Outflanking Immunodominance to Target Subdominant Broadly Neutralizing Epitopes

Outflanking immunodominance to target subdominant broadly neutralizing epitopes

Davide Angelettia,b,1, Ivan Kosika, Jefferson J. S. Santosa, William T. Yewdellc, Carolyn M. Boudreaud,e, Vamsee V. A. Mallajosyulaf, Madeleine C. Mankowskia, Michael Chambersg, Madhu Prabhakarang, Heather D. Hickmanh, Adrian B. McDermottg, Galit Alterd, Jayanta Chaudhuric, and Jonathan W. Yewdella,1

aLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; bDepartment of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, 40530 Gothenburg, Sweden; cImmunology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; dRagon Institute of MGH, MIT, and Harvard University, Cambridge, MA 02139; ePhD Program in Virology, Division of Medical Sciences, Harvard University, Boston, MA 02115; fInstitute for Immunity, Transplantation and Infection, Stanford University, Stanford, CA 94305; gVaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and hLaboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved May 29, 2019 (received for review September 20, 2018) A major obstacle to vaccination against antigenically variable viruses Results is skewing of antibody responses to variable immunodominant Stem-Only Immunogen Elicits a Robust GC B Cell Response. To better epitopes. For influenza virus hemagglutinin (HA), the immuno- understand the immunodominance of the HA head domain, we dominance of the variable head impairs responses to the highly immunized mice intramuscularly (i.m.; the typical route for human conserved stem. Here, we show that head immunodominance vaccination) with full-length H1 HA- or stem-only recombinant depends on the physical attachment of head to stem. Stem immuno- purified proteins (13, 14) in different combinations (Fig. 1A). genicity is enhanced by immunizing with stem-only constructs or by Twenty-one days after a single boost, we quantitated antigen-specific increasing local HA concentration in the draining lymph node. GC B cells in draining LNs, specific for HA head or HA stem, via Surprisingly, coimmunization of full-length HA and stem alters combinatorial flow cytometry staining using H1 HA and H5 HA (two stem-antibody class switching. Our findings delineate strategies proteins with different heads but semiconserved stems) (SI Appendix, for overcoming immunodominance, with important implications Fig. S1A). GCs formed in the ipsilateral draining LNs in similar INFLAMMATION

for human vaccination. numbers independent of the immunogen (SI Appendix, Fig. S1 B and IMMUNOLOGY AND C). As expected (5, 15, 16), HA i.m. immunization mostly induced influenza | immunodominance | Bcell| hemagglutinin | antibodies head-specific GC B cells. Importantly, stem immunization generated a stem-specific B cell response of similar magnitude to the head- specific response, demonstrating that our stem construct is not in- easonal influenza remains a significant public health burden, B with vaccines requiring frequent reformulation yet providing trinsically of low immunogenicity (Fig. 1 ). After immunization with S HA and stem in separate legs, immunogen-specific B cells developed limited protection (1, 2). Broadly neutralizing antibodies (Abs) only in the ipsilateral LN (Fig. 1C and SI Appendix, Fig. S1 D and E). binding viral hemagglutinin (HA) have sparked the hope of Notably, this lack of competition in the draining LN also occurred developing a universal influenza vaccine (3). Most Abs target the when we mixed intact HA and stem (Fig. 1C and SI Appendix, Fig. S1 – highly variable globular head of HA (3 5). The conserved HA D and E), strongly suggesting that head immunodominance results stem is much more cross-reactive between strains and a target for from naïve B cell competition for full-length HA. Immunization with protective Abs, but is poorly immunogenic following infection or vaccination (6). HA and other immunogens activate naïve B cells Significance present in lymph nodes (LNs) or spleen. Epitopes with sufficient avidity for B cell receptors (BCRs) trigger signaling events that The most promising target for a universal influenza A vaccine is lead to B cell seeding of germinal centers (GCs). Here, B cells the conserved hemagglutinin (HA) stem domain. However, in proliferate and experience somatic BCR hypermutation and infected or immunized individuals, the response to HA stem is class-switch recombination. B cell clones with increased BCR limited due to HA head immunodominance. To understand avidity for immunogen are selected for proliferation and can factors hampering the development of stem B cells, we have differentiate into antibody-secreting plasma cells and memory immunized mice with full-length HA and HA stem alone or in B cells (7). different combinations and shown that physical attachment of Although Abs can potentially bind to all surfaces of immunogenic HA head to stem severely hinders stem responses. By increasing proteins, Ab responses focus on a limited number of immuno- local antigen concentration of full-length HA, we were able to dominant antigenic sites. This phenomenon, termed immunodomi- rescue stem-specific B cells and serum antibodies. Our results nance, is just now being defined and mechanistically dissected at the provide a template for improving vaccination by overcoming level of serum Abs and B cell responses (8). Two recent studies immunodominance. suggest that B cell precursor frequency and BCR avidity contribute to the subdominance of conserved HIV GP160 epitopes (9, 10). Author contributions: D.A., I.K., C.M.B., G.A., and J.W.Y. designed research; D.A., I.K., J.J.S.S., W.T.Y., C.M.B., V.V.A.M., M.C.M., M.C., and M.P. performed research; V.V.A.M., Other studies suggest, however, that after initial B cell seeding, GCs M.C., M.P., H.D.H., A.B.M., G.A., and J.C. contributed new reagents/analytic tools; D.A., are more permissive than previously thought, allowing B cells with I.K., J.J.S.S., W.T.Y., C.M.B., M.C.M., and J.W.Y. analyzed data; and D.A. and J.W.Y. wrote BCRs of even 100-fold differences in avidity to emerge from the the paper. same LN (11, 12). The contribution of these factors to HA stem The authors declare no conflict of interest. subdominance in primary responses and how stem-specific Abs can This article is a PNAS Direct Submission. be efficiently induced with vaccination in the context of full-length Published under the PNAS license. HA remain to be determined. Here, we mechanistically dissect de- 1To whom correspondence may be addressed. Email: [email protected] or jyewdell@ terminants of B cell immunodominance to HA, providing evidence niaid.nih.gov. that stem subdominance is due to competition between head and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stem naïve B cells that can be overcome by simply increasing im- 1073/pnas.1816300116/-/DCSupplemental. munogen delivery to the draining lymph node.

