Abstract
The conserved region of influenza hemagglutinin (HA) stalk (or stem) has gained attention as a potent target for universal influenza vaccines1,2,3,4,5. Although the HA stalk region is relatively well conserved, the evolutionarily dynamic nature of influenza viruses6 raises concerns about the possible emergence of viruses carrying stalk escape mutation(s) under sufficient immune pressure. Here we show that immune pressure on the HA stalk can lead to expansion of escape mutant viruses in study participants challenged with a 2009 H1N1 pandemic influenza virus inoculum containing an A388V polymorphism in the HA stalk (45% wild type and 55% mutant). High level of stalk antibody titers was associated with the selection of the mutant virus both in humans and in vitro. Although the mutant virus showed slightly decreased replication in mice, it was not observed in cell culture, ferrets or human challenge participants. The A388V mutation conferred resistance to some of the potent HA stalk broadly neutralizing monoclonal antibodies (bNAbs). Co-culture of wild-type and mutant viruses in the presence of either a bNAb or human serum resulted in rapid expansion of the mutant. These data shed light on a potential obstacle for the success of HA-stalk-targeting universal influenza vaccines—viral escape from vaccine-induced stalk immunity.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
This work was supported by the intramural research program of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). We thank the Comparative Medicine Branch (NIAID, NIH) for assistance with the animal studies and S. Hunsberger (Biostatistics Research Branch, NIAID, NIH) for the helpful discussion of statistical analyses performed in this study. We also thank Janssen Pharmaceutica, R. Ahmed (Emory Vaccine Center) and A. Townsend (University of Oxford) for generously sharing monoclonal antibodies.
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J.-K.P., M.J.M., J.C.K. and J.K.T. conceived and designed the study. J.-K.P., Y.X., M.D.R., L.A.R., S.F., A.M.M., A.D.F., M.A.G., N.A.B., L.Q., X.Y. and K.S. generated the laboratory data. S.R., R.A., L.C., A.H. and M.J.M. designed and performed the primary clinical study. J.-K.P., A.H., Y.X., M.D.R., D.M.M., K.-A.W., M.J.M., J.C.K. and J.K.T. interpreted the data. J.-K.P., D.M.M., K.-A.W., M.J.M., J.C.K. and J.K.T. wrote the manuscript. All authors critically reviewed the paper and approved of the final version of the paper for submission.
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Extended data
Extended Data Fig. 1 A Single Nucleotide Polymorphism (SNP) assay using minor groove binder (MGB)-based TaqMan probes for detecting wild-type (A388) or mutant (V388) genes in various conditions.
A SNP assay was developed for detecting wild-type (A388) or mutant (V388) HA genes in various conditions utilizing a set of Minor Groove Binder (MGB)-based TaqMan probes: VIC-labeled probe detecting the wild-type (A388) and a FAM-labeled probe detecting the mutant (V388). The SNP assay was validated using a mixed viral genome from the 2009 H1N1pdm wild-type (A388) and the mutant (V388) viruses. Viral RNA was mixed in varying ratios from 10:0 (0% mutant virus) to 0:10 (100% mutant virus). For the validation of two-step SNP assay for analyzing nasal wash samples from the human challenge study, mixed viral RNA was diluted to represent varying viral loads of (a) 105.5 TCID50/ml, (b) 104.0 TCID50/ml, and (c) 103.0 TCID50/ml. Prepared viral RNA was analyzed by the two-step SNP assay (see Methods). Blue and red bars indicate the ∆Rn value of wild-type and mutant virus, respectively. Graphs show mean ∆Rn value and standard deviation from 4 independent experiments (a-c, n = 4). For the validation of one-step SNP assay used to analyze selection dynamics in vitro, mixed viral RNA was diluted to represent varying viral loads of (d) 107.0 TCID50/ml, (e) 105.5 TCID50/ml, and (f) 104.0 TCID50/ml. Prepared viral RNA was analyzed by the one-step SNP assay (see Methods). Blue bars indicate the threshold cycle (Ct) values representing the amount of wild-type (A388) virus. Red bars represent the amount of mutant (V388) virus. Graphs show mean Ct value and standard deviation from 3 independent experiments (d-f, n = 3). Results show that the SNP assay reliably detects minor population, either wild-type or mutant, existing as low as 10% across the various viral loads.
