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Published in final edited form as: Science. 2020 Jun 19;368(6497):1335–1340. doi: 10.1126/science.aaz5143

Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses

Nicholas C Wu 1,*, Andrew J Thompson 2,*, Juhye M Lee 3,4,5, Wen Su 6, Britni M Arlian 2, Jia Xie 7, Richard A Lerner 7,8, Hui-Ling Yen 6, Jesse D Bloom 3,4,9, Ian A Wilson 1,8,§
PMCID: PMC7412937  NIHMSID: NIHMS1613127  PMID: 32554590

Abstract

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype due to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains, and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.

ONE SENTENCE SUMMARY

Resistance to broadly neutralizing hemagglutinin stem antibodies varies substantially between influenza subtypes.

Introduction

The major surface antigen of influenza virus, the hemagglutinin (HA), is composed of a highly variable globular head domain that contains the receptor binding site and a highly conserved stem domain that contains the membrane fusion machinery (1). The major antigenic sites on HA are located on the globular head (2, 3), which is immunodominant over the stem (4). However, most antibodies to the head domain are strain-specific, whereas antibodies to the stem are harder to elicit but have considerably more breadth [reviewed in (5)]. The isolation, characterization and structure determination of broadly neutralizing human antibodies (bnAbs) to the HA stem since 2008 (6–9) have provided insights into immunogen design towards a universal influenza vaccine (8, 10–13), and have offered templates for design of small proteins, peptides and small molecules against influenza virus (14–18). Several stem bnAbs are also currently in clinical trials (19). Therefore, stem bnAbs open up multiple promising avenues to tackle this challenging global health problem.

Resistance mutations can be a major obstacle for antiviral and vaccine development, but it is unclear the extent to which this is a problem for stem bnAbs. Several studies have reported difficulty in selecting strong resistance mutations to stem bnAbs even after extensive virus passaging (20–22), or through deep mutational scanning (23), which is a comprehensive and unbiased approach (24). Nonetheless, other studies have obtained strong resistance mutations through virus passaging (6, 21, 25). Here, we systematically compare the extent to which resistance can emerge to stem bnAbs in HAs from the H3 and H1 subypes that represent the circulating influenza A strains.

Deep mutational scanning of the major HA stem epitope in H3/HK68 HA

CR9114 (26) and FI6v3 (27) are two prototypic bnAbs that bind the HA stem and represent those with the greatest breadth. Both FI6v3 and CR9114 neutralize group 1 and 2 influenza A viruses (26, 27), and CR9114 further cross-reacts with influenza B HA (26). Deep mutational scanning (24), which combines saturation mutagenesis and next-generation sequencing, has been applied to study how HA mutations affect influenza viral fitness (28–30), and to identify viral mutants that are resistant to anti-HA antibodies (31). Here, we employed deep mutational scanning of the HA stem of H3N2 A/Hong Kong/1/1968 (H3/HK68) to search for virus resistance mutations to CR9114 and FI6v3. Unlike previous studies that examined the entire HA (28–31), we focused on eight residues in HA2 (HA0 is cleaved during maturation into HA1 and HA2) of H3/HK68 HA in and around the main stem epitope (region recognized by antibody): namely Q42, I45, D46, Q47, I48, N49, L52, and T111 (Fig. 1, A to C). All except T111 are located on HA2 helix A, which is a common target for stem bnAbs. Residues 42, 45, 46, 48, 49, and 52 were chosen because they interact with CR9114 and FI6v3, and 47 because it interacts with FI6v3 (Fig. 1, D and E). Completely buried T111 was selected because its mutation in H5 HA enabled escape from CR6261 (6), which binds a similar epitope to CR9114 (9, 26).

Fig. 1. Epitopes of broadly neutralizing antibodies to the HA stem.

Fig. 1.

