Immunodominance is a poor predictor of vaccine-induced T follicular helper cell quality

Immunodominance is a poor predictor of vaccine-induced T follicular helper cell quality | 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 Immunodominance is a poor predictor of vaccine-induced T follicular helper cell quality HX Tan , MZM Zheng , K Wragg , L Murdiyarso , D Pilapitiya , A Kelly , R Esterbauer , C Gonelli , AK Wheatley , View ORCID Profile JA Juno doi: https://doi.org/10.1101/2025.07.21.666029 HX Tan 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site MZM Zheng 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site K Wragg 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site L Murdiyarso 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site D Pilapitiya 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site A Kelly 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site R Esterbauer 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site C Gonelli 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site AK Wheatley 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: a.wheatley{at}unimelb.edu.au jennifer.juno{at}unimelb.edu.au JA Juno 1 Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria 3000, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for JA Juno For correspondence: a.wheatley{at}unimelb.edu.au jennifer.juno{at}unimelb.edu.au Abstract Full Text Info/History Metrics Preview PDF Abstract Rational engineering of vaccine immunogens to focus B cell responses on potently neutralizing epitopes is a promising approach to improve the potency, breadth and durability of viral vaccines. Such strategies, however, can compromise vaccine immunogenicity through the unintended exclusion of CD4+ T cell epitopes, which are critical for the development of T follicular helper (TFH) cells and to support high affinity antibody production. Using a prototypic influenza HA stem immunogen lacking effective CD4+ T cell help in BL6 mice, we interrogated the minimal requirements for T cell help needed to drive serological responses to vaccination. We find that priming of naïve CD4 T cells is markedly efficient, however the immunodominance of a given CD4 T cell epitope is not predictive of the propensity to provide high quality help to antigen-specific B cells. In the context of soluble antigens, provision of a single MHC class II epitope is sufficient to drive robust germinal centre responses and serum IgG titres. However not all CD4 epitopes provide equivalent levels of B cell help, despite priming comparable numbers of antigen-specific CD4 T cells. Finally, we show multimerizing and arraying antigens on nanoparticle scaffolds unlocks highly subdominant, near-undetectable CD4 T cell helper responses to support a T-dependent antibody response. Our findings emphasize the importance of CD4+ T cell help for programing robust and durable humoral immunity, and provide crucial insights to guide the rational incorporation of favorable T cell epitopes into vaccines. Introduction The development of effective vaccines has fundamentally reshaped the ability of human populations to contain and control infectious diseases. While historically vaccines were derived from attenuated or killed whole pathogens, more recent vaccine development efforts have focused upon identification of the most protective “subunits” for inclusion as vaccine immunogens. This was extended during the COVID-19 pandemic, where rational vaccine design was used to engineer the SARS-CoV-2 spike immunogens in protein, mRNA or viral-based vaccines to structurally stabilize 1 , 2 and/or prevent cleavage 3 , 4 of the spike trimer. Analogous modifications of the F protein are incorporated into recently approved vaccines for RSV 5 . Further efforts to concentrate immunity upon conserved or potently neutralizing domains have seen re-engineering of vaccine immunogens to the level of isolated protein subdomains such as the SARS-CoV-2 RBD 6 , 7 or stem region of influenza HA 8 . While such small protein targets effectively focus immune recognition by B cells, increasing evidence suggests this might come at a cost to immunogenicity, seen in both pre-clinical models 9 , 10 and human clinical trials 11 , 12 . Outside of the stochasticity of vaccine delivery, the immunogenicity of vaccine antigens varies, likely reflecting a combination of protein intrinsic factors and host genetics. We and others have identified that a paucity of CD4 helper T cell epitopes is one important factor constraining vaccine immunogenicity 9 , 10 , 13 , 14 . Unlike B cells, whose immunoglobulin receptors scan and engage the near infinite diversity of conformational epitopes on protein surfaces, CD4+ T cells can only recognize linear peptide epitopes in the context of a host MHC-II. This heightened restriction limits the absolute number of T cell epitopes within any given immunogen, with distribution being uneven and individualistic 15 . Reducing the size of a vaccine immunogen therefore has the potential to concomitantly shrink the pool of available CD4+ T cell epitopes, leaving some antigens poorly recognized even in diverse human populations 16 - 18 . In extreme examples, exemplified by model HA stem 9 or HEL 19 immunogens in genetically inbred C57BL/6 mice, a lack of CD4 T cell epitopes can render immunization immunologically silent. Various strategies have been reported to augment CD4 T cell priming, T follicular helper cell (TFH) differentiation, and subsequent vaccine immunogenicity, but generalized applicability, comparative utility, and mechanisms of action remain unclear. Presentation of vaccine antigens on nanoparticle scaffolds enhances BCR recognition and signaling 20 , but can also augment CD4 T cell help via epitopes localized within the scaffold 21 . Similarly, covalent