{"paper_id":"07a36eb4-7cfb-49d0-a477-351aaf26e06d","body_text":"Title: Memory T and B cells with recognition of avian 1 \ninfluenza hemagglutinins are poorly responsive to 2 \nexisting seasonal influenza vaccines 3 \n 4 \nAuthors: Christopher A Gonelli1, Marios Koutsakos1, Robyn Esterbauer1, Ming Z M 5 \nZheng1, Yee-Chen Liu1, Amanda Zin1, Lara S U Schwab1, Danielle Tilmanis2, Malet 6 \nAban2, Aeron Hurt2, Stephen J Kent1, Jennifer A Juno1, Adam K Wheatley1 7 \n 8 \nAffiliations: 1Department of Microbiology and Immunology, The University of 9 \nMelbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, 10 \nVIC, Australia; 2WHO Collaborating Centre for Reference and Research on 11 \nInfluenza, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 12 \nAustralia. 13 \n 14 \n*Address correspondence to: Adam K Wheatley (a.wheatley@unimelb.edu.au) 15 \n  16 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nAbstract 17 \nImmunisation remains the most cost-effective mechanism to combat global influenza 18 \ninfection and is widely employed against seasonal influenza viruses. Zoonotic 19 \ntransmission of avian influenza A viruses represents a significant threat to human 20 \nhealth given the lack of population level immunity, which could translate into an 21 \ninfluenza pandemic. Therefore, there is a need to better understand pre-existing 22 \nhuman immunity against avian influenza strains. as highlighted by the recent rapid, 23 \nglobal spread of avian H5Nx clade 2.3.4.4b variants. Here, we sought to quantify the 24 \nfrequencies and specificities of B cells recognising avian hemagglutinin (HA) within 25 \nunexposed adults, and to characterise the ability of seasonal immunisation to boost 26 \ncross-reactive immune responses to H5Nx strains, including from clade 2.3.4.4b. Low 27 \nbut detectable serum antibody titres against H5 and H7 avian influenza HA were 28 \nobserved in donors. The frequency of memory B cells with cross-reactive recognition 29 \nof H5 and H7 HA was low and 2–5 fold lower than populations of seasonal H1N1 and 30 \nH3N2 HA-specific B cells. Boosting of B cell responses against H5Nx clade 2.3.4.4b 31 \nHA following seasonal immunisation were sporadic with only 3 out of 19 individuals 32 \nshowing an increased population of probe-positive cells. Cross-reactive B cells 33 \ngenerally expressed immunoglobulins drawn from variable heavy chain genes 34 \nassociated with recognition of the HA stem (VH6-1, VH1-69, VH1-18). CD4+ T cell 35 \nresponses towards H5 HA were also weakly boosted with little to no increase in 36 \ncirculating T follicular helper cell populations. These findings highlight the need for 37 \navian influenza-specific vaccine products to bolster immunity in human populations, 38 \nwith consideration for use in pre-pandemic preparedness to expand baseline 39 \nfrequencies of avian influenza-specific memory B and T lymphocytes. 40 \n 41 \n42 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nIntroduction  43 \nZoonotic transmission of avian influenza A viruses such as H5N1 and H7N9 is often 44 \nassociated with extremely high pathogenicity and case fatality rates in humans 1-5. Due 45 \nto a lack of population level immunity, cross-over from avian reservoirs represents a 46 \npressing and emergent threat to human health, with any global pandemic likely to be 47 \nassociated with large loss of life and significant social upheaval. While a variety of 48 \nmonovalent vaccines targeting H5N1 or H7N9 influenza have been produced and are 49 \nimmunogenic in humans 6-10, the unpredictable location and timing of any emergent 50 \npandemic makes antigenic mismatch between existing vaccines and/or vaccine seed 51 \nstocks highly likely. There is therefore a need to better understand pre-existing human 52 \nimmunity against avian influenza strains, particularly in light of the recent global spread 53 \nof avian H5Nx 2.3.4.4b variants11. 54 \n 55 \nThe existence within unexposed individuals of serum antibodies able to bind and/or 56 \nneutralise the hemagglutinin (HA) of avian influenza strains has been widely reported. 57 \nH5- and/or H7-reactivity is observed in both pooled intravenous immunoglobulin 58 \n(IVIG) preparations 12,13 and in serum samples from human cohorts 14-16, although 59 \nreported serological concentrations are generally very low. In addition, monoclonal 60 \nantibodies (mAbs) binding H5 or H7 isolates are readily isolated from subjects not 61 \ndirectly exposed to avian influenza by immunisation or infection 17-19 and can protect 62 \nagainst H5N1 or H7N9 challenge in mice 17,18,20. Notably, immune exposure to 63 \nantigenically divergent HA drives the preferential expansion of highly cross-reactive 64 \nantibody and memory B cell populations 21-24, including those expressing rare mAbs 65 \nable to neutralise and/or protect against both group 1 and group 2 influenza 25,26. Thus, 66 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nthe human immune system appears highly capable of targeting conserved epitopes 67 \nshared by seasonal and avian influenza strains. 68 \n 69 \nExisting H5-specific cellular responses are also present within populations of 70 \nunexposed individuals, as evidenced by CD4+ (and CD8+) memory T cell reactivity 71 \npredominantly recognising internal proteins of avian viruses 27-31. This cross-reactive 72 \nmemory is likely the product of epitope conservation between seasonal and avian 73 \nviruses32, and can be expanded following inactivated H5N1 virus immunisation 33. 74 \nStudies suggest that the immunogenicity of HA-based vaccines in humans is 75 \ndetermined, in part, by levels of pre-existing HA-specific CD4 memory T cells 34,35. In 76 \npopulations with low baseline H5- or H7-reactive T cell pools, vaccine immunogenicity 77 \nmay be improved by prior CD4 T cell priming 33 or by covalent coupling of novel HA 78 \nantigens to seasonal HA proteins36.  