Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin
- Fred Hutchinson Cancer Research Center, United States
- University of Washington, United States
- Perelman School of Medicine, University of Pennsylvania, United States
- University of Southern California, United States
- Section of Rheumatology, University of Chicago, United States
- WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Australia
- School of Medicine, University of Pittsburgh, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1
Mutational antigenic profiling quantifies the antigenic effect of all amino-acid mutations to HA.
We generate libraries of mutant viruses carrying all mutations to HA compatible with viral growth. We incubate the libraries with antibodies or serum, and infect cells with non-neutralized virus. …
Figure 2 with 2 supplements
Mutational antigenic profiling of monoclonal antibodies targeting HA.
(A) Maps of immune selection from two antibodies targeting near the receptor binding pocket. Line plots show the total immune selection at each site, and logo plots show mutations at strongly …
Figure 2—figure supplement 1
Percent of viral library retaining infectivity after antibody treatment during mutational antigenic profiling.
The percent of the mutant virus library that remained infectious after incubation with antibody was determined using qPCR for each replicate of the mutational antigenic profiling. Higher antibody …
Figure 2—figure supplement 2
Biological replicates of the mutational antigenic profiling are well correlated.
For each antibody, we performed two or three biological replicates of the mutational antigenic profiling. Each replicate used a fully independently generated mutant library. (A) Maps of mutations …
Figure 3 with 2 supplements
Mutational antigenic profiling of four human serum samples.
Plot titles indicate the year the serum was collected and the age of the individual at that time. (A) Line plots show the total immune selection at each site, and logo plots show mutations at …
Figure 3—figure supplement 1
Percent of viral library retaining infectivity after serum treatment during mutational antigenic profiling.
The percent of the mutant virus library that remained infectious after incubation with serum was determined using qPCR for each replicate of the mutational antigenic profiling. Higher serum …
Figure 3—figure supplement 2
Biological replicates of the mutational antigenic profiling are well correlated.
For each serum, we performed three biological replicates of the mutational antigenic profiling. Each replicate used a fully independently generated mutant library. (A) Maps of mutations selected by …
Figure 4 with 2 supplements
The maps of immune selection are stable over short time periods in the absence of vaccination or infection.
This figure re-displays the (A) map of immune selection and (B) neutralization curves from Figure 3 for the serum from the 53-year-old individual alongside comparable data generated using another …
Figure 4—figure supplement 1
Percent of viral library retaining infectivity after serum treatment during mutational antigenic profiling.
The percent of the mutant virus library that remained infectious after incubation with serum was determined using qPCR for each replicate of the mutational antigenic profiling. Higher serum …
Figure 4—figure supplement 2
Biological replicates of the mutational antigenic profiling are well correlated.
For each serum, we performed three biological replicates of the mutational antigenic profiling. Each replicate used a fully independently generated mutant library. Shown are the Pearson correlation …
Figure 5 with 2 supplements
Mutational antigenic profiling of sera from four humans pre- and post-vaccination.
Plot titles indicate the year the serum was collected, the age of the individual at that time, and the vaccination status. (A) Line plots show total immune selection at each site, and logo plots …
Figure 5—figure supplement 1
Percent of viral library retaining infectivity after serum treatment during mutational antigenic profiling.
The percent of the mutant virus library that remained infectious after incubation with serum was determined using qPCR for each replicate of the mutational antigenic profiling. Higher serum …
Figure 5—figure supplement 2
Biological replicates of the mutational antigenic profiling are well correlated.
For each serum, we performed three biological replicates of the mutational antigenic profiling. Each replicate used a fully independently generated mutant library. (A) Maps of mutations selected by …
Figure 6 with 2 supplements
Mutational antigenic profiling of sera from five ferrets.
Plot titles indicate the lab that performed the infection and if the infecting strain was Victoria/2011 rather than Perth/2009. For ferrets from Pittsburgh, both pre- and post-infection sera were …
Figure 6—figure supplement 1
Percent of viral library retaining infectivity after serum treatment during mutational antigenic profiling.
The percent of the mutant virus library that remained infectious after incubation with serum was determined using qPCR for each replicate of the mutational antigenic profiling. Higher serum …
Figure 6—figure supplement 2
Biological replicates of the mutational antigenic profiling are well correlated.
For each serum, we performed three biological replicates of the mutational antigenic profiling. Each replicate used a fully independently generated mutant library. (A) Maps of mutations selected by …
Figure 7 with 2 supplements
Mutational antigenic profiling of polyclonal human serum spiked with a monoclonal antibody.