coupling of poorly immunogenic proteins to carrier proteins or alternate sources of T cell help can supplement the available antigen-specific CD4 T cell pool 9 , 22 , analogous to the carrier proteins essential for driving antibody responses to pneumococcal carbohydrates in childhood vaccines 23 . However the impacts of epitope specificity or relative immunodominance of polyclonal CD4+ T cell populations upon the capacity to provide help to germinal centre (GC) B cells is poorly understood. Here, we made use of the HA stem-C57BL/6 model 9 to address the features of CD4 T cell immunity that drive potent humoral immune responses to vaccination. We find evidence for a spectrum of epitope-level regulation of GC initiation, with some CD4 T cell specificities unable to support antigen-specific B cell proliferation and IgG production. Critically, epitope immunodominance did not necessarily predict the quality of help provided by antigen-specific CD4 T cells: some immunodominant epitopes exhibited no helper capability, while subdominant responses provided effective help, with even CD4 T cells at near-undetectable levels successfully contributing to GC formation in the context of multimerized antigen. Overall, these data demonstrate that while vaccine immunogenicity is critically dependent on CD4 T cell availability, the capacity to support humoral responses is not universal to all CD4 T cell epitopes, with implications for the rational design of small, engineered vaccine immunogens. Results Multimeric display of HA stem antigens on self-assembling ferritin nanoparticles compensates for highly subdominant CD4 help We previously established primary vaccination of BL/6 mice with a soluble HA stem protein fails to elicit an antigen-specific GC B cell response or production of serum IgG due to a lack of available CD4 T cell epitopes 9 . Presentation of stem on self-assembling ferritin nanoparticles enhances its immunogenicity 24 , 25 but the source of T cell help in this system is unclear, as BL/6 mice are reported to lack ferritin-specific CD4 T cells 21 . We find that stem-ferritin nanoparticles (Stem-Fe) are robustly immunogenic, eliciting significantly higher stem IgG titres than soluble stem trimers (p=0.0001; Fig1A) and greater numbers of bulk and stem-specific GC B cells (p=0.0001 and p=0.02, respectively; Fig 1B-C , gating in Supplementary Fig 1 ). Using confocal imaging and flow cytometry, we confirmed the GL7 hi CD38 lo B cell populations in stem-Fe vaccinated mice reflected bona fide GC structures. GL7 expression was correctly localized to GC-like structures within the follicle ( Supplementary Fig 2A ), and canonical light zone and dark zone GC B cell populations were observed in both full-length HA (HA-FL) and stem-Fe animals ( Supplementary Fig 2B ), indicative of a T-dependent GC reaction. Download figure Open in new tab Figure 1. Nanoparticle display enhances stem immunogenicity in the absence of prominent CD4 T cell help. Mice were vaccinated with 5mg of full-length HA protein (HA-FL), HA-stem protein, or stem-ferritin nanoparticles (Stem-Fe) co-formulated 1:1 with Addavax adjuvant. Serum and draining LN were collected at day 14 post-vaccination. (A) Serum endpoint titres of stem-specific IgG (N=10/group). (B) Frequency of GL7 hi CD38 lo GC B cells, (C) number of stem-specific GC B cells, or (D) frequency of CXCR5 hi PD-1 hi TFH in the draining LN (N=10/group). (E) Representative staining and frequency of CD154 + stem or HA-specific memory CD4 T cells following in vitro stimulation. Frequencies are background subtracted based on the DMSO control (N=5/group). (F) CD4 + T cell proliferation following in vitro peptide stimulation with HA, stem or ferritin peptide pools. Splenocytes were harvested from vaccinated mice at day 14, or at day 79 after 3 vaccinations (N=5/group). Lines indicate median and IQR. Statistics assessed by Mann-Whitney test comparing Stem and Stem-Fe groups. *p<0.05, ****p=0.0001 Given the lack of identifiable CD4 T cell epitopes within the stem antigen and the limited impact of stem-Fe vaccination on total TFH frequency ( Fig 1D ), we assessed the magnitude of ferritin-specific responses. We observed limited CD4 T cell responsiveness upon in vitro re-stimulation with either stem or ferritin peptide pools ( Fig 1E ). Similarly, use of a proliferation assay for sensitive detection of any low-frequency populations of stem- or ferritin-specific T cells revealed only sporadic, low-level proliferation when compared to the vehicle control ( Fig 1F ). To further amplify antigen-specific T cell frequencies, we primed and then boosted mice twice on days 21 and 42. Three out of five Stem-Fe vaccinated animals exhibited clear proliferative responses to the ferritin peptide pool ( Fig 2F ), suggesting highly subdominant epitopes within ferritin could be the source of T cell help supporting Stem-Fe immunogenicity. Download figure Open in new tab Figure 2. Stem-Fe nanoparticle immunogenicity in SMARTA and BALB/c mice. Stem-specific (A) GC B cell frequencies, (B) GC B cell counts or (C) IgG titres at day 14 following Stem-Fe vaccination of SMARTA (N=4), WT BL6 (N=10) or BALB/c (N=5) mice. (D) Stem- or ferritin-specific CD4 + T cell responses in the draining LN of Stem-Fe vaccinated BALB/c mice at day 14 (n=4). (E) CD4 + T cell proliferation in BALB/c mice following in vitro peptide stimulation with DMSO, stem or ferritin peptide pools. Splenocytes were harvested from vaccinated mice at day 14 (N=5/group). Lines indicate median and IQR. If ferritin-specific CD4 help below the detection limit of conventional T cell assays was responsible for supporting a primary GC reaction, we reasoned that the Stem-Fe antigen should not be immunogenic in transgenic (tg) BL/6 mice that express a single TCR. Conversely, animals with high