Memory T and B cells with recognition of avian influenza hemagglutinins are poorly responsive to existing seasonal influenza vaccines

17 Immunisation remains the most cost-effective mechanism to combat global influenza 18 infection and is widely employed against seasonal influenza viruses. Zoonotic 19 transmission of avian influenza A viruses represents a significant threat to human 20 health given the lack of population level immunity, which could translate into an 21 influenza pandemic. Therefore, there is a need to better understand pre-existing 22 human immunity against avian influenza strains. as highlighted by the recent rapid, 23 global spread of avian H5Nx clade 2.3.4.4b variants. Here, we sought to quantify the 24 frequencies and specificities of B cells recognising avian hemagglutinin (HA) within 25 unexposed adults, and to characterise the ability of seasonal immunisation to boost 26 cross-reactive immune responses to H5Nx strains, including from clade 2.3.4.4b. Low 27 but detectable serum antibody titres against H5 and H7 avian influenza HA were 28 observed in donors. The frequency of memory B cells with cross-reactive recognition 29 of H5 and H7 HA was low and 2–5 fold lower than populations of seasonal H1N1 and 30 H3N2 HA-specific B cells. Boosting of B cell responses against H5Nx clade 2.3.4.4b 31 HA following seasonal immunisation were sporadic with only 3 out of 19 individuals 32 showing an increased population of probe-positive cells. Cross-reactive B cells 33 generally expressed immunoglobulins drawn from variable heavy chain genes 34 associated with recognition of the HA stem (VH6-1, VH1-69, VH1-18). CD4+ T cell 35 responses towards H5 HA were also weakly boosted with little to no increase in 36 circulating T follicular helper cell populations. These findings highlight the need for 37 avian influenza-specific vaccine products to bolster immunity in human populations, 38 with consideration for use in pre-pandemic preparedness to expand baseline 39 frequencies of avian influenza-specific memory B and T lymphocytes. 40 41 42 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint

43 Zoonotic transmission of avian influenza A viruses such as H5N1 and H7N9 is often 44 associated with extremely high pathogenicity and case fatality rates in humans 1-5. Due 45 to a lack of population level immunity, cross-over from avian reservoirs represents a 46 pressing and emergent threat to human health, with any global pandemic likely to be 47 associated with large loss of life and significant social upheaval. While a variety of 48 monovalent vaccines targeting H5N1 or H7N9 influenza have been produced and are 49 immunogenic in humans 6-10, the unpredictable location and timing of any emergent 50 pandemic makes antigenic mismatch between existing vaccines and/or vaccine seed 51 stocks highly likely. There is therefore a need to better understand pre-existing human 52 immunity against avian influenza strains, particularly in light of the recent global spread 53 of avian H5Nx 2.3.4.4b variants11. 54 55 The existence within unexposed individuals of serum antibodies able to bind and/or 56 neutralise the hemagglutinin (HA) of avian influenza strains has been widely reported. 57 H5- and/or H7-reactivity is observed in both pooled intravenous immunoglobulin 58 (IVIG) preparations 12,13 and in serum samples from human cohorts 14-16, although 59 reported serological concentrations are generally very low. In addition, monoclonal 60 antibodies (mAbs) binding H5 or H7 isolates are readily isolated from subjects not 61 directly exposed to avian influenza by immunisation or infection 17-19 and can protect 62 against H5N1 or H7N9 challenge in mice 17,18,20. Notably, immune exposure to 63 antigenically divergent HA drives the preferential expansion of highly cross-reactive 64 antibody and memory B cell populations 21-24, including those expressing rare mAbs 65 able to neutralise and/or protect against both group 1 and group 2 influenza 25,26. Thus, 66 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint the human immune system appears highly capable of targeting conserved epitopes 67 shared by seasonal and avian influenza strains. 