The antibody is spiked into the serum at a ‘low’ concentration (antibody alone less potent than serum alone), a ‘mid’ concentration (antibody similarly potent to serum), and a ‘high’ concentration …
Figure 7—figure supplement 1
Percent of viral library retaining infectivity after treatment with each serum+antibody mix.
The percent of the mutant virus library that remained infectious after incubation with serum and/or antibody was determined using qPCR for each replicate of the mutational antigenic profiling. The …
Figure 7—figure supplement 2
Biological replicates of the mutational antigenic profiling are well correlated.
For each serum-antibody mix, we performed three biological replicates of the mutational antigenic profiling. Each replicate used a fully independently generated mutant library. (A) Maps of mutations …
Figure 8
Frequencies of amino acids at key sites in human H3N2 influenza HA between 2007 and 2019.
There are 16 sites under strong immune selection in our mutational antigenic profiling with human sera, and this figure shows the nine of these sites for which a new amino acid rose to ≥5% …
Additional files
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Supplementary file 1
Classically defined antigenic regions of H3N2 HA and their relationship to sites of strong selection in our mapping experiments.
The first column of this Excel document lists all sites of relevance in H3 numbering. The second and third column indicates which sites have been assigned to antigenic regions A, B, C, D, or E according to Table 1 of Wiley et al. (1981) or SI Table 1 of Shih et al. (2007) (sites listed in those papers but not assigned a labeled antigenic region are not included in the columns). The remaining columns indicate which sites are under strong selection from any antibody/serum in each set.
- https://doi.org/10.7554/eLife.49324.023
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Supplementary file 2
The curve fit parameters for all neutralization curves shown in the figures.
The IC50 values are not extrapolated, and so are shown as upper or lower bounds if they fall outside the range of the measurements. For sera, the IC50s are the serum dilution; for antibodies they are the antibody concentration in μg/ml. This CSV file is also available at https://github.com/jbloomlab/map_flu_serum_Perth2009_H3_HA/blob/master/results/neutralization_assays/neut_assay_figs_fit_params.csv.
- https://doi.org/10.7554/eLife.49324.024
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Supplementary file 3
The serum dilution or antibody concentration used for each replicate of the mutational antigenic profiling.
For sera, the values indicate the dilution of serum. For antibodies, they are the concentration in μµg/ml. For serum/antibody mixes, they are the dilution of serum followed by the antibody concentration in µg/ml. These dilutions/concentrations were chosen to give the desired percent of viral infectivity remaining for the libraries after treatment (see Supplementary file 4). The dashed vertical lines in Figure 3B, Figure 4B, Figure 5, and Figure 6 indicate the average concentration of serum used across the replicates. This CSV file is also available at https://github.com/jbloomlab/map_flu_serum_Perth2009_H3_HA/blob/master/results/selection_tables/serum_dilution_table.csv.
- https://doi.org/10.7554/eLife.49324.025
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Supplementary file 4
The percent of the overall viral library that retained infectivity after incubation with serum or antibody.
- https://doi.org/10.7554/eLife.49324.026
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Supplementary file 5
HTML rendering of Jupyter notebook that analyzes the mutant virus libraries generated by reverse genetics.
- https://doi.org/10.7554/eLife.49324.027
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Supplementary file 6
A GenBank file providing the full sequence of the protein expression plasmid pHAGE2-EF1aInt-TCmut-P09-HA, which encodes for the wildtype Perth/2009 HA sequence.
- https://doi.org/10.7554/eLife.49324.028
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Supplementary file 7
Numerical values of the differential selection (immune selection) values for each amino-acid at each site after taking the median across replicates.
These are the values plotted in the line and logo plots in the main figures. This tidy-format CSV file is also available at https://github.com/jbloomlab/map_flu_serum_Perth2009_H3_HA/blob/master/results/avgdiffsel/avg_sel_tidy.csv.
- https://doi.org/10.7554/eLife.49324.029
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Supplementary file 8
Logo plots of the positive differential selection for all sites in HA for each serum and antibody selection.
The main figures in this paper just zoom in on the key sites of selection. These PDFs are also available at https://github.com/jbloomlab/map_flu_serum_Perth2009_H3_HA/tree/master/results/avgdiffsel/full_logo_plots.
- https://doi.org/10.7554/eLife.49324.030
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Supplementary file 9
Key resources table listing the most crucial reagents and computer software used in the study.
- https://doi.org/10.7554/eLife.49324.031
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Transparent reporting form
- https://doi.org/10.7554/eLife.49324.032