www.pnas.org/cgi/doi/10.1073/pnas.1816300116 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 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)Enu- meration 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.

unrelated protein or denatured HA elicited strong GC formation (SI Mixing Full-Length HA with Stem-Only Immunogen Affects the Quality Appendix,Fig.S1F) but no B cells specific to folded HA or stem (SI of the Serum Ab Responses. We next measured head- vs. stem-specific Appendix,Fig.S1G). To assess the contribution of N-linked glycans in serum Abs in four immunization groups (Fig. 2A and SI Appendix,Fig.S3 masking stem subdominance (since the stem-only immunogen is of bac- A and B). Stem was highly immunogenic as a standalone immunogen, but terial origin, it is not glycosylated), we removed glycans on full-length HA full-lengthHAfailedtoelicitastemresponsein11of12mice.StemAbs (SI Appendix,Fig.S2A) by endoglycosidase treatment. Despite increasing were not induced upon immunization with deglycosylated HA (SI Ap- accessibility of Abs to stem (SI Appendix,Fig.S2B), we still could not pendix,Fig.S2D). Mice generated an Ab response toward the His tag and/ detect stem-specific GC B cell responses (SI Appendix,Fig.S2C). or the foldon tag, albeit generally at much lower levels relative to the

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.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1816300116 Angeletti et al. Downloaded by guest on October 1, 2021 protection in mice acts via Fc-mediated functions (19), the Ab heavy- chain class is critical in anti-stem Ab-mediated protection. Re- markably, we found that head-specific Abs induced by HA immunization were biased to IgG1 while stem-induced Abs were IgG2-biased, a pattern that was maintained when immunizing with each immunogen in opposite legs (Fig. 2B). Importantly, however, stem Abs induced by HA/stem mixed immunization did not switch toward IgG2, providing an important potential caveat for mixing immunogens in a single site. Stem-induced sera were unable to neutralize PR8 in vitro (SI Appendix,Fig.S3G) (13, 19), despite binding cell-surface HA (SI Appendix,Fig.S3H). Rather, in vitro serum neutralization titers highly correlated with head-specific Ab ELISA titers (SI Appendix,Fig.S3I). We tested sera for Ab-dependent complement deposition (ADCD) activity and Ab-dependent cellular phagocytosis (ADCP) by primary monocytes (20). We found that stem-only immunization induced high levels of stem-dependent ADCD and ADCP, and that mixed immunization significantly reduced stem-mediated effector functions (Fig. 2 C and D). This revealed a positive correlation with IgG2 anti- stem titers but not total IgG titers (SI Appendix,Fig.S4A and B). When sera were tested on H5 HA-conjugated beads, ADCD activity mirrored the stem results (SI Appendix,Fig.S4C); however, H1 HA ADCD and ADCP results highlighted the important contribution of anti-head Abs (SI Appendix,Fig.S4D and E). Taken together, these findings indicate that mixing immunogens at the same immunization site or immunizing Fig. 3. Naïve B cell precursor frequency is similar between head and stem. at distant sites can influence the magnitude and quality of Ab responses. (A) Representative flow cytometry plot showing background stain of naïve B − − + cells (CD3 CD43 B220 ) on streptavidin (SA) conjugated to PE and APC and Stem Abs in Serum Are Durable and Not Influenced by Immunization 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 with Full-Length HA. Most of the human population has serum Abs − − + + low low high toward the variable head. We therefore wanted to test whether mice INFLAMMATION

follicular (FO) (CD3 CD43 B220 CD23 CD21 IgM IgD ) and mar- IMMUNOLOGY AND ginal zone (MZ) (CD3− CD43− B220+ CD23− CD21l+ IgMhigh IgDlow) B cells. with preexisting immunity to full-length HA would be able to mount Overlay shows IgM and IgD expression in FO vs. MZ B cells. (C) Precursor a sizable immune response after stem-only immunization. We also frequency as % of parent population for mature naïve, FO, and MZ B cells. wanted to determine the durability of stem-specific Ab responses (n = 8). Three independent experiments with 2 or 4 mice each. Bars represent after full-length HA challenge. Therefore, we primed mice with full- SEM; statistical analysis was performed using two-sided unpaired t test. length HA or stem constructs and boosted after 21 d with stem or full-length HA, respectively. In both cases, we obtained robust GC responses in the draining iliac LN, with the majority of B cells being SI Appendix C specific response to HA ( ,Fig.S3 ). Stem-induced Abs specific for the boosting protein (SI Appendix, Fig. S5A). Impor- bound to H5 HA (SI Appendix,Fig.S3D) and inhibited binding of two tantly, after stem priming and HA boosting, levels of stem-specific B well-characterized anti-stem mAbs (SI Appendix,Fig.S3E)(17,18).Ab cells in GCs were higher than after stem immunization. Remarkably, titers only partially correlated with GC B cell numbers (SI Appendix,Fig. stem Abs in serum were comparably high between the two groups (SI S3F), suggesting bottlenecks between GC B cells, plasmacell (PC) Appendix, Fig. S5B), albeit at a lower level compared with stem-only differentiation, and serum Ab production. Since anti-stem Ab-based immunization (see Fig. 2A for comparison). Taken together, these

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.

Angeletti et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 1, 2021 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.