frequencies of ferritin-specific T cells (i.e. BALB/c mice 21 ) should produce superior serological responses. Stem-Fe vaccination of SMARTA tg (BL/6 background), wild-type (WT) BL/6 and WT BALB/c animals demonstrated a striking immunogenicity gradient, with negligible numbers of stem-specific GC B cells ( Fig 2A-B ) or IgG ( Fig 2C ) detected in SMARTA mice, while responses in WT BALB/c were more than 20-fold higher compared to WT BL/6 ( Fig 2A-C ). We confirmed that ferritin-specific CD4 T cells were readily detectible in BALB/c mice by both standard restimulation ( Fig 2D ) and proliferation assays ( Fig 2E ), demonstrating that the ferritin core provides substantially more CD4 T cell help in BALB/c versus BL/6 mice. Overall, when stem is arrayed on the surface of a nanoparticle, the low level of ferritin-specific CD4 T cell help available in BL/6 mice becomes sufficient to support a stem-specific GC response that is otherwise absent following soluble stem vaccination. The TFH repertoire of full-length HA is limited in breadth and dominated by a single epitope The gradient of Stem-Fe immunogenicity across SMARTA, BL/6 and BALB/c mice strains suggests that availability of CD4 T cell help acts as a rheostat that directly tunes the magnitude of the GC and resultant serological response. While we have established the near-complete lack of CD4 help in stem, the breadth and specificity of epitopes that successfully support the immunogenicity of the full-length HA (HA-FL) protein requires detailed mapping. To maximize the number of TFH for screening, BL/6 mice were infected with a sublethal dose of PR8 influenza virus and mediastinal LN (mLN) which drain the site of infection were collected on day 14. LN cell suspensions were stimulated in vitro with a matrix of 20 peptide pools spanning the HA protein ( Supplementary Fig 3A ), with candidate immunogenic peptides selected by identifying pools that elicited a CD154 response greater than the DMSO control ( Supplementary Fig 3B ). Individual peptide screening identified 14 potential hits, which we subsequently tested for consistent recognition across multiple animals. Nine peptides elicited CD4 T cell responses in at least 3 of 5 mice, with 4 putative epitopes identified for the CXCR5 + PD-1 + CD4 T cell population (enriched for pre-TFH/TFH cells; Fig 3A ). HA 91-107 was identified as highly immunodominant, with a median of 2.6% of TFH specific for this single peptide. Three other epitopes elicited lower magnitude responses: HA 301-323 (two overlapping peptides spanning HA 301-317 and HA 307-323 ), HA 115-131 , and HA 523-539 ( Fig 3A ). A similar hierarchy was observed within the total CD4+ T cell population ( Supplementary Fig 3C ), with no immunogenic peptides located within the HA-stem domain as expected. Download figure Open in new tab Figure 3. Rescue of stem immunogenicity by genetically fused CD4 T cell epitopes. (A) Identification of immunogenic peptides following intranasal infection with PR8 virus. Mediastinal LN were collected on day 14 and restimulated in vitro with DMSO, ConA or indicated peptides (N=4-5 mice per peptide). Immunogenic peptides are labelled with the median response and number of responding mice. (B) Design of trimeric stem antigens covalently linked to HA-derived peptides or prototypic OVA 323 and GP 61 peptides. (C) Stem-specific IgG endpoint titres at day 14 post-vaccination with 5ug of HA-FL, stem, or stem-peptide antigens formulated with Addavax (N=7-10 per group). Statistics assessed by Kruskal-Wallis test with Dunn’s post-test compared to the stem control. (D) Number of stem-specific GC B cells (GL7 + CD38 lo ) at day 14 post-vaccination (N=4-5 per group). (E) Longitudinal tracking of stem-specific GC B cells in the draining LN at days 4, 5, 6, 10 and 14 post-vaccination (N=5 per group). (F) Durability of antigen-specific serum IgG following stem-GP 61 or HA-FL vaccination (N=5 per group). Symbols or lines indicate median with IQR. To confirm whether these same peptides were immunogenic in the context of vaccination, we intramuscularly immunized mice with 5μg of soluble HA-FL protein in Addavax adjuvant. HA 91 epitope was again immunodominant, accounting for 74% of the CD154 response to the full HA peptide pool (median of 7.85% CD154 + for HA pool versus 5.76% for HA 91 peptide) ( Supplementary Fig 3D ). Addition of an MHC class II epitope to soluble stem protein rescues stem-specific IgG Given the wide spectrum of TFH responses capable of modulating serological outcomes, from low-level ferritin-specific help for nanoparticle-driven GCs to the heavy dominance of HA 91 in the native HA-specific T cell pool, we further sought to clarify the intrinsic “helpfulness” of discrete CD4+ T cell epitopes. We tested whether restoration of a single epitope onto the stem vaccine antigen could prime cognate CD4+ T cells in vivo and thereby improve the stem-specific IgG response. Variants of the trimeric stem immunogen were developed with incorporation of a single peptide epitope sequence adjoined at the C-terminus of the T4 foldon domain ( Fig 3B ). We produced immunogens incorporating each peptide mapped from HA (HA 91 , HA 115 , HA 301 and HA 523 ) as well as prototypic IA b -restricted peptides OVA 323 (‘OTII’) and LCMV GP 61 (‘SMARTA’). WT BL/6 mice were vaccinated with a single 5μg dose of HA-FL, stem, or stem variant antigens formulated in Addavax. On day 14, stem IgG titres were assessed by ELISA and stem-specific GC B cells quantified in the dLN. Only two antigens, stem-HA 523 and stem-GP 61 , were capable of eliciting stem-specific IgG and GC B cells at levels above the stem control (p=0.006 and p<0.0001, respectively; Fig 3C-D ). The failure of the immunodominant HA 91 epitope to rescue the stem response prompted us to examine the biogenesis of the GC