Extended Data Fig. 2 Comparison of different parameters between study participants with different selection outcomes.
Different parameters were compared between groups to find possible correlates of the selection outcomes. a, Pre-challenge serum hemagglutination inhibition (HAI) titers were compared. Black horizontal lines and grey error bars represent geometric mean titer and 95% CI, respectively. Dashed line shows the detection limit. b, The fraction of HA stalk antibodies relative to total HA antibodies was compared. The ratio is a calculation of serum anti-stalk IgG and anti-full-length HA IgG titers measured by ELISA. c, Serum IgG titer data (anti-stalk and anti-total HA) generated in (b) were used to analyze the correlation between the stalk and total HA antibody titers in each selection group. A two-tailed Pearson’s correlation coefficient (Pearson’s r) was used for the analysis. The positive correlation between the stalk antibody titers and total HA titers may explain the lack of difference in stalk/total HA antibody ratio between groups seen in (b). To find a possible role of immunological imprinting on the selection outcomes, (d) age of the participants at the study enrollment and (e) birth year was compared. To find potential loss in viral fitness in humans caused by the A388V mutation, (f) days of symptoms, (g) number of symptoms, and (h) duration of shedding after the challenge infection were compared between groups. Black horizontal lines and grey error bars represent median value and 95% CI, respectively (b,d,e-h). A two tailed nonparametric Mann-Whitney test was used to compare parameters from the mutant selection group to other selection groups. Each symbol represents an individual study participant and their selection outcome (wild-type selection, n = 8; mixed shedding, n = 9; mutant selection, n = 12).
Extended Data Fig. 3 A significant conformational change to the HA stalk region induced by A388V mutation.
MDCK cells were infected with 1 multiplicity of infection (MOI) of wild-type (A388) or mutant (V388) H1N1pdm viruses generated by reverse genetics. 24 hours after infection, cells were harvested, and the expression of wild-type and mutant HA was measured by flow cytometry to evaluate the effect of the A388V mutation on the HA stalk epitopes. a, A representative example of the gating strategy. Dead cells and debris were excluded based on FSC/SSC cell dot plot. Anti-influenza nucleoprotein (NP) antibodies conjugated with allophycocyanin (APC) were used for gating. Only infected cells, expressing NP, were used for the analysis. Broadly neutralizing antibodies binding to the HA stalk, (b) CR6261, (c) CR9114, (d) FI6V3, (e) 70-1F02, (f) C179, and (g) CT149 were conjugated with fluorescein isothiocyanate (FITC). A monoclonal antibody that binds to the HA globular head, (h) EM-4C04, was conjugated with r-phycoerythrin (R-PE). Each stalk-binding antibody was mixed with the head-binding EM-4C04 antibody and NP antibody, and the antibody mixtures were used to stain cells expressing the wild-type or mutant HA. Histograms are colored differently to show different experimental groups: Blue - cells infected with wild-type (A388) virus; Pink - cells infected with mutant (V388) virus; Black - unstained cell control. Representative histograms from three independent experiments are shown. The summary table shows the average median fluorescence intensity (MFI) and standard error of mean (s.e.m.) of the three independent experiments.
Extended Data Fig. 4 Comparison between stalk-only and full-length HA construct.
Stalk-only constructs with or without the A388V mutation (See Extended Data Fig. 7b for the amino acid sequence) were produced to measure the level of antibodies recognizing the mutant stalk in human serum while excluding head-binding antibodies (Fig. 3j). To confirm that the stalk-only construct with A388V mutation appropriately represents the natural A388V stalk structure, the level of decrease in broadly neutralizing monoclonal antibodies (bNAbs) binding was compared between the stalk-only construct and the full-length HA. ELISA was performed using serially diluted (a,b) CR6261, (c,d) CR9114, (e,f) FI6V3, (g,h) 70-1F02, (I,j) C179, and (k,l) CT149. (m,n) Anti-StrepTag II antibody was used to show that equal amounts of wild-type and mutant antigen were used for the analysis. The AUC was calculated using GraphPad Prism8 (v.8.3.0). The AUC for the full-length HAs (b,d,f,h,j,l) was calculated using the data from Fig. 3. Graphs show mean and standard deviations from three independent measurements. The summary table shows the mean OD492 values and standard error of mean (s.e.m.) of the three independent measurements. The comparison result (summarized in the table) shows that the A388V stalk-only construct closely represents the natural stalk structure of the full-length A388V HA.
