Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity

Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity View ORCID Profile Akshita B. Gopal , Huibin Lv , View ORCID Profile Tossapol Pholcharee , View ORCID Profile Wenhao O. Ouyang , Qi Wen Teo , Yasha Luo , Yun Sang Tang , Mingyong Luo , Chris K. P. Mok , View ORCID Profile Nicholas C. Wu doi: https://doi.org/10.1101/2025.03.24.644842 Akshita B. Gopal 1 Department of Biochemistry, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Akshita B. Gopal Huibin Lv 1 Department of Biochemistry, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA 2 Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: huibinlv{at}illinois.edu nicwu{at}illinois.edu Tossapol Pholcharee 1 Department of Biochemistry, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tossapol Pholcharee Wenhao O. Ouyang 1 Department of Biochemistry, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wenhao O. Ouyang Qi Wen Teo 1 Department of Biochemistry, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA 2 Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yasha Luo 3 Department of Clinical Laboratory, Guangdong Women and Children Hospital , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yun Sang Tang 4 The Jockey Club School of Public Health and Primary Care, The Chinese University of Hong Kong , Hong Kong, China 5 Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong , Hong Kong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mingyong Luo 3 Department of Clinical Laboratory, Guangdong Women and Children Hospital , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chris K. P. Mok 4 The Jockey Club School of Public Health and Primary Care, The Chinese University of Hong Kong , Hong Kong, China 5 Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong , Hong Kong, China 6 S.H. Ho Research Centre for Infectious Diseases, The Chinese University of Hong Kong , Hong Kong, China 7 School of Biomedical Sciences, The Chinese University of Hong Kong , Hong Kong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicholas C. Wu 1 Department of Biochemistry, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA 2 Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA 8 Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA 9 Carle Illinois College of Medicine, University of Illinois Urbana-Champaign , Urbana, IL 61801, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicholas C. Wu For correspondence: huibinlv{at}illinois.edu nicwu{at}illinois.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT The conserved stem domain of influenza hemagglutinin (HA), which is classified into group 1 and group 2, is a target of broadly neutralizing antibodies. While many group 1 HA stem antibodies have been described, much less is known about group 2 HA stem antibodies. This study structurally characterizes two group 2 HA stem antibodies, 2F02 and AG2-G02, targeting the central stem epitope and the lower stem epitope, respectively. Unlike prototypic group 2 HA stem antibodies, 2F02 and AG2-G02 do not compete for binding. Both antibodies offer protection in vivo despite having minimal neutralization activity in vitro . We further demonstrate that the natural evolution of HA2 position 32 restricts the binding of AG2-G02 to recent human H3N2 HAs and influences the binding of human plasma samples. Overall, these findings advance our understanding of the antigenicity of HA stem, which has important implications for the development of broadly protective influenza vaccines. INTRODUCTION With seasonal influenza A viruses causing around 3–5 million cases of severe illness each year globally, they remain a persistent threat to public health 1 . Hemagglutinin (HA) and neuraminidase (NA) are the major surface glycoprotein antigens of the influenza A viruses. HA and NA are classified into 19 and 11 antigenic subtypes, respectively 1 , 2 . The 19 HA subtypes are further categorized into two groups. Group 1 HA includes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18 and H19, whereas group 2 HA includes H3, H4, H7, H10, H14, and H15. Among these, H1N1 and H3N2 are responsible for seasonal influenza epidemics in humans. H3N2 was introduced into the human population in 1968 after a reassortment event involving an avian H3 virus 3 . New antigenic variants of human H3N2 viruses emerge every 3–5 years, often resulting in antigenic mismatches between seasonal influenza vaccine strains and circulating strains 4 , 5 . Furthermore, spillover events of avian H3 viruses into humans have been reported 6 – 9 . In China, avian H3N8 viruses were linked to two confirmed human infections in 2022, followed by the first case of mortality in 2023 6 – 8 . Although seasonal influenza vaccines confer protection against the human H3N2 viruses, the human population lacks immunity to the zoonotic H3 strains 10 . HA is expressed as a single polypeptide chain (HA0), which forms a homotrimer. After protease cleavage, HA0 matures into HA1 and HA2 subunits. The immunodominant head domain, composed entirely of the HA1 subunit, sits atop the membrane-proximal stem domain, primarily composed of the HA2 subunit 11 . The HA head binds to sialylated host receptors and is highly variable, whereas the evolution of the HA stem is somewhat constrained as it possesses the membrane fusion machinery essential for viral entry. Since the HA stem is highly conserved compared to the HA head 12 , 13 , antibodies targeting the HA stem are often broadly neutralizing against multiple antigenically distinct strains or even subtypes 11 . Studies have also demonstrated that HA stem antibodies have fewer potential escape mutants as compared to the HA head antibodies 14 , 15 . Therefore, the discovery and characterization of HA stem antibodies have motivated the development of broadly protective influenza vaccines 16 – 19 , with some of these candidates showing promising results in phase I clinical trials 20 – 22 . While numerous stem-binding antibodies targeting group 1 HA or cross-react with both group 1 and 2 HAs have been identified, those specific to group 2 HA are much less explored 23 – 25 . Antibodies to group 2 HA stem are mapped to two slightly overlapping yet distinct epitopes 11 , 26 . The first one is known as the central stem epitope, which is centered at the helix A of the HA2 subunit 27 . The second one is known as the lower stem epitope, which is closer to the viral membrane compared to the central stem epitope and involves the basal β-sheet that is formed by both HA1 and HA2 subunits 28 , 29 . Many central stem antibodies acquire reactivity to group 2 HA from a group 1-specific ancestor 24 , 30 , 31 . As a result, most central stem antibodies targeting group 2 HA typically also cross-react with group 1 HA 24 , 28 , 30 – 32 , although exceptions exist 25 . By contrast, lower stem antibodies are specific to group 2 influenza virus HAs 26 , 33 . Due to their larger breadth, central stem antibodies have received a lot more attention than lower stem antibodies. As a result, the contribution of lower stem antibodies to human antibody responses against influenza virus remains largely elusive. Recently, we discovered dozens of HA stem antibodies from the literature using machine learning and high-throughput experimental screening 33 , 34 . Two of these HA stem antibodies, namely AG2-G02 35 and 240-14 IgA 2F02 36 (hereinafter abbreviated as 2F02), are group 2-specific. In this study, cryogenic electron microscopy (cryo-EM) analysis showed that AG2-G02 and 2F02 bound to the lower stem and central stem epitopes, respectively. We further demonstrated that AG2-G02 and 2F02 could bind concurrently to the HA stem. While 2F02 bound to all tested H3 HAs, AG2-G02 did not bind to HAs from the recent human H3N2 strains. We found that a natural mutation at HA2 position 32 of the recent human H3N2 strains abolished the binding of AG2-G02. Additionally, serological analysis indicated that the natural evolution of HA2 position 32 has altered the antigenicity of human H3N2 HA stem. Throughout this study, H3 numbering and Kabat numbering are used for HA residue positions and antibody residue positions, respectively, unless otherwise stated. RESULTS Identification of two non-competing antibodies to H3 HA stem We have previously curated a large dataset of human monoclonal HA antibodies, among which 4,469 did not have any epitope information 33 . From these 4,469 antibodies, we have further identified AG2-G02 35 and 2F02 36 , both of which were derived from the plasmablasts of vaccinees, as HA stem antibodies that target H3 HA but not H1 HA 33 , 34 . AG2-G02 is encoded by IGHV1-2/IGHD3-3/IGHJ5 and IGKV3-20/IGKJ3 33 , 35 , whereas 2F02 is encoded by IGHV3-23/IGHD3-3/IGHJ5 and IGLV2-11/IGLJ3 33 , 36 To probe the epitopes of AG2-G02 and 2F02, we performed a binding competition assay using an H3 stem construct designed based on H3N2 A/Finland/486/2004 HA 18 . Our competition assay also included two central stem antibodies, CR9114 and FI6v3, as well as two lower stem antibodies, CR8020 and CR8043 24 , 28 , 29 , 37 . AG2-G02 competed strongly with all the tested antibodies except 2F02 and FI6v3. Similarly, 2F02 competed strongly with all the tested antibodies except AG2-G02 ( Figure 1A ). This observation suggested that the epitopes of AG2-G02 and 2F02 were proximal to each other but did not overlap. Download figure Open in new tab Figure 1. AG2-G02 and 2F02 have non-overlapping epitopes. (A) Competition among the indicated antibodies for binding to H3 stem was measured by biolayer interferometry (BLI). All antibodies were in Fab format except for FI6v3, which was in IgG format. Competition index is shown as a heatmap. (B) Cryo-EM structures of AG2-G02 and 2F02 in complex with H3N8 A/mallard/Alberta/362/2017 HA are shown. HA1 and HA2 are in dark grey and light grey, respectively. The heavy and light chains of AG2-G02 are in dark pink and pale pink, respectively. The heavy and light chains of 2F02 are in dark green and pale green, respectively. (C) Epitopes of CR9114 37 (PDB 4FQY) 37 , FI6v3 32 (PDB 3ZTJ), 2F02 (this study), CR8020 28 (PDB 3SDY), CR8043 29 (PDB 4NM8), and AG2-G02 (this study) are compared. Epitope residues on HA1 and HA2 are in orange and yellow, respectively. (D) The binding of AG2-G02 (pink) and CR9114 (orange), 2F02 (green) and FI6v3 (blue) to HA (grey) is compared. (E) A close-up view illustrating a steric clash between AG2-G02 (pink) and CR9114 (orange). Such steric clash was not observed between AG2-G02 and 2F02 (green) or between AG2-G02 and FI6v3 (blue). (F) A top-down view of AG2-G02 (pink) and 2F02 (green) Fabs simultaneously in complex with an HA trimer (grey), which was determined to a resolution of 5.6 Å using cryo-EM. Structural analysis reveals distinct epitopes for AG2-G02 and 2F02 To characterize the epitopes of AG2-G02 and 2F02 and their molecular basis of binding, we determined the cryo-EM structures of AG2-G02 Fab and 2F02 Fab in complex with H3N8 A/mallard/Alberta/362/2017 HA to resolutions of 2.60 Å and 2.71 Å, respectively. Structural analysis revealed that AG2-G02 bound to the lower stem epitope, whereas 2F02 bound to the central stem epitope ( Figure 1B ). Although the epitope of AG2-G02 highly overlapped with that of CR8020 28 and CR8043 29 , it is shifted laterally with its complementarity determining region (CDR) H3 stretching into a groove along the basal β-sheet ( Figure 1C and S1A) . Notably, the epitope of AG2-G02 resembled that of another lower stem antibody, ADI-85666 ( Figure S1C-D) , as reported in a recent preprint 38 . Although 2F02, CR9114, and FI6v3 target a similar epitope ( Figure 1C ), their differences in the angles of approach explain why CR9114, but not 2F02 and FI6v3, competed with AG2-G02 ( Figure 1D-E ). Consistently, low-resolution cryo-EM analysis showed that three copies of AG2-G02 Fab and three copies of 2F02 Fab could concurrently bind to a single HA trimer ( Figure 1F ) . Both AG2-G02 and 2F02 use IGHD3-3 -encoded CDR H3 for binding Both AG2-G02 and 2F02 relied heavily on CDR H3 for binding to HA, with greater than 40% of the paratope buried surface area attributing to CDR H3 ( Figure 2A-B ) . Besides, their CDR H3s shared an IGHD3-3 -encoded DFW motif, albeit engaging HA very differently ( Figure 2C and Figure S2) . For the DFW motif (positions 98-100) in 2F02, the side chain of V H W100 H-bonded with the backbone oxygen of T318 HA1 and formed a T-shaped π-π stacking interaction with W21 HA2 . By contrast, for the DFW motif (positions 97-99) in AG2-G02, the side chain of V H W99 did not interact with HA. Instead, AG2-G02 relied on the side chain of V H F98 in the DFW motif for binding via a cation-π stacking interaction with R25 HA2 . Notably, neither AG2-G02 nor 2F02 used the side chain of the Asp in the DFW motif for binding. AG2-G02 and 2F02 also H-bonded with HA through the side chains of multiple other residues. AG2-G02 H-bonded with HA via the side chains of multiple tyrosines on the heavy chain, including V H Y32, V H Y96, and V H Y100c, whereas 2F02 H-bonded with HA via the side chains of V H S52, V H N100f, V L K52, and V L K53. These observations indicated that although AG2-G02 and 2F02 converged on the use of the IGHD3-3 -encoded DFW motif, they targeted distinct epitopes with different binding modes. Download figure Open in new tab Figure 2. Structural analysis of the binding of AG2-G02 and 2F02 to HA. (A) Interaction of CDRs (ribbon representation) of AG2-G02 and 2F02 with HA is shown. (B) Contributions from CDR H3 (red), non-CDR H3 V H (yellow), and V L (orange) to the paratope buried surface area (BSA). (C) A close-up view of the molecular interactions between the antibodies and HA. Key interacting residues are shown by sticks representation. Black dashed lines represent H-bonds and salt bridges. (A and C) The heavy and light chains of AG2-G02 are in pink and pale pink, respectively. The heavy and light chains of 2F02 heavy chain are in green and pale green, respectively. Epitope residues on HA1 and HA2 are in orange and yellow, respectively. AG2-G02 and 2F02 are protective antibodies that broadly react with H3 strains To examine the cross-reactive breadth of AG2-G02 and 2F02, we used an enzyme-linked immunosorbent assay (ELISA) to measure their binding activity against various group 2 HAs. We found that 2F02 bound to all tested H3 HAs and cross-reacted with an H15 HA ( Figure 3A ). By contrast, the reactivity breadth of AG2-G02 was restricted to H3 HAs. Additionally, AG2-G02 had no detectable binding to HAs from more recent human H3N2 strains, including H3N2 A/Switzerland/9715293/2013 and H3N2 A/Darwin/6/2021. Consistently, while AG2-G02 and 2F02 had similar binding activity to cells infected with H3N2 A/Moscow/10/1999, the binding activity of AG2-G02 to cells infected with the H3N2 A/Darwin/6/2021 virus was much weaker than that of 2F02 ( Figure S3A-B ). Although the reactivity breadth of 2F02 was broader than AG2-G02, none of