Antibodies, Memory B Cells, and Antigen Valency Reshape B Cell Responses to Drifted Influenza Virus Vaccination

influenza A virus; vaccination; germinal center; antibodies; memory B cells 19 20 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 2

21 Antigenic drift in influenza A virus hemagglutinin (HA) limits humoral protective immunity. 22 Here, we combine cell fate mapping with adoptive transfer of antigenic-site-specific antibodies 23 (Abs) and memory B cells (MBCs) with moderately drifted HA vaccination in mice to better 24 understand how this influences immune escape and protective responses. We demonstrate that 25 drift in vaccine antigen s affects MBC reactivation and naïve B cell responses in germinal 26 centers (GC) . Strikingly, passively transferred monoclonal and polyclonal Abs suppress 27 cognate epitope-specific GC B cell responses only when the vaccine antigen was multivalent 28 while responses to monovalent recombinant trimeric HA remain unaffected. Using MBC and 29 Abs co-transfer we unveil that antigenic site-specific suppression is more potent in blocking 30 MBC rather than naïve B cells entry into GC. In addition, we show that MBC hamper naïve B 31 cell recruitment to GC even in the absence of antibody transfer through local differentiation 32 and Ab release in the responding lymph node . Altogether, our study reveals that serum Ab 33 feedback depends on vaccine valency, while pre-existing MBC alone without Abs present can 34 reshape immunodominance of naïve B cells, with critical practical implications for rational 35 universal influenza vaccine design. 36

37 Human influenza A viruses (IA V) exhibit rapid antigenic evolution in their hemagglutinin (HA) 38 and neuraminidase (NA) virion surface glycoproteins. These “drifted” variants emerge to evade 39 existing neutralizing antibodies (Abs) induced by prior infection and vaccination 1, 2. Often, a 40 few mutations in critical sites of HA are suffi cient to evade protection, with an average of 41 0.0011 mutations per antigenic site per year3. Memory B cells (MBC) are often better equipped 42 than Abs to deal with drifted variants, owing to their wider B cell receptor (BCR) diversity than 43 Ab-secreting plasma cells 4, 5, 6 . Despite many studies, it has not been fully elucidated how 44 drifted viruses and vaccines, pre-existing Abs, and MBC, interact to reshape humoral immunity 45 at detailed epitope resolution . This is a central question in the quest for universal influenza 46 vaccination 7, 8. 47 After infection/vaccination, MBC can arise both from the rapid extrafollicular (EF) response 48 or the slower germinal center (GC) reaction 6, 9, 10 . Antigen-specific GC B cells experience 49 several rounds of BCR diversification and selection , ultimately differentiating into e ither 50 plasma cells (PC) or MBC 11. After exiting the GC, MBC either circulate or reside in infected 51 tissues: the respiratory tract in the case of IA V 8. Upon antigen re-encounter, MBC either enter 52 a secondary GC or quickly differentiate into antibody -secreting cells (ASC) 11, 12 . A better 53 understanding of factors affecting MBC fate decision upon antigen rechallenge will facilitate 54 designing viral vaccines that optimize MBC differentiation. 55 The fate of MBC during recall responses depends on many cell intrinsic factors, including 56 expression of specific surface molecules , antibody heavy chain class , and BCR affinity 57 dependent on somatic mutations introduced into the Ab variable region 12, 13, 14, 15, 16, 17, 18, 19, 20 . 58 Extrinsic factors, including the amount and specificity of pre-existing Abs and CD4 T cells as 59 well as antigenic distance between the priming and challenging antigens, also contribute to 60 MBC fate choice 21, 22, 23, 24, 25 . For instance, greater antigenic distance and more memory CD4 61 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 3 T cells increased MBC reentry into secondary GC, while pre-existing Abs favored ASC 62 generation. In addition, monoclonal Abs (mAbs) have been shown to influence de novo B cell 63 responses by suppressing differentiation of B cells specific for the mAb´s cognate antigenic site 64 and thereby instead promoting B cell responses towards non-cognate antigenic sites 24, 25, 26, 27, 65 28, 29, 30. 66 Most studies that have addressed these topics relied on model antigens with few antigenic 67 epitopes, transfer of mAbs, knock-in BCR mice with fixed antigen specificity or a combination 68 of them. How immune responses are affected in a more physiological setting where a drifted 69 antigen is used to challenge a host with a preexisting polyclonal Ab and/or MBC has not been 70 tested. 71 For all but unvaccinated children under three years of age 31, human IA V infection/vaccination 72 confronts a polyclonal Ab and MBC repertoire that dictates B cell responses 32 33. The majority 73 of protective Abs are specific for HA 29. For the H1 subtype of HA, most Abs bind five 74 overlapping canonical antigenic sites within the HA head (namely Sa, Sb, Ca1, Ca2 and Cb ; 75 Figure 1A) 34, 35, 36 . These are targeted hierarchically after intranasal ( i.n.) infection or 76 vaccination, with the early response focusing on Cb and the later responses on Sb 29. Abs to 77 non-canonical (n.c.) antigenic sites, including the receptor binding site (RBS) 37 or the stem 38, 78 are usually cross-reactive between IA V strains but are immunosubdominant in animals and 79 humans 2, 29, 39, 40, 41, 42 . Intriguingly, several studies revealed a polyclonal Ab response highly 80 focused on a single epitope, in approximately a fifth of individuals 43, 44, 45, 46 : serum Ab 81 recognition of HA in these people is easily abrogated by a single amino substitution in one of 82 the canonical antigenic sites. It is easy to imagine scenarios where polyclonal Ab responses are 83 more focused toward a single antigenic site while MBC target a wider array of epitopes , 84 reflecting the repertoire diversity between PC and MBC 4, 5, 6. In this case, how will the recall 85 response be influenced ? Will MBC reactivation and naïve B cells recruitment into GC be 86 impacted differently ? And how will it contribute to the phenomenon known as original 87 antigenic sin 47, the boosting of pre -existing antibodies weak ly cross-reactive with the 88 challenging immunogen? 89 Here, we use cell fate tracking models, adoptive transfers of polyclonal Abs and MBC, and a 90 panel of drifted mutants of IA V A/Puerto Rico/8/1934 (PR8) HA to dissect how pre-existing 91 humoral immunity redirects memory and de novo B cell responses upon IA V vaccination. 92

93 Antigen-specific MBC re-entry into secondary germinal centers is de pendent on drift of 94 vaccine antigen. 95 To track the fate of antigenic site-specific cells, we used a panel of escape mutant viruses and 96 corresponding HA based on the wildtype HA (wt) from the PR8 H1N1 influenza strain 29. Each 97 “D4 mutant” expresses just one of the five original canonical antigenic sites (Error! Reference 98 source not found.A). We focused on Cb and Sb antigenic sites as they represent early and late 99 immunodominant sites after infection that are spatially oriented to minimize steric hindrance 100 of simultaneous Ab binding 29. In addition, we used S12 as a fully drifted antigen that is poorly 101 recognized by anti-wt HA sera 48. The mutants used in this work represent a timeframe of two 102 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 4 to four influenza seasons 49, corresponding to an antigenic drift of up to 2.1% or £12 amino 103 acids (aa) (Error! Reference source not found.A) 29, a likely time frame for an individual to 104 be reinfected. These represent an improvement of the D4 panel used in one of our prior studies 105 29, as we have optimized the panel to abolish residual Ab binding to a cryptic epitope present 106 in the Sb site defined by residues 189 and 198 (H1 numbering)50. 