data show that serum stem Abs are durable and that after stem C). Consistent with their higher avidity, the median fluorescence + priming, stem-specific B cells are present in the GC after boosting intensity of HA cells was significantly higher for head-specific B with full-length HA. cells (Fig. 4D). By 3 wk postboosting, the avidity of both head- and stem-specific GC B cells had increased 5- to 20-fold, as expected Head- and Stem-Specific Naïve B Cells Exhibit Comparable Frequencies. from affinity maturation (SI Appendix, Fig. S6). Why does the physical attachment of the head to the stem suppress Ab responses to the stem? We hypothesized that head immunodominance Modifying the Immunization Route Overcomes Stem Subdominance. results from sequestration of full-length HA by naïve head-specific B Previous studies suggested that GCs are more permissive in allowing cells, preventing stem-specific B cell activation due to differences in entry of B cells with a wide range of affinities (11, 12). Importantly, naïve B cell numbers or BCR avidity (8, 9). We first measured the these findings derived from footpad (f.p.) immunization, while we frequencies of head- and stem-specific naïve B cells. Using a method used i.m. immunization. To determine how the route of immunization developed for quantitating naïve T cells (21), we detected naïve HA- influences antigen concentration in the draining LN, we immunized specific B cells at low frequencies, despite their low avidity. HA-specific mice i.m. or f.p. with 10 μg R-phycoerythrin (PE) in adjuvant and 24 h B cells were detected by double staining with the same probe labeled later measured the amount of fluorescent PE present in LN extracts separately with different fluorophores (Fig. 3A). Double staining (Fig. 5A). Strikingly, after f.p. immunization, we could detect PE in allowed for the exclusion of naïve B cells specific for the fluorophores. 78% of the popliteal LNs and 61% of the iliac LNs tested, while after This revealed that HA and stem precursor frequencies are similar: i.m. immunization, PE was detectable only in 22% of the LNs analyzed ∼250/300 cells per million naïve mature B cells (Fig. 3 B and C), or (for both inguinal and iliac) (P = 0.0022; Fisher’s exact test). The ∼15,000 cells per mouse, given a naïve B cell population of ∼5 × 107 average amount of PE detected (with our limit of detection being cells (22). 0.1 ng/mL) in the popliteal LN after f.p. immunization was at least 14- and 30-fold higher, respectively, than in draining inguinal and Early Head-Specific GC B Cells Have Higher-Avidity BCRs. We next iliac LNs after i.m. immunization. We next immunized mice with measured GC B cell avidity at day 10 after full-length HA or stem virion-derived H1 HA either f.p. or i.m. (with a dose of 10 μg), or immunization using the AC50 method (23). This was the first day i.m. with a dose of 25 μg followed by an additional 50 μg on day 1 and postimmunization with sufficient expansion of GC B cells to identify on day 2, to increase immunogen concentration in the LN (repeated antigen-specific class-switched (swIg) and IgM cells in draining LNs i.m.). Three weeks postimmunization, GC frequencies in the iliac LN (Fig. 4A). Consistent with our prior findings (23), head-specific B were similar but nodal cellularity significantly expanded with increasing cells exhibited AC50 values in the low-nanomolar range. Critically, antigen dose, with the average number of GC B cells per LN compared the avidity of stem-specific B cells was ∼10-fold lower (Fig. 4 B and with i.m. immunization nearly doubling or tripling, respectively, with f.p.