them reacted with H7 HAs, unlike CR8020 28 and CR8043 29 . Download figure Open in new tab Figure 3. In vitro and in vivo protection of AG2-G02 and 2F02. (A) The binding activities of AG2-G02 and 2F02 against r combinant HA proteins from the specified influenza strains were measured by ELISA. The absorbance values at 450 nm are shown as a heatmap. (B) The neutralization activity of AG2-G02 and 2F02 against recombinant H3N8 and different recombinant H3N2 viruses was measured by a microneutralization assay. The IC 50 values are shown as a heatmap. (A-B) Strain names are abbreviated as follows: H3N2 A/Hong Kong/1/1968 (HK/1968), H3N2 A/Philippines/2/1982 (Philippines/1982), H3N2 A/Beijing/109/1992 (Beijing/1992), H3N2 A/Wuhan/359/1995 (Wuhan/1995), H3N2 A/Moscow/10/1999 (Moscow/1999), H3N2 A/New York/55/2004 (NY/2004), H3N2 A/Wisconsin/67/2005 (Wisconsin/2005), H3N2 A/Uruguay/716/2007 (Uruguay/2007), H3N2 A/Switzerland/9715293/2013 (Switzerland/2013), H3N2 A/Darwin/6/2021 (Darwin/2021), H3N8 A/mallard/Alberta/362/2017 (Alberta/2017), H4N6 A/mallard/Alberta/455/2015 (Alberta/2015), H7N3 A/Canada/rv444/2004 (Canada/2004), H7N9 A/Hong Kong/125/2017 (HK/2017), H7N9 A/Anhui/1/2013 (Anhui/2013), H14N5 A/mallard/Astrakhan/263/1982 (Astra/1982), and H15N2 A/Australian shelduck/Western Australia/1756/1983 (WAus/1983). (C-E ) Four to five female BALB/c mice at six weeks old were injected intraperitoneally with 5 mg/kg of the indicated antibody four hours prior to challenge with 5× LD 50 of H3N2 Philippines/1982 (X-79). CR9114 was used as a positive control for protection. (C) The mean percentage of body weight change post-infection is shown. The humane endpoint was defined as a weight loss of 25% from the initial weight on day 0. (D) Kaplan-Meier survival curves are presented. (E) Lung viral titers on day 3 post-infection are shown (n = 3). We further measured the neutralization activity of AG2-G02 and 2F02. AG2-G02 only neutralized H3N2 A/Moscow/10/1999 among the five tested H3 strains, whereas 2F02 did not neutralize any ( Figure 3B ) . The inability of 2F02 to neutralize any tested strains was likely due to its weaker binding affinity to the H3 stem compared to AG2-G02 (Figure S3C-D) . As measured by biolayer interferometry, the binding affinity of AG2-G02 to the H3 stem (K D = 0.21 nM) was 100-fold stronger than that of 2F02 (K D = 22 nM). Studies have shown that antibodies with minimal neutralization activity may still confer protection in vivo due to Fc-effector functions 39 . Consistently, both AG2-G02 and 2F02 showed prophylactic protection in vivo at 5 mg/kg against a lethal challenge of H3N2 A/Philippines/2/1982 (X-79) virus, based on weight loss profiles ( Figure 3C ), survival analysis ( Figure 3D ), and lung viral titer at day 3 post-infection ( Figure 3E ) . Together, these results demonstrated that AG2-G02 and 2F02 are protective antibodies with broad reactivity against H3 HAs. The antigenicity of the lower stem epitope has evolved in human H3N2 viruses To investigate the molecular basis for the lack of binding of AG2-G02 to the recent human H3N2 HAs ( Figure 3A ), we examined the natural amino acid variants in the AG2-G02 epitope. We found that the amino acid variants at HA2 position 32 of H3 HAs correlated with AG2-G02 reactivity. H3 HAs that reacted with AG2-G02 either possessed a Thr or an Ile at HA2 position 32, whereas those that did not bind AG2-G02 had an Arg ( Figure 3A and Figure S4 ). When the H3N2 virus entered the human population in 1968, it carried T32 HA2 . During 2004-2005 influenza season, T32 HA2 was replaced by I32 HA2 , which was subsequently replaced by R32 HA2 in the 2008-2009 influenza season ( Figure 4A ) . Our cryo-EM structure of AG2-G02 in complex with H3 HA showed that T32 HA2 fitted tightly into a pocket in the AG2-G02 paratope. Structural modeling indicated that I32 HA2 , albeit slightly bulkier than T32 HA2 , would still fit into the pocket ( Figure 4B ). However, R32 HA2 would clash with AG2-G02. Consistently, biolayer interferometry analysis showed that AG2-G02 bound to the H3 stem with T32 HA2 (K D = 0.57 nM) and I32 HA2 (KD = 0.21 nM) with similar affinity but did not bind to the H3 stem with R32 HA2 ( Figure 4B and Figure S3C) . As a control, we demonstrated that the binding activity of 2F02 was minimally affected by these H3 stem variants ( Figure S3D ). These results suggested that mutations at HA2 position 32 could influence the antigenicity of the lower stem epitope. Download figure Open in new tab Figure 4. Evolution of HA2 position 32 influences the antigenicity of the lower stem epitope. (A) Occurrence of different amino acid variants at HA2 position 32 of human H3N2 strains from 1968 to the present was analyzed based on 33,000 human H3N2 HA sequences from the GSAID database 60 . The x-axis represents the years, while the y-axis shows the percentage of strains containing a particular amino acid at HA2 position 32 in a given year. Thr, Ile, and Arg are represented by orange, brown, and green lines, respectively. (B) The structures of AG2-G02 binding to HA stem with I32 HA2 and R32 HA2 are modelled. The binding affinity (K D ) of AG2-G02 Fab to different HA stem variants is indicated in parentheses. (C-E) The binding of plasma samples from (C) pre-2003 adults (n = 20), (D) post-2011 adults (n = 20), and (E) post-2011 infants (n = 15) to H3 stem with either T32 HA2 , I32 HA2 or R32 HA2 is measured by ELISA. The y-axis represents the area under the curve (AUC) of four serial 10-fold dilutions of serum (1:100, 1:1,000, 1:10,000, and 1:100,000). P-values were determined using a paired two-tailed Student’s t-test, where ns represents p-value > 0.05, * represents p-value ≤ 0.05, ** represents p-value ≤ 0.01, *** represents p-value ≤ 0.001, **** represents p-value ≤ 0.0001. To further analyze how the natural evolution of HA2 position 32 influenced the antigenicity of the HA stem, we measured the binding activity of human plasma samples to different H3 stem variants (T32 HA2 , I32 HA2 , and R32 HA2 ). Three groups of plasma samples were obtained. The first group was collected prior to 2003 from 20 adults aged between 20 and 52 (pre-2003 adults) who should have been infected by H3N2 strains with T32 HA2 but not I32 HA2 or R32 HA2 (Figure S5). The second group was collected after 2011 from 20 adults over the age of 60 (post-2011 adults), whose initial exposure to the human H3N2 virus likely involved strains with T32 HA2 . The third group included 15 infants born after 2011 (post-2011 infants), who, if previously been infected with the human H3N2 virus, would have encountered strains with R32 HA2 but not T32 HA2 or I32 HA2 . The binding activity of plasma samples from pre-2003 adults to H3 stem with T32 HA2 (median AUC = 11,044) was slightly, yet significantly, higher than that with I32 HA2 (median AUC = 7,007, p-value = 0.0001) or R32 HA2 (median AUC = 9,321, p-value = 0.03, Figure 4C ). Similarly, the binding activity of plasma samples from post-2011 adults to H3 stem with T32 HA2 (median AUC = 25,916) was also slightly, yet significantly, higher than that with I32 HA2 (median AUC = 17,988, p-value = 0.001, Figure 4D ). By contrast, the binding activity of plasma samples from post-2011 infants to H3 stem with R32 HA2 (median AUC = 21,083) was significantly higher than that with T32 HA2 (median AUC = 13,123, p-value = 0.008) or I32 HA2 (median AUC = 12,415, p-value = 0.0004, Figure 4E ). Notably, the central stem epitope was the same among all