79 \n 80 \nMost recently novel H5 viruses from clade 2.3.4.4.b have spread globally through wild 81 \nbird populations in four continents37, and with zoonotic outbreaks have reported within 82 \ndomestic cattle38-40, sea mammals41,42 and mustelids43. Disease course within mammals 83 \nvaries in severity, with a mild disease largely confined to mammary tissues reported in 84 \ncattle, while infection in cats 44 and experimentally infected naïve ferrets 45 and non-85 \nhuman primates46 can be highly pathogenic causing major lung pathology and/or death. 86 \nTo date, zoonotic infections in humans have been nearly universally mild, likely 87 \nreflecting a degree of cross-protective immunity within the population seeded by 88 \nseasonal exposure.  89 \n 90 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nHere we sought to quantify the frequencies and specificities of B cells recognising avian 91 \nHA within adults, and to determine the extent to which seasonal immunisation can 92 \nboost cross-reactive immune responses to H5 including lineages such as 2.3.4.4b. We 93 \nfind memory B cells recognising HA from avian H5N1 and H7N9 influenza strains are 94 \nwidely prevalent in healthy unexposed Australian adults and utilise stereotypic 95 \nimmunoglobulin sequences previously shown to be able to protect in animal models. 96 \nVaccination with seasonal inactivated influenza vaccine drives a modest, transient 97 \nexpansion of B cells and CD4+ T cells recognising avian influenza strains. Our results 98 \nsuggest that while population level immunity to H5N1 2.3.4.4b in the form of antibody 99 \nand memory lymphocyte populations is widespread, targeted vaccine strategies against 100 \nH5N1 will be required to markedly bolster immunity to emerging avian influenza 101 \nthreats. 102 \n 103 \nResults 104 \nAntibodies and B cells binding hemagglutinin from H5 and H7 avian influenza 105 \nstrains are widely prevalent in unexposed adults 106 \nConsistent with previous reports 14-16, we observed low but detectable serum antibody 107 \ntitres reactive against H5 and H7 avian influenza strains within a cohort of healthy, 108 \nunexposed Australian adults (N=18), a level 5- to 118-fold reduced compared with 109 \nendemic H3N2 and H1N1 influenza strains (Fig. 1A). Memory B cells recognising HA 110 \nfrom historical H5 (H5N1; A/Indonesia/05/2005) and H7 (H7N9; A/Shanghai/02/2013) 111 \navian influenza strains were quantified by flow cytometry using recombinant HA 112 \nprobes47. Distinct H5+ and H7+ memory B cell populations were detected in all 113 \nindividuals tested (Fig. 1B), with median respective frequencies of 0.054% (range 114 \n0.021 - 0.122) and 0.056% (range 0.031 - 0.086) of total class-switched (IgD-) B cells 115 \n(Fig. 1C). Cross-reactive memory B cells binding both H5 and H7 probes were 116 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \ncomparatively infrequent (0.008%; range 0.002 - 0.046), however notable populations 117 \nwere readily discernible in several subjects screened (Fig 1B-C). As a point of 118 \nreference, memory B cells specific for seasonally epidemic H1N1 (A/New 119 \nCaledonia/20/1999 and A/California/04/2009) and H3N2 (A/Hong Kong/1/1968 and 120 \nA/Victoria/361/2011) influenza strains were commonly observed at frequencies 2–5 121 \ntimes greater within the same individuals (Fig. 1D) (N=16 matched subjects). Memory 122 \nB cells specific for avian HA were phenotypically comparable to the parental memory 123 \nB cell population, displaying a similar distribution of surface immunoglobulin usage 124 \n(Supplementary Fig. 1A) and a predominantly resting memory phenotype (CD27+ 125 \nCD21-) (Supplementary Fig. 1B). There was a weak association between subject age 126 \nand the frequency of cross-reactive memory B cells, an observation that requires 127 \nclarification in larger cohorts (Supplementary Fig. 1C).  128 \nRecovered immunoglobulins recognise HA from diverse influenza subtypes 129 \nSingle memory B cells binding H5, H7 or both HA probes were sorted from two 130 \nindividuals and immunoglobulin genes sequences recovered as previously 131 \ndescribed47,48. A small panel of monoclonal antibodies was expressed (Fig. 2A) and 132 \nbinding to HA from diverse influenza strains was confirmed by ELISA using 133 \nrecombinant HA proteins (Fig. 2B). Consistent with reports of broadly cross-reactive 134 \nhuman antibodies, mAbs derived from H5 specific B cells primarily bound influenza A 135 \nviruses from Group 1, while those from H7-specific B cells bound group 2. Antibodies 136 \nfrom B cells with H5/H7 cross-binding activity bound more broadly and were generally 137 \ndrawn from IGHV6-1 and IGHV1-18 convergent classes previously described26,49. The 138 \nability of the mAbs to neutralise virus activity was assessed using hemagglutination 139 \ninhibition (HAI) and focus reduction assays (FRA) against a panel of influenza A and 140 \nB viruses (Fig. 2C). None of the isolated mAbs showed HAI activity against H1, H5, 141 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nH3 or H7 viruses nor influenza B viruses. However, most of the mAbs showed FRA 142 \nactivity against one or more HA subtypes, particularly for H5 among the H5/H7 cross-143 \nreactive mAbs. As HAI exclusively measures antibodies targeting the HA head domain 144 \nwhile FRA activity measures antibodies that inhibit virus spread (binding, fusion and 145 \nrelease), this suggests that as expected mAbs are specific for the stem region of HA. 146 \nThis is consistent with the positive control stem-binding mAb, CR9114, showing no 147 \nHAI activity while having broad FRA activity against the influenza A viruses 50. 148 \nOverall, pre-existing neutralising antibodies binding the HA stem domain are prevalent 149 \namong unexposed individuals and can exhibit neutralising and potentially protective 150 \nactivity against avian influenza strains. 151 \n 152 \nResponsiveness of memory B cells binding avian HA to vaccination or 153 \ninfection with seasonal influenza viruses 154 \nHighly cross-reactive serum antibody responses that bind avian influenza strains are 155 \nreported to be poorly elicited by inactivated seasonal influenza vaccines 17. However, 156 \nthe extent to which cross-reactive memory B cells are directly elicited by seasonal 157 \nvaccines remains unclear. Given the recent spread of avian Clade 2.3.4.4b H5N1 virus, 158 \nwe sought to characterise cross-reactive humoral responses to this H5 clade following 159 \nadministration of the 2017 Southern Hemisphere inactivated quadrivalent influenza 160 \nvaccine (IIV4).  