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1816300116 Angeletti et al. Downloaded by guest on October 1, 2021 or repeated i.m. immunization (SI Appendix,Fig.S7A–C). Interestingly, among the five major head antigenic sites (SI Appendix,Fig.S8B and + IgM GC B cells increased to nearly equal to switched Ig after repeated C). At the most extreme, Ca1-specific responses nearly doubled in f.p. i.m. immunization (SI Appendix,Fig.S7B). This suggests both sustained vs. i.m. immunization, consistent with our previous findings of route recruitment of B cells to the GC due to increased immunogen delivery dependence of immunodominance (16). Viral challenge studies with + and extended cycling of IgM GC B cells that are not able to receive heterologous virus, of animals immunized one single time, revealed a adequate signals from T follicular helper cells to exit the GC reaction. tendency toward higher protection after stem i.m. but no difference All immunization groups generated large numbers of head-specific between the other groups (SI Appendix,Fig.S8D), indicating the need + + IgG and IgM GC B cells on day 21. In contrast, i.m. immunization for further boosting to achieve protection. Finally, we determined the + + generated a low number of stem-specific IgG and IgM GC B cells and influence of f.p. vs. i.m. HA immunization on the ratio of T follicular undetectable stem-specific serum responses. F.p. immunization in- regulatory (Tfr) cells to T follicular helper (Tfh) cells, which has been creased stem-specific switched B cells 100-fold compared with i.m. (Fig. reported to regulate Ab responses (24). Although f.p. immunization 5 B and C and SI Appendix,Fig.S7D and E), comparable to stem-only generated higher frequencies of both Tfh and Tfr cells compared with immunization (Fig. 1C). Repeated i.m. immunization also increased i.m. (Fig. 6 C–F), the Tfr/Tfh ratio was doubled after i.m. immuni- + switched and IgM stem-specific B cells (Fig. 5 D and E). zation, suggesting an additional contribution to the suppression of We were unable to detect IgG or IgM anti-stem Abs in i.m. immu- stem-specific responses (Fig. 6D). nized mice (Fig. 5F). Footpad immunization generated stem-specific IgG and IgM serum responses in the ∼50% of mice with the highest Discussion levels of GC B cells (on average 20× more abundant than seronegative There is great hope for broad antiinfluenza vaccination based on mice; Fig. 5C). This is consistent with a functional threshold of GC inducing stem Abs, which cross-neutralize in vitro and cross-protect responses to generate sufficient numbers of plasma cells for a detectable animals. However, in humans, despite repeated seasonal infection serum Ab response. Based on a cell-surface phenotype by flow cytom- and immunization, anti-stem plasma cells are transient in blood and etry, head- and stem-specific GC B cells had a similar dark zone/light serum Abs only rarely achieve a likely functional threshold. Notably, zone distribution (Fig. 6 A and B). Consistent with the B cell analyses, anti-stem memory B cells seem to be common in humans but can repeated i.m. immunization elicited significant levels of H5 cross-reactive rarely outcompete anti-head memory B cells after vaccination (6). IgG in 3/12 mice, although 8/12 mice generated IgM responses. Although Interestingly, with the introduction of the H1N1 2009 pandemic, stem titers only partially correlated with H5 titers (SI Appendix,Fig.S8A), where the HA head was remarkably different from previous in- all H5 reactivity was removed by competition with anti-stem mAbs. Im- fluenza seasons, there was a more sustained reactivation of stem- munization route also shaped the GC B cell immunodominance hierarchy specific memory B cells (25). Here, we investigated the ability of