107 We first investigated the ability of drifted vaccines to reactivate a ntigenic-site specific MBC 108 elicited by a prior infection. We fate mapped GC B cells and their progeny generated during 109 the primary infection using S1pr2CreERT2-R26tdTomato mice (S1pr2-Tomato) 51 infected i.n. with 110 wt virus and treated with tamoxifen every second day after infection until rechallenge, to 111 irreversibly label GC B cells and their progeny (Figure 1B). At 28dpi, mice were vaccinated 112 with 10 µg recombinant HA vaccine (rHA) in the left footpad, thereby anatomically separating 113 the draining lymph nodes (dLN) targeted by the primary infection (the mediastinal lymph node, 114 medLN) and the secondary vaccination (the popliteal lymph node, pLN) (Figure 1B) . The 115 vaccine antigens used were either wt, Cb D4, Sb D4 or S12. Importantly, previous work has 116 primarily investigated prime-boost vaccination but rarely infection-induced priming, which is 117 physiologically relevant in most individuals 21, 22, 23. 118 Despite the relatively small changes between wt and D4 HAs, the total number of GC B cells 119 in the pLN was significantly lower after a homologous wt rHA challenge than Δ4 or S12 rHA 120 challenges (Figure 1C; Supplementary Figure 1B). This is consistent with previous prime and 121 boost studies, where h omologous vaccination generates small secondary GCs and low MBC 122 re-entry 21, 22, 23 . The increased GC response correlated with a greater total number but not 123 frequency of HA wt-binding (wt HA+) B cells in the response to drifted vs. wt rHA antigen 124 (Figure 1D; Supplementary Figure 1D), even though the analysis excluded naïve B cells 125 responding to drifted antigenic sites. Most wt HA+ cells exhibited a GC phenotype, especially 126 when mice were vaccinated using a drifted rHA (Supplementary Figure 1E), suggesting some 127 level of Ab -mediated feedback when antigens were more similar. While total Tomato+ fate 128 mapped MBC represented only 1-2% of vaccination-induced secondary GC B cells , in 129 agreement with previous results 21 (Figure 1E and Supplementary Figure 1 F), among wt HA+ 130 GC B cells the frequency of previously generated MBC re-entering the GC was much higher, 131 depending on vaccine antigen (Figure 1E and Supplementary Figure 1F). 132 These findings indicate that heterologous vaccinations with antigenically distant antigens 133 (SbD4 and S12) are better at inducing de novo responses, while homologous and less drifted 134 vaccines (wt PR8 and Cb D4) are more efficient in recalling infection -induced MBC to 135 secondary GCs. As expected, when analyzing ASC, the majority of wt HA+ plasmablasts within 136 the pLN were fate-mapped, regardless of challenging antigen (Figure 1F; Supplementary 137 Figure 1F), in accordance with a model where MBC can rapidly differentiate to ASC to produce 138 Abs 22. 139 Vaccine antigen dictates the immunodominance pattern of recalled and de novo B cell 140 responses. 141 To determine the antigenic site specificity of responding B cells , we employed simultaneous 142 D4 rHA staining with each probe bearing a different fluorophore (Figure 2A and Supplementary 143 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 5 Figure 1B). After a multistep gating strategy to identify the B cell subpopulation of interest 144 (Supplementary Figure 1B), two wt HA probes, conjugated to two distinct fluorochromes were 145 used to m inimize spurious binding. Next, binding to S12 HA defined specificity for non-146 canonical (n.c.) antigenic sites (grey). Among the wt HA+ S12- B cells we then defined specific 147 antigenic-site binders to Cb (green) and Sb (gold) by double staining with CbD4 HA and SbD4 148 HA probes. All wt HA+ cells that did not bind to S12 HA, CbD4 HA and SbD4 HA were defined 149 as “others” (purple) binding to Ca1, Ca2 or Sa (Figure 2A and Supplementary Figure 1B). 150 Alternatively, to define de novo reactivity to drifted sites, present only in the new variants 151 (blue), we directly compared wt together with the drifted HA immediately after B cell gating: 152 e.g. comparing the binding to wt HA and SbD4 HA with “new”, drifted reactivity defined as wt 153 HA- and SbD4 HA+ B cells (Figure 2B). 154 We first determined how vaccination with the drifted immunogens broadens the response 155 towards the antigen. We thus divided all wt HA+ cells according to antigenic site specificity 156 (Figure 2C, Supplementary Figure 2A-B). wt HA challenge generated relatively similar ASC, 157 MBC and GC responses equally distributed across the four antigenic groups (Cb, Sb, other 158 immunodominant sites and n.c.; Figure 2C). A drifted rHA challenge favored responses not 159 only to the conserved antigenic site but also increased the relative targeting of n.c. sites (grey) 160 which are also conserved between the prime and boost antigens (Figure 2C), but, importantly, 161 poorly immunogenic in primary immune responses 29, 50. This applied to B cells derived from 162 both fate-mapped recalled MBC (Figure 2C and Supplementary Figure 2A) and naïve B cells 163 (Figure 2C and Supplementary Figure 2B). The majority of these specific B cells exhibited a 164 GC phenotype suggesting ongoing clonal diversification (Supplementary Figure 2C). These 165 data support the concept that drifted immunogen boosting in a polyclonal setting extends B cell 166 responses to clones not involved in the primary response , shifting B cell immunodominance 167 towards sites less targeted in the primary Ab response. 168 By cell numbers, GC responses to the boosted antigenic site ( i.e. Cb for Cb D4 boost, Sb for 169 SbD4 boost and n.c. for S12 boost) were dominated by a de novo response and not re-entering 170 MBC (Figure 2D). However, the frequency of antigenic-site specific GC B cells was similar 171 across Tomato+ and Tomato- populations and increased according to antigenic distance to the 172 wt rHA prime (Figure 2D). The significance of this finding is that the boosted antigen site is 173 immunodominant both among the naïve- and the MBC-derived GC responses, with the de novo 174 responses dominating. 175 ASC generation demonstrated a more complex pattern. Total cell numbers of fate-mapped and 176 naïve B cell were similar in number after SbD4 and S12 but not CbD4 rHA vaccination groups 177 (Figure 2E). More fate mapped MBC became ASC after vaccination with SbD4 rHA compared 178 to CbD4 rHA (Figure 2E). Further, after SbD4 rHA vaccination, the fraction of Sb-specific ASC 179 was much higher in Tomato+ vs. Tomato- ASC indicating that only recalled MBC could become 180 Sb-specific ASCs, at this early time point (Figure 2E). This was not the case for Cb, similar to 181 what was observed upon primary PR8 infection 29. The res ults so far showed that minor 182 antigenic differences can lead to distinctive outcomes in MBC reactivation. 183 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 6 Vaccination with variant rHA introduces drifted antigenic sites (blue in Figure 1A, 2B and 2F-184 H). As the drifted sites are not present on wt HA, only naïve B cells are activated to respond to 185 them. For CbD4 rHA vaccination, GC response was similar between “old” and “new” antigenic 186 sites, but the ASC response almost exclusively target ed “old” antigenic sites ( Figure 2F and 187 Supplementary Figure 2D). Conversely, after either SbD4 rHA and S12 rHA vaccination, GC 188 responses were domina ted by cells specific for drifted sites (Figure 2 G-H). For ASC the 189 response changed depending on vaccine (Supplementary Figure 2D -F). As the Cb site is 190 immunodominant early after primary infection 29 it is reasonable to speculate that for SbD4 and 191 S12 rHA vaccinations, most of the GC response to the drifted antigenic sites would target the 192 “new”, drifted Cb site. This differential responses to drifted antigenic sites may contribute to 193 the variable prevalence of original antigenic sin in previous studies 47. 194 We next used cell fate tracking to explore how peripheral rHA vaccination alters ongoing 195 infection induced medLN GC reactions. Footpad vaccination with drifted rHA but not wt rHA 196 invigorated total GC B cell responses in lung draining medLN (Supplementary Figure 3A), 197 including wt HA specific responses (Supplementary Figure 3B). Interestingly, SbD4 rHA and 198 S12 rHA vaccination increased numbers of n.c. and drifted-epitope specific GC B cells in 199 comparison to CbD4 rHA or wt rHA challenges (Supplementary Figure 3C) and led to higher 200 number of ASC in medLN (Supplementary Figure 3D), including wt HA+ specific ASC 201 (Supplementary Figure 3E-F). 