161 \n 162 \nAs expected, serum HAI titres against A/Michi (vaccine component strain) 163 \nsignificantly increased 1 month after immunisation (Fig 3A). However, no measurable 164 \nHAI titres were detected against H5 (A/Fujian-Sanyuan/21099/2017) prior to or after 165 \nseasonal vaccination. The frequency of class-switched H1+, H5+, and H1+H5+ B cells 166 \nwas assessed in individuals (N=23) using B cell probes derived from H1 167 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n(A/Victoria/2570/2019) and H5 (A/Fujian-Sanyuan/21099/2017). H1+ responses 168 \nsignificantly increased at 1 month post-immunisation (p=0.0431) although the 169 \nmagnitude of the change in frequency was small (median 1.3-fold relative to baseline) 170 \n(Fig 3B). No significant changes in H5+ or H1+H5+ responses were observed up to 1 171 \nmonth post-vaccination, with only 3 individuals showing a notable increase in H5-172 \nbinding cells post-vaccination. The distribution of antibody isotypes and B cell subsets 173 \n(as defined by CD21 and CD27 expression) of H1 and H5 reactive B cells were broadly 174 \nsimilar to the total memory B cell population (Supplementary Fig 2).  175 \n 176 \nCD4+ T cell responses against avian H5 are expanded by seasonal vaccination 177 \nCD4+ T cell responses towards H1 and H5 were assessed via ex vivo restimulation and 178 \nactivation-induced marker (AIM) assay.  Vaccine-induced expansion of H1-specific 179 \nCD4+ cells was primarily observed in the cTFH (CD45RA−CXCR5+) compartment 180 \n(Supplementary Fig 3), with a 4.6-fold increase in median CD184−CD137+ 181 \nfrequency51 between baseline and week 1 (p=0.0632), before returning to baseline (Fig 182 \n4A). Limited responsiveness was observed within Tmem (CXCR5− and not 183 \nCCR7+CD45RA+) cells based upon either CD184−CD137+ or CD154 expression. H5-184 \nspecific CD184−CD137+ Tmem responses were 3.2-fold higher between baseline and 185 \n4 weeks post-vaccination (p=0.0419). However, there were no significant changes in 186 \ncTFH responses following IIV4 immunisation. 187 \n 188 \nDiscussion 189 \nThe rapid global spread of pathogenic avian strains such as H5 2.3.4.4b has highlighted 190 \nthe omnipresent threat of an influenza pandemic. Highly cross-reactive T cell 27-31 and 191 \nB cell14-16 populations have been described in influenza-exposed adults and may both a 192 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nsource of background immune protection in the face of a pandemic, or a potential target 193 \nfor expansion via vaccination to broaden immune protection. Here we show that 194 \nmemory B cells with cross-reactive recognition of avian H5 and H7 influenza viruses 195 \nare detectable in unexposed Australian adults and express immunoglobulins capable of 196 \nheterosubtypic HA recognition, some with a degree of neutralising activity. As 197 \nexpected and consistent with prior reports, B cells with broadly cross-reactive 198 \nrecognition of influenza A likely to target the HA stem region and are generally drawn 199 \nfrom well characterised stereotypic classes (e.g. VH6-1 26,49, VH1-6952-54, VH1-1826).  200 \nAt high concentrations, such antibodies have shown an ability to protect against 201 \npathogenic infection in pre-clinical challenge settings 17,55-57 including against H5 202 \n2.3.4.4b58. 203 \n 204 \nThe protective potential against avian influenza offered by routine seasonal vaccination 205 \nappears relatively limited, based upon low titres of serum antibody recognising HA 206 \nfrom avian strains, no baseline HAI activity, and low frequencies of HA-specific B and 207 \nT cells. While some individuals show evidence of limited cross-reactive immunity to 208 \nthese unencountered HA antigens, confirming previous reports of limited baseline 209 \nimmunity at a population level59-62. Upon administration of IIV4, we observed transient 210 \nboosts in the frequency of H1-specific B and CD4+ T cells as expected. However, this 211 \nwas not recapitulated with regards to H5-specific responses, where expansion of H5 212 \n2.3.4.4b responses was limited.  213 \n 214 \nSimilarly, our data demonstrate negligible frequencies of H5 2.3.4.4b HA-specific CD4 215 \nmemory at baseline in a healthy adult cohort, with only minimal augmentation by 216 \nseasonal vaccination. T cell cross-reactivity between seasonal and avian influenza 217 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nstrains is likely to be greater for conserved internal proteins such as NP or M than for 218 \nHA, which may constitute a degree of baseline protection in the event of an outbreak.  219 \nHA-based vaccines (whether split, recombinant protein, or mRNA/LNP), however, rely 220 \nentirely on T cell help derived from the HA antigen. Our data and others suggest the 221 \npool of cross-reactive CD4 T cells established by seasonal influenza exposure is 222 \nlimited, and that pre-priming is required to support the immunogenicity of H5 223 \nvaccines33. Recently, covalent linkage of H5 HA to seasonal HA proteins successfully 224 \naugmented H5 antibody responses in human tonsil organoids by “borrowing” existing 225 \nCD4 memory36. Establishment of broad population immunity against pre-pandemic H5 226 \nstrains may thus require multiple vaccine doses to establish a sufficient pool of T cell 227 \nhelp, or rational design of novel vaccines that maximise availability of preexisting CD4 228 \nT cell immunity. 229 \n 230 \nOverall, our findings highlight the need for avian influenza-specific vaccine products 231 \nto bolster immunity in human populations. Vaccines targeting avian influenza strains 232 \nwith pandemic potential have been developed and are immunogenic in humans 7,22,63. 233 \nHowever better pre-pandemic preparedness might necessitate consideration of 234 \nprophylactic immunisation of human populations prior to an outbreak to expand 235 \nbaseline frequencies of H5- and H7-specific memory T and B lymphocytes, albeit with 236 \nthe understanding that strain matching to the strain that facilitates sustained human-to-237 \nhuman transmission might be imperfect. 