68 69 Existing H5-specific cellular responses are also present within populations of 70 unexposed individuals, as evidenced by CD4+ (and CD8+) memory T cell reactivity 71 predominantly recognising internal proteins of avian viruses 27-31. This cross-reactive 72 memory is likely the product of epitope conservation between seasonal and avian 73 viruses32, and can be expanded following inactivated H5N1 virus immunisation 33. 74 Studies suggest that the immunogenicity of HA-based vaccines in humans is 75 determined, in part, by levels of pre-existing HA-specific CD4 memory T cells 34,35. In 76 populations with low baseline H5- or H7-reactive T cell pools, vaccine immunogenicity 77 may be improved by prior CD4 T cell priming 33 or by covalent coupling of novel HA 78 antigens to seasonal HA proteins36. 79 80 Most recently novel H5 viruses from clade 2.3.4.4.b have spread globally through wild 81 bird populations in four continents37, and with zoonotic outbreaks have reported within 82 domestic cattle38-40, sea mammals41,42 and mustelids43. Disease course within mammals 83 varies in severity, with a mild disease largely confined to mammary tissues reported in 84 cattle, while infection in cats 44 and experimentally infected naïve ferrets 45 and non-85 human primates46 can be highly pathogenic causing major lung pathology and/or death. 86 To date, zoonotic infections in humans have been nearly universally mild, likely 87 reflecting a degree of cross-protective immunity within the population seeded by 88 seasonal exposure. 89 90 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint Here we sought to quantify the frequencies and specificities of B cells recognising avian 91 HA within adults, and to determine the extent to which seasonal immunisation can 92 boost cross-reactive immune responses to H5 including lineages such as 2.3.4.4b. We 93 find memory B cells recognising HA from avian H5N1 and H7N9 influenza strains are 94 widely prevalent in healthy unexposed Australian adults and utilise stereotypic 95 immunoglobulin sequences previously shown to be able to protect in animal models. 96 Vaccination with seasonal inactivated influenza vaccine drives a modest, transient 97 expansion of B cells and CD4+ T cells recognising avian influenza strains. Our results 98 suggest that while population level immunity to H5N1 2.3.4.4b in the form of antibody 99 and memory lymphocyte populations is widespread, targeted vaccine strategies against 100 H5N1 will be required to markedly bolster immunity to emerging avian influenza 101 threats. 102 103

104 Antibodies and B cells binding hemagglutinin from H5 and H7 avian influenza 105 strains are widely prevalent in unexposed adults 106 Consistent with previous reports 14-16, we observed low but detectable serum antibody 107 titres reactive against H5 and H7 avian influenza strains within a cohort of healthy, 108 unexposed Australian adults (N=18), a level 5- to 118-fold reduced compared with 109 endemic H3N2 and H1N1 influenza strains (Fig. 1A). Memory B cells recognising HA 110 from historical H5 (H5N1; A/Indonesia/05/2005) and H7 (H7N9; A/Shanghai/02/2013) 111 avian influenza strains were quantified by flow cytometry using recombinant HA 112 probes47. Distinct H5+ and H7+ memory B cell populations were detected in all 113 individuals tested (Fig. 1B), with median respective frequencies of 0.054% (range 114 0.021 - 0.122) and 0.056% (range 0.031 - 0.086) of total class-switched (IgD-) B cells 115 (Fig. 1C). Cross-reactive memory B cells binding both H5 and H7 probes were 116 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint comparatively infrequent (0.008%; range 0.002 - 0.046), however notable populations 117 were readily discernible in several subjects screened (Fig 1B-C). As a point of 118 reference, memory B cells specific for seasonally epidemic H1N1 (A/New 119 Caledonia/20/1999 and A/California/04/2009) and H3N2 (A/Hong Kong/1/1968 and 120 A/Victoria/361/2011) influenza strains were commonly observed at frequencies 2–5 121 times greater within the same individuals (Fig. 1D) (N=16 matched subjects). Memory 122 B cells specific for avian HA were phenotypically comparable to the parental memory 123 B cell population, displaying a similar distribution of surface immunoglobulin usage 124 (Supplementary Fig. 1A) and a predominantly resting memory phenotype (CD27+ 125 CD21-) (Supplementary Fig. 1B). There was a weak association between subject age 126 and the frequency of cross-reactive memory B cells, an observation that requires 127 clarification in larger cohorts (Supplementary Fig. 1C). 