202 Overall, our fine epitope mapping of B cell specificities demonstrates that recall and de novo 203 responses target similar antigenic sites . However, the ability of targeting “new” , drifted 204 antigenic sites was also on vaccine drift. These differences may be caused by relative exposure 205 of the conserved antigenic sites, its immunodominance and/ or by relative abundance of 206 epitope-specific, pre-existing MBC and Abs. 207 Antigenic-site specific boosting of Ab responses varies according to vaccine antigen 208 Which factors exactly do reshape the specificity of rHA boost-induced B cell responses ? A 209 likely suspect is serum IgG, previously shown to modify B cell responses 22, 23, 24, 25, 30, 52 . To 210 assess the HA antigenic site specificities of anti -HA IgG, we measured IgG titers against wt, 211 CbD4, SbD4 and S12 rHA 1 day pre- and 7 days post vaccination (dpv) (Supplementary Figure 212 4A-D) form the same experiment (Figure 1B) , plotting the area under the curve ( AUC) 213 differences (Figure 3A-D). 214 Relative to mock vaccination, each vaccination condition enhanced wt HA specific IgG titers 215 with homologous challenge showing the greatest increase, as expected (Figure 3A). A similar 216 pattern was observed for Cb-specific Abs upon CbD4 rHA vaccination but not for Sb-specific 217 Abs upon SbD4 rHA vaccination, consistent with the fate-mapped ASC data (Figure 3B-C and 218 Supplementary Figure 4C-D). Intriguingly, n.c.-specific Ab titers did not increase with any rHA 219 boost, contrary to the n.c.-specific GC B cell dominance observed after CbD4 or SbD4 220 vaccination and among ASC after S12 vaccination (Figure 3C; Supplementary Figure 4A-C). 221 This suggests that n.c.-specific B cells remain in GCs, preferentially become MBC or ASC at 222 later time point after vaccination. Alternatively, ASC may secrete low avidity Abs not detected 223 by our standard ELISA conditions. These findings demonstrate that different heterologous 224 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 7 vaccinations alter the balance of antigenic site -specific Abs, with site -specific differences 225 influenced by pre-existing MBC and Abs. 226 Collectively, challenge induced serum Ab changes mostly paralleled B cell changes: CbD4 rHA 227 induced Abs showed a greater “primary addiction” than SbD4 HA Abs, despite nearly identical 228 antigenic distance from wt HA 29. We hypothesized that pre-existing Cb and Sb specific 229 antibodies have distinct effects on secondary Ab responses to rHA immunization. 230 Transferred monoclonal and polyclonal antibodies do not inhibit naïve B cell responses 231 following vaccination with HA protein 232 The effects of pre-existing antibodies on B cell fates have been explored by mAb transfer 26, 27, 233 28, 53, 54, 55 in humans and animals. We examined the effect of mAbs targeting Cb (mAb H9-D3, 234 green) or Sb (mAb H28-E23, yellow) on primary responses to rHA (Figure 4A)35. To equalize 235 the effective dose, we transferred a standardized wt HA-binding Ab dose based on their ELISA 236 maximum binding capacity (Bmax) (Supplementary Figure 5A), using biotinylated mAbs to 237 enable their removal from sera in subsequent analysis of their effects on antibody responses to 238 vaccination. We transferred 0.1xBmax (between 0.002 and 0.005 ug per dose) , 1xBmax 239 (between 16 and 18 ug per dose) of H9D3 and H28E23 intraperitoneally (i.p.) and 100µg (5.6x 240 Bmax) of H28E23 (Figure 4A; Supplementary Figure 5A). Both 1x Bmax and 100µg doses are 241 in the range of previous studies, with 100µg being the highest amount used by others and 242 employed here as a positive control 26, 27, 28. Four hours post transfer, we vaccinated mice in the 243 left footpad with wt rHA. Mice were sacrificed 14 days after vaccination to enable a proper GC 244 response to develop (Figure 4A). 245 We enumerated total and antigen -site specific ASC, GC and switched MBC in the draining 246 pLN (Figure 4B -C; Supplementary Figure 5B). Surprisingly, despite using up to saturating 247 doses of high-avidity mAbs, these had no significant effects on B-cell responses compared to 248 PBS transfer, including total and antigen-specific GC B cells and changes in the antigenic site-249 specific immunodominance profile (Figure 4B-C). ELISA analysis extended status quo 250 maintenance to serum Ab titers (Figure 4D). 251 We hypothesized that polyclonal Abs may be more effective in modulating B cell responses via 252 Ab feedback 24, 30, 52, 56, 57. We purified polyclonal IgG (pIgG) from mouse serum 28 d post-i.n. 253 PR8 or D4 virus infection using Melon Gel, which retains most non-IgG serum proteins , 254 resulting in ~ 90% purification of IgG, mainly IgG2b and IgG2c isotypes and lacking IgM 255 (Figure 4E, Supplementary Figure 5C). To distinguish transferred from host Abs, we 256 biotinylated pIgG before transfer (Figure 4E) and used rHA ELISA to determine the Bmax and 257 titer of all anti-HA pIgG pools generated (Supplementary Figure 5D). Four h ours post-i.p. 258 transfer we detected biotinylated pIgG in sera by its binding to goat anti-mouse IgG in ELISA 259 (Supplementary Figure 5E). 260 We then repeated the mAb (Figure 4E) experiment with CbD4- and SbD4-speific pIgG with 261 near identical results. (Figure 4F-G), even when using pIgG to wt HA at high concentration (8x 262 Bmax; 100µg) (Supplementary Figure 5F). Host serum anti-HA Ab binding remained virtually 263 unchanged in transferred vs non transferred mice (Figure 4H). Transferred pIgG alone did not 264 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 8 induced an anti-HA immune response, ruling out anti-Ig response (Supplementary Figure 5G). 265 Using pIgG purified by protein G gave similar results (Supplementary Figure 5H). 266 Our data show that mAb and polyclonal Ab, at the concentrations used, do not affect B cell and 267 Ab responses after vaccination with wt rHA, implicating that rather MBCs influenced the B 268 cell and Ab differences we detailed in Figures 1 and 2. 269 Immunogen valency dictates Ab modulation of naive B cell responses 270 The Ab transfer results are nevertheless surprising as numerous laboratories have reported that 271 transferred mAbs or sera interfere with antigen specific de novo B cell responses 24, 26, 27, 28, 52, 272 55, 58 . We noted that in most studies the challenging immunogen expressed multiple antigen 273 copies on the surface of cells, virions, or artificial particles. r HA, as a monomeric (though 274 homotrimeric) vaccine antigen, cannot easily crosslink the BCR on B cells 59. Furthermore, 275 immune complex (IC) formation is disfavored using homotrimeric rHA, as a single Ab can 276 most likely only bind one epitope per antigenic particle 60, 61, 62, 63. Focusing on GC and serum 277 Ab responses (most studied previously ) we examined the influence of antigen valency (and 278 thus, indirectly, avidity) after passive Ab transfer, using three vaccine antigens of increasing 279 valency (Figure 5A), in addition to rHA: 1) Streptavidin (SA) tetramerized biotinylated HA; 280 2) Split virion vaccines (following the protocol used for human influenza vaccines) consisting 281 of HA present in a mixture of monovalent and multivalent HA aggregated by their hydrophobic 282 tails, with up to 50 HA trimers per aggregate 64; and, 3) UV-inactivated virions, which have 283 500 or more HA trimers per virion 65. 284 Two weeks post vaccination, in animals with no passively transferred Abs, split or inactivated 285 virus vaccines (but not SA-HA) increased GC size and numbers of wt HA+ specific GC B cells 286 in dLN, relative to rHA vaccination (Figure 5B). This could be explained by increased valency 287 but also by the extra adjuvanticity from residual viral components. Virion-based vaccines were 288 better at inducing Cb- and Sb- binding GC B cells while generally retaining Cb dominance 289 (Figure 5C). Similarly, all multivalent vaccine antigens were better than monovalent rHA at 290 inducing serum anti-HA Ab responses (Figure 5D). Surprisingly, HA-titers were comparatively 291 lower when vaccinating with UV-inactivated whole virus, possibly due to competition from NP 292 and chicken host component (Figure 5D). 293 Having established baseline responses to the four vaccines, we transferred mAb (1x Bmax) or 294 pIgG (0.1x Bmax) specific for either Cb or Sb sites, vaccinated mice with each of the vaccines, 295 and analyzed B cell immunity two weeks post-immunization. Overall, transferred Abs had 296 limited effects on the total magnitude of GC responses with few exceptions (Figure 5E). 