Angeletti et al., 'Defining B cell immunodominance to viruses' (Nature Immunology 18:456-463, 2017), established reproducible but dynamic antibody hierarchies across the five antigenic sites of the influenza A virus hemagglutinin (HA) head in mice and left four interlocking open issues in its Discussion: (i) how much of the inter-strain and inter-individual variability in the hierarchy (C57BL/6 versus BALB/c mice; humans) reflects germline immunoglobulin gene differences versus other host genetic or environmental factors such as the microbiota; (ii) the structural and steric rules by which epitope position on the HA trimer dictates the neutralization efficiency of polyclonal antibodies (hemagglutination-inhibition and virus-neutralization activity per unit binding titer), including the unexplained divergence between HI and VN activity at one site; (iii) the quantitative rules by which the route, order and timing of repeated exposures combine to produce memory recall dominance (original antigenic sin) versus antibody-mediated suppression of previously targeted sites, and whether the strength of original antigenic sin is governed by the balance between circulating antibodies and their cognate memory B cells; and (iv) whether deliberately masking immunodominant head epitopes with antibody or antibody-like blocking molecules can focus responses onto other domains — in particular the conserved HA stem — beyond the single-immunogen setting in which the source demonstrated such focusing.

Background

B cell immunodominance and its measurement in the influenza system. When a host is infected or vaccinated with a complex antigen such as a virus, the antibody response is not spread evenly over the many available epitopes (the molecular surfaces recognized by antibodies): it focuses hierarchically on a subset, a phenomenon called immunodominance (ID). ID was studied for decades for CD8+ T cells, but B cell and antibody ID became a central vaccinology problem once 'universal' vaccines against antigenically variable viruses — influenza A virus (IAV) and HIV-1 — were conceived around conserved epitopes: such vaccines must overcome the immune system's tendency to focus on variable, immunodominant epitopes.

Influenza A virus hemagglutinin (HA) is the main target of infection-blocking antibodies. HA is a trimer; each subunit has a membrane-distal globular head that binds sialic-acid-containing receptors on host cells and a conserved stem (stalk) domain that drives membrane fusion. For H1-subtype HAs the head carries five classically defined antigenic sites (Sa, Sb, Ca1, Ca2, Cb), originally mapped by monoclonal antibody (mAb) escape selection in the laboratories of Gerhard and Caton. The head sites mutate rapidly ('antigenic drift') and dominate the response; the conserved stem is the target of broadly neutralizing antibodies but is immunologically subdominant. Standard readouts: ELISA measures total binding antibody irrespective of function; the hemagglutination-inhibition (HI) assay measures the capacity of serum antibodies to block HA-mediated agglutination of red blood cells and is the licensed-vaccine correlate of protection; virus-neutralization (VN) assays measure blockage of infection in culture. HI or VN activity per unit ELISA titer ('specific activity') differs strongly between antibodies to different antigenic sites even at similar binding affinity — the puzzle at the heart of one open question below. The antibodies that mediate long-term humoral immunity come from B cells that enter germinal centers (GCs), the microanatomical sites where B cells proliferate and mutate their immunoglobulin (Ig) genes to raise antigen affinity (affinity maturation), with help from T follicular helper (Tfh) cells.

The source study. Angeletti et al. (2017) quantified site-specific responses using 'Δ4' mutant PR8 (A/Puerto Rico/8/1934, H1N1) viruses: each mutant had been selected with mAbs so that four of the five head sites carried escape mutations and only one retained native antigenicity, allowing the response to each site to be read from serum panels. They found a highly reproducible but dynamic hierarchy in C57BL/6 mice (the Cb site dominated early after intranasal infection, with Sb, Sa and Ca2 rising over weeks; Sa dominated after intramuscular immunization with adjuvant), a different early hierarchy in BALB/c mice, hierarchy changes with route and antigen formulation, and no effect of CD4+ T cell depletion on the hierarchy's shape. Site-specific polyclonal sera showed very different functional specific activities (Sa high, Cb low in HI and VN) that could not be attributed to antibody affinity, and Sb-specific antibodies showed high VN but low HI activity, unexplained by existing mAb data. Passive antibody transfer and sequential infection produced 'original antigenic suppression': antibodies selectively suppressed memory responses to the antigenic site they bound — the mirror image of classical original antigenic sin (OAS), the doctrine articulated by Francis in 1960 that exposure to a new antigenic variant preferentially recalls antibody memory for the epitopes of the priming virus.

