Outflanking immunodominance to target subdominant broadly neutralizing epitopes

Abstract

A major obstacle to vaccination against antigenically variable viruses is skewing of antibody responses to variable immunodominant epitopes. For influenza virus hemagglutinin (HA), the immunodominance of the variable head impairs responses to the highly conserved stem. Here, we show that head immunodominance depends on the physical attachment of head to stem. Stem immunogenicity is enhanced by immunizing with stem-only constructs or by increasing local HA concentration in the draining lymph node. Surprisingly, coimmunization of full-length HA and stem alters stem-antibody class switching. Our findings delineate strategies for overcoming immunodominance, with important implications for human vaccination.

Keywords: B cell; antibodies; hemagglutinin; immunodominance; influenza.

Fig. 1.

Immunodominance of the B cell responses depends on physical attachment of the HA head to stem. (A) Schematic of the immunization strategy. Group 1 was immunized with full-length HA in the left hind leg, group 2 with stem in the left hind leg, group 3 with full-length HA and stem in equimolar amounts in the left hind leg, and group 4 with full-length HA in the left hind leg and stem in the right hind leg. (B) Representative flow cytometry plot showing swIg GC B cells, gated as live CD3⁻ B220⁺ GL7⁺ CD38⁻ IgD⁻ IgM⁻, and ability to bind HA head (H1 single positive), and HA stem (H1⁺H5⁺). (C) Enumeration of head vs. stem swIg GC B cells for the four different groups 21 d after challenge. Three independent experiments with 4 mice each (pooled for the first experiment; individual for the other 2 experiments) (n = 9). Bars represent mean ± SEM; statistical analysis was performed using two-way ANOVA with Holm–Sidak’s multiple comparison test. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 2.

Serum immune responses of immunized mice are impaired upon mixed immunization. (A and B) Antibody end point titers (A) and IgG subclass response (B) to HA head (following stem Ab absorption) and to stem for the different immunization groups (n = 12 for groups 1, 2, and 4; n = 10 for group 3). Bars represent mean ± SEM; statistical analysis was performed using two-way ANOVA with Holm–Sidak’s multiple comparison test. (C) Sera were tested for the ability to induce ADCD on stem-conjugated beads. Data are presented as the area under the curve (AUC) of geometrical mean fluorescence intensity (GMFI) of 1:5 and 1:10 dilutions, and each data point is the mean of two technical replicates. (D) Ability of the sera to induce ADCP on stem-conjugated beads by primary monocytes. Each data point is the mean of 2 technical replicates. Three independent experiments with four mice each (n = 12 for groups 1, 2, and 4; n = 10 for group 3). Bars represent mean ± SEM; statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Naïve B cell precursor frequency is similar between head and stem. (A) Representative flow cytometry plot showing background stain of naïve B cells (CD3⁻ CD43⁻ B220⁺) on streptavidin (SA) conjugated to PE and APC and used in combination and staining of the same cells with H1 HA. (B) Gating strategy for total mature naïve B cell population (CD3⁻ CD43⁻ B220⁺), and follicular (FO) (CD3⁻ CD43⁻ B220⁺ CD23⁺ CD21^low IgM^low IgD^high) and marginal zone (MZ) (CD3⁻ CD43⁻ B220⁺ CD23⁻ CD21^l+ IgM^high IgD^low) B cells. Overlay shows IgM and IgD expression in FO vs. MZ B cells. (C) Precursor frequency as % of parent population for mature naïve, FO, and MZ B cells. (n = 8). Three independent experiments with 2 or 4 mice each. Bars represent SEM; statistical analysis was performed using two-sided unpaired t test.

Fig. 4.