297 However, wt HA+ GC B cell responses induced by UV -virus vaccine were significantly 298 decreased by all Ab treatment s (Figure 5F). The wt HA+ GC B cell responses after split 299 influenza vaccine were also inhibited by mAbs, but not pIgG, possibly due to the lower avidity 300 (determined by resistance to elution by 6M urea in ELISA) or dose of polyclonal IgG (Figure 301 5F and Supplementary Figure 5 I). Of note, Cb -specific pIgG had a higher avidity index 302 compared to Sb pIgG (Supplementary Figure 5I). Effect on GC B cell immunodominance was 303 observed within the same groups, with passively transferred Abs potently blocking responses 304 to the cognate antigenic site (Figure 5F-G). The cognate inhibition was even observed for Sb-305 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 9 specific responses after Sb-mAb administration and SA-HA vaccine (Figure 5G). Total serum 306 Abs to wt HA were generally only affected by mAb but not pIgG administration (Figure 5H). 307 Similarly, we observed a more potent antigenic -site specific suppression when using mAbs , 308 independently of the vaccine (Figure 5I). 309 Taken together, these finding indicate that antigen valency is a major determinant of Ab -310 mediated inhibition of B cell and Ab responses. While mAbs and pIgG had little effects on rHA 311 vaccination, they blocked overall and antigenic site-specific B cells when vaccine valency was 312 increased. 313 MBC suppress germinal center recruitment of antigen-specific naïve B cell. 314 The experiments above demonstrated that while previous antigen exposure influenced recall 315 responses markedly, Abs alone were unable to reshape humoral immunity to low valency 316 antigens. In a more physiological scenario, memory responses will take place in the presence 317 of both Abs and MBC, and the antigen would be multivalent. We hypothesized that since MBC 318 can rapidly differentiate into ASC secreting Abs in situ 13, 66 , they may be able to limit naïve B 319 cell recruitment into dLN GCs more efficiently than circulating Abs, due to high local 320 concentrations. Consistent with this idea, Abs were readily detected in pLN of i.n. infected, 321 footpad vaccinated mice, 7 days after boosting (Figure 6A). 322 To address the relative contribution of MBC and pIgG to the results observed in the rechallenge 323 experiment (Figures 1-3), we set up a system where both Abs and MBC were transferred into 324 recipient mice. We used C57BL/6 mice, expressing the congenic marker CD45.2 on immune 325 cells and secreting Abs of the Igh[b] allotype, as donors and B6 CD45.1 x Igha mice, expressing 326 the congenic marker CD45.1 on cells and secreting Abs of the Igh[a] allotype, as recipients 327 (Figure 6B). Total MBC that had specificities to all antigenic sites were purified from mice i.n. 328 infected with wt PR8 before transfer (Figure 6B-C; Supplementary Figure 6A). For transfer of 329 pIgG we either used pIgG purified from mice previously infected with CbD4 virus (thus only 330 carrying Ab against the the Cb site of wt PR8 HA) or a n equimolar amount of pooled pIgG 331 from mice that had been individually infected with all 5 viruses carrying different D4 mutations 332 (thus targeting all wt PR8 HA sites) (Figure 6C). As vaccines we used either wt PR8 or DCb 333 (carrying all canonical antigenic sites, except for Cb). 334 We focused the experiments on UV virus vaccine, as this was the most sensitive to inhibition 335 by mAb and pIgG transfers in the previous experiments (Figure 5F-G), and on the response to 336 the Cb site because of its immunodominance 29. When setting up the experiments, we noted 337 that recipient mice had to be pre -infected i.n. with J1, a reassortant PR8 virus expressing H3 338 HA (Supplementary Figure 6B-D) to enable transferred MBC to survive. J1 infected mice did 339 not develop detectable Abs reactive against wt PR8 HA that could influence MBC responses 340 against this antigen (Supplementary Figure 6D). Pre-infection was possibly needed to generate 341 CD4 T cells targeting conserved internal viral antigens 67, 68 or cross-reactive HA peptides69. 342 Whatever the case , as J1 priming was an essential step to support transferred MBC survival 343 and involvement in the recall response all experiments were performed in primed recipient 344 mice (see Methods and Supplementary Figure 6B-D). Four weeks later, MBC and/or pIgG were 345 transferred and a day later mice were vaccinated i.p with UV -inactivated virus (Figure 6B). 346 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 10 Seven days post vaccination, both donor and recipient GC and ASC could be identified in 347 spleen and medLN (Figure 6D and Supplementary Figure 6 E). In this setup, engraftment was 348 observed in the majority of transferred mice and detection of transferred B cells was strictly 349 dependent on vaccination, demonstrating antigen -dependent expansion of MBC after 350 immunization (Supplementary Figure 7A). 351 First, we determined the effect of MBC transfer alone on immune response. When compared 352 to no transfer, the MBC transfer resulted in a general increase in overall GC and ASC cellularity 353 (Supplementary Figure 7B-C). However, while the total number of activated cells increased, 354 the number of wt HA+ naïve-derived GC and ASC cells decreased drastically in MBC -355 transferred vs non-transferred mice (Figure 6 E and Supplementary Figure 7D-E), 356 demonstrating that MBC alone could inhibit antigen-specific naïve B cell differentiation. We 357 hypothesize that this observation was due to high levels of Ab being secreted by differentiated 358 donor MBC in situ (Figure 6A). 359 Subsequently, we analyzed the humoral response of mice receiving both MBC and pIgG . 360 Groups were designed so that MBC repertoire was broad and Ab response narrow 44. Overall, 361 recipient cells dominated the response, but transferred cells were also detected (Figure 6 F-G 362 and Supplementary Fig ure 7F-G). Consistent with our other data above (Figure 5), the total 363 numbers of responding GC and ASC cells were not influenced by co -transfer of Abs (Figure 364 6F-G and Supplementary Figure 7F -G). However, when we defined antigen specificity as in 365 previous experiments (Figure 2A), we observed subtle differences in wt HA specific donor 366 MBC that could re -enter the GC reaction, depending on the presence of transferred pIgG 367 (Figure 6H). Specifically, we found that pIgG blocked donor B cell response to the homologous 368 antigenic site they targeted (e.g. Cb-specific Abs decreased the MBC -derived response to Cb 369 after PR8 wt virus infection) (Figure 6H-I). Interestingly, this was not the case for de novo 370 responding B cells, as recipient (naïve-derived) wt HA+ GC B cells were largely Cb -site 371 specific to the same extent even after CbD4 pIgG transfer, albeit the total response was lower 372 when compared to no pIgG -transfer (Figure 6 J-K). Similarly, Cb -specific, donor ( MBC-373 derived) ASC were more efficiently inhibited by Cb-reactive pIgG when compared to recipient 374 (naïve-derived) (Supplementary Figure 7H-K). However, as noted above, the presence of MBC 375 alone also reshaped the immunodominance patterns of naïve -derived B cells (Supplementary 376 Figure 7 D-E). The overall results suggest differential Ab feedback and MBC regulation of 377 naïve B cells vs MBC. 378 We finally measured Abs secreted by recipient (Igh[a]) vs donor (Igh[b]) ASC (Figure 6L-M). 379 At this early time point (day 7 post vaccination), donor Ab titers (Igh[b]) were higher than the 380 recipient Ab titers (Igh[a]) (Figure 6L), showing that early secondary Ab responses from ASC 381 are dominated by activation of antigen experienced MBC. When analyzing immunodominance 382 in donor cells the pattern was similar across conditions, consistent with a general reactivation 383 of all MBC, with few exceptions (Figure 6M). For example, when DCb vaccine was used 384 without Ab presence, there was very limited production of Cb-specific Abs, as expected; and a 385 combination of all pIgG blocked differentiation of almost all MBC to ASC. 386 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 11 Based on these findings, we conclude that pre -existing Abs impact MBC and naïve cells in 387 different ways and that MBC themselves also affect the magnitude of total and antigen specific 388 de novo B cell responses, likely through rapid differentiation and local Ab secretion. 389