238 \n  239 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nMaterials and Methods  240 \nParticipant recruitment and sample collection 241 \nStudy protocols were approved by the University of Melbourne Human Research Ethics 242 \nCommittee (Projects 432/14 and 11395), and all associated procedures were carried out 243 \nin accordance with the approved guidelines. All participants provided written informed 244 \nconsent in accordance with the Declaration of Helsinki. Participants were not 245 \ncompensated for their participation. 246 \n 247 \nPeripheral blood samples were collected from a cohort of 18 healthy adults as well as a 248 \ngroup of 23 healthy individuals who provided samples at baseline, 1 week, and 4 weeks 249 \nafter immunisation with the 2017 Southern Hemisphere inactivated quadrivalent 250 \ninfluenza vaccine (IIV4). Whole blood was collected in sodium heparin anticoagulant. 251 \nPlasma was collected and stored at -80C. Peripheral blood mononuclear cells (PBMC) 252 \nwere collected by Ficoll-Paque separation, washed, and cryopreserved in 10% 253 \nDMSO/90% fetal calf serum (FCS). PBMC were stored in liquid nitrogen until use.  254 \n 255 \nHA-specific probes and flow cytometry 256 \nThe design and purification of fluorescently labelled recombinant HA probes with 257 \nablated sialic acid binding activity has been previously described 47. HA-specific B cells 258 \nwere identified within cryopreserved PBMC samples by co-staining with relevant 259 \ncombinations of: H7 (A/Shanghai/01/2013), H1 (A/California/04/2009), H1 (A/New 260 \nCaledonia/20/1999), H3 (A/Hong Kong/1/1968), H3 (A/Victoria/361/2011)  H5 261 \n(A/Indonesia/05/2005) probes conjugated to streptavidin-PE or -APC (Life 262 \nTechnologies, New York, NY) respectively. B cells were characterised using the 263 \nfollowing: CD3-QD655, CD14-QD800, CD27-QD605 (Invitrogen), CD19-ECD 264 \n(Beckman Coulter), IgM-Cy5.5-PerCP, IgG-FITC (BD Pharmingen).  265 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n 266 \nFor the seasonal influenza vaccine cohort, B cells were co-stained with H1 267 \n(A/Victoria/2570/2019) and H5 (A/Fujian-Sanyuan/21099/2017) probes conjugated to 268 \nstreptavidin-APC or -PE, respectively. The staining panel included IgM BUV395 (G20-269 \n127), CD21 BUV737 (B-ly4), IgG BV786 (G18-145), IgD PE-Cy7 (IA6-2) (BD), 270 \nCD27 BV605 (O323; BioLegend), CD19 ECD along with BV510 dump makers 271 \n(CD14, M5E2; CD3, OKT3; CD8α, RPA-T8; CD16, 3G8; CD10, HI10a; all from 272 \nBioLegend) and unconjugated streptavidin-BV510 (BD). For all samples, cell viability 273 \nwas assessed using Aqua Live/Dead amine-reactive dye (Invitrogen). 1–2 million 274 \nevents were collected on an LSR II instrument (BD Immunocytometry Systems) or a 275 \nFACSymphony A5 SE (BD). Analysis was performed using FlowJo software version 276 \n9.5.2 or 10.10 (TreeStar).  277 \n 278 \nActivation Induced Marker (AIM) assay 279 \nCryopreserved PBMC samples were thawed and rested for 2–4 hr at 37°C in RPMI-280 \n1640 supplemented with penicillin/streptomycin/L-glutamate and 10% FCS (Sigma) 281 \n(RF10). Cells were cultured at 1–2 million cells per well in 200μL in 96-well plates 282 \n(Corning) and stimulated for 20 hr with 5μg/mL of protein (BSA, 283 \nA/Victoria/2570/2019 HA or A/Fujian-Sanyuan/21099/2017 HA). Small pools of 284 \nselected donors were also stimulated with SEB (5μg/mL) as a positive control. 285 \nFollowing stimulation, cells were washed and stained with monoclonal antibodies 286 \n(mAbs) CD183 PE-Dazzle594 (G02H57, BioLegend), CD184 BUV395 (12G5, BD), 287 \nand CD185 PE-Cy7 (MU5UBEE, ThermoFisher) for 30 min at 37°C. Cells were then 288 \nwashed, stained with Live/Dead Aqua viability dye (ThermoFisher) and incubated with 289 \nmAbs CD3 BUV805 (SK7), CD20 BV510 (2H7), CD154 PE (TRAP1), CD45RA R718 290 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n(HI100) (BD), CD137 BV421 (4B4-1), CD14 BV510 (M5E2), CD4 BV605 (RPA-T4), 291 \nCD196 BV785 (G034E3), CD134 PerCP-Cy5.5 (ACT35), CD197 Ax647 (G043H7) 292 \n(BioLegend) for 30min at 4°C. Cells were washed, fixed with 1% formaldehyde and 293 \nacquired on a BD FACS Symphony A5 SE and analysis was performed using FlowJo 294 \nSoftware v10.10 (BD). 295 \nSequencing, cloning and expression of B cell immunoglobulins 296 \nThe sequencing and cloning of BCRs from single B cells was performed as previously 297 \ndescribed48,64. Plasmids expressing heavy and light immunoglobulin chains were 298 \ntransfected into Expi293F cells using ExpiFectamine (Invitrogen). Recombinant 299 \nmonoclonal antibodies were purified from culture supernatants using Protein-A or G 300 \n(Pierce) as per the manufacturer’s instructions.  301 \n 302 \nEnzyme-linked immunosorbant assay (ELISA) 303 \nAntibody binding to HA was tested by ELISA. 96-well Immunosorp plates (Nunc) were 304 \ncoated overnight at 4 °C with 2 μg/mL recombinant HA either expressed in house in 305 \nExpi293 cells or sourced commercially (Sino Biological). After blocking with 1% fetal 306 \ncalf serum (FCS) in PBS, duplicate wells of monoclonal antibodies (starting at 307 \n10 μg/mL, four times serial dilutions) or human sera (1:100, four times serial dilutions) 308 \nwere added and incubated for one hour at room temperature. Plates were washed prior 309 \nto incubation with 1:20 000 dilution of HRP-conjugated anti-human IgG (KPL) for 1 h 310 \nat room temperature. Plates were washed and developed using 3,3′,5,5′-311 \nTetramethylbenzidine (TMB) substrate and read at 450 nm. HA-binding activity of 312 \nmonoclonal antibodies was calculated as the antibody concentration giving half-313 \nmaximal signal (EC50) using a fitted curve (4 parameter log regression). For serum 314 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nsamples, endpoint titres were using a fitted curve (4 parameter log regression) and a 315 \ncutoff of two times background. 