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(A) The location of residues of interest in this study on the HA structures. All residues of interest are on HA2. One protomer of the trimer is shown in light gray and the other two protomers in dark gray and a detailed view of the location of the residues of interest is shown in the inset. (B-C) Epitopes of (B) CR9114 Fab in complex with H1 HA (PDB 4FQI) (26) and (C) FI6v3 in complex with H3 HA (PDB 3ZTJ) (27) are colored in yellow and green, and residues of interest colored in green. The arrows indicate the positions of HA2 residues 42 and 45, which are in the center of the bnAb epitopes. Antibody paratopes (CDRs and FR regions) are shown in tube representation and labeled accordingly. Blue: heavy chain. Pink: light chain. (D-E) The relative solvent accessibility (RSA) of each residue of interest is shown. Black bar: apo form. Gray bar: Fab-bound form.

By considering mutations to all amino acids at each position, we quantified the in vitro viral fitness of 147 of 152 possible single mutants and 6,234 of 10,108 possible double mutants across the eight residues of interest in H3/HK68 under five conditions: no antibody, 2 μg/mL CR9114 IgG, 10 μg/mL CR9114 IgG, 0.3 μg/mL FI6v3 IgG, and 2.5 μg/mL FI6v3 IgG (fig. S1). Many mutants have a relative fitness [proxy for replication fitness (30)] similar to wild type (WT), which was set as 1 (Fig. 2, A and B), indicating that the HA stem region can tolerate many mutations.

Fig. 2. Fitness and resistance profile of H3/HK68 HA2 single and double viral mutants.

Fig. 2.

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(A-B) Based on the deep mutational scanning experiment, the relative fitness of (A) single and (B) double mutants are shown with wild type (WT) set as 1. In (A) and (B), mutants with a next-generation sequencing read count of less than 20 from the plasmid mutant library are excluded and shown as grey. (C-D) Relative resistance for (C) each single and (D) each double mutant against 10 μg/mL CR9114 antibody or 2.5 μg/mL FI6v3 antibody is shown. Relative resistance for WT is set as 1. In (C) and (D), mutants with a relative fitness of less than 0.5 are shown as grey. Residues correspond to WT sequence are boxed. In (B) and (D), each color point in the heatmap represents a double mutant. Of note, some mutants with an increased sensitivity to antibody are shown in green on the relative resistance color scale.

We further quantified the relative resistance (normalized to WT) of each viral mutant by measuring relative fitness (normalized to WT) with or without antibody (fig. S2). Many resistance mutations to CR9114 and FI6v3 were observed (Fig. 2, C and D) and most were located at HA2 residues 42 and 45, which are important in the binding interface with CR9114 and FI6v3 (Fig. 1, D and E). The double mutants also showed high relative resistance if one mutation exhibited high relative resistance even if the other did not (fig. S3A). Consistently, the relative resistance of double mutants can generally be explained by additivity of their corresponding single mutants (fig. S3B). These results demonstrate the prevalence of H3/HK68 resistance mutations to stem bnAbs and identify cross-resistance mutations to both CR9114 and FI6v3, which are encoded by different germline genes and have very different angles of approach to the HA (5).

Validation of resistance mutations

We then individually constructed and tested 18 single and 6 double HA mutants of the H3/HK68 virus spanning a range of relative resistance against CR9114 and FI6v3 IgGs (Fig. 3A). The minimum inhibitory concentration (MIC) in a microneutralization assay strongly correlated with the relative resistance from deep mutational scanning (Spearman’s rank correlation > 0.8, Fig. 3B) with several mutants showing strong cross-resistance to both CR9114 and FI6v3. For example, the MICs of CR9114 and FI6v3 against mutants I45Y/S/N/F/W were all >100 and ≥20 μg mL−1, respectively, compared to 3.1 and 0.2 μg mL−1 for WT. This validation experiment substantiates our finding that strong resistance mutations are prevalent in H3/HK68.

Fig. 3. Characterization of antibody-resistant mutants.

Fig. 3.

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(A) The minimum inhibitory concentration (MIC) of CR9114 and FI6v3 to individual viral mutants are shown. The MIC of CR9114 is in red and represented by the y-axis on the left. The MIC of FI6v3 is in blue and represented by the y-axis on the right. (B) The Spearman’s rank correlations (ρ) between the MIC measured from individual mutants and the relative resistance (against 10 μg/mL CR9114 antibody or 2.5 μg/mL FI6v3 antibody) computed from the profiling experiment (screening) are shown. The green dashed line represents the upper detection limit in A and B. Wild type is represented by the red “X”.