Early B cell affinity is different for head vs. stem. (A) Representative flow cytometry plot showing total (CD3⁻ B220⁺ GL7⁺ CD38⁻), swIg (CD3⁻ B220⁺ GL7⁺ CD38⁻ IgD⁻ IgM⁻), and IgM (CD3⁻ B220⁺ GL7⁺ CD38⁻ IgD⁻ IgM⁺) GC B cells and the gating selection for the AC₅₀ at 10 d post HA or stem i.m. immunization. (B) Pooled iliac LNs from three mice at 10 d after immunization with full-length H1 HA (orange) or stem (blue) were stained with graded amounts of H1 HA (orange) or H5 HA (blue) and plotted against the frequency of GC B cells stained (n = 3). (C) Nanomolar concentration of HA giving half-maximal binding (AC₅₀) was derived from individual experimental curves (n = 3). (D) MFI of HA⁺ B cells at the highest concentration (66 nM) at 10 d postimmunization. Three independent experiments with 3 pooled LNs each. Bar graphs represent mean and bars represent SEM; statistical analysis was performed using two-sided unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Stem subdominance can be subverted by increasing local antigen concentration. (A) Amount of PE detected in draining LNs following f.p. or i.m. immunization (n = 17–18). Boxplot showing median and min to max. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. (B and D) Representative flow cytometry plots showing swIg GC B cells (gated as live CD3⁻ B220⁺ GL7⁺ CD38⁻ IgD⁻ IgM⁻) (B) and IgM GC B cells (gated as live CD3⁻ B220⁺ GL7⁺ CD38⁻ IgD⁻ IgM⁺) (D), and ability to bind HA head (H1 HA single positive) and HA stem (H1⁺H5⁺) after i.m., footpad, or repeated i.m. immunization. (C and E) Enumeration of head vs. stem swIg GC B cells (C) and IgM GC B cells (E) for the three different groups 21 d after immunization (n = 12 for i.m. and repeated i.m.; n = 24 for f.p.). (F) Antibody end point titers for total Ig, IgG, and IgM to H1-PR8 or stem for the different immunization groups. For f.p., red symbols indicate mice that are stem-seropositive, while open pink indicates stem-seronegative (“low”). Three independent experiments with 4 mice each for i.m. and repeated i.m., and 5 independent experiments with 5 or 4 mice each for f.p. immunization. Bars represent mean ± SEM; statistical analysis was performed using two-way ANOVA with Holm–Sidak’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6.

Footpad immunization does not shift stem GC B cells to DZ but alters Tfr/Tfh ratio. (A) Representative flow cytometry plot showing dark zone (DZ) (CD3⁻ B220⁺ GL7⁺ CD38⁻ IgD⁻ IgM⁻ IgG⁺ CXCR4⁺ CD86⁻) vs. light zone (LZ) (CD3⁻ B220⁺ GL7⁺ CD38⁻ IgD⁻ IgM⁻ IgG⁺ CXCR4⁻ CD86⁺) distribution of head- or stem-specific GC B cells at 11 d after f.p. immunization. (B) Quantification of the frequency of head or stem GC B cells in the DZ (n = 9). Two independent experiments with 5 and 4 mice each. Bars represent SEM; statistical analysis was performed using two-way unpaired t test. (C) Gating strategy for T follicular (Tf) cell population (B220⁻ CD4⁺ CXCR5⁺ PD1⁺) and further classification into T follicular helper (Tfh) and T follicular regulatory (Tfr) cells based on FoxP3 expression. (D) Quantification of Tfr contribution to total Tf (CD4⁺ CXCR5⁺ PD1⁺) (Tfr/Tfh ratio) in draining LNs (iliac for i.m. and popliteal for f.p.) 8 d postimmunization. (E and F) Tfh (E) and Tfr (F) expressed as frequency of CD4⁺ T cells (n = 10 for i.m.; n = 7 for f.p.). Two independent experiments with 5 mice each for i.m. and with 4 and 3 mice for f.p. immunization. Bars represent mean ± SEM; statistical analysis was performed using two-sided unpaired t test. *P < 0.05, **P < 0.01, ****P < 0.0001.