316 \nHAI Assay 317 \nHAI was performed according to the WHO Global Influenza Surveillance Network 318 \nprotocols65 with the exception that volumes were reduced to 25 µL of ferret sera, 25 µL 319 \nof antigen (4 HA units), and 25 µL of 1% turkey erythrocytes (0.33% final 320 \nconcentration). Samples were treated with receptor-destroying enzyme (Denka Sieken) 321 \nat a 1:3 ratio for 18 hours at 37oC, heat inactivated at 56oC for 30min and adsorbed with 322 \n5% erythrocytes before testing. The A/Michigan/45/2015 (H1N1) virus was propagated 323 \nin day-10 embryonated chicken eggs. For H5-ferritin nanoparticles, genes expressing 324 \nthe ectodomain of H5 A/Fujian-Sanyuan/21099/2017 HA were synthesised (IDT-325 \nDNA) and cloned into mammalian expression vector allowing the expression of ferritin 326 \nnanoparticles as described previously66. H5-ferritin nanoparticles were expressed using 327 \nExpi293F cells (ThermoFisher) and purified using HiTrap Anion exchange and 328 \nCaptoCore chromatography. Purified nanoparticles were diluted to 4 HA units for use 329 \nin HAI assays. 330 \n 331 \nAssessment of HAI activity of recombinant mAbs was assessed using 1% turkey 332 \nerythrocytes in a WHO standardised assay. Briefly, mAbs were diluted to 100 μg/mL 333 \nin PBS prior to incubation with ether treated influenza viruses from strains 334 \nA/California/07/2009, A/Indonesia/5/2005/PR8-IBDC-RG2, NIBRG-268 335 \nA/Anhui/1/2013, A/Hong Kong/4801/2014, B/Phuket/3073/2013 and 336 \nB/Brisbane/60/2008. HAI titres are reported as the reciprocal of the highest dilution 337 \nwhere hemagglutination was completely inhibited. 338 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nFRA Assay 339 \nNeutralisation activity of recombinant mAbs against A/California/07/2009, 340 \nA/Indonesia/5/2005/PR8-IBDC-RG2, NIBRG-268 A/Anhui/1/2013, A/Hong 341 \nKong/4801/2014, B/Phuket/3073/2013 and B/Brisbane/60/2008 was examined using 342 \nfocus reduction assays as previously described 67. The neutralisation titre is expressed 343 \nas the reciprocal of the highest dilution of a 1 mg/mL mAb stock at which virus 344 \ninfection is inhibited by ≥50%. 345 \n 346 \nStatistical Analyses 347 \nData is generally presented as median +/− IQR. All statistical analyses were 348 \nperformed using GraphPad Prism v5.0 or v10.4.2 (GraphPad Software Inc.).  349 \nCompeting interests 350 \nThe authors declare no competing interests. 351 \nAuthors' contributions 352 \nC.A.G., J.A.J. and A.K.W. designed the study and experiments. C.A.G., M.K., R.E., 353 \nM.Z.M.Z., Y.L., A.Z., L.S.U.S., D.T., M.A. and A.H. performed experiments. S.J.K. 354 \nprovided unique samples. C.A.G., M.K., J.A.J. and A.K.W. analysed the experimental 355 \ndata. C.A.G., J.A.J. and A.K.W. wrote the manuscript. All authors have read and 356 \napproved the manuscript. 357 \nAcknowledgements  358 \nThe authors express gratitude towards the study participants for their provision of 359 \nsamples. We acknowledge the Melbourne Cytometry Platform for provision of flow 360 \ncytometry services. J.A.J., S.J.K. and A.K.W. were awarded Australian National 361 \nHealth and Medical Research Council Investigator Grants. J.A.J. was the recipient of 362 \na Viertel Senior Medical Research Fellowship.  363 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nReferences 364 \n1 Chen, Z.  et al. Asymptomatic, mild, and severe influenza A(H7N9) virus 365 \ninfection in humans, Guangzhou, China. Emerg Infect Dis 20, 1535-1540 366 \n(2014). doi:10.3201/eid2009.140424 367 \n2 Kandun, I. N.  et al. Three Indonesian clusters of H5N1 virus infection in 2005. 368 \nN Engl J Med 355, 2186-2194 (2006). doi:10.1056/NEJMoa060930 369 \n3 Chen, Y.  et al. Human infections with the emerging avian influenza A H7N9 370 \nvirus from wet market poultry: clinical analysis and characterisation of viral 371 \ngenome. Lancet 381, 1916-1925 (2013). doi:10.1016/S0140-6736(13)60903-4 372 \n4 Chotpitayasunondh, T.  et al. Human disease from influenza A (H5N1), 373 \nThailand, 2004. Emerg Infect Dis 11, 201-209 (2005). 374 \ndoi:10.3201/eid1102.041061 375 \n5 Gao, H. N.  et al. Clinical findings in 111 cases of influenza A (H7N9) virus 376 \ninfection. N Engl J Med 368, 2277-2285 (2013). 377 \ndoi:10.1056/NEJMoa1305584 378 \n6 Bart, S. A.  et al. A cell culture-derived MF59-adjuvanted pandemic A/H7N9 379 \nvaccine is immunogenic in adults. Sci Transl Med 6, 234ra255 (2014). 380 \ndoi:10.1126/scitranslmed.3008761 381 \n7 Ehrlich, H. J.  et al. A clinical trial of a whole-virus H5N1 vaccine derived 382 \nfrom cell culture. N Engl J Med 358, 2573-2584 (2008). 383 \ndoi:10.1056/NEJMoa073121 384 \n8 Ledgerwood, J. E.  et al. DNA priming and influenza vaccine immunogenicity: 385 \ntwo phase 1 open label randomised clinical trials. Lancet Infect Dis 11, 916-386 \n924 (2011). doi:10.1016/s1473-3099(11)70240-7 387 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n9 van der Velden, M. V.  et al. Safety and immunogenicity of a vero cell culture-388 \nderived whole-virus influenza A(H5N1) vaccine in a pediatric population. J 389 \nInfect Dis 209, 12-23 (2014). doi:10.1093/infdis/jit498 390 \n10 van der Velden, M. V.  et al. Safety and immunogenicity of a vero cell culture-391 \nderived whole-virus H5N1 influenza vaccine in chronically ill and 392 \nimmunocompromised patients. Clin Vaccine Immunol 21, 867-876 (2014). 393 \ndoi:10.1128/cvi.00065-14 394 \n11 Peacock, T. P.  et al. The global H5N1 influenza panzootic in mammals. 395 \nNature 637, 304-313 (2025). doi:10.1038/s41586-024-08054-z 396 \n12 Sullivan, J. S.  et al. Heterosubtypic anti-avian H5N1 influenza antibodies in 397 \nintravenous immunoglobulins from globally separate populations protect 398 \nagainst H5N1 infection in cell culture. J Mol Genet Med 3, 217-224 (2009). 399 \ndoi:10.4172/1747-0862.1000038 400 \n13 Jegaskanda, S.  et al. Cross-reactive influenza-specific antibody-dependent 401 \ncellular cytotoxicity in intravenous immunoglobulin as a potential therapeutic 402 \nagainst emerging influenza viruses. J Infect Dis 210, 1811-1822 (2014). 403 \ndoi:10.1093/infdis/jiu334 404 \n14 Sui, J.  et al. Wide prevalence of heterosubtypic broadly neutralizing human 405 \nanti-influenza A antibodies. Clin Infect Dis 52, 1003-1009 (2011). 