Natural occurrence of resistance mutations

Next, we explored whether these resistance mutations can be found in naturally circulating strains and identified a few of these strong resistance mutations at low frequency in human H3N2 isolates (32), including I45T, I45M and N49D (Fig. 4A), although most have not been observed yet in naturally circulating strains. I45T is also observed in human H3N2 isolates sequenced without any passaging (fig. S4A), implying that its presence is not due to a passaging artifact (33). Moreover, strong cross-resistance mutation I45F was found in all human H2N2 viruses that circulated from 1957 to 1968 (Fig. 4B, fig. S4B), while almost all avian H2N2 viruses have Ile45 (Fig. 4C). This particular mutation in human H2N2 viruses explains why it is more difficult for some stem bnAbs to bind or neutralize this subtype compared to other subtypes as found in previous studies (6, 26, 34). Thus, resistance mutations to stem bnAbs already do indeed occur in circulating strains.

Fig. 4. In vivo characterization and natural occurrence of antibody-resistant mutants.

Fig. 4.

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(A) The natural occurrence frequencies of different amino-acid variants at the residues of interest in human H3 HAs are shown as a heatmap. (B-C) The natural occurrence frequencies of different amino-acid variants at the residues of interest in (B) human H2N2 HA or (C) avian H2N2 HA are shown as sequence logos. (D-G) Prophylactic protection experiments were performed with different doses of CR9114 against (D) WT, (E) HA2 I45T mutant, (F) HA2 I45M mutant, and (G) HA2 I45F mutant. Recombinant H3/HK68 (7:1 on H1/PR8 backbone) viruses were used. Lethal doses (25 mLD50) of WT or mutant viruses were used. Kaplan-Meier survival curves are shown. Paired analysis of each treatment group, relative to control, was conducted using Log-rank (Mantel-Cox) tests. *** indicates p-value ≤ 0.001; ** indicates p-value ≤ 0.01; * indicates p-value ≤ 0.05; ns (not significant) indicates p-value > 0.05.

In vivo pathogenicity and escape of resistance mutations

Given their relevance to circulating strains, we further tested the in vivo viral pathogenicity of HA2 mutants I45T, I45M, and I45F. The weight loss profiles in mice after infection by HA2 mutant and WT viruses were comparable (fig. S5), indicating that these resistance mutations do not reduce in vivo pathogenicity. We further demonstrated that I45T, I45M, and I45F were able to escape in vivo prophylactic protection. While mice infected with WT were completely protected by CR9114 IgG at all tested doses (1, 4, and 10 mg kg−1), mutants I45T, I45M, and I45F were lethal even at the highest dose of CR9114 IgG (Fig. 4, D to G).

Resistance mutations decrease affinity to bnAbs

To dissect the resistance mechanism, we tested binding of H3/HK68 I45T, I45M, and I45F recombinant HAs to CR9114 and FI6v3, and to another stem bnAb 27F3 (34), which utilizes the same VH1–69 germline as CR9114 and similarly neutralizes group 1 and 2 influenza A viruses. Binding (Kd) of CR9114 Fab, CR9114 IgG, 27F3 Fab, 27F3 IgG, and FI6v3 IgG was diminished against the HA mutants (Table 1 and fig. S6), particularly with I45F, where binding was undetectable to CR9114 and 27F3 Fab and IgG. In contrast, binding of these stem Fabs and IgGs to the HA mutant N49T, which did not exhibit resistance against CR9114 and FI6v3 (Fig. 2C, Fig. 3A), was comparable to the WT (Table 1). As a control, we also tested binding of bnAb S139/1 that targets the receptor-binding site far from the stem epitope (35, 36). S139/1 IgG affinities against those HA mutants (Kd = 1.8 nM to 3.1 nM) were similar to WT (Kd = 2.1 nM). Thus, virus resistance to stem bnAbs correlated with a decrease in binding affinity to the mutant HAs.

Table 1.

Binding affinity of IgGs and Fabs against HAs of WT and different H3/HK68 HA2 mutants.