390 Using cell fate tracking experiments and transfer of epitope specific Abs and MBC, we revealed 391 how each component influences MBC and naïve B cell responses to vaccination with drifted 392 antigens in naïve and infection-experienced animals. We have made 4 key discoveries: 1) Even 393 a few amino acids substitutions in immunodominant vaccine antigenic sites, comparable to 394 normal antigenic drift, can reshape naïve B cell recruitment into GC and secondary B cell 395 responses, 2) Ab-mediated regulation o f de novo B cell responses depends on antigen 396 multivalency, 3) MBC can suppress naïve B cell entry into GC in an antigen-specific manner, 397 and 4) pre-existing Abs differently regulate MBC and naïve B cells. 398 Schiepers et al. used drifted monovalent rHAs representing various IA V strains to examine 399 secondary MBC responses with protein priming and boosting in opposite footpad. They found 400 that boosting with drifted, but not identical, rHA resulted in increased numbers of HA-binding 401 GC B cells and increased frequency of MBCs re-entering into secondary GCs in dLNs 23. The 402 boosting rHAs were, however, extremely drifted from priming rHA, carrying 10%-20% amino-403 acid substitutions, which represents decades of normal antigenic drift in influenza. To create a 404 closer to a real-life human scenario we here used engineered D4 PR8 rHAs to mimic the typical 405 circumstance of being challenged with an IA V strain (by infection or vaccination) that has only 406 drifted a few years from viral strains used for prior infection. Despite this and our use of i.n. 407 infection for priming, we also observed that secondary GCs are mostly populated by de novo 408 responses, with the proportion of GC cells derived from MBC re-entry increasing up to ~20% 409 if only considering HA-specific cells. However, we may be underestimating the memory 410 contribution, as early GC-independent, and thus non-fate mapped MBC, may also participate 411 in recall responses 10. We also found that m ost fate-mapped MBC rapidly differentiate d into 412 ASC in the dLN after vaccination. Even so, we acknowledge that the number of HA-specific 413 ASC likely were even higher, as terminally differentiated IgG producing PC mostly lack 414 surface BCR 70. Importantly, the degree of drift in boosting vaccine rHA governed the 415 magnitude of both the MBC response to the boosted site but also the naïve B cell responses to 416 the novel antigenic sites , resulting from drift . This dependency on the degree and nature of 417 antigenic drift likely explain some of the contradictory results in prior original antigenic sin 418 studies 47. 419 Several recent studies examined the effects of mAbs, pIgG and whole ser a on de novo 420 activation of B cells specific for the same epitope targeted by the Ab upon vaccination 24, 26, 27, 421 28, 30, 52, 53, 54, 56, 57, 71. The results from these studies should be reevaluated in light of our finding 422 that Ab feedback inhibition depends not only on the nature of the antigen but also on its valency. 423 GC responses to r HA vaccination were mostly unperturbed by transferred Abs but likely 424 affected by MBC, which we expect to act by intranodal Ab secretion after rapid differentiation 425 to ASC. This implies that transferred Abs possibly do not achieve sufficient concentration in B 426 cell follicles to affect B cell activation, unless multivalent vaccine antigens are used, likely due 427 to the increase in functional Ab avidity in this case. Furthermore, ICs preferentially accumulate 428 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 12 on FDCs when formed with multivalent antigens 72, thereby allowing for sustained antigen 429 availability within GC to amplify B cell responses (Figure 5B). This provides a possible 430 mechanistic explanation to our findings: low-valency antigens do not form sufficiently stable 431 ICs to deposit on FDCs, and circulating antibodies remain too diluted in the follicle. In contrast, 432 multivalent vaccines generate high-avidity ICs that can saturate epitopes, concentrate on FDCs, 433 and effectively mask antigens from competing B cells. 434 Lack of Ab inhibition of monovalent vaccination echoes a recent study from Termote et al.20, 435 but contrasts with Dvorscek et al. 26 who found that transferred mAbs could block responses 436 to the monovalent protein hen egg lysozyme given that they had sufficient affinity. Differences 437 between our studies is that while we examined responses from endogenous polyclonal B cells 438 of varying specificity and avidity, Dvorscek et al. used identical mAb and fixed monoclonal B 439 cells competing with each other for antigen access. Furthermore, the affinity of OV A-specific 440 Abs 26 is overall higher than avidity of B cells and Abs to HA 73. The discrepancies highlight 441 that this is a tightly regulated system, where modulation of antigen valency as well as affinity 442 may help to overcome Ab-dependent inhibition. Finally, timing of Ab administration may also 443 be crucial as immune serum (containing also IgM) transferred few days after immunization 444 was also able to modulate immunity to monovalent antigens 30, 52. 445 Our experimental design enabled precise characterization of epitope-specific MBC activation 446 and dissection of differences in specificity between naïve B cells vs reactivated MBC as well 447 as their secreted Abs. While Schiepers “primary addiction” model 22 is consistent with some 448 of our findings, we find that monovalent rHA vaccines, even if differing from priming HA by 449 only 5 amino acids , can profoundly reshape secondary B cell responses ( Figure 1-3 and 6). 450 Importantly, our MBC transfer experiments (Figure 6E) establish that that MBC play a critical 451 role in B cell fate and the resulting immunodominance of booster vaccine even for de novo 452 responses initiated after vaccination. This effect can likely be explained by rapid differentiation 453 of MBC to ASC with the local secretion of enormous amounts of blocking Abs (5 – 10x107 454 molecules per cell per hour 74, 75) in situ. Interestingly, our model is consistent with results from 455 Schiepers et al. where, by deleting Prdm1 in GC-derived cells (including MBC), they found 456 decreased Ab-mediated suppression 23. 457 Our transfer studies highlight the complex interplay between polyclonal Abs and B cells. Using 458 MBC and Abs with a range of avidities and specificities we recreated complex scenarios of 459 pre-immunity similar to what occurs with IA V , CoVs and other drifting human viruses 43, 44, 45. 460 In this context, we found that, using a multivalent antigen vaccine, epitope-specific MBC are 461 more likely to re -enter a GC in the absence of pre -existing Abs (Figure 6H) 23. This Ab-462 mediated inhibition was more potent in blocking GC entry of MBC as compared to naïve B 463 cells, thus explaining results from the infection/vaccination experiment (Figure 1) and from 464 previous studies 21, 22, 23. These scenarios unveiled how pre-existing MBC, Abs and challenge 465 antigen interact to determine MBC and naïve cells activation and magnitude as well as 466 antigenic site specificity of the overall response. 467 A limitation of our study is that, w hile it considered multiple factors simultaneously and 468 allowed a more faithful recapitulation of real life scenarios 43, 44, 45 , by design, it could not 469 resolve the detailed affinity/avidity and epitope -specificity of Abs vs B cells, as was possible 470 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 13 in some previous studies 22, 23, 24, 26, 27, 28 . Furthermore, to capture reactivation of MBC we 471 conducted our experiments at 7 days post vaccination. Future studies need to be conducted to 472 gain further insight into earlier and later timepoints to illustrate how secondary B cell responses 473 are initiated and persist. 474 In summary, our findings illuminate the complex regulation of secondary B cell responses and 475 original antigenic sin phenomenon and should be taken into consideration when designing 476 monovalent and multivalent immunogens for vaccination. 477 ACKNOWLEDGMENTS 478 We thank all members of the Angeletti lab for helpful discussion and helpful interactions. 