Extended Data Fig. 5 AUC calculation using wile-type and mutant stalk-only construct.
Raw ELISA data used to generate Fig. 3j are shown. Stalk-only constructs with or without the A388V mutation (See Extended Data Fig. 7b for the amino acid sequence) were used to measure changes in the level of antibodies recognizing the wild-type (A388) of mutant (V388) stalk in human serum. Twenty-nine pre-challenge serum samples from the influenza human challenge study participants were serially diluted and used for the ELISA. The AUC was calculated using GraphPad Prism8 (v.8.3.0) with the baseline value of 0.1 (approximately 2 times the OD492 value from the control wells). Blue lines and numbers show the ELISA data and the AUC, respectively, obtained using the wild-type (A388) stalk construct. Red lines and numbers show the ELISA data and the AUC, respectively, obtained using the mutant (V388) stalk construct. Graphs show mean and standard deviations from three independent measurements.
Extended Data Fig. 6 Association between the stalk antibody titers and selection pressure measured in vitro.
Selection pressure placed by individual human sera was measured using pre-challenge serum from the influenza human challenge study participants. a, An equal mixture of the wild-type (A388) and mutant (V388) virus was cultured with 1:50 diluted serum samples. Sera from Q1, Q2, Q3 and Q4 from Fig. 1 (n = 7 per quartile) were used. Culture supernatants were collected at 48 and 72 hours after infection followed by viral RNA extraction. The selection dynamics were measured using a Single Nucleotide Polymorphism (SNP) assay utilizing a set of Minor Groove Binder (MGB)-based TaqMan probes; VIC-labeled probe detects the wild-type (A388); FAM-labeled probe detects the mutant (V388). Data are presented as the threshold cycle (Ct) value from the SNP assay. Dashed lines show the Ct value limit (Ct 40) of the SNP assay. A Ct value of 41 was given to undetected signals to generate graphs. Error bars represent standard deviations from three independent experiments. The final dilution is noted on the individual graph if higher than 1:50. b, Mutant selection index was calculated based on data from (a) by ∆∆Ct method using controls cultured without serum (see Methods). A mutant selection index higher than 0 (pink area) indicates a serum sample selected for the mutant virus. An index lower than 0 (blue area) indicates a serum sample selected for the wild-type virus. Horizontal lines show median values and error bars represent 95% CI. The indexes between samples from different quartiles were compared using nonparametric one-way analysis of variance (Kruskal-Wallis test) and Dunn’s test as a post-hoc test. c, Correlation between the anti-stalk serum IgG titer and the selection index of 29 sera samples were analyzed by calculating two-tailed Spearman’s rank correlation coefficient (Spearman r). The best-fit line was plotted using simple linear regression analysis. Statistical analyses were performed using GraphPad Prism8 (v.8.3.0).
Extended Data Fig. 7 Sequence of wild-type and mutant full-length HA or HA stalk-only constructs.
Amino acid sequences of (a) full-length HA and (b) stalk-only proteins with or without A388V mutation are shown. A388V mutation, HA trimerization domain, and StrepTag II sequence are highlighted. Consensus amino acid sequences are shown in dots.
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Park, JK., Xiao, Y., Ramuta, M.D. et al. Pre-existing immunity to influenza virus hemagglutinin stalk might drive selection for antibody-escape mutant viruses in a human challenge model. Nat Med 26, 1240–1246 (2020). https://doi.org/10.1038/s41591-020-0937-x
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DOI: https://doi.org/10.1038/s41591-020-0937-x
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