406 \ndoi:10.1093/cid/cir121 407 \n15 Garretson, T. A.  et al. Immune history shapes human antibody responses to 408 \nH5N1 influenza viruses. Nat Med (2025). doi:10.1038/s41591-025-03599-6 409 \n16 Le Sage, V.  et al. Influenza A(H5N1) Immune Response among Ferrets with 410 \nInfluenza A(H1N1)pdm09 Immunity. Emerg Infect Dis 31, 477-487 (2025). 411 \ndoi:10.3201/eid3103.241485 412 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n17 Corti, D.  et al. Heterosubtypic neutralizing antibodies are produced by 413 \nindividuals immunized with a seasonal influenza vaccine. J Clin Invest 120, 414 \n1663-1673 (2010). doi:10.1172/jci41902 415 \n18 Henry Dunand, C. J.  et al. Preexisting human antibodies neutralize recently 416 \nemerged H7N9 influenza strains. J Clin Invest 125, 1255-1268 (2015). 417 \ndoi:10.1172/jci74374 418 \n19 Throsby, M.  et al. Heterosubtypic neutralizing monoclonal antibodies cross-419 \nprotective against H5N1 and H1N1 recovered from human IgM+ memory B 420 \ncells. PLoS One 3, e3942 (2008). doi:10.1371/journal.pone.0003942 421 \n20 Thomson, C. A.  et al. Pandemic H1N1 Influenza Infection and Vaccination in 422 \nHumans Induces Cross-Protective Antibodies that Target the Hemagglutinin 423 \nStem. Front Immunol 3, 87 (2012). doi:10.3389/fimmu.2012.00087 424 \n21 Ellebedy, A. H.  et al. Induction of broadly cross-reactive antibody responses 425 \nto the influenza HA stem region following H5N1 vaccination in humans. Proc 426 \nNatl Acad Sci U S A 111, 13133-13138 (2014). doi:10.1073/pnas.1414070111 427 \n22 Nachbagauer, R.  et al. Induction of broadly reactive anti-hemagglutinin stalk 428 \nantibodies by an H5N1 vaccine in humans. J Virol 88, 13260-13268 (2014). 429 \ndoi:10.1128/jvi.02133-14 430 \n23 Wheatley, A. K.  et al. H5N1 Vaccine-Elicited Memory B Cells Are 431 \nGenetically Constrained by the IGHV Locus in the Recognition of a 432 \nNeutralizing Epitope in the Hemagglutinin Stem. J Immunol 195, 602-610 433 \n(2015). doi:10.4049/jimmunol.1402835 434 \n24 Wrammert, J.  et al. Broadly cross-reactive antibodies dominate the human B 435 \ncell response against 2009 pandemic H1N1 influenza virus infection. J Exp 436 \nMed 208, 181-193 (2011). doi:10.1084/jem.20101352 437 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n25 Andrews, S. F.  et al. Preferential induction of cross-group influenza A 438 \nhemagglutinin stem-specific memory B cells after H7N9 immunization in 439 \nhumans. Sci Immunol 2 (2017). doi:10.1126/sciimmunol.aan2676 440 \n26 Joyce, M. G.  et al. Vaccine-Induced Antibodies that Neutralize Group 1 and 441 \nGroup 2 Influenza A Viruses. Cell 166, 609-623 (2016). 442 \ndoi:10.1016/j.cell.2016.06.043 443 \n27 Jameson, J., Cruz, J., Terajima, M. & Ennis, F. A. Human CD8+ and CD4+ T 444 \nlymphocyte memory to influenza A viruses of swine and avian species. 445 \nJournal of Immunology (Baltimore, Md.: 1950) 162, 7578-7583 (1999).  446 \n28 Lee, L. Y.-H.  et al. Memory T cells established by seasonal human influenza 447 \nA infection cross-react with avian influenza A (H5N1) in healthy individuals. 448 \nThe Journal of Clinical Investigation 118, 3478-3490 (2008). 449 \ndoi:10.1172/JCI32460 450 \n29 Roti, M.  et al. Healthy human subjects have CD4+ T cells directed against 451 \nH5N1 influenza virus. Journal of Immunology (Baltimore, Md.: 1950) 180, 452 \n1758-1768 (2008). doi:10.4049/jimmunol.180.3.1758 453 \n30 Cusick, M. F., Wang, S. & Eckels, D. D. In vitro responses to avian influenza 454 \nH5 by human CD4 T cells. Journal of Immunology (Baltimore, Md.: 1950) 455 \n183, 6432-6441 (2009). doi:10.4049/jimmunol.0901617 456 \n31 Noisumdaeng, P.  et al. T cell mediated immunity against influenza H5N1 457 \nnucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors 458 \nand non-H5N1 subjects. PeerJ 9, e11021 (2021). doi:10.7717/peerj.11021 459 \n32 Sidney, J.  et al. Targets of influenza human T-cell response are mostly 460 \nconserved in H5N1. mBio, e0347924 (2024). doi:10.1128/mbio.03479-24 461 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n33 Nayak, J. L., Richards, K. A., Yang, H., Treanor, J. J. & Sant, A. J. Effect of 462 \ninfluenza A(H5N1) vaccine prepandemic priming on CD4+ T-cell responses. 463 \nThe Journal of Infectious Diseases 211, 1408-1417 (2015). 464 \ndoi:10.1093/infdis/jiu616 465 \n34 DiPiazza, A., Richards, K., Poulton, N. & Sant, A. J. Avian and Human 466 \nSeasonal Influenza Hemagglutinin Proteins Elicit CD4 T Cell Responses That 467 \nAre Comparable in Epitope Abundance and Diversity. Clin Vaccine Immunol 468 \n24 (2017). doi:10.1128/CVI.00548-16 469 \n35 Nayak, J. L.  et al. CD4+ T-cell expansion predicts neutralizing antibody 470 \nresponses to monovalent, inactivated 2009 pandemic influenza A(H1N1) virus 471 \nsubtype H1N1 vaccine. J Infect Dis 207, 297-305 (2013). 472 \ndoi:10.1093/infdis/jis684 473 \n36 Mallajosyula, V.  et al. Coupling antigens from multiple subtypes of influenza 474 \ncan broaden antibody and T cell responses. Science 386, 1389-1395 (2024). 475 \ndoi:10.1126/science.adi2396 476 \n37 Plaza, P. I., Gamarra-Toledo, V., Euguí, J. R. & Lambertucci, S. A. Recent 477 \nChanges in Patterns of Mammal Infection with Highly Pathogenic Avian 478 \nInfluenza A(H5N1) Virus Worldwide. Emerg Infect Dis 30, 444-452 (2024). 479 \ndoi:10.3201/eid3003.231098 480 \n38 Burrough, E. R.  et al. Highly Pathogenic Avian Influenza A(H5N1) Clade 481 \n2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 482 \n2024. Emerg Infect Dis 30, 1335-1343 (2024). doi:10.3201/eid3007.240508 483 \n39 Hu, X.  et al. Genomic characterization of highly pathogenic avian influenza A 484 \nH5N1 virus newly emerged in dairy cattle. Emerg Microbes Infect 13, 485 \n2380421 (2024). doi:10.1080/22221751.2024.2380421 486 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n40 Caserta, L. C.  et al. Spillover of highly pathogenic avian influenza H5N1 virus 487 \nto dairy cattle. Nature 634, 669-676 (2024). doi:10.1038/s41586-024-07849-4 488 \n41 de Carvalho Araujo, A.  et al. Mortality in sea lions is associated with the 489 \nintroduction of the H5N1 clade 2.3.4.4b virus in Brazil October 2023: whole 490 \ngenome sequencing and phylogenetic analysis. BMC Vet Res 20, 285 (2024). 