128 Recovered immunoglobulins recognise HA from diverse influenza subtypes 129 Single memory B cells binding H5, H7 or both HA probes were sorted from two 130 individuals and immunoglobulin genes sequences recovered as previously 131 described47,48. A small panel of monoclonal antibodies was expressed (Fig. 2A) and 132 binding to HA from diverse influenza strains was confirmed by ELISA using 133 recombinant HA proteins (Fig. 2B). Consistent with reports of broadly cross-reactive 134 human antibodies, mAbs derived from H5 specific B cells primarily bound influenza A 135 viruses from Group 1, while those from H7-specific B cells bound group 2. Antibodies 136 from B cells with H5/H7 cross-binding activity bound more broadly and were generally 137 drawn from IGHV6-1 and IGHV1-18 convergent classes previously described26,49. The 138 ability of the mAbs to neutralise virus activity was assessed using hemagglutination 139 inhibition (HAI) and focus reduction assays (FRA) against a panel of influenza A and 140 B viruses (Fig. 2C). None of the isolated mAbs showed HAI activity against H1, H5, 141 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint H3 or H7 viruses nor influenza B viruses. However, most of the mAbs showed FRA 142 activity against one or more HA subtypes, particularly for H5 among the H5/H7 cross-143 reactive mAbs. As HAI exclusively measures antibodies targeting the HA head domain 144 while FRA activity measures antibodies that inhibit virus spread (binding, fusion and 145 release), this suggests that as expected mAbs are specific for the stem region of HA. 146 This is consistent with the positive control stem-binding mAb, CR9114, showing no 147 HAI activity while having broad FRA activity against the influenza A viruses 50. 148 Overall, pre-existing neutralising antibodies binding the HA stem domain are prevalent 149 among unexposed individuals and can exhibit neutralising and potentially protective 150 activity against avian influenza strains. 151 152 Responsiveness of memory B cells binding avian HA to vaccination or 153 infection with seasonal influenza viruses 154 Highly cross-reactive serum antibody responses that bind avian influenza strains are 155 reported to be poorly elicited by inactivated seasonal influenza vaccines 17. However, 156 the extent to which cross-reactive memory B cells are directly elicited by seasonal 157 vaccines remains unclear. Given the recent spread of avian Clade 2.3.4.4b H5N1 virus, 158 we sought to characterise cross-reactive humoral responses to this H5 clade following 159 administration of the 2017 Southern Hemisphere inactivated quadrivalent influenza 160 vaccine (IIV4). 161 162 As expected, serum HAI titres against A/Michi (vaccine component strain) 163 significantly increased 1 month after immunisation (Fig 3A). However, no measurable 164 HAI titres were detected against H5 (A/Fujian-Sanyuan/21099/2017) prior to or after 165 seasonal vaccination. The frequency of class-switched H1+, H5+, and H1+H5+ B cells 166 was assessed in individuals (N=23) using B cell probes derived from H1 167 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint (A/Victoria/2570/2019) and H5 (A/Fujian-Sanyuan/21099/2017). H1+ responses 168 significantly increased at 1 month post-immunisation (p=0.0431) although the 169 magnitude of the change in frequency was small (median 1.3-fold relative to baseline) 170 (Fig 3B). No significant changes in H5+ or H1+H5+ responses were observed up to 1 171 month post-vaccination, with only 3 individuals showing a notable increase in H5-172 binding cells post-vaccination. The distribution of antibody isotypes and B cell subsets 173 (as defined by CD21 and CD27 expression) of H1 and H5 reactive B cells were broadly 174 similar to the total memory B cell population (Supplementary Fig 2). 