Kd in nM WT HA2-I45T HA2-I45M HA2-I45F HA2-N49T
CR9114 Fab 43.2 ± 0.4 779.6 ± 50.0 319.1 ± 12.1 n.b. 11.3 ± 0.4
27F3 Fab 122.6 ± 3.0 n.b. n.b. n.b. 105.0 ± 1.0
CR9114 IgG < 0.1 164.8 ± 2.3 75.0 ± 1.0 n.b. < 0.1
27F3 IgG 1.7 ± 0.1 n.b. n.b. n.b. 1.9 ± 0.9
FI6v3 IgG < 0.1 7.8 ± 0.2 6.7 ± 0.1 62.0 ± 0.2 < 0.1
S139/1 IgG 2.1 ± 0.1 3.1 ± 0.3 1.9 ± 0.1 1.8 ± 0.1 2.8 ± 0.1
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n.b.: no binding

To evaluate the structural basis of the resistance, we determined crystal structures of HAs with HA2 mutations I45T, I45M, and I45F to 2.1 to 2.5 Å resolutions (table S1 and fig. S7A). Compared to WT (Ile45), the shorter side chain of I45T would create a void when CR9114 is bound (fig. S7B) that would be energetically unfavorable. In contrast, the longer flexible side chain of I45M would likely clash with CR9114 (fig. S7B), but CR9114 is still able to bind the I45M mutant, albeit with much lower affinity than WT (Table 1). The I45F mutant, however, would clash more severely with CR9114 and no binding was detected (Table 1, fig. S7B). Similar observations for FI6v3 (fig. S7C) explain the sensitivity of CR9114 and FI6v3 to mutations at HA2 residue 45.

Resistance to HA stem bnAbs is subtype specific

We further examined whether mutations that conferred strong resistance in H3/HK68 would have the same phenotypes in other H3 strains. Consequently, we examined I45T, I45M, and I45F mutations on H3N2 A/Wuhan/359/95 (H3/Wuhan95) virus. These mutants had WT-like titers after viral rescue and passaging (fig. S8A), showed similar plaque size as WT (fig. S8B), conferred strong resistance to FI6v3 (fig. S8, C and D), and, therefore, have similar phenotypes in H3/HK68 and H3/Wuhan95 viruses. This result led us to hypothesize that strong resistance mutants to stem bnAbs are readily attainable in human H3 strains that span a wide range of years, as well as explore whether the same phenomenon can be observed in the H1 subtype, which is the other currently circulating influenza A subtype in the human population.

In contrast to our findings for H3/HK68, deep mutational scanning found that resistance mutants of H1N1 A/WSN/33 (H1/WSN) virus to FI6v3 were rare and had only very small effects (23). To further investigate the difference between H3 and H1 strains, we performed four additional deep mutational scanning experiments – three with H1N1 strains, namely A/Solomon Islands/3/2006 (H1/SI06) against FI6v3, A/Michigan/45/2015 (H1/Mich15) against FI6v3, H1/WSN against CR9114, and one with H3N2 strain A/Perth/16/2009 (H3/Perth09) against FI6v3. The H1/SI06 and H1/Mich15 HA mutant virus libraries contain all possible single substitutions at HA2 residues 42, 45, 46, 47, 48, 49, 52, and 111, whereas H1/WSN and H3/Perth09 HA mutant virus libraries both contain all possible single substitutions across the entire HA and were constructed previously (37, 38). We also analyzed the previously published dataset on H1/WSN against FI6v3 (23).