three H3 stem variants in this experiment. Therefore, the impact of mutations at HA2 position 32 on the antigenicity of the lower stem epitope should be larger than what was measured in this experiment, due to the contribution of central stem antibodies to the binding signal. Overall, these observations further substantiated that the antigenicity of the lower stem epitope in human H3N2 HA has changed over time due to the natural evolution of HA2 position 32. DISCUSSION Although the discovery of HA stem antibodies has motivated the development of broadly protective influenza vaccines, most known HA stem antibodies to date are specific to group 1 HA or cross-react with both group 1 and 2 HAs. By contrast, group 2-specific HA stem antibodies are not as well characterized. Through analyzing two non-competing group 2-specific HA stem antibodies, namely the lower stem antibody AG2-G02 and the central stem antibody 2F02, this study advances our understanding of broadly reactive antibody responses to influenza A viruses. In the HA stem, the central stem epitope represents the most conserved epitope and is targeted by several known cross-group antibodies 11 . By contrast, the anchor epitope is mainly targeted by group 1-specific antibodies 40 , whereas the lower stem epitope is only known to be targeted by group 2-specific antibodies 19 , 28 , 29 . Since central stem antibodies have larger cross-reactivity breadth but weaker neutralization potency compared to lower stem antibodies 26 , an ideal broadly protective influenza vaccine against group 2 HA should elicit antibodies to both epitopes. At the same time, lower stem antibodies often compete with central stem antibodies, as shown by this study and others 37 . Besides, antibody responses are influenced by pre-existing immunity due to competition between circulating antibodies and B cell receptors 41 – 43 . Therefore, a high level of circulating antibodies targeting the lower stem epitope would most likely interfere with the B cell responses against the central stem epitope and vice versa . Nevertheless, this study showed that the competition between lower stem and central stem antibodies is not universal, as exemplified by the lack of competition between AG2-G02 and 2F02 or FI6v3. This observation suggests the possibility of minimizing the interference between antibody responses targeting lower stem and central stem epitopes, which should be explored in future immunogen design. A major highlight in this study is identifying the altered antigenicity of the lower stem epitope in human H3N2 virus due to the natural evolution of HA2 position 32. Similarly, a recent study has also suggested that sequence differences at HA2 position 32 across different group 2 HA subtypes restrict the cross-reactivity of lower stem antibodies 44 . In fact, mutations at this position have been known to reduce the binding affinity of one of the classic lower stem antibodies, CR8043 26 , 29 , as well as 9H10 45 , which is a murine lower stem antibody. Furthermore, a deep mutational scanning study has shown that HA2 position 32 has high mutation tolerance in vitro 46 , suggesting minimal fitness costs for altering the antigenicity of the lower stem epitope, although further characterization in vivo is needed. At the same time, lower stem antibodies have higher neutralization potency than central stem antibodies 44 . Therefore, while the lower stem epitope remains an attractive target for the development of broadly protective influenza vaccines 44 , potential resistance mutations as well as natural sequence variations at HA2 position 32 need to be considered. Ancestral human H3N2 strains carry T32 HA2 , whereas more recent ones carry R32 HA2 . Consistently, we showed that people who were born after 2011, have weaker pre-existing immunity against the HA stem of H3N2 strains with T32 HA2 . By contrast, such pre-existing immunity was stronger in older people who were born when T32 HA2 was prevalent in human H3N2 strains. Since most avian H3 strains have T32 HA2 ( Figure S6A ), our observation suggested that older people could generate a better recall B cell response against the HA stem of zoonotic H3 strains. Consistently, our preliminary result indicated that plasma samples from older people had better cross-reactivity than those from young people to an avian H3 HA ( Figure S6B ), although head antibodies may also contribute to such difference. As human H3N2 strains with R32 HA2 continue the circulate, we anticipate that pre-existing immunity to the lower stem epitope of zoonotic H3 strains would diminish over time at the population level. This may potentially increase the public health risks of spillover of zoonotic H3 strains, provided that the lower stem epitope is a major cross-neutralizing epitope in the H3 subtype 26 . Multiple broadly protective influenza vaccine candidates that are designed based on group 1 HA stem have been shown to elicit antibody responses against group 1 HAs but not group 2 HAs 20 – 22 . On the contrary, a recently completed phase I clinical trial of an H10 stem-based vaccine has demonstrated its ability to induce antibody responses against group 2 HAs ( ClinicalTrials.gov identifier: NCT04579250 ) 44 . These observations indicate the complementarity of group 1 and group 2 HA stem-based influenza vaccines. Nevertheless, our previous study has suggested that resistance mutations against HA stem antibodies are much easier to arise in H3 HA than in H1 HA 4 , 47 , indicating that the development of broadly protective influenza vaccines against group 2 HA might be more challenging than for those against group 1 HA. Given the importance of antibody characterization in vaccine development, an optimal design of group 2 HA stem-based vaccines would benefit from continued study of group 2 HA stem antibodies in the future. MATERIALS AND METHODS Sample collection Plasma samples were collected from elderly individuals (ages 59-65) and young individuals (ages 17-25) between January and March 2020 at the Red Cross in Hong Kong. Plasma samples from infants were collected between January and March 2022 in Guangzhou, China. These samples were obtained from ethylenediaminetetraacetic acid (EDTA)-anticoagulated peripheral blood. Peripheral blood samples were centrifuged at 3000 × g for 10 minutes at room temperature to isolate plasma, which was then stored at −80°C until needed. The study received approval from the Human Research Ethics Committee at Guangdong Women and Children Hospital (Approval number: 202101231) and The Chinese University of Hong Kong (IRB: 2020.229). Plasma samples from adults that were collected before 2003 were purchased from BioCollections Worldwide Inc. Cell lines MDCK-SIAT1 cells (Madin-Darby canine kidney cells engineered for stable human 2,6-sialyltransferase expression, female, Sigma-Aldrich) and HEK 293T cells (human embryonic kidney cells, female) were grown at 37°C with 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) enriched with high glucose (Thermo Fisher Scientific), along with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific), 1× penicillin-streptomycin at final concentrations of 100 U mL −1 of penicillin and 100 μg mL −1 of streptomycin (Thermo Fisher Scientific), as well as 1× GlutaMax (Thermo Fisher Scientific). MDCK-SIAT1 and HEK 293T cells were passaged every 3 to 4 days using a 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Thermo Fisher Scientific). Expi293F cells (human embryonic kidney