175 176 CD4+ T cell responses against avian H5 are expanded by seasonal vaccination 177 CD4+ T cell responses towards H1 and H5 were assessed via ex vivo restimulation and 178 activation-induced marker (AIM) assay. Vaccine-induced expansion of H1-specific 179 CD4+ cells was primarily observed in the cTFH (CD45RA−CXCR5+) compartment 180 (Supplementary Fig 3), with a 4.6-fold increase in median CD184−CD137+ 181 frequency51 between baseline and week 1 (p=0.0632), before returning to baseline (Fig 182 4A). Limited responsiveness was observed within Tmem (CXCR5− and not 183 CCR7+CD45RA+) cells based upon either CD184−CD137+ or CD154 expression. H5-184 specific CD184−CD137+ Tmem responses were 3.2-fold higher between baseline and 185 4 weeks post-vaccination (p=0.0419). However, there were no significant changes in 186 cTFH responses following IIV4 immunisation. 187 188

189 The rapid global spread of pathogenic avian strains such as H5 2.3.4.4b has highlighted 190 the omnipresent threat of an influenza pandemic. Highly cross-reactive T cell 27-31 and 191 B cell14-16 populations have been described in influenza-exposed adults and may both a 192 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint source of background immune protection in the face of a pandemic, or a potential target 193 for expansion via vaccination to broaden immune protection. Here we show that 194 memory B cells with cross-reactive recognition of avian H5 and H7 influenza viruses 195 are detectable in unexposed Australian adults and express immunoglobulins capable of 196 heterosubtypic HA recognition, some with a degree of neutralising activity. As 197 expected and consistent with prior reports, B cells with broadly cross-reactive 198 recognition of influenza A likely to target the HA stem region and are generally drawn 199 from well characterised stereotypic classes (e.g. VH6-1 26,49, VH1-6952-54, VH1-1826). 200 At high concentrations, such antibodies have shown an ability to protect against 201 pathogenic infection in pre-clinical challenge settings 17,55-57 including against H5 202 2.3.4.4b58. 203 204 The protective potential against avian influenza offered by routine seasonal vaccination 205 appears relatively limited, based upon low titres of serum antibody recognising HA 206 from avian strains, no baseline HAI activity, and low frequencies of HA-specific B and 207 T cells. While some individuals show evidence of limited cross-reactive immunity to 208 these unencountered HA antigens, confirming previous reports of limited baseline 209 immunity at a population level59-62. Upon administration of IIV4, we observed transient 210 boosts in the frequency of H1-specific B and CD4+ T cells as expected. However, this 211 was not recapitulated with regards to H5-specific responses, where expansion of H5 212 2.3.4.4b responses was limited. 213 214 Similarly, our data demonstrate negligible frequencies of H5 2.3.4.4b HA-specific CD4 215 memory at baseline in a healthy adult cohort, with only minimal augmentation by 216 seasonal vaccination. T cell cross-reactivity between seasonal and avian influenza 217 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint strains is likely to be greater for conserved internal proteins such as NP or M than for 218 HA, which may constitute a degree of baseline protection in the event of an outbreak. 219 HA-based vaccines (whether split, recombinant protein, or mRNA/LNP), however, rely 220 entirely on T cell help derived from the HA antigen. Our data and others suggest the 221 pool of cross-reactive CD4 T cells established by seasonal influenza exposure is 222 limited, and that pre-priming is required to support the immunogenicity of H5 223 vaccines33. Recently, covalent linkage of H5 HA to seasonal HA proteins successfully 224 augmented H5 antibody responses in human tonsil organoids by “borrowing” existing 225 CD4 memory36. Establishment of broad population immunity against pre-pandemic H5 226 strains may thus require multiple vaccine doses to establish a sufficient pool of T cell 227 help, or rational design of novel vaccines that maximise availability of preexisting CD4 228 T cell immunity. 