479 Additionally, we want to thank the staff or the animal facility Experimental Biomedicine, 480 especially Pernilla Ahlgren, for excellent upkeeping and care of animals used in this study. We 481 also want to thank the Protein Production Sweden (Gothenburg, Sweden), especially M alin 482 Bäckström and Mikael Andersson, for production of rHA proteins. We thank Tomohiro 483 Kurosaki (WPI Immunology Frontier Research Center, University of Osaka, Japan) for kindly 484 providing S1pr2-ERT2-cre-TdTomato mice 51 and Masaru Kanekiyo (VRC, NIAD, NIH) for 485 the plasmids for recombinant S12 HA expression. 486 The study was supported by grants from the European Research Council (ERC -StG, B -487 DOMINANCE, grant no. 850638 to DA); the Swedish Research Council (grant no. 2017-488 01439, 2021-01164, 2021-01165 to DA); the Knut and Alice Wallenberg Foundation (grant no 489 2021.0033 to DA), the Jeanssons Foundation (grant nos. JS2018 -0011 and JS2019-0038), the 490 Claes Groschinsky Foundation (grant no. M18237). NRM is supported by Svenska Sällskapet 491 för Medicinsk Forskning post -doctoral grant (grant no: PD20 -0017). BioRender was used to 492 generate images and graphics. 493 AUTHOR CONTRIBUTIONS 494 L.R. Performed the majority of experimental work ; K.S. produced virus, handled animal 495 breeding, performed TCID50 assays as well as some vaccinations and ELISA; D.A. performed 496 flow cytometric comparison of multivalent va ccines; N.R.M. started optimization work for 497 MBC transfer ; D.F.B. contributed to protein production ; D.A., I.K., J.S.G., M.C.M were 498 involved in development of new HA escape mutants ; J.W.Y . provided key reagents; D.A. 499 conceptualized the study; L.R., D.A. and M.B. designed all experiments and interpreted data; 500 L.R. and D.A. wrote the first draft of the manuscript with critical input from J.W.Y . and M.B.. 501 All authors reviewed, edited and approved the manuscript. 502 DECLARATION OF INTEREST 503 The authors declare no competing financial interest or personal relationships that could have 504 appeared to influence the work reported in this paper. 505 506 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 14

507 Animals 508 Wild type C57BL/6 mice were obtained from Janvier Labs. S1pr2CreERT2 animals were kindly 509 provided by Tomohiro Kurosaki 51 and crossed with Ai14 R26 Tom ato to obtain S1pr2CreERT2-510 R26tdTomato. Recipients for MBC transfer, PepBoyIgha, were bred onsite from 511 B6.CgGpi1aThy1a Igha/J and B6.SJLPtprcaPepcb/BoyJ animals obtained from Jackson 512 Laboratory. Animals were kept in a specific pathogen free facility under Biosafety -Level 2 513 conditions. Breeding was conducted under the ethical permit 3307/20 and experiments under 514 the ethical permits 1666/19, 2230/21 and 38/23. All ethical permits were approved by the 515 Swedish Board of Agriculture. 516 For cell fate tracking experiments , S1pr2 -ERT2-cre-TdTomato mice were infected with 25 517 TCID50 PR8. Every second day, mice received an oral gavage of 2mg tamoxifen per 100ul of 518 corn oil. At 26 days post infection (dpi), treatment was stopped, at 27dpi animals were bled 519 from vena saphena and at 28dpi animals were challenged as indicated. 520 7 days post vaccination (dpv), animals were sacrificed. Mediastinal lymph node (medLN), 521 popliteal lymph node (pLN) and blood for serum were harvested. Tissue was processed for 522 flow cytometric staining and serum used for ELISA. 523 Viruses and proteins 524 Viruses were grown in 10 day s old embryonated chicken eggs or propagated in MDCK cell 525 culture and purified using ultracentrifugation on a sucrose gradient. Viruses used were 526 A/PR/8/34 (PR8), its Δ4 and D1 escape mutants. For the new Δ4 selection, we reselected the 527 original Δ4 viruses using a mixture of 2 mAbs targeting the Sb cryptic epitopes defined by 528 residues 189 and 198 (H1 numbering )50, according to the same protocol previously described 529 29 . Resulting viruses had the same mutations as described 29 but also carried the following extra 530 mutations: Sa Δ4 K189E, Ca1 Δ4 E198G, Ca2Δ4 E198G and CbΔ4 Y201S. Escape was 531 confirmed by ELISA. DCb was a precursor of Δ4 viruses and defined by the following 532 mutations: L75P, V77M, R78K, E124G . J1, is a PR8 reassortant virus expressing an H3N 1 533 instead of the PR8 HA. Viral titers were determined by TCID50. 534 Virus was UV inactivated on ice for 20-30 min and stored at -20°C. HAU was determined from 535 UV inactivated virus by hemagglutination inhibition (HI) assay as described previously 29. 536 Briefly, a 1% solution of chicken red blood cells was incubated in a 96-well round bottom plate 537 with a serial titration of influenza A virus for 30min at RT. HAU titer was determined 538 subsequently. 539 Recombinant HA (rHA) proteins were produced by Protein Production Sweden as previously 540 described 76. All rHA included the Y98F mutation to abrogate sialic -acid binding. Protein 541 biotinylating was performed using BirA -mediated enzymatic reaction, according to 542 manufacturer´s instructions (Avidity LLC). 543 544 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 15 Infections and immunizations 545 For i.n. infection, animals were anesthetized and 25 -100 TCID50 of virus per 25 µl 546 HBSS/0.1%BSA were injected in one nostril. Animals were monitored for signs of severe 547 disease and sacrificed if too sick. Animals were kept for 28 days to allow sufficient B cell 548 memory and Ab titers to develop. To check titers, animals were bled 26 days post vaccination 549 from the Vena Saphena. 550 For rHA immunizations, 10µg of rHA was mixed with AddaVax™ (MF59®- like squalene oil-551 in-water adjuvant, InvivoGen) in a 1:1 v/v ratio to 30 µl and injected subcutaneous (s.c.) into 552 the left foot hock. 553 For SA-HA immunization: biotinylated HA was tetramerized with 4 fold molar excess of SA 554 on ice and stored at 4 °C. 10µg of the complex was mixed with AddaVax ™ (MF59®- like 555 squalene oil-in-water adjuvant, InvivoGen) in a 1:1 v/v ratio to 30ul and injected s .c. into the 556 left foot hock. 557 For split virus vaccine: purified virus was fractionated by incubation with an equal volume of 558 15% octyl-β-glucoside. After addition of PBS, the solution was spun at 50,000g for 2 h at 4 °C. 559 The supernatant, containing HA was collected and quantified using a Bradford assay. HA 560 presence and amount was confirmed using immunoblot with HA 2-specific mAb. 8µg of split 561 vaccine was mixed with AddaVax™ (MF59®- like squalene oil-in-water adjuvant, InvivoGen) 562 in a 1:1 v/v ratio to 30ul and injected s.c. into the left foot hock. 563 For UV inactivated virus immunizations, 2000 HAU were injected either intraperitoneal (i.p.) 564 100ul in PBS or s.c. in the foot hock 30µl in PBS. 565 ELISA with serum, purified Abs or LN supernatant 566 96 half-well plates for high protein binding were coated with rHA, recombinant Neuraminidase 567 (rNA) from PR8, recombinant Nucleoprotein from PR8 (rNP) or UV-inactivated J1 over night 568 (o/n) or up to 1 week. mAbs, Fabs and pIgG were diluted to a defined concentration and sera 569 were diluted 1:100 in PBS-T. Diluted samples were titrated 2-fold down from the first row and 570 incubated for 1.5h. After that, a secondary antibody coupled to peroxidase was used, incubating 571 for 1h. Plates were finally developed, adding TMB for 5min and stopping the reaction with 2M 572 H2SO4. Pates were read at 450nm within 30min of development. 573 For avidity ELISA, plates were coated with wt HA and pIgG was used in duplicate at 1xBmax. 574 Following incubation, unbound pIgG was removed by treatment with either 6 M urea or PBS 575 for 5 min to disrupt low -avidity interactions. Secondary antibody incubation and plate 576 development were performed as described above. The avidity index was calculated as the ratio 577 of absorbance in urea-treated wells to that in ÅBS-treated controls. 578 Preparation of HA probes and staining of tissue samples for flow cytometry 579 rHA was biotinylated with the Biotin Protein Ligase standard reaction kit (Avidity), excess 580 biotin was removed using a 30kDa MWCO protein concentrator. Biotinylated rHA was 581 conjugated to streptavidin (SA) coupled to fluorescent molecules to create HA probes. Once 582 conjugated, rHA was stored at 4°C in the dark. 583 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 16 Harvested tissue was either grinded on a 70 µm filter or smashed directly in the tube. After 584 washing with FACS buffer, samples were stained with fluorescent labelled mAbs in FACS 585 buffer. Following a wash in PBS -EDTA, samples were stained for viability using 586 LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit or LIVE/DEAD ™ Fixable Far Red Dead 587 Cell Stain Kit (Thermo Fisher). Subsequently, samples were fixed using 1.5% PFA in PBS and 588 stored at 4°C in the dark until acquisition (maximum 1 week). Just before acquisition, 10µl of 589 counting beads were added (SONY). 