response from days 4-14 post-vaccination. Stem-HA 91 vaccinated animals failed to form a stem-specific GC B cell population at any timepoint, while stem-GP 61 -immunised animals exhibited a robust stem-specific GC response evident from day 6 onwards ( Fig 3E ). At peak, antigen-specific GC B cell numbers were 33-fold higher in Stem-GP 61 vaccinated animals compared to HA-FL, with the serological response demonstrating similar durability for both antigens ( Fig 3F ). HA 91 -specific TFH cell fail to populate the germinal centre Despite HA 91 dominance in the native CD4+ T cell response to HA-FL vaccination and PR8 infection, incorporation of the HA 91 peptide failed to rescue stem immunogenicity. Possible mechanisms could include a peptide processing defect in the engineered stem immunogen that prevented or reduced naïve CD4 T cell priming by DCs, or alternatively a qualitative defect in TFH differentiation and/or T cell:B cell interactions. To address this, we produced an IA b /HA 91 tetramer that facilitated tracking of antigen-specific T cells. In both PR8 infection and HA vaccination, staining of the draining LN with tetramer confirmed the expansion of a prominent CD44 hi epitope-specific CD4 T cell population ( Supplementary Fig 4A ). A similar tetramer successfully identified GP 61 -specific T cells in dLN following LCMV GP immunization ( Supplementary Fig 4A ). Using the HA 91 and GP 61 tetramers, we compared epitope-specific CD4 T cell numbers and phenotypes at days 4, 5, 6, 10 or 14 post-vaccination with either stem-HA 91 or stem-GP 61 (gating in Supplementary Fig 4B ). Both antigens readily primed CD4 T cells, evidenced by the expansion of a tetramer + CD44 hi population ( Fig 4A ) with progressive maturation from a CD62L hi to CD62L lo phenotype ( Supplementary Fig 4C ). Total numbers of HA 91 - or GP 61 -specific T cells in the dLN were comparable, although the stem-HA 91 antigen primed significantly more tetramer + cells at day 5 compared to HA-FL protein ( Fig 4A ). Nevertheless, these populations followed different TFH differentiation trajectories over the subsequent 11 days. The small numbers of CXCR5 hi PD-1 hi TFH-like GP 61 -specific cells seen on day 4 became a substantial TFH population by day 6, when median numbers of antigen-specific TFH were 4.2-fold greater for GP 61 compared to HA 91 ( Fig 4B ). Download figure Open in new tab Figure 4. Differential GC recruitment of HA 91 and GP 61 -specific TFH populations. (A) Longitudinal tracking of total, (B) TFH (CXCR5 hi PD-1 hi ) or (C) GC resident TFH (CD90 lo ) antigen-specific CD4 + T cell numbers in the draining LN using IA b tetramers. (D) Proportion of tetramer + TFH with CD90 lo phenotype at days 5, 6 and 10 post-vaccination. Symbols indicate median and IQR (n=5 per group). Recent work has shown that the identification of TFH based on high CXCR5 and PD-1 expression includes cells located both within and adjacent to the GC 26 . Downregulation of surface marker CD90 delineates a population of GC-resident TFH that emerge at day 5 post-immunisation and are dependent on MHCII-expressing B cells for their development and maintenance. Based on CD90 expression, we found significantly higher numbers of GC-resident TFH in stem-GP 61 compared to stem-HA 91 vaccinated animals from days 5-10 ( Fig 4C ). While this was partially driven by the overall greater number of GP 61 -specific TFH, the dynamics of CD90 downregulation also differed between HA 91 and GP 61 TFH populations. From day 5 to 10, the proportion of GP 61 TFH with a GC-resident phenotype increased from a median of 18.0% to 31.4% ( Fig 4D ). In contrast, the proportion of HA 91 TFH recruited into the GC declined from 6.9% on day 5 to 2.3% on day 10 ( Fig 4D ). As sustained GC B cell presentation of pMHCII is required to maintain CD90 lo TFH populations 26 , these data suggested that suboptimal class-II presentation of the HA 91 peptide by B cells may limit the development of GC-resident TFH. Broader B cell presentation of HA 91 supports the Stem-HA 91 GC response If differential B cell peptide-MHCII presentation controls the recruitment of ag-specific TFH into the GC, then altering B cell antigen presentation should rescue stem-HA 91 immunogenicity ( Fig 5A ). Prior studies have suggested that defects in TFH differentiation can be overcome when DC antigen presentation is extended by boosting a protein immunization with cognate peptide three days later 27 . However, vaccination with 5ug of stem-HA 91 supplemented with 5ug of free HA 91 peptide at either day 0 or day 3 failed to rescue stem antibody titres ( Fig 5B ). Download figure Open in new tab Figure 5. Modulation of stemHA 91 immunogenicity through via altered B cell antigen presentation. (A) Overview of immunisation timeline and groups. ( B) Stem IgG titres at day 14 post-vaccination for groups shown in panel A. N=3-4 per group from either one or two independent experiments. (C) Frequencies of total, TFH, or GC TFH HA 91 Tet+ T cells at day 6 post-vaccination with 5ug stem-HA 91 + 5ug OVA-HA 91 (light blue) or 5ug stem-HA 91 + 5ug OVA + 0.2ug HA 91 (dark blue). N=5 per group. Lines indicate median and IQR. We next considered if the small pool of stem-specific naïve B cells may limit the presentation of pMHCII complexes, and contribute to the poor immunogenicity of stem-HA 91 compared to HA-FL. We therefore immunised mice with 5ug of stem-HA 91 co-formulated with 5ug of OVA-HA 91 to increase the potential pool of naïve B cells presenting IA b /HA 91 . As controls for the OVA-induced GC response and increased dose of HA 91 peptide, mice were vaccinated with 5ug of stem-HA 91 co-formulated with 5ug OVA and an equimolar amount (0.2ug) of free HA 91 . Only animals vaccinated with stem-HA 91 and OVA-HA 91 exhibited stem-specific IgG at day 14 ( Fig 5B ), suggesting that covalent linkage of HA 91 to an additional B cell antigen was necessary for T cell-B cell interactions that supported a productive stem-specific GC. Both groups exhibited similar HA 91 -specific T cell frequencies ( Fig 5C ). While frequencies of bulk HA 91 TFH were slightly higher in the stem-HA 91 +OVA-HA 91 group (median 0.17% of CD4 versus 0.11% for stem-HA 91 +OVA+HA 91 ), CD90 lo GC resident TFH were 1.75-fold more frequent (0.021% versus 0.012%) ( Fig 5C ). Collectively, these data suggest that the poor performance of the HA 91 epitope as a sole source of CD4 T cell help is at least partially attributable to a failure to establish or maintain an antigen-specific CD90 lo GC-resident TFH population, which can be mitigated by expanding the pool of B cells capable of presenting IA b /HA 91 complexes. Antigen dose differentially impacts CD4 priming and TFH differentiation If differences in stem-HA 91 and stem-GP 61 immunogenicity were driven by pMHCII presentation in the GC, we next asked what the impact would be of reducing GP 61 peptide availability. To titrate GP 61 dose without changing the total amount of stem antigen available for B cell uptake, we vaccinated mice with varying doses of the stem-GP 61 antigen supplemented with unmodified stem, such that the total dose of protein remained 5ug ( Fig 6A ). Draining lymph nodes were collected at day 6 post-vaccination, the timepoint of maximal GP 61 -specific T cell expansion and the appearance of stem-specific GC B cells. All immunization doses elicited a similar number of total GP 61 -specific CD44 hi CD4 T cells ( Fig 6B ), highlighting the marked efficiency of T cell priming by DCs. Download figure Open in new tab Figure 6. Modulation of stem immunogenicity through via altered B cell antigen presentation. (A) Antigen doses for titration of GP 61 peptide availability. (B) Numbers of total and TFH GP 61 Tet+ cells at day 6 post-vaccination (N=5 per group). (C) Number of stem-specific GC B cells at day 6 post-vaccination (N=5 per group). Symbols indicate median and IQR. (D) Correlation between number of Tet+ TFH or (E) total Tet + cells with stem-specific GC B cells. Lymph nodes with no detectible stem-specific B GC B cells were arbitrarily assigned a value of 0.5 (marked with a dashed line) for graphical display. Statistics assessed by Spearman correlation. In contrast, we observed a dose-dependent decrease in GP 61 -specific TFH differentiation ( Fig 6B ) that paralleled a dose-dependent decline of stem-specific GC B cells ( Fig 6C ). Accordingly, the number of stem+ GC B cells correlated with the number of ag-specific TFH but not total numbers of ag-specific CD4 T cells ( Fig 6D-E ), reinforcing that when T cell priming and TFH differentiation are decoupled, GC formation is controlled by TFH availability. Collectively, these data suggest that vaccine dosing sufficient to establish efficient B cell presentation of pMHC to pre-TFH cells is markedly higher than doses required for DC-priming of naïve T cells, and that differences in B cell presentation of HA 91 and GP 61 peptides may underpin the differential immunogenicity of stem-HA 91 and stem-GP 61 antigens. Discussion Engineering vaccine immunogens to reduce inclusion of B cell epitopes with weak or narrow protective capability is an attractive approach for maximizing durable protection against challenging pathogens such as influenza, coronaviruses, HIV, and malaria. While such strategies facilitate highly tailored B cell engagement, increasing evidence suggests that a concomitant loss of CD4 T cell epitopes can hinder overall vaccine immunogenicity in vivo . Here, we assessed how CD4 T cell help supports productive GC reactions toward multimerized or soluble protein antigens, and found that T cell epitope specificity, rather than immunodominance, was a determining factor in TFH differentiation and vaccine immunogenicity. Intrinsic protein immunogenicity is likely determined by the complex interplay of multiple factors including B cell precursor frequencies, antigen size, glycosylation, and the ability to elicit an effective CD4 T helper cell response. In a polyclonal system with soluble viral antigens, gross immunogenicity is likely to reflect the totality of available CD4 T cell help, with large antigens generally containing an increased number of CD4 epitopes than small antigens. This is consistent with the observed immunogenicity gradient of stem-ferritin nanoparticles across SMARTA, BL/6 and BALB/c mice; the broad ferritin-specific CD4 T cell pool in BALB/c mice was associated with 10-fold higher stem antibody titres compared to BL/6 animals. This appears to be generally consistent in genetically diverse human cohorts as well: large glycoproteins such as SARS-CoV-2 spike contain sufficient epitopes restricted by a broad array of HLA alleles to support immunogenicity in diverse populations. Small antigens like HA stem, SARS-CoV-2 RBD, HIV env or Plasmodium CSP, however, contain reduced numbers of CD4 cell epitopes that begin to restrict the quantity of help available in certain individuals or populations 15 - 18 . At this point, vaccine immunogens are at higher risk of failure, particularly for pathogens with high antigenic diversity 16 . Considering these factors, it becomes essential to understand the minimal requirements for CD4 T cell help to support a robust GC reaction. Our data identify distinct scenarios under which vaccination can succeed or fail to generate a serological response. In the context of multimeric arrayed antigens, highly subdominant T cell help becomes sufficient to drive a vaccine-specific serological response. Despite using multiple assays, the frequency of stem- or ferritin-specific CD4 T cells elicited by primary vaccination of BL6 mice appears to be below our limit of