To compare H1/SI06, H1/Mich15, H1/WSN and H3/Perth09 to H3/HK68, we computed the relative resistance of mutations at HA2 residues 42, 45, 46, 47, 48, 49, 52, and 111 (Fig. 5, A to E). Similar to H3/HK68, resistance mutations are highly prevalent in H3/Perth09 (Fig. 5C). Conversely, resistance mutations were rare in H1/SI06, H1/Mich15, and H1/WSN (Fig. 5, A to E). We further calculated the fraction surviving (23) for each viral mutant across the entire H1/WSN and H3/Perth09 HAs during antibody selection. Fraction surviving is a quantitative measure for the resistance and normalized across deep mutational scanning experiments (23). For example, fraction surviving values of 1, 0.5, and 0.1 indicate that the replication fitness in the presence of antibody selection is 100%, 50%, and 10% of that without antibody selection. The fraction surviving values of H1/WSN mutants against CR9114 were all very small, similar to previous observations of H1/WSN against FI6v3 (Fig. 5F, fig. S10). In stark contrast, many mutants of H3/Perth09 were identified with a large fraction surviving value (Fig. 5F). Consistent with the relative resistance profile of H3/HK68 (Fig. 2C), a number of H3/Perth09 mutants with a large fraction surviving value were found at HA2 residues 42 and 45 (Fig. 5F and fig. S11A). Moreover, mutations at HA2 residue 53, which were not examined in H3/HK68 (Fig. 2), had high fraction surviving in H3/Perth09 against FI6v3 (fig. S11, A and B). In H3 HA, mutation of HA2 residue 53 would abolish a hydrogen bond to FI6v3 (fig. S11B). Together, these results suggest that the prevalence of resistance mutations to stem bnAbs is a general phenomenon for the H3 subtype, but not the H1 subtype.

Fig. 5. Relative resistance profile of single mutants in multiple strains of influenza virus.

Fig. 5.

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(A-E) Relative resistance is measured for each single viral mutant at HA2 residues 42, 45, 46, 27, 48, 49, 52, and 111 of (A) H1/SI06 against 300 ng/mL FI6v3 antibody, (B) H1/Mich15 against 300 ng/mL FI6v3 antibody, (C) H1/WSN against 100 ng/mL CR9114 antibody, (D) H1/WSN against 200 ng/mL FI6v3 antibody (data are from (23)), and (E) H3/Perth09 against 15 μg/mL FI6v3 antibody. Relative resistance for WT is set as 1. Mutants with a relative fitness of less than 0.5 are excluded and shown as grey. Residues correspond to WT sequence are boxed. (F) Fraction surviving for all single mutants across the HA protein are shown. Each data point represents one mutant. Fraction surviving was computed as previously described (23). Assuming no antibody-mediated enhancement of virus replication, the theoretical upper limit for the fraction surviving is 1, which indicates that the replication fitness is the same with and without antibody selection (in practice, fraction surviving values slightly > 1 can sometimes be obtained due to the experimental error in qPCR and next-generation sequencing). Data for H1/WSN against FI6v3 were from a previous study (23). Data points that represent mutations at residues 42 and 45 are colored in blue and red, respectively, and mutations at other residues are in grey. (G) The crystal structures of H3 HA in complex with FI6v3 (PDB 3ZTJ) (27) and H1 HA in complex with FI6v3 (PDB 3ZTN) (27) were compared by aligning their heavy chain variable domains. Since the structure of CR9114 with H1 HA is not available, a similar analysis was not performed on CR9114.

Subtype-specific differences in the HA stem

We next aimed to elucidate the mechanism that underlies the lower genetic barrier to resistance to stem bnAbs in H3 HA as compared to H1 HA. Many mutations at HA2 residue 45 have a high fitness cost in H1/SI06 (fig. S9A), which can increase the genetic barrier to resistance. However, most mutations at HA2 residue 45 have no fitness cost in H1/Mich15. In addition, the mutational fitness profiles of H1/SI06 and H1/Mich15 (fig. S9, A and B) show that many mutations can be tolerated in the HA stem, similar to H3/HK68 (Fig. 2A, fig. S9, C to F). Thus, the difference in genetic barrier to resistance to stem bnAbs between H1 and H3 subtypes cannot be fully explained by their ability to tolerate mutations (i.e. fitness cost of mutations).

We therefore further compared the structures of FI6v3 in complexes with H1 HA and H3 HA (27). The orientation of Tyr100c (Kabat numbering (39)) on the third complementarity determining region of the antibody heavy chain (CDR H3) of FI6v3 differs when binding to H1 or H3 HAs (Fig. 5G). The position of HA2 Ile45 also differs between H1 and H3 HAs when FI6v3 is bound. As a result, Tyr100c of FI6v3 packs tighter to HA2 Ile45 of H3 than of H1 HA. Thus, a bulkier substitution at residue 45 of HA2 will disrupt binding of FI6v3 to H3 HA more than to H1 HA. Therefore, the low genetic barrier to resistance to stem bnAbs in the H3 subtype can be partly attributed to both high mutational tolerance in the HA stem and subtype-specific structural features, but it is likely that the latter plays a more critical role in determining subtype-specific differences. While a number of subtype-specific structural features are known in the stem region (40), how they influence the genetic barriers for resistance to stem bnAbs remain to be addressed in future studies.