The open questions. The paper's discussion closed with four interlocking issues that together define its program: (1) the mechanistic basis of inter-individual and inter-strain variability in the hierarchy — specifically how much of the C57BL/6 versus BALB/c difference reflects differences in the Ig genes encoding the B cell repertoire versus other host genetic or epigenetic factors such as the microbiota, a question the authors proposed to attack with collaborative cross mice (a genetically diverse mouse reference population derived from eight founder strains); (2) the structural and steric rules by which epitope position on the HA trimer determines neutralization efficiency — the authors' 'simple explanation', proximity of an epitope to the sialic-acid receptor binding site, was unproven, and the Sb HI/VN discrepancy unresolved; (3) the quantitative rules governing whether repeated exposures produce recall dominance (OAS) or antibody-mediated suppression — the paper noted that administration route, order of virus administration and timing all play a key role in OAS induction, and hypothesized that the strength of OAS 'may be governed by the balance of Abs versus their cognate memory B cells', requiring systematic, longitudinal, multi-exposure experiments; and (4) the generality of the paper's concluding finding that, 'when using a single immunogen, responses can be focused on various domains by including the appropriate antibody or antibody like molecule that sterically prevents Ab access to a given antigenic site if relevant memory cells are present' — deliberate focusing whose extension to the conserved stem, the target the paper's Introduction identifies for universal vaccines based on conserved regions, and to settings beyond a single immunogen, was not established (neither of the paper's masking approaches induced detectable anti-stem titers). Ndifon, Wingreen and Levin had already argued mathematically in 2009 that HA epitopes differ intrinsically in neutralization efficiency because of steric interference between bound antibodies, giving the structural question a theoretical frame.

Problem Statement

What defines — and to what extent can one deliberately set — the B cell immunodominance hierarchy to influenza A virus hemagglutinin? Building on the reproducible, dynamic, antigenic-site-specific hierarchies established by Angeletti et al. (2017), the source's Discussion leaves four interlocking open issues, stated here as the source left them:

  1. Determinants of inter-individual variability: attribute, mechanistically and quantitatively, the differences in antigenic-site hierarchies between individuals and strains (C57BL/6 versus BALB/c mice; the predicted variability among humans) to specific causes — differences in the germline immunoglobulin genes encoding the B cell repertoire versus other host genetic factors and environmental or epigenetic factors such as the microbiota.

  2. Structural rules of site-specific antibody function: establish or refute the structural and steric rules by which epitope position on the HA trimer determines the functional activity of polyclonal antibody responses — hemagglutination-inhibition and virus-neutralization activity per unit binding titer — including the source's proximity-to-sialic-acid-receptor-binding-site hypothesis and the unexplained divergence between HI and VN activity observed for Sb-specific polyclonal antibodies.

  3. Quantitative multi-exposure rules: determine how the route, order and timing of successive exposures combine to produce memory recall dominance (original antigenic sin) versus antibody-mediated suppression of responses to previously targeted antigenic sites, testing the source's hypothesis that the strength of original antigenic sin is governed by the balance between circulating antibody levels and the numbers of their cognate memory B cells — high antibody levels suppressing responses, low levels allowing memory B cells to dominate naive B cells.

  4. Controlled redirection: determine whether, and under what conditions, deliberately masking immunodominant head antigenic sites with antibody or antibody-like blocking molecules can focus responses onto other domains — in particular the conserved HA stem, the target of broadly neutralizing antibodies — beyond the single-immunogen demonstration of the source, in which it required that the relevant memory cells be present.

These issues share the framing with which the source opens its Discussion: 'The ability to rationally focus vaccine-induced Ab responses onto targeted antigenic sites requires deeper understanding of Ab immunodominance.' The objective uniting them is therefore a mechanistic account of what sets the anti-HA immunodominance hierarchy and how it can be deliberately directed.