detection. Nonetheless, the lack of stem-ferritin immunogenicity in SMARTA transgenic mice and ferritin-specific proliferative responses evident upon repeated boosting clearly support the contribution of low frequency or low affinity antigen-specific T cells to vaccine immunogenicity. Mechanistically, the augmented BCR crosslinking and NF-kB signaling induced by nanoparticle vaccines 20 may potentially reduce the magnitude and/or quality of CD4 T cell help required to initiate a stem-specific GC B cell response. In the context of soluble non-arrayed antigens, we find that a single CD4 epitope is sufficient to support an antigen-specific GC reaction, but perhaps more importantly, find that only some epitopes drive CD4 T cell responses capable of providing suitable B cell help. The potent immunogenicity of the stem-GP 61 protein clearly demonstrates how even a comparatively restricted CD4 T cell repertoire can support robust GC activity, as the naive repertoire of GP 61 -specific T cells is heavily biased toward TRAV14/TRAV14D and TRBV13/TRBV31 gene usage 28 . Our data therefore suggest that a highly diverse TFH pool may not be required for vaccine efficacy; rather, the rational selection of a small number of high-quality class II epitopes could be sufficient to support robust humoral immunity. Collectively, our data support the development of GC TFH at an epitope-specific level by two distinct antigen presentation events: naïve T cell priming by DCs which drives immunodominance but not TFH selection, and subsequent T cell-B cell interactions that down select T cell specificities to populate the GC TFH pool. During the first 5 days post-vaccination, the kinetics of T cell expansion were comparable between HA 91 and GP 61 epitopes, likely reflecting naïve T cell precursor frequencies which are known to be high for GP 61 compared to other epitopes such as OTII 28 . Interestingly, early DC priming was markedly efficient, as all doses of stem-GP61 antigen tested (0.25ug to 5ug) were saturating for T cell effector frequency, even when they compromised TFH numbers. It is currently unclear whether this is driven by differential antigen processing and presentation in B cells versus DCs, more efficient acquisition by DCs due to immunogen draining patterns in the LN, or a selective threshold imposed by low-frequency CD4 T cell/B cell interactions at the T:B border. It is well known that the major selective event for TFH differentiation and GC residence is recognition of peptide:MHC complexes presented by B cells. However, our data suggest that even in a polyclonal context, there are stark differences in the capacity of epitope-specific T cell populations to provide B cell help. Mechanistically, this appears to be governed by a low propensity of some polyclonal populations (like HA 91 -reactive T cells) to undergo differentiation into CD90lo GC-resident TFH. Our panel of stem-peptide immunogens identified some ‘immunocapable’ epitopes (GP 61 and HA 523 ), which support GC responses using the endogenous T cell and B cell repertoire in BL/6 mice. Other epitopes, such as HA 91 , required manipulation of B:T interactions (e.g. expansion of the peptide-presenting B cell pool) to effectively drive GC. Possible mechanisms underpinning these observations include peptide-intrinsic differences in presentation/processing by B cells (but not DCs) or epitope-specific differences in TCR/MHCII avidity at an aggregate, polyclonal level. The linear relationship between dose of stem-GP 61 and GP 61 TFH differentiation suggest the absolute densities of IAb/GP 61 on the surface of stem-specific B cells dictates a propensity to recruit GP 61 T cells into the GC, potentially favouring a deterministic role for B cell peptide presentation in controlling TFH composition. Additional study is required to extend these observations into human cohorts. Despite the incredible diversity provided by HLA polymorphism and TCR repertoires, mounting evidence suggests that the human TFH repertoire may also be relatively restricted, with individual epitopes or class II alleles exerting substantial impacts on the outcome of vaccination 22 , 29 . Antigens such as HIV env, Plasmodium CSP and SARS-CoV-2 RBD frequently contain only 1-2 epitopes that are recognized on an individual level 15 - 18 . The qualitative differences between HA 91 and GP 61 highlight the potential pitfalls of relying on few, endogenous epitopes for consistent vaccine immunogenicity. Further work is required to clarify whether multiple, subdominant epitopes provide additive or synergistic support for GC B cell development. These data also underscore the need to accurately predict the quality of B cell help, rather than just immunogenicity or immunodominance, of any given epitope and responding T cell population to aid vaccine design efforts. Methods Mouse immunizations and infection C57BL/6, BALB/c, and SMARTA-transgenic (C57BL/6 background) mice were bred in-house under specific pathogen-free conditions in the animal facility at the Peter Doherty Institute of Infection and Immunity, University of Melbourne, Australia. Mouse studies were carried out in accordance with the University of Melbourne Animal Ethics Committee (no. 22954). All mice were female and aged 6 to 12 weeks at the time experiments commenced. A total of 5μg (unless otherwise indicated) of protein, peptide, or nanoparticle were formulated in phosphate buffered saline (PBS) at a 1:1 ratio with Addavax (InvivoGen, cat#INV-vac-adx-10) adjuvant in a total volume of 100μL. Mice were anesthetized by isoflurane inhalation with oxygen flow at 2L/min and isoflurane vaporizer set to 3 (Stinger Anesthetic Machine), prior to 50μl intramuscular injections at the left and right quadriceps using a 29G needle. For influenza infections, mice were anesthetized as above