229 230 Overall, our findings highlight the need for avian influenza-specific vaccine products 231 to bolster immunity in human populations. Vaccines targeting avian influenza strains 232 with pandemic potential have been developed and are immunogenic in humans 7,22,63. 233 However better pre-pandemic preparedness might necessitate consideration of 234 prophylactic immunisation of human populations prior to an outbreak to expand 235 baseline frequencies of H5- and H7-specific memory T and B lymphocytes, albeit with 236 the understanding that strain matching to the strain that facilitates sustained human-to-237 human transmission might be imperfect. 238 239 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint

240 Participant recruitment and sample collection 241 Study protocols were approved by the University of Melbourne Human Research Ethics 242 Committee (Projects 432/14 and 11395), and all associated procedures were carried out 243 in accordance with the approved guidelines. All participants provided written informed 244 consent in accordance with the Declaration of Helsinki. Participants were not 245 compensated for their participation. 246 247 Peripheral blood samples were collected from a cohort of 18 healthy adults as well as a 248 group of 23 healthy individuals who provided samples at baseline, 1 week, and 4 weeks 249 after immunisation with the 2017 Southern Hemisphere inactivated quadrivalent 250 influenza vaccine (IIV4). Whole blood was collected in sodium heparin anticoagulant. 251 Plasma was collected and stored at -80C. Peripheral blood mononuclear cells (PBMC) 252 were collected by Ficoll-Paque separation, washed, and cryopreserved in 10% 253 DMSO/90% fetal calf serum (FCS). PBMC were stored in liquid nitrogen until use. 254 255 HA-specific probes and flow cytometry 256 The design and purification of fluorescently labelled recombinant HA probes with 257 ablated sialic acid binding activity has been previously described 47. HA-specific B cells 258 were identified within cryopreserved PBMC samples by co-staining with relevant 259 combinations of: H7 (A/Shanghai/01/2013), H1 (A/California/04/2009), H1 (A/New 260 Caledonia/20/1999), H3 (A/Hong Kong/1/1968), H3 (A/Victoria/361/2011) H5 261 (A/Indonesia/05/2005) probes conjugated to streptavidin-PE or -APC (Life 262 Technologies, New York, NY) respectively. B cells were characterised using the 263 following: CD3-QD655, CD14-QD800, CD27-QD605 (Invitrogen), CD19-ECD 264 (Beckman Coulter), IgM-Cy5.5-PerCP, IgG-FITC (BD Pharmingen). 265 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint 266 For the seasonal influenza vaccine cohort, B cells were co-stained with H1 267 (A/Victoria/2570/2019) and H5 (A/Fujian-Sanyuan/21099/2017) probes conjugated to 268 streptavidin-APC or -PE, respectively. The staining panel included IgM BUV395 (G20-269 127), CD21 BUV737 (B-ly4), IgG BV786 (G18-145), IgD PE-Cy7 (IA6-2) (BD), 270 CD27 BV605 (O323; BioLegend), CD19 ECD along with BV510 dump makers 271 (CD14, M5E2; CD3, OKT3; CD8α, RPA-T8; CD16, 3G8; CD10, HI10a; all from 272 BioLegend) and unconjugated streptavidin-BV510 (BD). For all samples, cell viability 273 was assessed using Aqua Live/Dead amine-reactive dye (Invitrogen). 1–2 million 274 events were collected on an LSR II instrument (BD Immunocytometry Systems) or a 275 FACSymphony A5 SE (BD). Analysis was performed using FlowJo software version 276 9.5.2 or 10.10 (TreeStar). 277 278 Activation Induced Marker (AIM) assay 279 Cryopreserved PBMC samples were thawed and rested for 2–4 hr at 37°C in RPMI-280 1640 supplemented with penicillin/streptomycin/L-glutamate and 10% FCS (Sigma) 281 (RF10). Cells were cultured at 1–2 million cells per well in 200μL in 96-well plates 282 (Corning) and stimulated for 20 hr with 5μg/mL of protein (BSA, 283 A/Victoria/2570/2019 HA or A/Fujian-Sanyuan/21099/2017 HA). Small pools of 284 selected donors were also stimulated with SEB (5μg/mL) as a positive control. 