Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr 11;9(1):1386.
doi: 10.1038/s41467-018-03665-3.

How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin

Michael B Doud  1   2   3 , Juhye M Lee  1   2   3 , Jesse D Bloom  4   5
Affiliations

How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin

Michael B Doud et al. Nat Commun. .

Abstract

Influenza virus can escape most antibodies with single mutations. However, rare antibodies broadly neutralize many viral strains. It is unclear how easily influenza virus might escape such antibodies if there was strong pressure to do so. Here, we map all single amino-acid mutations that increase resistance to broad antibodies to H1 hemagglutinin. Our approach not only identifies antigenic mutations but also quantifies their effect sizes. All antibodies select mutations, but the effect sizes vary widely. The virus can escape a broad antibody to hemagglutinin's receptor-binding site the same way it escapes narrow strain-specific antibodies: via single mutations with huge effects. In contrast, broad antibodies to hemagglutinin's stalk only select mutations with small effects. Therefore, among the antibodies we examine, breadth is an imperfect indicator of the potential for viral escape via single mutations. Antibodies targeting the H1 hemagglutinin stalk are quantifiably harder to escape than the other antibodies tested here.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Quantifying the fraction of virions with each mutation that escape antibody neutralization. This figure shows hypothetical data for four viral variants. a Virions with the V1K mutation (orange) completely survive an antibody concentration where most other virions are neutralized. b This resistance is manifested by a large shift in V1K’s neutralization curve. c For each dotted vertical line drawn through the neutralization curves in b, we calculate the fraction of virions with that mutation that survive the antibody, and indicate this fraction by the height of the letter corresponding to that amino acid at that site. d–f Similar data to the first three panels, but now V1K has only a small antigenic effect, and modestly increases the fraction of virions that survive antibody treatment
Fig. 2
Fig. 2
Epitopes and breadth of broad and narrow antibodies targeting HA. a Crystal structures of the broad antibodies and sites of escape mutations selected by the narrow ones superimposed on the structure of the HA trimer (PDB 1RVX). S139/1 (PDB 4GMS) targets residues in the receptor-binding pocket; C179 (PDB 4HLZ) and FI6v3 (PDB 3ZTN) target the stalk. The sites of escape mutations for H17-L19, H17-L10, and H17-L7 are those mapped by Doud et al. b A phylogenetic tree of HA subtypes. Circles (broad antibodies) and squares (narrow antibodies) denote reported antibody binding or neutralization activity against that subtype. Not all antibodies have been tested against all subtypes
Fig. 3
Fig. 3
Neutralization of wildtype virus by each antibody, and the fraction of mutant library virions surviving at each concentration used in our experiments. The curves show neutralization of the wildtype A/WSN/1933 virus. Each point represents the mean and standard deviation of three measurements. The vertical dotted lines show the concentrations of antibody that were then used in the mutant virus library selections, and the tables give the overall fraction of the mutant virus libraries that survived at each concentration, determined by qRT-PCR. As described in the text, the antibody concentrations were chosen to give similar fractions of the mutant virus libraries that survive, rather than to fall at uniform positions on the neutralization curves of the wildtype virus
Fig. 4
Fig. 4
Strain-specific and anti-receptor-binding-site antibodies select mutations with large antigenic effects, but anti-stalk antibodies only select small-effect mutations. The excess fraction of virions with a mutation at each site that survive the antibody, averaging across all amino-acid mutations at each site (see Eq. 10). There are multiple sites of large-effect mutations for H17-L19, H17-L10, H17-L7, and S139/1, but none for FI6v3 and C179. Supplementary Fig. 2 shows the excess fraction surviving for the largest-effect mutation at each site. Supplementary Figs. 3, 4, 5, 6, 7, and 8. show all mutations using logo plots. Sites are labeled in H3 numbering
Fig. 5
Fig. 5
Mutations selected by broad and narrow antibodies. a Logo plots show sites where mutations have the largest effect. Letter heights are proportional to the excess fraction of virions with that mutation that survive antibody, as indicated by the scale bars. Structures are colored white to red by the excess fraction surviving for the largest-effect mutation at each site, with each antibody scaled separately. b Sites of selection from anti-stalk antibodies, with the same coloring scale for both antibodies. Selection for serine or threonine at sites 280 and 291 introduces glycosylation sites at 278 and 289, respectively. c Cladogram of group 1 HA subtypes. The amino acid at site 38 is indicated. Colors indicate whether a subtype has been reported in the literature to be bound or neutralized by C179
Fig. 6
Fig. 6
The mutations selected by FI6v3 increase neutralization resistance, but the effects are small. a Neutralization curves of individual viral mutants with FI6v3. The mutations K280S, K280T, N291S, G47R (HA2), and K(-8)T are all expected to increase neutralization resistance based on the mutational antigenic profiling (Fig. 5a), whereas K280A, M17L (HA2), P80D, and V135T are not expected to affect neutralization (Supplementary Fig 6). All neutralization curves in this panel were performed in triplicate on the same day. This panel shows the average of the replicates; Supplementary Fig. 9 shows the curves for each replicate individually and performs statistical testing of whether the IC50s for mutants are significantly different than for wildtype. b,c In contrast to FI6v3, mutations selected by narrow antibodies have very large effects on neutralization. Neutralization curves for representative escape mutants from H17-L19 and H17-L7 taken from Doud et al. are shown. Points indicate mean and standard error of three replicates
Fig. 7
Fig. 7
Mutational tolerance of HA sites in the antibody-binding footprints. These plots show all HA sites within 4 Å of the antibody in the crystal structure, plus any additional sites (marked with a *) where we identified antigenic mutations. The logo plots at bottom show the preference of each HA site for each amino acid under selection for viral replication as measured by Doud and Bloom. For instance, site 153 only tolerates tryptophan, so W occupies the entire height of the preference logo stack. In contrast, site 156 tolerates many amino acids, all of which contribute to the height of the preference logo stack. Above the preference logo stacks are logo plots showing the excess fraction surviving antibody treatment as measured in the current study. Note that scale for these antigenic effects is 10× smaller for FI6v3 and C179 than for S139/1
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Birrer MJ, Udem S, Nathenson S, Bloom BR. Antigenic variants of measles virus. Nature. 1981;293:67–69. doi: 10.1038/293067a0. - DOI - PubMed
    1. Ter Meulen V, Löffler S, Carter MJ, Stephenson JR. Antigenic characterization of measles and SSPE virus haemagglutinin by monoclonal antibodies. J. General. Virol. 1981;57:357–364. doi: 10.1099/0022-1317-57-2-357. - DOI - PubMed
    1. Crainic R, et al. Natural variation of poliovirus neutralization epitopes. Infect. Immun. 1983;41:1217–1225. - PMC - PubMed
    1. Diamond DC, et al. Antigenic variation and resistance to neutralization in poliovirus type 1. Science. 1985;229:1090–1093. doi: 10.1126/science.2412292. - DOI - PubMed
    1. Drexler JF, et al. Robustness against serum neutralization of a poliovirus type 1 from a lethal epidemic of poliomyelitis in the Republic of Congo in 2010. Proc. Natl Acad. Sci. USA. 2014;111:12889–12894. doi: 10.1073/pnas.1323502111. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

-