References

  1. Angeletti D, Gibbs JS, Angel M, Kosik I, Hickman HD, Frank GM, Das SR, Wheatley AK, Prabhakaran M, Leggat DJ, McDermott AB, Yewdell JW. Defining B cell immunodominance to viruses. Nature Immunology 18(4):456-463 (2017). DOI: 10.1038/ni.3680. https://doi.org/10.1038/ni.3680 (source paper)
  2. Angeletti D, Yewdell JW. Understanding and manipulating viral immunity: antibody immunodominance enters center stage. Trends in Immunology 39(7):549-561 (2018). DOI: 10.1016/j.it.2018.04.008. https://doi.org/10.1016/j.it.2018.04.008
  3. Liu STH, Behzadi MA, Sun W, Freyn AW, Liu WC, Broecker F, Albrecht RA, Bouvier NM, Simon V, Nachbagauer R, Krammer F, Palese P. Antigenic sites in influenza H1 hemagglutinin display species-specific immunodominance. Journal of Clinical Investigation 128(11):4992-4996 (2018). DOI: 10.1172/JCI122895. https://doi.org/10.1172/JCI122895
  4. Broecker F, Liu STH, Sun W, Krammer F, Simon V, Palese P. Immunodominance of antigenic site B in the hemagglutinin of the current H3N2 influenza virus in humans and mice. Journal of Virology 92(20):e01100-18 (2018). DOI: 10.1128/JVI.01100-18. https://doi.org/10.1128/JVI.01100-18
  5. Sánchez-de Prada L, Sanz-Muñoz I, de Lejarazu RO, Eiros JM, García-Sastre A, Aydillo T. Immunodominance hierarchy after seasonal influenza vaccination. Emerging Microbes & Infections 11(1):2670-2679 (2022). DOI: 10.1080/22221751.2022.2135460. https://doi.org/10.1080/22221751.2022.2135460
  6. Paparoditis PCG, Fruehwirth A, Bevc K, Low JS, Jerak J, Terzaghi L, Foglierini M, Fernandez B, Jarrossay D, Corti D, Sallusto F, Lanzavecchia A, Cassotta A. Site-specific serology unveils cross-reactive monoclonal antibodies targeting influenza A hemagglutinin epitopes. European Journal of Immunology 54(10):e2451045 (2024). DOI: 10.1002/eji.202451045. https://doi.org/10.1002/eji.202451045
  7. Noll KE, Whitmore AC, West A, McCarthy MK, Morrison CR, Plante KS, Hampton BK, Kollmus H, Pilzner C, Leist SR, Gralinski LE, Menachery VD, Schäfer A, Miller D, Shaw G, Mooney M, McWeeney S, Pardo-Manuel de Villena F, Schughart K, Morrison TE, Baric RS, Ferris MT, Heise MT. Complex genetic architecture underlies regulation of influenza-A-virus-specific antibody responses in the collaborative cross. Cell Reports 31(4):107587 (2020). DOI: 10.1016/j.celrep.2020.107587. https://doi.org/10.1016/j.celrep.2020.107587
  8. Vieira MC, Palm AE, Stamper CT, Tepora ME, Nguyen KD, Pham TD, Boyd SD, Wilson PC, Cobey S. Germline-encoded specificities and the predictability of the B cell response. PLoS Pathogens 19(8):e1011603 (2023). DOI: 10.1371/journal.ppat.1011603. https://doi.org/10.1371/journal.ppat.1011603
  9. Sangesland M, Li N, Tsybovsky Y, Rodgers MD, Han J, Rodriguez AJ, Ferguson JA, Henry AR, Smith SC, Roberts-Torres J, Gillespie RA, Liu C, Merriam JS, Stephens T, Williams C, Walker E, Corcoran M, Ravichandran M, Creanga A, Andrews SF, Pierson TC, Karlsson Hedestam GB, Schramm CA, Reed DS, Douek DC, Zhou T, Ward AB, Kanekiyo M. Functional, immunogenetic, and structural convergence in influenza immunity between humans and macaques. Science Translational Medicine 17(815):eady3570 (2025). DOI: 10.1126/scitranslmed.ady3570. https://doi.org/10.1126/scitranslmed.ady3570