cells, female, Thermo Fisher Scientific) were cultured at 37°C with 8% CO 2 in Expi293 Expression Medium (Thermo Fisher Scientific). Sf9 cells ( Spodoptera frugiperda ovarian cells, female, ATCC) were grown in Sf-900 II SFM medium (Thermo Fisher Scientific). Mice The animal experiments were performed in accordance with protocols approved by UIUC Institutional Animal Care and Use Committee (IACUC). Six-week-old female BALB/c mice (Jackson Laboratory) were used for all animal experiments. Influenza virus The recombinant influenza H3N2 A/Darwin/6/2021 virus and H3N2 A/Moscow/10/1999 virus were generated based on the A/PR/8/34 (PR8) eight-plasmid reverse genetic system 48 . The PR8 backbone was used to generate 7:1 reassortants, with the entire HA coding region being replaced by those from H3N2 strains. Transfection was performed in HEK 293T/MDCK-SIAT1 cells co-culture (ratio of 6:1) at 60% confluence using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. At 24 hours post-transfection, cells were washed twice with phosphate-buffered saline (PBS), and cell culture medium was replaced with OPTI-MEM medium supplemented with 1 μg mL −1 tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin. The virus was harvested at 72 hours post-transfection. TCID 50 (tissue culture infectious dose 50) was used to quantify the viral titer. Briefly, serial dilutions of the virus sample were made and added to MDCK-SIAT1 cells supplemented with 1 μg mL −1 TPCK-trypsin. After incubation for 72 hours, the cells were examined for cytopathic effect (CPE). The dilution that caused infection in 50% of the wells was used to calculate the TCID 50 . The reed-Muench method was used to estimate the viral titer in terms of TCID 50 mL −1 . The following strains of influenza virus were obtained from BEI Resources ( https://www.beiresources.org/ ): H3N2 A/Philippines/2/1982 (cat #: NR-28649), H3N2 A/Wisconsin/67/2005 (cat #: NR-41800). Mouse-adapted H3N2 A/Philippines/2/1982 (X-79, 6:2 A/PR/8/34 reassortant) virus was grown in 10-day old embryonated chicken eggs at 37°C for 48 hours and were cooled at 4°C overnight. Cell debris was removed by centrifugation at 4000 × g for 20 minutes at 4°C. HA proteins The H1 and H3 stems as well as their mutants, along with the HAs from H3N2 A/Darwin/6/2021, H3N8 A/mallard/Alberta/362/2017 and H4N6 A/mallard/Alberta/455/2015 HA, were produced in the baculovirus expression system in the lab. HA proteins from the following strains were obtained from BEI Resources ( https://www.beiresources.org/ ): H3N2 A/New York/55/2004 (cat #: NR-19241), H3N2 A/Uruguay/716/2007 (cat #: NR-15168), H7N9 A/Hong Kong/125/2017 (cat #: NR-51367), H7N9 A/Anhui/1/2013 (cat #: NR-44081),H7N3 A/Canada/rv444/2004 (cat #: NR-43740). HA proteins of the following strains were purchased from SinoBiological: H3N2 A/HongKong/1/1968, H3N2 A/Beijing/109/1992, H3N2 A/Wuhan/359/1995, H3N2 A/Moscow/10/1999, H3N2 A/Switzerland/9715293/2013, H14N5 A/mallard/Astrakhan/263/1982, and H15N2 A/Australian shelduck/Western Australia/1756/1983. Expression and purification of Fabs and IgGs Plasmids encoding the heavy and light chains of IgG and Fabs were generated by cloning into the phCMV3 vector with a mouse kappa signal peptide. These plasmids were transfected into Expi293F cells using the ExpiFectamine 293 transfection kit (Gibco) in a 2:1 mass ratio of heavy to light chains, following the manufacturer’s protocol. Cells were cultured at 37°C, 8% CO₂, and 125 rpm. After six days, the supernatant was harvested and centrifuged at 4,000 × g for 30 minutes at 4°C. Purification was performed by adding CaptureSelect CH1-XL beads (Thermo Fisher Scientific) to the supernatant, followed by overnight shaking at 4°C. The beads were eluted with 60mM glycine at pH 4.0, neutralized with 1 M Tris at pH 8.0, and then buffer-exchanged three times to Phosphate-Buffered Saline (PBS) using 50 kDa molecular weight cutoff (MWCO) concentrators (Millipore) for IgGs and 30 kDa MWCO concentrators for Fabs. The antibody concentrations were determined using a Nanodrop One (Thermo Fisher Scientific) at 280 nm, and the antibodies were stored at 4°C. Expression and purification of HA recombinant proteins The H1 18 and H3 49 stem constructs, along with the H3N2 A/Darwin/6/2021 HA, H4N6 A/mallard/Alberta/455/2015 HA, and H3N8 A/mallard/Alberta/362/2017 HA ectodomain, were cloned into a customized baculovirus transfer vector. Each construct included an N-terminal gp67 signal peptide, a BirA biotinylation site, a thrombin cleavage site, a trimerization domain, and a C-terminal 6×His-tag. Recombinant bacmid DNA was created using the Bac-to-Bac system (Thermo Fisher Scientific) following the manufacturer’s instructions. Baculovirus was generated by transfecting the purified bacmid DNA into adherent Sf9 cells with Cellfectin reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. The baculovirus was then amplified by passaging in adherent Sf9 cells at a multiplicity of infection (MOI) of 1. Recombinant HA stem constructs were expressed in 1 L of suspension Sf9 cells at an MOI of 1. Three days post-infection, the cells were harvested by centrifugation at 4,000 × g for 25 minutes. Soluble recombinant H1 and H3 stem constructs were purified from the supernatant using Ni Sepharose Excel resin (Cytiva) for affinity chromatography, followed by size exclusion chromatography on a HiLoad 16/100 Superdex 200 prep grade column (Cytiva) in 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The purified protein was concentrated using an Amicon spin filter (Millipore Sigma) and filtered through a 0.22 μm centrifuge Tube Filter (Costar). Protein concentration was quantified by measuring A280 absorbance using a Nanodrop One (Thermo Fisher Scientific). ELISA for human plasma samples Assays were performed using Nunc MaxiSorp plates (Thermo Fisher Scientific), which were coated overnight with 100 ng per well of recombinant HA protein in phosphate-buffered saline (PBS). The following day, plates were washed three times with PBS containing 0.1% Tween 20. The plates were then blocked with 100 μL of Chonblock blocking/sample dilution ELISA buffer (Chondrex Inc., Redmond, US) and incubated at room temperature for 1 hour. For human plasma samples, each sample was diluted to an initial concentration of 1:100, followed by ten-fold dilutions (1:1,000, 1:10,000, 1:100,000) prepared in Chonblock blocking/sample dilution ELISA buffer and added to the antigen wells. Plates were incubated for 2 hours at 37°C. After washing the plates three times with PBS containing 0.1% Tween 20, each well was incubated with 100 μL of anti-human IgG secondary antibody (1:5000, Thermo Fisher Scientific) for 1 hour at 37°C. After six washes with 1× PBS containing 0.05% Tween 20, 100 μL of 1-Step Tetramethylbenzidine (TMB) ELISA Substrate Solution (Thermo Fisher Scientific) was added to each well. After incubation for 10 minutes, the reaction was stopped with 50 μL of 2 M H 2 SO 4 solution, and absorbance values were measured at 450 nm using a BioTek Synergy HTX Multimode Reader (Agilent). Monoclonal antibody binding using ELISA Assays used Nunc MaxiSorp plates (Thermo Fisher Scientific) coated overnight with 100 ng recombinant HA protein in phosphate-buffered saline (PBS). The next day, plates were washed three times with PBS and 0.1% Tween 20. Blocking was carried out with 5% nonfat dry milk for 2 hours at room temperature. An initial concentration of 100 µg mL −1 was used to calculate the area under the curve (AUC) and 10-fold dilutions were done. For end-point ELISA experiments, the purified recombinant antibodies were diluted to 10 μg mL −1 in 5% Nonfat dry milk, added to the plates, and incubated for 2 hours at 37°C. Following three washes with PBS containing 0.1% Tween 20, each well was incubated with 100 μL of anti-human IgG secondary antibody (1:5000, Thermo Fisher Scientific) for one hour at 37°C. After six washes with 1× PBS containing 0.05% Tween 20, 100 μL of 1-Step TMB ELISA Substrate Solution (Thermo Fisher Scientific) was added to each well. After a 10-minute incubation, the reaction was stopped with 50 μL of 2 M H 2 SO 4 solution, and absorbance values were measured at 450 nm using a BioTek Synergy HTX Multimode Reader (Agilent). Biolayer interferometry binding assay Binding assays were performed using biolayer interferometry (BLI) on an Octet RED96e instrument (Sartorius). For the measurement of K D , His-tagged HA protein (20 μg mL −1 ) in 1× kinetics buffer (1× PBS, pH 7.4, with 0.002% Tween 20) was loaded onto HIS1K biosensors and incubated with Fabs at concentrations of 300 nM, 100 nM, and 33.3 nM. The assay consisted of five steps: baseline (60 seconds in 1× kinetics buffer), loading (60 seconds with His-tagged HA protein), a second baseline (60 seconds in 1× kinetics buffer), association (60 seconds with Fabs), and dissociation (60 seconds in 1× kinetics buffer). K D values were estimated using a 1:1 binding model. For competition assays, H3 stem was first loaded onto HIS1K biosensors for 120 seconds. Antibody binding was measured by exposing the sensors to 100 nM of the first antibody in 1× kinetics buffer for 120 seconds. The degree of additional binding was then assessed by exposing the sensors to 100 nM of a second antibody in the presence of the first antibody (100 nM) for another 120 seconds. The competition index (CI) for an antibody Ab1 competing with a pre-bound antibody Ab2 was calculated as: where “max response without Ab2 pre-bound ” is the maximum response of Ab1 in the absence of Ab2 pre-bound and “max response with Ab2 pre-bound ” is the maximum response of Ab1 in the presence of Ab2 pre-bound. Cryo-EM sample preparation and data collection The purified H3N8 full-length, uncleaved protein was mixed with each Fab at a 1:4 molar ratio and incubated at 4 degrees overnight before size exclusion chromatography. The peak fraction of the Fab-HA complex was eluted in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl and concentrated to around 3 mg mL −1, and mixed with n-octyl-β-D-glucoside (Anagrade) at a final concentration of 0.1% w/v for cryo-EM sample preparation. Cryo-EM grids were prepared using a Vitrobot Mark IV machine. An aliquot of 3 μL sample was applied to a 300-mesh Quantifoil R1.2/1.3 Cu grid pre-treated with glow-discharge. Excess liquid was blotted away using filter paper with blotting force 0 and blotting time 3 seconds. The grid was plunge-frozen in liquid ethane. Data collection was done on a Titan Krios microscope equipped with Gatan detector. Images were recorded at 81,000× magnification, corresponding to a pixel size of 0.53 Å/pix at the super-resolution mode of the camera. A defocus range of −0.8 μm to −3 μm was used with a total dose of 57.35 e-/Å 2 . Cryo-EM data processing Data processing was conducted using CryoSPARC Live (version 4.5) 50 . Movies were subjected to motion correction and contrast transfer function (CTF) estimation, and particles picked with CryoSPARC blob picker followed by 2D classification. The best classes identified by the blob picker served as templates for CryoSPARC template pickers. The resulting particles underwent multiple rounds of 2D classification to ensure thorough cleanup before proceeding to ab initio reconstruction. The most effective class from the ab initio reconstruction was then subjected to homogeneous refinement, reference-based motion correction, followed by an additional round of homogeneous refinement, local and global CTF estimation, and non-uniform refinement. Finally, the map was sharpened using DeepEMhancer 51 . Low resolution cryo-EM analysis For the H3N8 complex with AG2-G02 and 2F02 Fabs, data were collected using a Glacios 2 Cryo-TEM, equipped with a Falcon 4i Direct Electron Detector. Images were recorded at a magnification of 150,000×, with a pixel size of 0.96 Å/pix. A defocus range of −0.3 μm to −3 μm was applied, along with a total dose of 60 e-/Ų. Data processing was conducted using CryoSPARC Live (version 4.5) 50 as described above. Model building and refinement An initial model for the cryo-EM maps was built using ModelAngelo 52 , an automated atomic model-building program. This model was then fitted into the cryo-EM density map using UCSF Chimera 53 , followed by manual adjustments in Coot 54 and refinement with the Phenix real-space refinement program 55 . Refinement was repeated iteratively until no further significant improvements were observed. Sequence conservation analysis Full-length human H3N2 HA and avian H3 protein sequences from different subtypes were downloaded from the Global Initiative for Sharing Avian Influenza Data (GISAID; https://gisaid.org ). Sequences corresponding to HA2 were extracted. We then calculated the percentage of each character at HA2 position 32 across the strains per year. The percentages were written to a CSV file to generate a graph depicting the frequencies of each amino acid variant over time. Structural analysis of HA-antibody complexes Buried surface areas upon binding and paratope residues of AG2-G02, 2F02, CR9114 (PDB 4FQY) 37 , FI6v3 (PDB 3ZTJ) 32 , CR8020 (PDB 3SDY) 28 , CR8043 (PDB 4NM8) 29 , (PDB 4KVN), 222-1C06 (PDB 7T3D) 40 , and ADI-85666 (PDB 9BDF) 38 were analyzed using PDBePISA 56 . The CDR regions and germline gene usage of each antibody was annotated using IgBLAST 57 . The molecular interactions of AG2-G02 and 2F02 in complex with H3N8 HA were analyzed and visualized using PyMOL (Schrödinger). Microneutralization assay For the microneutralization assay, MDCK-SIAT1 cells were seeded in 96-well plates. After reaching 100% confluency, MDCK-SIAT1 cells were washed once with 1× PBS. Minimal essential media (Gibco) containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Gibco) was then added to the cells. Monoclonal antibodies were serially diluted 10-fold starting from 100 μg mL −1 and mixed with 100 TCID 50 (median tissue culture infectious dose) of viruses at equal volume and incubated at 37°C for 1 hour. Subsequently, the mixture was inoculated into cells and incubated at 37°C for another hour. Cell supernatants were discarded and replaced with minimal essential media containing 25 mM HEPES, and 1 μg mL − 1 TPCK-trypsin (Sigma). Plates were incubated at 37°C for 48 hours, and virus presence was detected by monitoring the cytopathic effects (CPE) to determine the MN 50 titers. HAI assay Briefly, 50 µl of H3N2 A/Darwin/6/2021 virus was mixed with 2-fold serial dilutions of plasma samples and incubated for one hour. After incubation, 50 µl of 1% turkey red blood cells were added to the wells. The highest dilution of the serum that prevented hemagglutination was recorded, and the HI titer was calculated. Flow cytometry analysis MDCK-SIAT1 cells were seeded in 12-well plates. At 100% confluency, cells were washed with 1× PBS and infected with H3N2 A/Darwin/6/2021 virus and H3N2 A/Moscow/10/1999 virus at an MOI of 0.1 in minimal essential media (Gibco) containing 25 mM HEPES, and 1 μg mL −1 TPCK-trypsin (Sigma). At 36 hours