491 \ndoi:10.1186/s12917-024-04137-1 492 \n42 Uhart, M. M.  et al. Epidemiological data of an influenza A/H5N1 outbreak in 493 \nelephant seals in Argentina indicates mammal-to-mammal transmission. Nat 494 \nCommun 15, 9516 (2024). doi:10.1038/s41467-024-53766-5 495 \n43 Aguero, M.  et al. Highly pathogenic avian influenza A(H5N1) virus infection 496 \nin farmed minks, Spain, October 2022. Euro Surveill 28 (2023). 497 \ndoi:10.2807/1560-7917.ES.2023.28.3.2300001 498 \n44 Chothe, S. K.  et al. Marked neurotropism and potential adaptation of H5N1 499 \nclade 2.3.4.4.b virus in naturally infected domestic cats. Emerg Microbes 500 \nInfect 14, 2440498 (2025). doi:10.1080/22221751.2024.2440498 501 \n45 Belser, J. A., Sun, X., Pulit-Penaloza, J. A. & Maines, T. R. Fatal Infection in 502 \nFerrets after Ocular Inoculation with Highly Pathogenic Avian Influenza 503 \nA(H5N1) Virus. Emerg Infect Dis 30, 1484-1487 (2024). 504 \ndoi:10.3201/eid3007.240520 505 \n46 Rosenke, K.  et al. Pathogenesis of bovine H5N1 clade 2.3.4.4b infection in 506 \nmacaques. Nature (2025). doi:10.1038/s41586-025-08609-8 507 \n47 Whittle, J. R.  et al. Flow cytometry reveals that H5N1 vaccination elicits 508 \ncross-reactive stem-directed antibodies from multiple Ig heavy-chain lineages. 509 \nJ Virol 88, 4047-4057 (2014). doi:10.1128/JVI.03422-13 510 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n48 Tiller, T.  et al. Efficient generation of monoclonal antibodies from single 511 \nhuman B cells by single cell RT-PCR and expression vector cloning. J 512 \nImmunol Methods 329, 112-124 (2008). doi:10.1016/j.jim.2007.09.017 513 \n49 Kallewaard, N. L.  et al. Structure and Function Analysis of an Antibody 514 \nRecognizing All Influenza A Subtypes. Cell 166, 596-608 (2016). 515 \ndoi:10.1016/j.cell.2016.05.073 516 \n50 Dreyfus, C.  et al. Highly conserved protective epitopes on influenza B viruses. 517 \nScience 337, 1343-1348 (2012). doi:10.1126/science.1222908 518 \n51 Zheng, M. Z. M.  et al. Deconvoluting TCR-dependent and -independent 519 \nactivation is vital for reliable Ag-specific CD4(+) T cell characterization by 520 \nAIM assay. Sci Adv 11, eadv3491 (2025). doi:10.1126/sciadv.adv3491 521 \n52 Ekiert, D. C.  et al. Antibody recognition of a highly conserved influenza virus 522 \nepitope. Science 324, 246-251 (2009). doi:10.1126/science.1171491 523 \n53 Lingwood, D.  et al. Structural and genetic basis for development of broadly 524 \nneutralizing influenza antibodies. Nature 489, 566-570 (2012). 525 \ndoi:10.1038/nature11371 526 \n54 Pappas, L.  et al. Rapid development of broadly influenza neutralizing 527 \nantibodies through redundant mutations. Nature 516, 418-422 (2014). 528 \ndoi:10.1038/nature13764 529 \n55 Paules, C. I.  et al. The Hemagglutinin A Stem Antibody MEDI8852 Prevents 530 \nand Controls Disease and Limits Transmission of Pandemic Influenza Viruses. 531 \nJ Infect Dis 216, 356-365 (2017). doi:10.1093/infdis/jix292 532 \n56 Sutton, T. C.  et al. In Vitro Neutralization Is Not Predictive of Prophylactic 533 \nEfficacy of Broadly Neutralizing Monoclonal Antibodies CR6261 and 534 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nCR9114 against Lethal H2 Influenza Virus Challenge in Mice. J Virol 91 535 \n(2017). doi:10.1128/JVI.01603-17 536 \n57 Beukenhorst, A. L.  et al. A pan-influenza monoclonal antibody neutralizes H5 537 \nstrains and prophylactically protects through intranasal administration. Sci Rep 538 \n14, 3818 (2024). doi:10.1038/s41598-024-53049-5 539 \n58 Kanekiyo, M.  et al. Pre-exposure antibody prophylaxis protects macaques 540 \nfrom severe influenza. Science 387, 534-541 (2025). 541 \ndoi:10.1126/science.ado6481 542 \n59 Galli, G.  et al. Adjuvanted H5N1 vaccine induces early CD4+ T cell response 543 \nthat predicts long-term persistence of protective antibody levels. Proceedings 544 \nof the National Academy of Sciences of the United States of America 106, 545 \n3877-3882 (2009). doi:10.1073/pnas.0813390106 546 \n60 Matsuda, K.  et al. Prolonged evolution of the memory B cell response induced 547 \nby a replicating adenovirus-influenza H5 vaccine. Science Immunology 4, 548 \neaau2710 (2019). doi:10.1126/sciimmunol.aau2710 549 \n61 Matsuda, K.  et al. A replication-competent adenovirus-vectored influenza 550 \nvaccine induces durable systemic and mucosal immunity. The Journal of 551 \nClinical Investigation 131, e140794,-140794 (2021). doi:10.1172/JCI140794 552 \n62 Moris, P.  et al. H5N1 influenza vaccine formulated with AS03 A induces 553 \nstrong cross-reactive and polyfunctional CD4 T-cell responses. J Clin 554 \nImmunol 31, 443-454 (2011). doi:10.1007/s10875-010-9490-6 555 \n63 Ledgerwood, J. E.  et al. Prime-boost interval matters: a randomized phase 1 556 \nstudy to identify the minimum interval necessary to observe the H5 DNA 557 \ninfluenza vaccine priming effect. J Infect Dis 208, 418-422 (2013). 558 \ndoi:10.1093/infdis/jit180 559 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \n64 Scheid, J. F.  et al. A method for identification of HIV gp140 binding memory 560 \nB cells in human blood. J Immunol Methods 343, 65-67 (2009). 561 \ndoi:10.1016/j.jim.2008.11.012 562 \n65 World Health Organization. Manual for the laboratory diagnosis and 563 \nvirological surveillance of influenza.  (Geneva: World Health Organization, 564 \n2011). 565 \n66 Kanekiyo, M.  et al. Self-assembling influenza nanoparticle vaccines elicit 566 \nbroadly neutralizing H1N1 antibodies. Nature 499, 102-106 (2013). 567 \ndoi:10.1038/nature12202 568 \n67 van Baalen, C. A.  et al. ViroSpot microneutralization assay for antigenic 569 \ncharacterization of human influenza viruses. Vaccine 35, 46-52 (2017). 