  10. Ndifon W, Wingreen NS, Levin SA. Differential neutralization efficiency of hemagglutinin epitopes, antibody interference, and the design of influenza vaccines. Proceedings of the National Academy of Sciences USA 106(21):8701-8706 (2009). DOI: 10.1073/pnas.0903427106. https://doi.org/10.1073/pnas.0903427106
  11. Williams JA, Gui L, Hom N, Mileant A, Lee KK. Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments. Journal of Virology 92(6):e02006-17 (2018). DOI: 10.1128/JVI.02006-17. https://doi.org/10.1128/JVI.02006-17
  12. Zost SJ, Wu NC, Hensley SE, Wilson IA. Immunodominance and antigenic variation of influenza virus hemagglutinin: implications for design of universal vaccine immunogens. Journal of Infectious Diseases 219(Suppl 1):S38-S45 (2019). DOI: 10.1093/infdis/jiy696. https://doi.org/10.1093/infdis/jiy696
  13. Linnik J, Syedbasha M, Hollenstein Y, Halter J, Egli A, Stelling J. Model-based inference of neutralizing antibody avidities against influenza virus. PLoS Pathogens 18(1):e1010243 (2022). DOI: 10.1371/journal.ppat.1010243. https://doi.org/10.1371/journal.ppat.1010243
  14. Zarnitsyna VI, Lavine J, Ellebedy A, Ahmed R, Antia R. Multi-epitope models explain how pre-existing antibodies affect the generation of broadly protective responses to influenza. PLoS Pathogens 12(6):e1005692 (2016). DOI: 10.1371/journal.ppat.1005692. https://doi.org/10.1371/journal.ppat.1005692
  15. Bergström JJ, Xu H, Heyman B. Epitope-specific suppression of IgG responses by passively administered specific IgG: evidence of epitope masking. Frontiers in Immunology 8:238 (2017). DOI: 10.3389/fimmu.2017.00238. https://doi.org/10.3389/fimmu.2017.00238
  16. Goodwin E, Gibbs JS, Yewdell JW, Eisenlohr LC, Hensley SE. Influenza virus antibodies inhibit antigen-specific de novo B cell responses in mice. Journal of Virology 98(9):e0076624 (2024). DOI: 10.1128/jvi.00766-24. https://doi.org/10.1128/jvi.00766-24
  17. Mesin L, Schiepers A, Ersching J, Barbulescu A, Cavazzoni CB, Angelini A, Okada T, Kurosaki T, Victora GD. Restricted clonality and limited germinal center reentry characterize memory B cell reactivation by boosting. Cell 180(1):92-106.e11 (2020). DOI: 10.1016/j.cell.2019.11.032. https://doi.org/10.1016/j.cell.2019.11.032
  18. Schiepers A, van 't Wout MFL, Greaney AJ, Zang T, Muramatsu H, Lin PJC, Tam YK, Mesin L, Starr TN, Bieniasz PD, Pardi N, Bloom JD, Victora GD. Molecular fate-mapping of serum antibody responses to repeat immunization. Nature 615(7952):482-489 (2023). DOI: 10.1038/s41586-023-05715-3. https://doi.org/10.1038/s41586-023-05715-3
  19. Schiepers A, Van't Wout MFL, Hobbs A, Mesin L, Victora GD. Opposing effects of pre-existing antibody and memory T cell help on the dynamics of recall germinal centers. Immunity 57(7):1618-1628.e4 (2024). DOI: 10.1016/j.immuni.2024.05.009. https://doi.org/10.1016/j.immuni.2024.05.009
  20. Tas JMH, Koo JH, Lin YC, Xie Z, Steichen JM, Jackson AM, Hauser BM, Wang X, Cottrell CA, Torres JL, Warner JE, Kirsch KH, Weldon SR, Groschel B, Nogal B, Ozorowski G, Bangaru S, Phelps N, Adachi Y, Eskandarzadeh S, Kubitz M, Burton DR, Lingwood D, Schmidt AG, Nair U, Ward AB, Schief WR, Batista FD. Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses. Immunity 55(10):1856-1871.e6 (2022). DOI: 10.1016/j.immuni.2022.07.020. https://doi.org/10.1016/j.immuni.2022.07.020