Implications for escape from a universal vaccine or therapeutic stem bnAbs

Prior studies of influenza bnAbs have not considered whether different subtypes might have different abilities to generate resistance mutations against bnAbs. We find here that H3 HA has a much lower genetic barrier to resistance to two of the broadest bnAbs, CR9114 and FI6v3, as compared to H1 HA, consistent with reports of strong resistance to other human HA stem antibodies in the H3 subtype (21, 25, 41) versus none (21) to weak resistance (22, 23) in the H1 subtype. Therefore, it may be easier for stem bnAbs to suppress H1 rather than H3 subtype viruses.

Since the HA stem is immunosubdominant to the head domain, immunological pressure on the HA stem may not yet be sufficient to impact the evolution of circulating human viruses (42). However, several stem bnAbs are currently in clinical trials (19) and some recently developed influenza vaccine immunogens have focused on targeting the HA stem (5, 8). If stem bnAbs begin to be distributed on a global scale, the immunological pressure on the HA stem will increase. Our findings here indicate that resistance mutations could emerge, at least in the H3 subtype.

Although resistance mutations to stem bnAbs are still rare in currently circulating influenza strains (Fig. 4A), it is important to evaluate the potential impact of such mutations since many vaccine strategies aim to elicit anti-stem antibodies. We were not able to overcome some key resistance mutations (I45T, I45M, and I45F) by in vitro evolution of CR9114 (fig. S12). Nonetheless, the best strategy to prevent or overcome such resistance may involve delivery or elicitation of a combination of antibodies with different resistance profiles. The discovery and characterization of bnAbs with different escape profiles will continue to be key to broaden our arsenal against influenza virus. For example, human H2N2 virus, which carries a Phe at HA2 residue 45, often has low reactivity with stem bnAbs (6, 26, 27, 34, 43), although a very few can have high potency against human H2N2 (44–46). Future studies on anti-stem responses against human H2N2 and emerging viruses, such as H5N1 and H7N9, may provide further insights into how to overcome potential resistance should immune pressure become focused on the HA stem.

Supplementary Material

Supplementary Material

ACKNOWLEDGEMENTS

We thank Wenli Yu and Geramie Grande for technical support in protein expression, Steven Head, Jessica Ledesma and Padmaja Natarajan at TSRI Next Generation Sequencing Core and Fred Hutch Genomics Core for next-generation sequencing, Matthew Haynes and Brian Seegers of the TSRI Flow Cytometry Core Facility for performing FACS, Alfred Ho, Eva-Maria Strauch, and Barney Graham for insightful discussions, and James Paulson for his continual support.

Funding: We acknowledge support from the Bill and Melinda Gates Foundation OPP1170236 (to I.A.W.), NIH K99 AI139445 (to N.C.W.), F30 AI136326 (to J.M.L.), R01 AI127893 (to J.D.B.), R56 AI127371 (to I.A.W.), and R01 AI114730 (to J.C.P.) This work was partially supported through a NIAID Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract (75N93019C00051).

J.M.L. was supported in part by the Center for Inference and Dynamics of Infectious Diseases (CIDID), funded by NIH U54 GM111274. J.D.B. is an Investigator of the Howard Hughes Medical Institute.

Footnotes

Competing interests: The authors declare no competing interests.

Data and materials availability: Raw sequencing data have been submitted to the NIH Short Read Archive under accession number: BioProject PRJNA510654, PRJNA493101, and PRJNA510700. X-ray coordinates and structure factors are deposited at the RCSB Protein Data Bank under accession codes: 6NHP, 6NHQ, and 6NHR. Computer codes and processed data are deposited at Zenodo (https://doi.org/10.5281/zenodo.3660467, https://doi.org/10.5281/zenodo.3660739, and https://doi.org/10.5281/zenodo.3660731). All other data that support the conclusions are included in the manuscript or supplementary materials.

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