590 Samples were acquired on a BD LSR Fortessa X-20 (BD Biosciences) or ID7000 (SONY) and 591 analysed using FlowJo V .10 software (BD Biosciences). 592 Table 1: Fluorescent labelled antibodies used for flow cytometric staining. 593 Specificity Conjugate Clone Manufacturer Cat.no. B220/CD45R BUV395 RA3-6B2 BD Biosciences 563793 BUV737 RA3-6B2 BD Biosciences 612838 BV605 RA3-6B2 Biolegend 103243 BV650 RA3-6B2 Biolegend 103241 CD3e BUV395 145-2C11 BD Biosciences 563565 PacificBlue 17A2 Biolegend 100213 BV510 145-2C11 BD Biosciences 563024 AF700 17A2 Biolegend 100216 CD19 BUV395 1D3 BD Biosciences 563557 BV650 6D5 Biolegend 115541 AF700 6D5 Biolegend 115528 CD38 BV786 90/CD38 BD Biosciences 740887 CD45.1 FITC A20 Biolegend 110706 PE A20 Biolegend 110707 CD45.2 BUV395 104 BD Biosciences 564616 FITC 104 Biolegend 109806 CD138 PE-Cy7 281-2 Biolegend 142514 GL7 Per-CPCy5.5 GL7 Biolegend 144610 FITC GL7 Biolegend 144603 IgD APC-Cy7 11-26c.2a Biolegend 405716 IgM BV421 RMM-1 Biolegend 406518 BV750 II/41 BD Biosciences 747333 FITC RMM-1 Biolegend 406506 NK1.1 BV510 PK136 Biolegend 108738 NP AF488 H16-L10-4R5 (HB65) BioXcell + conjugated with kit from Thermo Fisher BE0159 + 10701154 Streptavidin BUV496 - BD Biosciences 612961 BUV563 - BD Biosciences 612935 BUV737 - BD Biosciences 612775 BV421 - BD Biosciences 563259 BV650 - BD Biosciences 563855 BV711 - BD Biosciences 563262 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 17 AF594 - Jackson Immuno 016-580-084 PE - Invitrogen S866 APC - Biolegend/Invitrogen 405243/ 10125012 594 Preparation of Abs and transfer 595 To obtain polyclonal IgG (pIgG), female C57BL/6 mice were infected with 50 TCID50 of PR8 596 virus. At 28dpi, animals were sacrificed with a terminal bleed during ketamine anesthesia. Sera 597 were pooled and IgG was purified using a Melon™ Gel (Thermo Fisher) column on the ÄKTA 598 Start chromatography system (Cytvia) according to manufacturer’s instructions. Obtained IgG 599 was concentrated using a 50kDa MWCO protein concentrator. 600 To be able to identify transferred Abs quickly and to be able to remove them from a mix with 601 donor Abs, they were biotinylated. The EZ -Link™ Sulfo-NHS-LC-Biotin, No -Weigh™ 602 Format kit (Thermo Fisher) was used according to manufacturer’s instructions and residual 603 biotin was removed using a 50kDa MWCO protein concentrator. 604 Biotinylated pIgG, mAbs and Fabs were titrated using the ELISA method described above. 605 Secondary IgG(H+L) was used for pIgG and mAbs, secondary IgG -kappa was used for Fabs. 606 Maximum binding capacity (1xBmax) was determined, and corresponding concentration was 607 calculated by extrapolation. This way, 0.1xBmax and 10xBmax can also be determined with 608 respective concentrations. 609 To transfer Abs, desired concentration (0.1xBmax, 1xBmax or 10xBmax) was mixed in PBS 610 to 200µl and were transferred i.p. into recipients. 4 hours post transfer (4hpt), animals were 611 challenged as indicated. 14dpv, animals were sacrificed. Depending on challenging site, spleen 612 or popliteal lymph node (pLN) and blood for serum were harvested. Tissue was processed for 613 flow cytometric staining and serum was used for ELISA. 614 Adoptive MBC transfer 615 C57BL/6 donors were infected with 25 TCID50 PR8, PepBoyIgha recipients with 100 TCID50 616 J1. At 27dpi, recipients were bled from Vena Saphena. Donors were sacrificed at 28dpi, spleen 617 and medLN were harvested. Tissue was disrupted through a 70 µm mesh filter and cells were 618 washed. MBC were isolated using the EasySep ™ Mouse B Cell Isolation Kit (Stemcell) 619 according to manufacturer’s instructions, with exception of adding 5 µg/sample of anti -IgD 620 biotinylated and anti-GL7 biotinylated mAbs (both Biolegend) to the Abs mix and using 10µl 621 of extra magnetic spheres. After a final wash, isolated MBC are counted using Countess ™ 3 622 Automated Cell Counter (Thermo Fisher) and staining with Acridine Orange (abcam). Some 623 volume of isolated MBC was saved for isolation purity check via flow cytometry. 624 Priming was necessary to create an environment that supports a robust recall response as 625 transferred MBC did not survive (Supplementary Figure 6A), regardless of CD4 transfer . 626 Additionally, no wt HA-specific donor IgG2a (Igh [b]) were detected in naïve mice receiving 627 MBC (Supplementary Figure 6B). Primed J1 mice showed an Ab response that was specific 628 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 18 for J1 virus but not for wt HA, with only ~10% of mice having a very low but above background 629 response (Supplementary Figure 6C). 630 1-5x106 MBC were injected intravenously (i.v.) into recipients in the tail vein. pIgG was 631 transferred at 1xBmax to 200ul with PBS i.p. simultaneously. Animals were left to rest o/n and 632 challenged with UV inactivated virus at 2000 HAU in 100ul i.p. At 7 days post vaccination /8 633 days post transfer they were sacrificed and spleen, medLN and blood for serum were harvested. 634 Tissue was processed for flow cytometric staining and serum was used for ELISA. For ELISA, 635 secondary biotinylated mAbs IgG2a[a], clone 8.3, and IgG2a[b], clone 5.7, and avidin -HRP 636 (Thermo Fisher) were used. We only considered successful transfers those where at least 50 wt 637 HA specific donor B cells (IgM MBC+ switched MBC+ASC+ GC B cells) could be detected 638 after challenge. 639 Statistics 640 Statistic calculations (t-tests, one -way and two -way ANOV A) were performed using the 641 GraphPad Prism Software V .10. Asterisks correspond to: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001 642 in all figures. All data are represented as mean ± SD. 643 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 19

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 25 FIGURES 891 892 Figure 1. A drifted rHA challenge reshapes secondary GC responses in the LN draining 893 the challenge site. A: Simplified graphic of wt HA with canonical antigenic sites and D4 escape 894 mutants: Cb in green, Sb in yellow, Sa in purple, Ca1 in red, Ca2 in orange. Escaped/drifted 895 sites are in dark blue and non -canonical sites in grey (rest of protein). B: Experimental setup 896 of cell fate tracking using the S1pr2-Tomato mouse model. C: Total count of GC B cells in 897 pLN at 7 days post vaccination (dpv). Challenge groups are listed from left to right by antigenic 898 distance from wt (left). Representative flow plots of GC B cells (lin- B220+ CD138low/- CD19+ 899 CD38- GL7+) in pLN at 7dpv (right). D: Total count of wt HA+ GC B cells in pLN at 7dpv. 900 (left). Representative flow plots of wt HA+ GC B cells (lin - B220+ CD138low/- CD19+ CD38- 901 GL7+ wt-BV711+ wt-BUV496+) in pLN at 7dpv (right). E: Frequency of fate mapped (S1pr2-902 TdTomato+) cells among GC B cells and F: ASC (lin- B220+ CD138high). Light red = whole 903 GC B cell or ASC, dark red = wt HA+ GC B cells or ASC. p-values were calculated by unpaired 904 t tests (E, F) or one-way ANOV A with all groups compared to PR8 wt HA vaccination using 905 (C, D). (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). Lowest p value indicated if no significance 906 detected. Figures represent data from two experiments with 3 -7 mice per challenge group per 907 experiment. At least 2 experiments/group were performed. All data presented as mean ± SD. 908 Animals without any detectable anti-wt HA IgG serum titer at -1dpv were excluded from data 909 analysis. 910 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 26 911 Figure 2. A drifted rHA challenge reshapes immunodominance patterns in draining LN. 912 A: Gating strategy for antigenic site specificit y that s tart from wt HA+ GC B cells to define 913 non-canonical (S12 HA+), Cb+ (S12 HA- SbD4 HA- CbD4 HA+), Sb+ (S12 HA- CbD4 HA- SbD4 914 HA+), other+ (S12 HA- CbD4 HA- SbD4 HA-). Shown is a sample from medLN, 7dpv after wt 915 rHA challenge in foot hock. B: Gating strategy for antigenic site specificity of “new” antigenic 916 sites (dark blue) with example for Sb D4 stainining. The same gating was used for Cb D4 and 917 S12 binding. It start from total GC B cells or ASC, and “new” antigenic sites + (wt PR8 HA- 918 SbD4 or CbD4 or S12 HA +) and, respectively, Sb or Cb and/or n.c.