post-infection, cells were fixed with 4% paraformaldehyde overnight at 4°C. On the next day, paraformaldehyde was removed, and the cells were blocked in blocking buffer (1× PBS with 2% FBS and 0.1% BSA) for 30 minutes. Cells were incubated with 20 μg mL −1 of the indicated antibody in blocking buffer at 4°C for 1 hour. Subsequently, the cells were washed twice with blocking buffer and incubated with 2 μg mL −1 of PE anti-human IgG Fc (BioLegend, catalog #: 410708) at 4°C for 1 hour. The cells were then washed thrice and resuspended in 1× PBS for flow cytometry analysis using a FACSymphony A1 (BD Biosciences). Data were analyzed using FlowJo v10.10 Software (BD Life Sciences). Prophylactic protection experiments Female BALB/c mice at 6 weeks old (n = 4-5 per group) were anesthetized with isoflurane and intranasally infected with 5× lethal dose (LD 50 ) of H3N2 A/Philippines/2/1982 (X-79, 6:2 A/PR/8/34 reassortant, mouse-adapted) virus. Mice were given the indicated antibody at a dose of 5 mg/kg intraperitoneally at 4 hours before infection. Weight loss was monitored daily for 14 days. The humane endpoint was defined as a weight loss of 25% from initial weight at day 0. While our BALB/c mice were not modified to facilitate interaction with human IgG1, human IgG1 could interact with mouse Fc gamma receptor 58 , 59 . To determine the lung viral titers at day 3 post-infection, the lungs of infected mice were harvested and homogenized in 1 mL of minimal essential media using a gentleMACS C Tube (Miltenyi Biotec). Subsequently, virus titers were measured by TCID 50 assay. AUTHOR CONTRIBUTIONS A.B.G., H.L., W.O.O., and N.C.W. conceived and designed the study. A.B.G., H.L., T.P., Q.W.T., and W.O.O. performed the experiments. Y.L., M.L., C.K.P.M., Y.S.T., and H.L. collected the human samples. A.B.G., H.L., and N.C.W. wrote the paper, and all authors reviewed and edited the paper. DECLARATION OF INTERESTS N.C.W. consults for HeliXon. The authors declare no other competing interests. DATA AVAILABILITY Cryo-EM maps have been deposited to the Electron Microscopy Data Bank under accession codes: EMD-48873 and EMD-48874. The refined models have been deposited to the RCSB Protein Data Bank under accession codes: 9N4E and 9N4F. ACKNOWLEDGEMENTS We thank Kristen Flatt and the Materials Research Laboratory Central Research Facilities at University of Illinois Urbana-Champaign, as well as Frank Vago and the Cryo-EM Facility at Purdue University, for access to cryo-EM instrumentation and data collection. We also thank Disha Bhavsar and Florian Krammer for providing the H3N2 A/Philippines/2/1982 (X-79) virus, and Florian Krammer for helpful comments on the manuscript. This work is supported by the Searle Scholars Program (N.C.W.), the Vallee Scholars Program (N.C.W.), Howard Hughes Medical Institute Emerging Pathogens Initiative (N.C.W.), UIUC William T. and Lynn Jackson Graduate Student Fellowship (W.O.O.), and Carl R. Woese Institute for Genomic Biology Postdoctoral Fellowship (H.L.). REFERENCES 1. ↵ Krammer , F. et al. Influenza . Nat Rev Dis Primers 4 , 3 ( 2018 ). OpenUrl CrossRef PubMed 2. ↵ Fereidouni , S. et al. Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses . Emerg Microbes Infect 12 , ( 2023 ). 3. ↵ Jester , B. J. , Uyeki , T. M. & Jernigan , D. B . Fifty years of influenza A(H3N2) following the pandemic of 1968 . Am J Public Health 110 , 669 – 676 ( 2020 ). OpenUrl CrossRef PubMed 4. ↵ Bedford , T. et al. Global circulation patterns of seasonal influenza viruses vary with antigenic drift . Nature 523 , 217 – 220 ( 2015 ). OpenUrl CrossRef PubMed 5. ↵ Petrova , V. N. & Russell , C. A . The evolution of seasonal influenza viruses . Nat Rev Microbiol 16 , 47 – 60 ( 2018 ). OpenUrl CrossRef PubMed 6. ↵ Zhuang , Y. et al. First known human death after infection with the avian influenza A/H3N8 Virus: Guangdong province, China, March 2023 . Clinical Infectious Diseases 78 , 646 – 650 ( 2024 ). OpenUrl CrossRef PubMed 7. Yang , R. et al. Human infection of avian influenza A H3N8 virus and the viral origins: a descriptive study . Lancet Microbe 3 , e824 – e834 ( 2022 ). OpenUrl CrossRef 8. ↵ Bao , P. et al. Human infection with a reassortment avian influenza A H3N8 virus: an epidemiological investigation study . Nat Commun 13 , 6817 ( 2022 ). OpenUrl CrossRef PubMed 9. ↵ Yang , J. et al. Evolution of avian influenza virus (H3) with spillover into humans, China . Emerg Infect Dis 29 , ( 2023 ). 10. ↵ Sun , H. et al. Airborne transmission of human-isolated avian H3N8 influenza virus between ferrets . Cell 186 , 4074 – 4084 .e11 ( 2023 ). OpenUrl CrossRef PubMed 11. ↵ Wu , N. C. & Wilson , I. A . Influenza hemagglutinin structures and antibody recognition . Cold Spring Harb Perspect Med 10 , a038778 ( 2020 ). OpenUrl Abstract / FREE Full Text 12. ↵ Wu , N. C. & Wilson , I. A . A perspective on the structural and functional constraints for immune evasion: Insights from influenza virus . J Mol Biol 429 , 2694 – 2709 ( 2017 ). OpenUrl CrossRef PubMed 13. ↵ Kirkpatrick , E. , Qiu , X. , Wilson , P. C. , Bahl , J. & Krammer , F . The influenza virus hemagglutinin head evolves faster than the stalk domain . Sci Rep 8 , 10432 ( 2018 ). OpenUrl CrossRef PubMed 14. ↵ Roubidoux , E. K. et al. Mutations in the hemagglutinin stalk domain do not permit escape from a protective, stalk-based vaccine-induced immune response in the mouse model . mBio 12 , ( 2021 ). 15. ↵ Doud , M. B. , Lee , J. M. & Bloom , J. D . How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin . Nat Commun 9 , 1386 ( 2018 ). OpenUrl CrossRef PubMed 16. ↵ Krammer , F. , Pica , N. , Hai , R. , Margine , I. & Palese , P . Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies . J Virol 87 , 6542 – 6550 ( 2013 ). OpenUrl Abstract / FREE Full Text 17. Yassine , H. M. et al. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection . Nat Med 21 , 1065 – 1070 ( 2015 ). OpenUrl CrossRef PubMed 18. ↵ Impagliazzo , A. et al. A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen . Science (1979) 349 , 1301 – 1306 ( 2015 ). OpenUrl Abstract / FREE Full Text 19. ↵ Wu , N. C. & Wilson , I. A . Structural insights into the design of novel anti-influenza therapies . Nat Struct Mol Biol 25 , 115 – 121 ( 2018 ). OpenUrl CrossRef PubMed 20. ↵ Widge , A. T. et al. An influenza hemagglutinin stem nanoparticle vaccine induces cross-group 1 neutralizing antibodies in healthy adults . Sci Transl Med 15 , ( 2023 ). 21. Andrews , S. F. et al. An influenza H1 hemagglutinin stem-only immunogen elicits a broadly cross-reactive B cell response in humans . Sci Transl Med 15 , ( 2023 ). 22. ↵ Nachbagauer , R. et al. A chimeric hemagglutinin-based universal influenza virus vaccine approach induces broad and long-lasting immunity in a randomized, placebo-controlled phase I trial . Nat Med 27 , 106 – 114 ( 2021 ). OpenUrl CrossRef PubMed 23. ↵ Padilla-Quirarte , H. O. , Lopez-Guerrero , D. V. , Gutierrez-Xicotencatl , L. & Esquivel-Guadarrama , F . Protective antibodies against influenza proteins . Front Immunol 10 , ( 2019 ). 24. ↵ Kallewaard , N. L. et al. Structure and function analysis of an antibody recognizing all influenza A subtypes . Cell 166 , 596 – 608 ( 2016 ). OpenUrl CrossRef PubMed 25. ↵ Cheung , C. S.-F. et al. Structure of an influenza group 2-neutralizing antibody targeting the hemagglutinin stem supersite . Structure 30 , 993 – 1003 .e6 ( 2022 ). OpenUrl CrossRef 26. ↵ Mantus , G. E. et al. Vaccination with different group 2 influenza subtypes alters epitope targeting and breadth of hemagglutinin stem–specific human B cells . Sci Transl Med 17 , ( 2025 ). 