285 Following stimulation, cells were washed and stained with monoclonal antibodies 286 (mAbs) CD183 PE-Dazzle594 (G02H57, BioLegend), CD184 BUV395 (12G5, BD), 287 and CD185 PE-Cy7 (MU5UBEE, ThermoFisher) for 30 min at 37°C. Cells were then 288 washed, stained with Live/Dead Aqua viability dye (ThermoFisher) and incubated with 289 mAbs CD3 BUV805 (SK7), CD20 BV510 (2H7), CD154 PE (TRAP1), CD45RA R718 290 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint (HI100) (BD), CD137 BV421 (4B4-1), CD14 BV510 (M5E2), CD4 BV605 (RPA-T4), 291 CD196 BV785 (G034E3), CD134 PerCP-Cy5.5 (ACT35), CD197 Ax647 (G043H7) 292 (BioLegend) for 30min at 4°C. Cells were washed, fixed with 1% formaldehyde and 293 acquired on a BD FACS Symphony A5 SE and analysis was performed using FlowJo 294 Software v10.10 (BD). 295 Sequencing, cloning and expression of B cell immunoglobulins 296 The sequencing and cloning of BCRs from single B cells was performed as previously 297 described48,64. Plasmids expressing heavy and light immunoglobulin chains were 298 transfected into Expi293F cells using ExpiFectamine (Invitrogen). Recombinant 299 monoclonal antibodies were purified from culture supernatants using Protein-A or G 300 (Pierce) as per the manufacturer’s instructions. 301 302 Enzyme-linked immunosorbant assay (ELISA) 303 Antibody binding to HA was tested by ELISA. 96-well Immunosorp plates (Nunc) were 304 coated overnight at 4 °C with 2 μg/mL recombinant HA either expressed in house in 305 Expi293 cells or sourced commercially (Sino Biological). After blocking with 1% fetal 306 calf serum (FCS) in PBS, duplicate wells of monoclonal antibodies (starting at 307 10 μg/mL, four times serial dilutions) or human sera (1:100, four times serial dilutions) 308 were added and incubated for one hour at room temperature. Plates were washed prior 309 to incubation with 1:20 000 dilution of HRP-conjugated anti-human IgG (KPL) for 1 h 310 at room temperature. Plates were washed and developed using 3,3′,5,5′-311 Tetramethylbenzidine (TMB) substrate and read at 450 nm. HA-binding activity of 312 monoclonal antibodies was calculated as the antibody concentration giving half-313 maximal signal (EC50) using a fitted curve (4 parameter log regression). For serum 314 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint samples, endpoint titres were using a fitted curve (4 parameter log regression) and a 315 cutoff of two times background. 316 HAI Assay 317 HAI was performed according to the WHO Global Influenza Surveillance Network 318 protocols65 with the exception that volumes were reduced to 25 µL of ferret sera, 25 µL 319 of antigen (4 HA units), and 25 µL of 1% turkey erythrocytes (0.33% final 320 concentration). Samples were treated with receptor-destroying enzyme (Denka Sieken) 321 at a 1:3 ratio for 18 hours at 37oC, heat inactivated at 56oC for 30min and adsorbed with 322 5% erythrocytes before testing. The A/Michigan/45/2015 (H1N1) virus was propagated 323 in day-10 embryonated chicken eggs. For H5-ferritin nanoparticles, genes expressing 324 the ectodomain of H5 A/Fujian-Sanyuan/21099/2017 HA were synthesised (IDT-325 DNA) and cloned into mammalian expression vector allowing the expression of ferritin 326 nanoparticles as described previously66. H5-ferritin nanoparticles were expressed using 327 Expi293F cells (ThermoFisher) and purified using HiTrap Anion exchange and 328 CaptoCore chromatography. Purified nanoparticles were diluted to 4 HA units for use 329 in HAI assays. 330 331 Assessment of HAI activity of recombinant mAbs was assessed using 1% turkey 332 erythrocytes in a WHO standardised assay. Briefly, mAbs were diluted to 100 μg/mL 333 in PBS prior to incubation with ether treated influenza viruses from strains 334 A/California/07/2009, A/Indonesia/5/2005/PR8-IBDC-RG2, NIBRG-268 335 A/Anhui/1/2013, A/Hong Kong/4801/2014, B/Phuket/3073/2013 and 336 B/Brisbane/60/2008. HAI titres are reported as the reciprocal of the highest dilution 337 where hemagglutination was completely inhibited. 