stem-only immunogens to elicit functional B cell and Ab responses INFLAMMATION and mechanistically dissected the subdominance of the stem in the IMMUNOLOGY AND context of full-length HA immunization. The stem-only construct we used elicited a robust anti-stem GC B cell and Ab response, suggesting that stem subdominance in HA is dependent on its physical attachment to the globular domain. Using deglycosylated HA, we show that the lack of glycosylation of the bacterially synthesized stem construct is not responsible for its greatly enhanced immunogenicity vs. stem in full-length HA (SI Appendix, Fig. S2). This is consistent with reports that stem immunogenicity is enhanced by blocking head epitopes with glycans (26) but not by removing stem glycans from intact HA (27). Although mixing stem with HA does not interfere with head-specific responses, it reduces stem-specific polyclonal titers 10-fold and inter- feres with class switching, decreasing anti-stem ADCD and ADCP. This suggests possible competition for help from T follicular cells (28) and follicular dendritic cells (29) essential for PC differentiation. Diminished immunogenicity from mixing of viral vaccines (30, 31) or following immunization in different sites (32) clearly occurs in humans, but had not previously been mechanistically dissected. We show that head- and stem-specific B cells are present at similar precursor frequencies and the major detectable difference lies in B cell avidity which, due to limitations in B cell numbers, we can first measure at 10 d postimmunization. Recent reports showed that responding GC B cells can secrete mAbs with 10- to 100-fold differences in avidity for immunogen (11, 12), which is at odds with our findings. This discrepancy may be due to differences in meth- Fig. 6. Footpad immunization does not shift stem GC B cells to DZ but alters odology in the studies. First, while we directly measure BCR avidity, Tfr/Tfh ratio. (A) Representative flow cytometry plot showing dark zone (DZ) the other studies use soluble mAbs. Therefore, BCR multivalency − + + − − − + + − (CD3 B220 GL7 CD38 IgD IgM IgG CXCR4 CD86 ) vs. light zone (LZ) − + + − − − + − + might contribute to the higher avidity we measure. Second, by im- (CD3 B220 GL7 CD38 IgD IgM IgG CXCR4 CD86 ) distribution of head- munizing with stem only, we allow stem B cells to affinity mature or stem-specific GC B cells at 11 d after f.p. immunization. (B) Quantification of without competition for T cell help, which may allow them to attain a = the frequency of head or stem GC B cells in the DZ (n 9). Two independent higher affinity. Further, a study shows that only higher-affinity pre- experiments with 5 and 4 mice each. Bars represent SEM; statistical analysis cursors can enter GCs for monomeric antigen (as the ones used was performed using two-way unpaired t test. (C) Gating strategy for T fol- − + + + here) and, even for multimeric antigen, a 30-fold naïve avidity licular (Tf) cell population (B220 CD4 CXCR5 PD1 ) and further classification into T follicular helper (Tfh) and T follicular regulatory (Tfr) cells based on FoxP3 difference was sufficient to exclude low-avidity B cells from GCs + + + expression. (D) Quantification of Tfr contribution to total Tf (CD4 CXCR5 PD1 ) (9). Finally, we cannot completely exclude the influence of compe- (Tfr/Tfh ratio) in draining LNs (iliac for i.m. and popliteal for f.p.) 8 d post- tition events at early GC stages, related to competition for limited immunization. (E and F)Tfh(E)andTfr(F) expressed as frequency of CD4+ T cells T cell help. (n = 10 for i.m.; n = 7 for f.p.). Two independent experiments with 5 mice each While elegant previous studies used transgenic mice to evaluate for i.m. and with 4 and 3 mice for f.p. immunization. Bars represent mean ± SEM; the contribution of B cell precursor frequency and avidity to B cell statistical analysis was performed using two-sided unpaired t test. *P < 0.05, maturation (9, 10), they did not measure serum Abs, the key func- **P < 0.01, ****P < 0.0001. tional output of the humoral immune response. Here, after footpad