+ ( wt PR8 HA+ SbD4 or 919 CbD4 or S12 HA+) C: Sunburst plots at 7 days after vaccination (dpv) of wt HA+cells divided 920 by B cell phenotype (lined=ASC, dotted=GC, switched MBC=crossed lines), antigenic site 921 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 27 specificity (Cb=green, Sb=yellow, other=purple, n.c. = grey) and fate mapping (white=not fate 922 mapped, red=fate mapped/Tomato+) in pLN. Shown are plots for homologous/wt challenge as 923 well as Cb D4, SbD4 and S12 challenge. D: Total count and % per all wt-HA+ of GC B cells 924 and E: ASC of challenge homologous antigenic site. Data is for Cb+ for CbD4 challenge, Sb+ 925 for SbD4 challenge and n.c.+ for S12 challenge. Fate mapped indicated in red, non-fate mapped 926 in black. F: Tomato- Cb+ + n.c. + and “new” antigenic site + (wt HA- CbD4 HA+) with 927 representative plot from pLN at 7dpv, G: Sb+ + n.c.+ and “new” antigenic site + (wt HA- SbD4 928 HA+) with representative plot from pLN at 7dpv, H: n.c.+ and “new” antigenic site + (wt HA- 929 S12 HA+) with representative plot from pLN at 7dpv of GC B cells total count per pLN. p-930 values were calculated by unpaired t test (F, G, H) or two-way ANOV A (D, E). (∗p < 0.05; ∗∗p 931 < 0.01; ∗∗∗∗p < 0.0001). Figures represent data from two experiments with 3 -7 mice per 932 challenge group per experiment. All data as mean ± SD. Animals without any anti-wt HA IgG 933 serum titers at -1dpv were excluded from data analysis. 934 935 936 937 938 Figure 3: Immunodominance of serum Abs is influenced by drift in vaccine antigen. AUC 939 of OD450nm difference between day -1 and 7 days post vaccination of IgG from serum. Plates, 940 from left to right, were c oated with rHA: A: wt, B: CbD4, C: SbD4, D: S12. p-values were 941 calculated by one-way ANOV A, with all groups compared to wt HA challenge (homologous) 942 or mock challenge (A-F). (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). Lowest p -value indicated 943 if no significance detected. Figures represent data from two experiments with 3 -7 mice per 944 challenge group per experiment. At least two experiments per group were performed. All data 945 as mean ± SD. Animals without any anti-wt HA IgG serum titers at -1dpv were excluded from 946 data analysis. 947 948 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 28 949 Figure 4: Transfer of mAbs and pIgG does not affect B cell responses to rHA vaccination. 950 A: Experimental workflow for mAb transfer with rHA challenge are shown. B: Total cells per 951 pLN at 14 days post vaccination ( dpv) in ASC, GC B cells and switched MBC after mAb 952 transfer. C: Total wt HA+ ASC, GC B cells, switched MBC and immunodominance patterns at 953 14dpv. The mAb transferred is indicated in color of plot background (green = a -Cb/H9D3; 954 yellow = a-Sb/H28E23) D: AUC OD450nm values of titrated sera at 14dpv are shown, with 955 wt HA used for coating (left), Cb D4 and Sb D4 used for coating (right) as mAb homologous 956 antigens (Cb D4 as H9D3 homologous antigen, Sb D4 as H28E23 homologous antigen). E: 957 Experimental workflow of pIgG transfer with rHA challenge are shown. F: Total cells per pLN 958 at 14dpv in ASC, GC B cells and switched MBC after pIgG transfer with rHA challenge. G: 959 Total wt HA+ ASC, GC B cells, switched MBC and immunodominance patterns at 14dpv. pIgG 960 transferred is indicated in color of plot background (green = a -Cb; yellow = a -Sb). H: AUC 961 OD450nm of sera at 14dpv are shown, with wt HA used for coating (left), CbD4 and SbD4 used 962 for coating (right) as pIgG homologous antigens (CbD4 as a-Cb homologous antigen, SbD4 as 963 a-Sb homologous antigen). p-values were calculated by ordinary one -way ANOV A, with all 964 groups compared to PBS/mock transfer (B, D, F, H). (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). 965 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 29 Lowest p-value indicated if no significance was detected. At least 2 experiments per group 966 were performed. Figures represent data from two experiments with 3-5 mice per transfer group 967 per experiment. All data are shown as mean ± SD. 968 969 970 Figure 5: Ab modulation of B cell responses depends on antigen valency. A: Scheme 971 displaying vaccines used in this work. B: Total GC B cells and PR8 wt HA+ GC B cells per 972 dLN at 14 days post vaccination (dpv) after PBS (mock) transfer are shown. C: Total Cb+ and 973 Sb+ GC B cells per dLN at 14dpv after PBS (mock) transfer are shown. D: AUC OD450nm 974 determination of anti-wt HA+ IgG in serum at 14dpv after PBS (mock) transfer. Data for rHA 975 vaccination is the same already reported in Figure 4 and shown again here for comparison. E: 976 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 30 Total GC B cells per dLN at 14 dpv with or without mAb or pIgG transfer and for different 977 vaccines are shown. F: Total wt HA+ GC B cells and immunodominance patterns at 14 days 978 post vaccination with or without mAb or pIgG transfer and for different vaccines are shown. 979 The mAb/pIgG transferred is indicated by color of plot background (green = a-Cb; yellow = a-980 Sb). G: Cb+ and Sb+ HA+ GC B cells at 14 dpv with or without mAb or pIgG transfer and for 981 different vaccines are shown. H: AUC OD450nm of sera at 14 days post vaccination with or 982 without mAb or pIgG transfer and for different vaccines. wt HA was used for coating. I: 983 Calculated AUC OD450nm of sera at 14 dpv with or without mAb or pIgG transfer and for 984 different vaccines are shown. CbD4 was used for coating to define Cb -specificity while SbD4 985 was used for coating to define Sb -specificity. p-values were determined by one -way ANOV A 986 (B, D, E, F, F), unpaired t-test (G, I) or two-way ANOV A (C). (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 987 0.001; ∗∗∗∗p < 0.0001). Figures represent data from two experiments with 3-5 mice per transfer 988 group per experiment. All data as mean ± SD. The dose used for transfer was 1x Bmax for mAb 989 and 0.1x Bmax for pIgG. 990 991 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 31 992 Figure 6: Differential effect of antibodies on naïve B cells and MBC fate and 993 immunodominance and MBC-mediated suppression of antigen -specific naïve B cell. A: 994 Local Ab at 7 days post vaccination (dpc) in the pLN as measured by ELISA. B: Experimental 995 scheme of MBC + pIgG adoptive transfer into recipients are shown . C: Groups of pIgG and 996 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint 32 vaccination combinations used are demonstrated. D: Representative FACS plots of single, live, 997 CD3- NK1.1- B220+ cells showing identification of donor and recipient cells among ASC (top) 998 and GC B cells (bottom). E: Total count of recipient wt HA+ GC B cells in spleen with and 999 without MBC transfer are shown. F: Total count of donor and recipient GC B cells in spleen 1000 and G: ASC in spleen. H: Immunodominance patterns of donor wt HA+ GC B cells and I: 1001 frequency of Cb -specific GC B cells, among wt HA+ cells in spleen at 7 dpc. J: 1002 Immunodominance patterns of recipient wt HA+ GC B cells and K: frequency of Cb-specific 1003 GC B cells, among wt HA+ cells in spleen at 7 days post vaccination. L: AUC OD450nm of 1004 recipient (Igh[b]) and donor (Igh[a]) wt HA specific IgG2a. M: Immunodominance patterns 1005 among wt HA binding IgG2a of donor antibodies. p-values were calculated by one -way 1006 ANOV A (A, I, J, K) or unpaired t test ( E, L). (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). For I 1007 and K statistical differences are indicated by compact letter display (cld), where groups with 1008 different letters are statistically different. Figures represent data from n=2 experiments/group 1009 with 3-7 mice per challenge group per experiment. All data shown as mean ± SD. Animals with 1010 £50 donor wt HA+ B cells (ASC+IgM MBC+switched MBC+ GC B cells) per spleen were 1011 excluded from the analysis. 1012 1013 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 8, 2025. ; https://doi.org/10.1101/2025.11.07.687210doi: bioRxiv preprint