  21. Barbulescu A, Bilanovic J, Langelaar T, Teetz AK, Urnavicius L, Hobbs A, Shen JJ, Abrahamse NH, de Carvalho RVH, Mesin L, Ferreira CL, Bortolatto J, Victora GD. Antibody-mediated feedback modulates interclonal competition in the germinal center. Immunity 59(3):734-745.e5 (2026). DOI: 10.1016/j.immuni.2026.01.008. https://doi.org/10.1016/j.immuni.2026.01.008 (preprint: bioRxiv 2025.11.06.686519)
  22. Kosikova M, Li L, Radvak P, Ye Z, Wan XF, Xie H. Imprinting of repeated influenza A/H3 exposures on antibody quantity and antibody quality: implications for seasonal vaccine strain selection and vaccine performance. Clinical Infectious Diseases 67(10):1523-1532 (2018). DOI: 10.1093/cid/ciy327. https://doi.org/10.1093/cid/ciy327
  23. Arevalo P, McLean HQ, Belongia EA, Cobey S. Earliest infections predict the age distribution of seasonal influenza A cases. eLife 9:e50060 (2020). DOI: 10.7554/eLife.50060. https://doi.org/10.7554/eLife.50060
  24. Meade P, Kuan G, Strohmeier S, Maier HE, Amanat F, Balmaseda A, Ito K, Kirkpatrick E, Javier A, Gresh L, Nachbagauer R, Gordon A, Krammer F. Influenza virus infection induces a narrow antibody response in children but a broad recall response in adults. mBio 11(1):e03243-19 (2020). DOI: 10.1128/mBio.03243-19. https://doi.org/10.1128/mBio.03243-19
  25. Lee J, Paparoditis P, Horton AP, Frühwirth A, McDaniel JR, Jung J, Boutz DR, Hussein DA, Tanno Y, Pappas L, Ippolito GC, Corti D, Lanzavecchia A, Georgiou G. Persistent antibody clonotypes dominate the serum response to influenza over multiple years and repeated vaccinations. Cell Host & Microbe 25(3):367-376.e5 (2019). DOI: 10.1016/j.chom.2019.01.010. https://doi.org/10.1016/j.chom.2019.01.010
  26. Fox A, Zhu Z, Sánchez-Ovando S. The dual role of preexisting immunity in influenza: protective recall versus constraint of novel responses. Immunological Reviews 341(1):e70146 (2026). DOI: 10.1111/imr.70146. https://doi.org/10.1111/imr.70146
  27. Tan HX, Jegaskanda S, Juno JA, Esterbauer R, Wong J, Kelly HG, Liu Y, Tilmanis D, Hurt AC, Yewdell JW, Kent SJ, Wheatley AK. Subdominance and poor intrinsic immunogenicity limit humoral immunity targeting influenza HA stem. Journal of Clinical Investigation 129(2):850-862 (2019). DOI: 10.1172/JCI123366. https://doi.org/10.1172/JCI123366
  28. Kim D, Loeffler K, Hu Y, Arsiwala A, Frey S, Murali S, Hariharan V, Moreno A, Kane RS. Tethered antigenic suppression shields the hemagglutinin head domain and refocuses the antibody response to the stalk domain. Chemistry 7(1):12 (2025). DOI: 10.3390/chemistry7010012. https://doi.org/10.3390/chemistry7010012
  29. Folschweiller N, Vanden Abeele C, Chu L, Van Damme P, García-Sastre A, Krammer F, Nachbagauer R, Palese P, Solórzano A, Bi D, David MP, Friel D, Innis BL, Koch J, Mallett CP, Rouxel RN, Salaun B, Vantomme V, Verheust C, Struyf F. Reactogenicity, safety, and immunogenicity of chimeric haemagglutinin influenza split-virion vaccines, adjuvanted with AS01 or AS03 or non-adjuvanted: a phase 1-2 randomised controlled trial. Lancet Infectious Diseases 22(7):1062-1075 (2022). DOI: 10.1016/S1473-3099(22)00024-X. https://doi.org/10.1016/S1473-3099(22)00024-X
  30. Abbott RK, Crotty S. Factors in B cell competition and immunodominance. Immunological Reviews 296(1):120-131 (2020). DOI: 10.1111/imr.12861. https://doi.org/10.1111/imr.12861
  31. Lindahl G. Subdominance in antibody responses: implications for vaccine development. Microbiology and Molecular Biology Reviews 85(1):e00078-20 (2020). DOI: 10.1128/MMBR.00078-20. https://doi.org/10.1128/MMBR.00078-20