27. ↵ Andrews , S. F. , et al. Preferential induction of cross-group influenza A hemagglutinin stem–specific memory B cells after H7N9 immunization in humans . Sci Immunol 2 , ( 2017 ). 28. ↵ Ekiert , D. C. et al. A highly conserved neutralizing epitope on group 2 influenza A viruses . Science (1979) 333 , 843 – 850 ( 2011 ). OpenUrl Abstract / FREE Full Text 29. ↵ Friesen , R. H. E. et al. A common solution to group 2 influenza virus neutralization . Proceedings of the National Academy of Sciences 111 , 445 – 450 ( 2014 ). OpenUrl Abstract / FREE Full Text 30. ↵ Wu , N. C. et al. Convergent evolution in breadth of two VH6-1-encoded influenza antibody clonotypes from a single donor . Cell Host Microbe 28 , 434 – 444 .e4 ( 2020 ). OpenUrl CrossRef PubMed 31. ↵ Pappas , L. et al. Rapid development of broadly influenza neutralizing antibodies through redundant mutations . Nature 516 , 418 – 422 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 32. ↵ Corti , D. et al. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins . Science (1979) 333 , 850 – 856 ( 2011 ). OpenUrl Abstract / FREE Full Text 33. ↵ Wang , Y. et al. An explainable language model for antibody specificity prediction using curated influenza hemagglutinin antibodies . Immunity 57 , 2453 – 2465 .e7 ( 2024 ). OpenUrl CrossRef PubMed 34. ↵ Ouyang , W. O. et al. Rapid synthesis and screening of natively paired antibodies against influenza hemagglutinin stem via oPool + display . Preprint at doi: 10.1101/2024.08.30.610421 ( 2024 ). OpenUrl Abstract / FREE Full Text 35. ↵ Henry , C. et al. Influenza virus vaccination elicits poorly adapted B cell responses in elderly individuals . Cell Host Microbe 25 , 357 – 366 .e6 ( 2019 ). OpenUrl CrossRef PubMed 36. ↵ Dugan , H. L. et al. Preexisting immunity shapes distinct antibody landscapes after influenza virus infection and vaccination in humans . Sci Transl Med 12 , ( 2020 ). 37. ↵ Dreyfus , C. et al. Highly conserved protective epitopes on influenza B viruses . Science (1979) 337 , 1343 – 1348 ( 2012 ). OpenUrl Abstract / FREE Full Text 38. ↵ Maurer , D. P. et al. Conserved sites on the influenza H1 and H3 hemagglutinin recognized by human antibodies . Preprint at doi: 10.1101/2024.10.22.619298 ( 2024 ). OpenUrl Abstract / FREE Full Text 39. ↵ Chandler , T. L. , Yang , A. , Otero , C. E. , Permar , S. R. & Caddy , S. L . Protective mechanisms of nonneutralizing antiviral antibodies . PLoS Pathog 19 , e1011670 ( 2023 ). OpenUrl CrossRef PubMed 40. ↵ Guthmiller , J. J. et al. Broadly neutralizing antibodies target a haemagglutinin anchor epitope . Nature 602 , 314 – 320 ( 2022 ). OpenUrl CrossRef PubMed 41. ↵ Cyster , J. G. & Wilson , P. C . Antibody modulation of B cell responses—Incorporating positive and negative feedback . Immunity 57 , 1466 – 1481 ( 2024 ). OpenUrl CrossRef PubMed 42. Schiepers , A. et al. Molecular fate-mapping of serum antibody responses to repeat immunization . Nature 615 , 482 – 489 ( 2023 ). OpenUrl CrossRef PubMed 43. ↵ Lu , X. et al. Antibody-mediated suppression regulates the humoral immune response to influenza vaccination in humans . J Infect Dis 229 , 310 – 321 ( 2024 ). OpenUrl CrossRef PubMed 44. ↵ Mantus , G. E. et al. Vaccination with different group 2 influenza subtypes alters epitope targeting and breadth of hemagglutinin stem–specific human B cells . Sci Transl Med 17 , ( 2025 ). 45. ↵ Tan , G. S. et al. Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin . J Virol 88 , 13580 – 13592 ( 2014 ). OpenUrl Abstract / FREE Full Text 46. ↵ Lee , J. M. et al. Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants . Proceedings of the National Academy of Sciences 115 , ( 2018 ). 47. ↵ Wu , N. C. et al. Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses . Science (1979) 368 , 1335 – 1340 ( 2020 ). OpenUrl Abstract / FREE Full Text 48. ↵ Neumann , G. et al. Generation of influenza A viruses entirely from cloned cDNAs . Proceedings of the National Academy of Sciences 96 , 9345 – 9350 ( 1999 ). OpenUrl Abstract / FREE Full Text 49. ↵ Corbett , K. S. et al. Design of nanoparticulate group 2 influenza virus hemagglutinin stem antigens that activate unmutated ancestor B cell receptors of broadly neutralizing antibody lineages . mBio 10 , ( 2019 ). 50. ↵ Punjani , A. , Rubinstein , J. L. , Fleet , D. J. & Brubaker , M. A . cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination . Nat Methods 14 , 290 – 296 ( 2017 ). OpenUrl CrossRef PubMed 51. ↵ Sanchez-Garcia , R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing . Commun Biol 4 , 874 ( 2021 ). OpenUrl CrossRef PubMed 52. ↵ Jamali , K. et al. Automated model building and protein identification in cryo-EM maps . Nature 628 , 450 – 457 ( 2024 ). OpenUrl CrossRef PubMed 53. ↵ Pettersen , E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis . J Comput Chem 25 , 1605 – 1612 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 54. ↵ Emsley , P. & Cowtan , K . Coot : model-building tools for molecular graphics . Acta Crystallogr D Biol Crystallogr 60 , 2126 – 2132 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 55. ↵ Afonine , P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography . Acta Crystallogr D Struct Biol 74 , 531 – 544 ( 2018 ). OpenUrl CrossRef PubMed 56. ↵ Krissinel , E. & Henrick , K . Inference of macromolecular assemblies from crystalline state . J Mol Biol 372 , 774 – 797 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 57. ↵ Ye , J. , Ma , N. , Madden , T. L. & Ostell , J. M . IgBLAST: an immunoglobulin variable domain sequence analysis tool . Nucleic Acids Res 41 , W34 – W40 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 58. ↵ Dekkers , G. et al. Affinity of human IgG subclasses to mouse Fc gamma receptors . MAbs 9 , 767 – 773 ( 2017 ). OpenUrl CrossRef PubMed 59. ↵ Overdijk , M. B. et al. Crosstalk between human IgG isotypes and murine effector cells . The Journal of Immunology 189 , 3430 – 3438 ( 2012 ). OpenUrl CrossRef PubMed 60. ↵ Shu , Y. & McCauley , J . GISAID: Global initiative on sharing all influenza data – from vision to reality . Eurosurveillance 22 , ( 2017 ). View the discussion thread. Back to top Previous Next Posted March 27, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity Akshita B. Gopal , Huibin Lv , Tossapol Pholcharee , Wenhao O. Ouyang , Qi Wen Teo , Yasha Luo , Yun Sang Tang , Mingyong Luo , Chris K. P. Mok , Nicholas C. Wu bioRxiv 2025.03.24.644842; doi: https://doi.org/10.1101/2025.03.24.644842 Share This Article: Copy Citation Tools Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity Akshita B. Gopal , Huibin Lv , Tossapol Pholcharee , Wenhao O. Ouyang , Qi Wen Teo , Yasha Luo , Yun Sang Tang , Mingyong Luo , Chris K. P. Mok , Nicholas C. Wu bioRxiv 2025.03.24.644842; doi: https://doi.org/10.1101/2025.03.24.644842 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Immunology Subject Areas All Articles Animal Behavior and Cognition (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41970) Biophysics (21463) Cancer Biology (18605) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19911) Epidemiology (2067) Evolutionary Biology (24329) Genetics (15615) Genomics (22514) Immunology (17743) Microbiology (40424) Molecular Biology (17194) Neuroscience (88650) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4827) Physiology (7648) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)