer_T198 31503-31605 Sentence denotes Buried surface area calculation Solvent accessibility was computed by DSSP (Kabsch and Sander, 1983) .
TextSentencer_T199 31606-31732 Sentence denotes Buried surface area (BSA) was calculated by subtracting the solvent accessibility of the bound form from that of the apo form.
TextSentencer_T200 31733-31805 Sentence denotes HA residues that had a non-zero BSA were identified as epitope residues.
TextSentencer_T201 31806-32019 Sentence denotes Analysis of natural HA variants A total of 103,301 full-length HA protein sequences from different subtypes were downloaded from the Global Initiative for Sharing Avian Influenza Data (GISAID; https://gisaid.org).
TextSentencer_T202 32020-32155 Sentence denotes To avoid temporal sampling bias, we sampled at most 20 sequences per year per subtype, which resulted in a total of 6,984 HA sequences.
TextSentencer_T203 32156-32278 Sentence denotes Multiple sequence alignment of those 6,984 HA sequences was performed by MAFFT version 7.157b (Katoh and Standley, 2013) .
TextSentencer_T204 32279-32343 Sentence denotes Sequence logos were generated by WebLogo (Crooks et al., 2004) .
TextSentencer_T205 32344-32379 Sentence denotes Sequence entropy was calculated by:
TextSentencer_T206 32380-32491 Sentence denotes where P i is the fraction of residues of amino acid type i, and M is the number of amino acid types (i.e., 20).
TextSentencer_T207 32492-32645 Sentence denotes In Figure 2B , sequence entropy was calculated based on the alignment of 20 representative strains from different types and subtypes shown in Figure S4 .
TextSentencer_T208 32646-32888 Sentence denotes Analysis of published antibody sequences CDR H3 sequences recovered from H3 and H5 cross-reactive memory B cells of six H5N1 DNA/MIV-prime-boost influenza vaccinated subjects were retrieved from Table S3 in Joyce et al. (Joyce et al., 2016) .
TextSentencer_T209 32889-33070 Sentence denotes Germline usage of H1/H3/H7 cross-reactive B cells from six H7N9 DNA/MIV-prime-boost influenza vaccine subjects was retrieved from Table S7 in Andrews et al. (Andrews et al., 2017) .
TextSentencer_T210 33071-33263 Sentence denotes Germline usage of H1 and H3 cross-reactive memory B cells from six subjects immunized with trivalent influenza vaccine was retrieved from Figure S4 in McCarthy et al. (McCarthy et al., 2018) .
TextSentencer_T211 33264-33449 Sentence denotes Germline usage of HA stem-specific memory B cells from subjects immunized with trivalent influenza vaccine was retrieved from Tables S3 and S4 in Andrews et al. (Andrews et al., 2015) .
TextSentencer_T212 33450-33633 Sentence denotes Germline usage of HA stem-specific memory B cells from a single subject immunized with seasonal influenza vaccine was retrieved from Figure S1 in Pappas et al. (Pappas et al., 2014) .
TextSentencer_T213 33634-33823 Sentence denotes CDR H3 sequences from memory B cells of three healthy donors and their occurrence frequency information were retrieved from Supplemental Dataset 1 in DeKosky et al. (DeKosky et al., 2015) .
TextSentencer_T214 33824-34203 Sentence denotes CDR H3 sequences from the IgG + memory B cell repertoires from four donors after administration of two Meningococcal vaccines (Galson et al., 2015) , and the IgG + memory B cell repertoire from three time points that were one to two years apart in an HIV-infected patient (Huang et al., 2016) , were downloaded from the Observed Antibody Space database (Kovaltsuk et al., 2018) .
TextSentencer_T215 34204-34298 Sentence denotes Nucleotide sequences of HIV bnAbs were retrieved from bNAber database (Eroshkin et al., 2014).
TextSentencer_T216 34299-34364 Sentence denotes Germline usages were identified using IgBlast (Ye et al., 2013) .
TextSentencer_T217 34365-34515 Sentence denotes S9-3-37-like CDR H3s were defined as those CDR H3s that utilized the D3-9 gene segment and encoded a LXYFXWL motif, where X represents any amino acid.
TextSentencer_T218 34516-34759 Sentence denotes 31.b.09-like CDR H3s were defined as those CDR H3s that utilized the D3-9 gene segment and encoded an ILTG motif with one mismatch in the non-underlined residue (i.e., no variation was allowed for the Leu at second position of the ILTG motif).
TextSentencer_T219 34760-34813 Sentence denotes Statistical analysis was not performed in this study.
TextSentencer_T220 34814-34967 Sentence denotes The X-ray coordinates and structure factors of S9-3-37 in complex with H5 HA have been deposited in the RCSB Protein Data Bank under accession code 6E3H.
TextSentencer_T221 34968-35154 Sentence denotes Custom python scripts for sequence analyses have been deposited to https://github.com/wchnicholas/ S9-3-37. (Andrews et al., 2017) , and potentially binds to the same epitope as S9-3-37.
TextSentencer_T222 35155-35215 Sentence denotes The D3-9-encoded regions of S9-3-37 and 27-1C08 are aligned.
TextSentencer_T223 35216-35335 Sentence denotes Despite the difference in CDR H3 conformation, the D3-9-encoded region in 27-1C08 is highly similar to that in S9-3-37.
TextSentencer_T224 35336-35384 Sentence denotes Regions corresponding to CDR H3 are highlighted.
TextSentencer_T225 35385-35493 Sentence denotes A zoom-in view displays the tip of the CDR H3, with side chains of interest shown in sticks representations.
TextSentencer_T226 35494-35641 Sentence denotes The apo structure of 27-1C08 is used here (PDB 5WCA) (Andrews et al., 2017) , since the structure of 27-1C08 in complex with HA was not determined.
TextSentencer_T227 35642-35754 Sentence denotes On the top, the germline line sequence of D3-9 is aligned with the CDR H3 D gene segment of S9-3-37 and 27-1C08.
TextSentencer_T228 35755-35788 Sentence denotes Somatic mutations are underlined.
TextSentencer_T229 35789-35887 Sentence denotes Nucleotides from N-regions are shown in lower case. (B) D3-9-encoded region in S9-3-37 is in blue.
TextSentencer_T230 35888-35963 Sentence denotes The designed small peptide P7 (PDB 5W6T) (Kadam et al., 2017) is in orange.
TextSentencer_T231 35964-36020 Sentence denotes All side chains in P7 are shown in stick representation.
TextSentencer_T232 36021-36206 Sentence denotes Side chains of the contacting residues in the D3-9-encoded region of S9-3-37 (LGYFDWL) are also shown in stick representation and are labelled. (C) HA1 Asn21 is an N-glycosylation site.
TextSentencer_T233 36207-36281 Sentence denotes HA1 Asn21 is colored in orange on structure of HA in complex with S9-3-37.
TextSentencer_T234 36282-36420 Sentence denotes HA1 is in dark gray, HA2 is in light gray, and S9-3-37 is in blue. (D) The location of HA1 Asn21 relative to the S9-3-37 epitope is shown.
TextSentencer_T235 36421-36454 Sentence denotes The S9-3-37 epitope is in yellow.
TextSentencer_T236 36456-36477 Sentence denotes N-region 1 IGHD3-9*01