and intranasally infected in a volume of 50μl with a sublethal dose of 50 TCID 50 of A/Puerto Rico/8/34 (PR8). At experimental endpoints, mice were killed using CO 2 asphyxiation using a delivery of 50% chamber volume per minute. Protein expression Stem and Stem-Ferritin immunogens were prepared in-house, as previously described 9 , 30 . Briefly, stabilized HA stem proteins were engineered for A/Puerto Rico/08/1934 using methods established previously for the design of Gen6 HA stem in Yassine et al. 24 . Stem-Ferritin nanoparticles were expressed by transient transfection of Expi293F (Life Technologies, Thermo Fisher Scientific) suspension cultures and purified using ion exchange chromatography with HiTrap Q HP column (GE Healthcare) and exclusion chromatography. For stem proteins conjugated to CD4 T cell epitopes, expression constructs substituting the original Avitag for peptide sequences for OTII (ISQAVHAAHAEINEAG), HA 91 (RSWSYIVETPNSENGIC), HA 115 (YEELREQLSSVSSFERF), HA 301 (AINSSLPYQNIHPVTIG), HA 523 (SMGIYQILAIYSTVASS) and GP 61 (GLKGPDIYKGVYQFKSVEFD) were synthesized (GeneArt), cloned into mammalian expression vectors and expressed via transient transfection of Expi293 suspension cultures (Life Technologies, Thermo Fisher Scientific). Proteins were purified by polyhistadine-tag affinity chromatography and gel filtration. Enzyme-linked immunosorbent assay (ELISA) Blood samples for serum isolation were collected either by submandibular bleed or terminal cardiac puncture bleed using a 26G needle. 96-well MaxiSorp plates (ThermoFisher, cat# 3442404) were coated with 2μg/mL recombinant stem protein overnight at 4°C. Plates were washed with 0.05% (v/v) Tween20 (Sigma, cat# P1379) + PBS and blocked with 1% (v/v) FCS in PBS for 1 hr, room temperature, before incubation with serial dilutions of sera for 2 hr. Plates were washed and horseradish peroxidase-conjugated anti-mouse IgG (1:15 000; Seracare, cat# 5450-0011) was added for 1 hr. After washing, plates were developed with 3,3′,5,5′-Tetramethylbenzidine (TMB; ThermoFisher, cat#SB02), stopped with 0.16M sulfuric acid (Sigma, cat# 84727) and the absorbance measured at 450nm on FLUOstar Omega microplate reader (BMG Labtech). Curves were fitted (four-parameter log regression) and end-point titrations calculated as the reciprocal serum dilution yielding 2x background using GraphPad Prism version 10. Generation of B cell probes and peptide MHC II tetramers Recombinant biotinylated stem protein biotinylated using BirA (Avidity) and stored at –80°C. Conjugation was performed by sequential addition of streptavidin-PE or -APC (Life Technologies, cat#S866 and S868) or streptavidin-BV711 (BD, cat# 563262). Mouse H2-IAb RSWSYIVETPNSENGI PE-conjugated tetramer (IAb/HA 91 ) was generated by ProImmune. Biotinylated mouse H2-IAb DIYKGVYQFKSV monomer (IAb/GP 61 ; ProImmune) was tetramerized by sequential addition of streptavidin-PE (Life Technologies, cat# S866). Detection of antigen-specific B and T cells ex vivo Iliac and inguinal vaccine-draining lymph nodes were passed through a 70μm filter, centrifuged (500 g , 7 min) and washed with PBS prior to viability staining with live/dead red for 3 min (Invitrogen, cat# L34972). Cells were Fc blocked with anti-CD16/32 antibody (BioLegend, cat# 101302) for 10 min and stained with surface antibodies of interest for 30 min at 4°C. Cells were then washed and fixed with 1% (v/v) formaldehyde (BD, cat# 554655) before acquisition on a BD Symphony or Fortessa flow cytometer. For detection of antigen-specific B cells, PE, APC or BV711 conjugated stem probes were included in the surface staining antibody cocktail. For detection of antigen-specific T cells, single-cell suspensions were pre-incubated at 37°C with 50nM dasatinib (Sigma, cat# SML2589) in 2% (v/v) FCS in PBS containing 1μg/mL anti-TCRý (BD, cat# 553167). After 30 min, 4μg/mL of IAb/HA 91 or IAb/GP 61 tetramers were added for a further 3 hr, before proceeding with viability and surface staining. Data were analyzed using FlowJo (v10.10, BD Biosciences). Data collection and analysis were not performed blind to the conditions of the experiments. Activation Induced Marker assays Individual HA, stem (BEI Resources) or ferritin (GenScript) peptides (15-mer with 11 amino acid overlap) were pooled and used to detect antigen-specific CD4+ T cell responses in vitro. LN single cell suspensions were cultured in 96-well round-bottom plates in RPMI-1640 (Thermo Fisher Scientific) with 10% (v/v) FCS and 2% (v/v) penicillin-streptomycin (RF10) media containing 20mM A438079 (Santa Cruz, cat# 203788), anti-mouse CD154 antibody and peptide pool of interest (2mg/mL/peptide) or an equivalent volume of DMSO (Sigma, cat# D1435). Following 18hr of stimulation, cells were washed in PBS, stained for viability (3 minutes at room temperature) and stained with surface antibodies of interest. T cell proliferation assay To generate single-cell suspensions, spleens were passed through a 70μm filter and centrifuged (500xg, 7 min). Red blood cells were lysed using 1x BD PharmLyse (Cat #555899) for 3 min, and the reaction quenched with 1x PBS. Splenic single-cell suspensions were labelled with 2.5mM of CellTrace Violet (CTV; Invitrogen, cat#C34557) for 10 min and washed twice (500xg, 7 min) with RF10. 