570 \ndoi:10.1016/j.vaccine.2016.11.060 571 \n 572 \n 573 \n574 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nFigures 575 \n  576 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\na\nb\nFSC-H\nFSC-A FSC-H CD19-ECD IgD-Cy7PE\nSSC-A\nAQUA/DUMP\nSA-BB515\nH5 (IN05) - PE\nH7 (SH13) - APC\nH7+\nH5+\nH7+H5+\nNo probes\n SA-alone\n0\n0.05\n0.15\nH7+ H5+\n% of CD19+IgD- B cells\n0.10\nH7+H5+\n0\n0.8% of CD19+IgD- B cells\n0.6\n0.4\n0.2\nc d\n104\nReciprocal serum dilution (EC50)\nH1\n103\n102\n101\n100\nH3 H5 H7\nPR/34\nNC/99\nCA/09\nHK/68\nWY/03\nPE/09\nSW/13\nVN/04\nIN/05\nNE/03\nAN/13\nSH/13\n10-1\nH3H1\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nFigure 1: Serum antibody and memory B cells recognising avian influenza strains 577 \nare widely prevalent 578 \n(A) Plasma samples from healthy volunteers (N=18) were screened by ELISA for 579 \nreactivity against HA from diverse influenza A strains. Reciprocal serum dilutions 580 \nyielding half maximal binding (EC50) for each antigen are shown. (B) Staining of 581 \ncryopreserved PBMCs allows identification of CD19+ IgD- B cells not binding a 582 \nstreptavidin-BB515 decoy. Co-staining with recombinant HA probes derived from 583 \nH7N9 A/Shanghai/01/2013 (SH13) and H5N1 A/Indonesia/5/2005 (IN05) delineates 584 \nsingle- and cross-reactive memory B cell populations. (C) Frequencies of H5+, H7+ or 585 \nH7+H5+ memory B cells in healthy volunteers (N=18). (D) Frequencies of memory B 586 \ncells binding seasonal H1N1 (NC99 and CA09) and H3N2 (HK68 and VI11) influenza 587 \nstrains were measured in healthy volunteers (N=16). Lines indicate median and IQR. 588 \nPR/34, A/Puerto Rico/8/1934; NC/99, A/New Caledonia/20/1999; CA/09, 589 \nA/California/04/2009; HK/68, A/Hong Kong/1/1968; WY/03, A/Wyoming/3/2003; 590 \nPE/09, A/Perth/16/2009; SW/13, A/Switzerland/9715293/2013; VN/04, 591 \nA/Vietnam/1203/2004; IN/05, A/Indonesia/5/2005; NE/03, A/Netherlands/219/2003; 592 \nAN/13, A/Anhui/01/2013; SH/13, A/Shanghai/01/2013; VI/II, A/Victoria/361/2011 593 \n 594 \n  595 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\na\nb\nHemagglutination inhibition (HAI) Focus reduction assay (FRA)\nmAb 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\nH5 specific\nN98-H03 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10\nN98-E12 <10 <10 <10 <10 <10 <10 80 <10 <10 <10 <10 <10\nN98-F11 <10 <10 <10 <10 <10 <10 80 <10 <10 <10 <10 <10\nJ26-A05 <10 <10 <10 <10 <10 <10 160 80 <10 <10 <10 <10\nH7 specific\nN98-B10 <10 <10 <10 <10 <10 <10 80 <10 <10 <10 <10 <10\nN98-E05 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10\nJ26-D05 <10 <10 <10 <10 <10 <10 <10 <10 80 <10 <10 <10\nH5/H7 cross-reactive\nN98-G09 <10 <10 <10 <10 <10 <10 160 80 80 <10 <10 <10\nN98-2F05 <10 <10 <10 <10 <10 <10 80 20 40 <10 <10 <10\nN98-H07 <10 <10 <10 <10 <10 <10 160 40 40 <10 <10 <10\nJ26-B03 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10\nControl\nCR9114 <10 <10 <10 <10 <10 <10 1280 320 40 80 <10 <10\nVRC01 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10\nc\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nFigure 2.  Characteristics of monoclonal antibodies derived from avian HA-596 \nspecific B cells 597 \n(A) Gene usage, CDR-H3 sequence, and mutation loads for 14 monoclonal antibodies 598 \nderived from sorted H5+, H7+ or H7+H5+ memory B cells from two healthy 599 \nvolunteers. (B) Binding activity of each monoclonal antibody to a panel of recombinant 600 \nHA proteins derived from influenza A and influenza B strains. Values denote binding 601 \nlog10(EC50) determined by ELISA. HA and mAb combinations not tested are indicated 602 \nby “n.d.” (C) Neutralisation activity determined by hemagglutination inhibition 603 \n(HAI) or focus reduction assay (FRA) against a panel of influenza A and B viruses. 604 \nPR/34;A/Puerto Rico/8/1934, NC/99; A/New Caledonia/20/1999, CA/09; 605 \nA/California/04/2009, HK/68; A/Hong Kong/1/1968, WY/03; A/Wyoming/3/2003, 606 \nPE/09; A/Perth/16/2009, SW/13; A/Switzerland/9715293/2013, VN/04; 607 \nA/Vietnam/1203/2004, IN/05; A/Indonesia/5/2005, NE/03; A/Netherlands/219/2003, 608 \nAN/13; A/Anhui/01/2013, SH/13; A/Shanghai/01/2013 609 \n 610 \n  611 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n% of CD19+IgD- B cells\nB\nA\nHAI titre\nBL 4w\n16\n64\n256\n1,024\n4,096\n16,384\nH5\n<10\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nFigure 3: Cross-reactive serum antibodies not induced by seasonal vaccination but 612 \nsporadic induction of cross-reactive H5 memory B cells 613 \n(A) Plasma samples from healthy volunteers (N=19) were assayed for HA inhibition 614 \n(HAI) against the vaccine matched H1N1 strain (H1/Mic) and clade 2.3.4.4b H5N1 615 \nstrain (H5/Fuj). Baseline (BL) and 4 weeks post immunisation (4w) timepoints were 616 \nassayed. The lowest plasma dilution assayed was 1:10, with samples not achieving 617 \ninhibition at this dilution shown as “<10”. The horizontal black line represent the 618 \nmedian, and error bars equal the IQR. Significance was determined by Wilcoxon test 619 \nbetween BL and 4w. ( B) Frequencies of H1+ (H1/Vic), H5+ (H5/Fuj) and H1+H5+ 620 \nmemory B cells in healthy volunteers (N=19) at BL, 1- and 4-weeks post vaccination 621 \n(1w and 4w). Donor responses are each timepoint are linked by lines. Significance was 622 \ndetermined by Kruskal-Wallis test with Dunn ’s post-test comparing each post-623 \nimmunisation timepoint to baseline (p values corrected for multiple comparisons). 624 \n 625 \n  626 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\nB\nA\nCD184− CD137+ of\nCD4MEM (%)\nCD184− CD137+ of\n cTFH (%)\nCD154+ of cTFH (%)\np=0.0419\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint \n\n \nFigure 4: T cell responses against H1 and H5 HA following seasonal vaccination   627 \nPBMCs collected from healthy volunteers (N=23) at baseline (BL; black circle) and 628 \nfollowing seasonal vaccination at 1 week (1w; open circle) and 4 weeks (4w; half-filled 629 \ncircle) post-immunisation were tested via AIM assay against H1/Vic and H5/Fuj HA 630 \nprotein. Antigen-specific CD4+ memory T cells (CD4MEM) and circulating T follicular 631 \nhelper cells (cTFH) were identified via ( A) CD184−CD137+ or ( B) CD154+ gating. 632 \nIndividual donor responses are plotted and linked by lines between timepoints. 633 \nSignificance was determined by Kruskal-Wallis testing with Dunn ’s post-test 634 \ncomparing each post-immunisation timepoint to baseline (p values corrected for 635 \nmultiple comparisons). 636 \n 637 \n  638 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted April 29, 2025. ; https://doi.org/10.1101/2025.04.28.651131doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}