338 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint FRA Assay 339 Neutralisation activity of recombinant mAbs against A/California/07/2009, 340 A/Indonesia/5/2005/PR8-IBDC-RG2, NIBRG-268 A/Anhui/1/2013, A/Hong 341 Kong/4801/2014, B/Phuket/3073/2013 and B/Brisbane/60/2008 was examined using 342 focus reduction assays as previously described 67. The neutralisation titre is expressed 343 as the reciprocal of the highest dilution of a 1 mg/mL mAb stock at which virus 344 infection is inhibited by ≥50%. 345 346 Statistical Analyses 347 Data is generally presented as median +/− IQR. All statistical analyses were 348 performed using GraphPad Prism v5.0 or v10.4.2 (GraphPad Software Inc.). 349 Competing interests 350 The authors declare no competing interests. 351 Authors' contributions 352 C.A.G., J.A.J. and A.K.W. designed the study and experiments. C.A.G., M.K., R.E., 353 M.Z.M.Z., Y.L., A.Z., L.S.U.S., D.T., M.A. and A.H. performed experiments. S.J.K. 354 provided unique samples. C.A.G., M.K., J.A.J. and A.K.W. analysed the experimental 355 data. C.A.G., J.A.J. and A.K.W. wrote the manuscript. All authors have read and 356 approved the manuscript. 357

358 The authors express gratitude towards the study participants for their provision of 359 samples. We acknowledge the Melbourne Cytometry Platform for provision of flow 360 cytometry services. J.A.J., S.J.K. and A.K.W. were awarded Australian National 361 Health and Medical Research Council Investigator Grants. J.A.J. was the recipient of 362 a Viertel Senior Medical Research Fellowship. 363 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint

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Vaccine 35, 46-52 (2017). 570 doi:10.1016/j.vaccine.2016.11.060 571 572 573 574 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint Figures 575 576 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint a b FSC-H FSC-A FSC-H CD19-ECD IgD-Cy7PE SSC-A AQUA/DUMP SA-BB515 H5 (IN05) - PE H7 (SH13) - APC H7+ H5+ H7+H5+ No probes SA-alone 0 0.05 0.15 H7+ H5+ % of CD19+IgD- B cells 0.10 H7+H5+ 0 0.8% of CD19+IgD- B cells 0.6 0.4 0.2 c d 104 Reciprocal serum dilution (EC50) H1 103 102 101 100 H3 H5 H7 PR/34 NC/99 CA/09 HK/68 WY/03 PE/09 SW/13 VN/04 IN/05 NE/03 AN/13 SH/13 10-1 H3H1 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint Figure 1: Serum antibody and memory B cells recognising avian influenza strains 577 are widely prevalent 578 (A) Plasma samples from healthy volunteers (N=18) were screened by ELISA for 579 reactivity against HA from diverse influenza A strains. Reciprocal serum dilutions 580 yielding half maximal binding (EC50) for each antigen are shown. (B) Staining of 581 cryopreserved PBMCs allows identification of CD19+ IgD- B cells not binding a 582 streptavidin-BB515 decoy. Co-staining with recombinant HA probes derived from 583 H7N9 A/Shanghai/01/2013 (SH13) and H5N1 A/Indonesia/5/2005 (IN05) delineates 584 single- and cross-reactive memory B cell populations. (C) Frequencies of H5+, H7+ or 585 H7+H5+ memory B cells in healthy volunteers (N=18). (D) Frequencies of memory B 586 cells binding seasonal H1N1 (NC99 and CA09) and H3N2 (HK68 and VI11) influenza 587 strains were measured in healthy volunteers (N=16). Lines indicate median and IQR. 588 PR/34, A/Puerto Rico/8/1934; NC/99, A/New Caledonia/20/1999; CA/09, 589 A/California/04/2009; HK/68, A/Hong Kong/1/1968; WY/03, A/Wyoming/3/2003; 590 PE/09, A/Perth/16/2009; SW/13, A/Switzerland/9715293/2013; VN/04, 591 A/Vietnam/1203/2004; IN/05, A/Indonesia/5/2005; NE/03, A/Netherlands/219/2003; 592 AN/13, A/Anhui/01/2013; SH/13, A/Shanghai/01/2013; VI/II, A/Victoria/361/2011 593 594 595 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint a b Hemagglutination inhibition (HAI) Focus reduction assay (FRA) mAb H1 CA/09 H5 IN/05 H3 HK/14 H7 AN/13 B BR/08 B PH/13 H1 CA/09 H5 IN/05 H3 HK/14 H7 AN/13 B BR/08 B PH/13 H5 specific N98-H03 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 N98-E12 <10 <10 <10 <10 <10 <10 80 <10 <10 <10 <10 <10 