Angeletti et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 1, 2021 immunization, we show that while all of the mice demonstrated a PR8 (14) were used for the initial immunization studies. Groups 1 and 4 sizable stem-specific GC B cell population, not all exhibited de- received 10 μgofHA(0.8μM) in the left hind leg, group 2 received 4 μgof tectable levels of stem Abs in sera. In mice, we could bypass affinity- stem (0.8 μM) in the left hind leg, while group 4 received the same amount based head immunodominance by immunizing in a small anatomic in the right hind leg. Group 3 received an equimolar mixture of the two space (the footpad) draining to a single lymph node. In humans, proteins in the left hind leg. Other animals were immunized i.m. or via the practical realities would dictate either single immunization with a hock f.p. with 10 μg of virion-derived HA (0.8 μM) mixed with adjuvant. For subdominant immunogen, immunization in different sites, or engi- repeated i.m. immunization, animals were immunized i.m. with 25 μgofHA neering subdominant immunogens to increase LN delivery, either by with adjuvant on day 0, followed by 50 μg of HA without adjuvant on day 1 modifying delivery (e.g., needle patches or slow-release vaccines) or and 50 μg of HA without adjuvant on day 2. attaching immunogens to particles that enhance delivery to B cell areas of draining LNs (33). Statistical Analysis. Prism (GraphPad Software) was used for statistical anal- In summary, our findings demonstrate that the route of immuni- ysis. For comparison between two groups, two-sided unpaired Student’s t test zation governs the immunodominance of the head vs. the stem re- was performed. For comparison of one variable between multiple groups, gion. Stem subdominance is driven by differential B cell affinity one-way ANOVA with Tukey’s multiple comparison test was performed. For under conditions of limiting immunogen. To circumvent immuno- comparison between multiple variables across multiple groups, two-way domination, immunizing with a subdominant protein is a promising ANOVA with Holm–Sidak’s multiple comparison test was performed. When approach for universal vaccination. data did not pass the normality test, they were log-transformed. For all fig- ures, data points indicate individual mice. *P < 0.05, **P < 0.01, ***P < 0.001, Materials and Methods ****P < 0.0001. Detailed methods can be found in SI Appendix, Text. ACKNOWLEDGMENTS. We thank the NIAID Comparative Medicine Branch Animals. C57BL/6 mice were purchased from Taconic Farms. For all experi- for maintaining the mice used in this study, and Raghavan Varadarajan (Molecular Biophysics Unit, Indian Institute of Science) for assistance with ments, female 8- to 12-wk-old mice were used and randomly assigned to stem protein design and production. This work is supported by the Division experimental groups. All mice were held under specific pathogen-free con- of Intramural Research, National Institute of Allergy and Infectious Diseases. ditions. All animal procedures were approved and performed in accordance D.A. was partially supported by a grant from the Swedish Research Council with National Institute of Allergy and Infectious Diseases (NIAID) Animal Care (Vetenskapsrådet 2017-01439) and the Institute of Biomedicine at the Univer- and Use Committee guidelines. sity of Gothenburg. W.T.Y. was supported by a Special Fellow award from the Leukemia & Lymphoma Society and an NIH T32 training grant (CA009149). J.C. Proteins and Immunization. Recombinant A/Puerto Rico/8/34 (PR8) HA with the was supported by grants from the National Institutes of Health (1R01AI072194, Y98F mutation (18) and H1HA10-foldon stem-only construct derived from 1R01AI124186, and P30CA008748).

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