4×10 5 CTV-labelled cells were cultured in 96-well round-bottom plates in RF10 with 0.5mg/mL HA, stem or ferritin peptide pools, or anti-CD3/CD28 Dynabeads (Gibco, cat # 11456D) for 4 days at 37°C, prior to staining for flow cytometric analysis. Immunofluorescent microscopy Fresh tissues were snap-frozen in Tissue-Tek O.C.T. compound (Sakura Finetek USA) and stored at -80°C. 7 um sections were cut using the Leica CM3050S cryostat (Leica Biosystems). Prior to staining, sectioned tissues were fixed in cold acetone solution (Sigma) for 10mins, rehydrated with PBS for 10mins, and then blocked in 5% (w/v) bovine serum albumin (Sigma) and 2% (v/v) normal goat serum (Jackson ImmunoResearch). A cocktail of antibodies including GL7 AF488 (clone GL7, BioLegend) and B220 BV421 (clone RA3-6B2, BioLegend) were added for 1 hr at RT. Slides were mounted with ProLong Diamond Antifade Mountant (Life Technologies). Tiled z-stack images at 20x magnification and 1 airy unit were acquired on a LSM780 microscope (ZEISS) and analyzed with Fiji software 31 . Statistical analysis Data are presented as median ± interquartile range. All statistical analysis was performed in Prism 10 (GraphPad) using nonparametric statistical tests as indicated (making no assumptions about data normality). P<0.05 was considered statistically significant. Competing interests The authors declare no competing interests. Supplementary Figures Download figure Open in new tab Supplementary Figure 1. Gating strategy for GC B cell and TFH populations. (A) Lymphocyte populations are gated through a Dump (F4/80 and live/dead) vs Time plot, then identified by forward and side scatter, with exclusion of doublets (FSC-A vs FSC-H) and selection of CD45+ cells. Antibody secreting cells are defined as CD3 -CD4-cells that co-express CD138 and CD98. Class-switched B cells are defined as non-ASCs that are B220+ and IgD-. Germinal centre B cells are gated as GL7+CD38lo, and antigen specificity determined by staining with recombinant protein tetramers. (B) CD4+ T cells are identified as B220- and CD3+CD4+. Antigen-experienced cells are gated as CD44hi, and TFH are defined as CXCR5+PD-1+ cells. Download figure Open in new tab Supplementary Figure 2. Germinal centre morphology following stem-Fe vaccination. (A) Confocal imaging of vaccine draining lymph nodes showing localisation of B220 + B cells (yellow), GL7 + GC B cells (green), and CD35+ follicular dendritic cells (magenta). (B) Identification of light zone (CXCR4 lo CD86 hi ) and dark zone (CXCR4 hi CD86 lo ) GC B cells in stem-Fe (top) or HA-FL (bottom) vaccinated animals. Data are representative of 5 individual animals in each group. Download figure Open in new tab Supplementary Figure 3. Identification of immunogenic peptides in PR8 HA. (A) Representative staining of CD154 upregulation on CXCR5 + PD-1 + CD4 + T cells in response to in vitro stimulation with peptide pools covering the PR8 HA protein. (B) Frequencies of peptide-specific or ConA-responsive cells among CXCR5 + (left) or total (right) CD4 T cells following PR8 infection. (C) Frequencies of peptide-specific bulk CD4 + T cell responses in the mediastinal LN at day 14 post-PR8 infection. Each dot represents a single mouse (n=4-5 per group). (D) Peptide- or pool-specific TFH responses in draining LN following intramuscular vaccination with soluble PR8 HA antigen. Download figure Open in new tab Supplementary Figure 4. Gating strategy for longitudinal tracking of antigen-specific CD4 + T cells. (A) Representative staining of IA b /HA 91 and IA b /GP 61 tetramers in draining lymph nodes at day 14 following HA-FL vaccination, PR8 infection or LCMV GP vaccination. (B) Antigen-specific cells were defined as Live, F4/80-CD45+B220-CD3+CD4+CD44hi tetramer+ lymphocytes. (C) Expression of CD62L on Tet + cells from days 4-14 post-vaccination. Graph indicates median and IQR, N=5 per group. Acknowledgements This work was funded by an NHMRC Ideas grant to JAJ. HXT, AKW and JAJ are supported by NHMRC Investigator Grants, and JAJ is supported by the Sylvia and Charles Viertel Senior Medical Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Funder Information Declared National Health and Medical Research Council, https://ror.org/011kf5r70 Sylvia and Charles Viertel Charitable Foundation, https://ror.org/05539km59 Footnotes Small corrections to references, missing graph in supplementary figure References 1. ↵ Wrapp D , Wang N , Corbett KS , et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation . Science 2020 ; 367 ( 6483 ): 1260 – 3 . OpenUrl Abstract / FREE Full Text 2. ↵ Corbett KS , Edwards DK , Leist SR , et al. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness . Nature 2020 ; 586 ( 7830 ): 567 – 71 . 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Fiji: an open-source platform for biological image analysis . Nat Methods 2012 ; 9 ( 7 ): 676 - 82 . OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted July 23, 2025. Download PDF 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 Immunodominance is a poor predictor of vaccine-induced T follicular helper cell quality 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 Immunodominance is a poor predictor of vaccine-induced T follicular helper cell quality HX Tan , MZM Zheng , K Wragg , L Murdiyarso , D Pilapitiya , A Kelly , R Esterbauer , C Gonelli , AK Wheatley , JA Juno bioRxiv 2025.07.21.666029; doi: https://doi.org/10.1101/2025.07.21.666029 Share This Article: Copy Citation Tools Immunodominance is a poor predictor of vaccine-induced T follicular helper cell quality HX Tan , MZM Zheng , K Wragg , L Murdiyarso , D Pilapitiya , A Kelly , R Esterbauer , C Gonelli , AK Wheatley , JA Juno bioRxiv 2025.07.21.666029; doi: https://doi.org/10.1101/2025.07.21.666029 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 (7640) Biochemistry (17706) Bioengineering (13902) Bioinformatics (41978) Biophysics (21465) Cancer Biology (18611) Cell Biology (25528) Clinical Trials (138) Developmental Biology (13387) Ecology (19920) Epidemiology (2067) Evolutionary Biology (24332) Genetics (15615) Genomics (22519) Immunology (17747) Microbiology (40424) Molecular Biology (17194) Neuroscience (88662) Paleontology (667) Pathology (2838) Pharmacology and Toxicology (4827) Physiology (7650) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9826) Zoology (2271)