N98-F11 <10 <10 <10 <10 <10 <10 80 <10 <10 <10 <10 <10 J26-A05 <10 <10 <10 <10 <10 <10 160 80 <10 <10 <10 <10 H7 specific N98-B10 <10 <10 <10 <10 <10 <10 80 <10 <10 <10 <10 <10 N98-E05 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 J26-D05 <10 <10 <10 <10 <10 <10 <10 <10 80 <10 <10 <10 H5/H7 cross-reactive N98-G09 <10 <10 <10 <10 <10 <10 160 80 80 <10 <10 <10 N98-2F05 <10 <10 <10 <10 <10 <10 80 20 40 <10 <10 <10 N98-H07 <10 <10 <10 <10 <10 <10 160 40 40 <10 <10 <10 J26-B03 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 Control CR9114 <10 <10 <10 <10 <10 <10 1280 320 40 80 <10 <10 VRC01 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 c .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint Figure 2. Characteristics of monoclonal antibodies derived from avian HA-596 specific B cells 597 (A) Gene usage, CDR-H3 sequence, and mutation loads for 14 monoclonal antibodies 598 derived from sorted H5+, H7+ or H7+H5+ memory B cells from two healthy 599 volunteers. (B) Binding activity of each monoclonal antibody to a panel of recombinant 600 HA proteins derived from influenza A and influenza B strains. Values denote binding 601 log10(EC50) determined by ELISA. HA and mAb combinations not tested are indicated 602 by “n.d.” (C) Neutralisation activity determined by hemagglutination inhibition 603 (HAI) or focus reduction assay (FRA) against a panel of influenza A and B viruses. 604 PR/34;A/Puerto Rico/8/1934, NC/99; A/New Caledonia/20/1999, CA/09; 605 A/California/04/2009, HK/68; A/Hong Kong/1/1968, WY/03; A/Wyoming/3/2003, 606 PE/09; A/Perth/16/2009, SW/13; A/Switzerland/9715293/2013, VN/04; 607 A/Vietnam/1203/2004, IN/05; A/Indonesia/5/2005, NE/03; A/Netherlands/219/2003, 608 AN/13; A/Anhui/01/2013, SH/13; A/Shanghai/01/2013 609 610 611 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint % of CD19+IgD- B cells B A HAI titre BL 4w 16 64 256 1,024 4,096 16,384 H5 <10 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint Figure 3: Cross-reactive serum antibodies not induced by seasonal vaccination but 612 sporadic induction of cross-reactive H5 memory B cells 613 (A) Plasma samples from healthy volunteers (N=19) were assayed for HA inhibition 614 (HAI) against the vaccine matched H1N1 strain (H1/Mic) and clade 2.3.4.4b H5N1 615 strain (H5/Fuj). Baseline (BL) and 4 weeks post immunisation (4w) timepoints were 616 assayed. The lowest plasma dilution assayed was 1:10, with samples not achieving 617 inhibition at this dilution shown as “<10”. The horizontal black line represent the 618 median, and error bars equal the IQR. Significance was determined by Wilcoxon test 619 between BL and 4w. ( B) Frequencies of H1+ (H1/Vic), H5+ (H5/Fuj) and H1+H5+ 620 memory B cells in healthy volunteers (N=19) at BL, 1- and 4-weeks post vaccination 621 (1w and 4w). Donor responses are each timepoint are linked by lines. Significance was 622 determined by Kruskal-Wallis test with Dunn ’s post-test comparing each post-623 immunisation timepoint to baseline (p values corrected for multiple comparisons). 624 625 626 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint B A CD184− CD137+ of CD4MEM (%) CD184− CD137+ of cTFH (%) CD154+ of cTFH (%) p=0.0419 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint Figure 4: T cell responses against H1 and H5 HA following seasonal vaccination 627 PBMCs collected from healthy volunteers (N=23) at baseline (BL; black circle) and 628 following seasonal vaccination at 1 week (1w; open circle) and 4 weeks (4w; half-filled 629 circle) post-immunisation were tested via AIM assay against H1/Vic and H5/Fuj HA 630 protein. Antigen-specific CD4+ memory T cells (CD4MEM) and circulating T follicular 631 helper cells (cTFH) were identified via ( A) CD184−CD137+ or ( B) CD154+ gating. 632 Individual donor responses are plotted and linked by lines between timepoints. 633 Significance was determined by Kruskal-Wallis testing with Dunn ’s post-test 634 comparing each post-immunisation timepoint to baseline (p values corrected for 635 multiple comparisons). 636 637 638 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint