Molecular features of the serological IgG repertoire elicited by
egg-based, cell-based, or recombinant haemagglutinin-based seasonal influenza
vaccines: a comparative, prospective, observational cohort study
Seasonal influenza vaccination has been shown to attenuate the severity of symptomatic illness; however, the overall vaccine effectiveness of licensed influenza vaccines remains suboptimal, with only 35·7% average vaccine effectiveness in the past 10 years in the USA, according to the US Centers for Disease Control and Prevention. Among the influenza A viruses, the A/H3N2 strain is of particular importance due to its higher rate of antigenic drift than the A/H1N1 strain and associated lower vaccine effectiveness. Since the first report on growing influenza virus in embryonated chicken eggs in the 1930s, egg-produced viruses have been used to produce influenza vaccines. Drawbacks of egg-based inactivated quadrivalent seasonal influenza vaccine (eIIV4) include allergic responses to egg products and, separately, the need to introduce mutations around the haemagglutinin (HA) receptor binding site needed for propagation in chicken eggs and binding to avian receptors, namely α-2,3 linked sialic acids, as opposed to α-2,6 linked sialic acids in humans. Egg adaptation mutations in the eIIV4 vaccine can elicit antibodies that are unable to bind human influenza strains. , More recently, we and others have shown that eIIV4 immunisation directs part of the antibody response towards avian antigens, such as sulfated type Galβ1–4GalNAcβ avian glycans, which are prevalent in egg allantoic fluid. , To overcome these limitations, two alternate vaccine production platforms have been developed: inactivated subunit vaccines from virus grown in Madin–Darby Canine Kidney cells (cell culture-based inactivated quadrivalent seasonal influenza vaccine [ccIIV4]) and recombinant HA vaccines produced in insect cells (recombinant HA-based quadrivalent seasonal influenza vaccine [RIV4]), with ccIIV4 approved by the US Food and Drug Administration in November, 2012, and RIV4 in January, 2013. Some advantages of mammalian and insect cell tissue culture systems are their shorter vaccine production timelines and the lack of egg-adapted mutations and egg antigens. Similar to eIIV4, ccIIV4 vaccines use a standard dose of 15 μg HA per strain, whereas RIV4 vaccines are exclusively formulated with three times higher dose of HA (45 μg) per strain. Although eIIV4, ccIIV4, and RIV4 have been in use for over 10 years, detailed comparative studies of vaccine effectiveness and serological responses are scarce. Izurieta and colleagues , reported that ccIIV4 showed only minor improvement in relative vaccine effectiveness compared with eIIV4, and other studies found a similar titre of neutralising serum antibodies by ccIIV4 and eIIV4 immunisation against all four vaccine strains. , Recently, Dawood and colleagues reported that RIV4 elicited higher neutralising antibody titres to A/H1N1, A/H3N2, and B/Yamagata strains than did standard-dose eIIV4 and, similarly, Wang and colleagues and Gouma and colleagues reported that RIV4 elicits broader H3N2 neutralisation breadth than either eIIV4 or ccIIV4. Importantly, a large clinical study of vaccine efficacy of more than 8000 adults aged 50 years or older (which provided key evidence used for licensure) revealed 30% higher protection against RT-PCR-confirmed influenza infections for RIV4 than for standard-dose eIIV4 during the H3N2-predominant 2014–15 season. Although it could be argued that the higher vaccine efficacy reported with RIV4 might be a consequence of the three times higher concentration of HA in this vaccine formulation (45 μg per strain) than in the standard-dose eIIV4 and ccIIV4 (15 μg per strain), this is not likely to be the case in light of the fact that with eIIV4, the Fluzone High-Dose Quadrivalent vaccine approved for the elderly (aged >65 years) has a four times higher HA dose (60 μg per strain), yet it does not result in higher neutralising antibody titres. To better understand how different vaccine platforms affect antibody clonal compositions and their respective quantities and qualities in the serum response, in this study we aimed to comprehensively profile the sequence identity, abundance, and binding affinity of H3/HA-specific circulating antibodies that comprise the polyclonal IgG serological repertoire in three vaccine cohorts who received either RIV4, eIIV4, or ccIIV4 during the 2018–19 influenza season.   Study design and participants This comparative, prospective, observational cohort study is a preplanned exploratory analysis of the original randomised, open-label trial ( NCT03722589 ) involving 727 US health-care workers. We selected 15 female (mean age 47·6 years [SD 8]) trial participants who received either RIV4 (Flublok Quadrivalent by Sanofi Pasteur, Swiftwater, PA, USA; 45 μg of HA per strain), eIIV4 (Fluzone Quadrivalent by Sanofi Pasteur; 15 μg of HA per strain), or ccIIV4 (Flucelvax Quadrivalent by Seqirus, Holly Springs, NC, USA; 15 μg of HA per strain; n=5 per cohort) during the 2018–19 influenza season at Baylor Scott & White Health, Temple, TX, USA. Participants were excluded if they had experienced any previous hypersensitivity to influenza vaccines or received any vaccination within 4 weeks before and after the initial visit. Eligible individuals were selected based on comparable day 28 serum microneutralisation titres and similar vaccination history. This strategy for selecting individuals allows for a direct comparison of molecular features in anti-H3/HA serum repertoires induced by different vaccine platforms while mitigating confounding effects that could arise from comparing individuals with large variations in post-vaccination titres. Another consideration in the inclusion of individuals was the availability of sufficient amounts of sera and peripheral blood mononuclear cells, as required for B-cell receptor sequencing (BCR-Seq) and immunoglobulin sequencing (Ig-Seq). 15 was the maximum number of individuals we could analyse, given sample availability and the cost of immunoglobulin sequencing experiments. Participants provided written informed consent before enrolment and trial participation. Baseline characteristics for all individuals were collected from electronic medical records. Investigators were blinded to vaccine groups until the completion of the study. Participants had serum (day 0 and day 28) and peripheral blood mononuclear cells (day 0 and day 7) collected before and after vaccination ( p 25). Procedures Serum microneutralisation assays were performed using cell-grown A/Singapore/INFIMH-16–0019/2016 viruses propagated in MDCK-SIAT1 cells (MilliporeSigma, Burlington, MA, USA; p 3). The microneutralisation titres were measured and reported by Dawood and colleagues as primary endpoints, which in turn guided donor selection in our preplanned exploratory analysis. Serum IgG binding titres were determined using ELISA to recombinant A/Singapore/INFIMH-16–0019/2016 HA ( p 3). Circulating T-follicular helper cells (CD4 + CXCR5 + PD1 + CD25 − ) were identified by multiparametric fluorescence-activated cell sorting using fluorescent-labelled antibodies ( p 3). We used the serum proteomics workflow, Ig-Seq, which capitalises on liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based serum proteomics combined with subject-specific, natively paired sequencing of variable heavy chains (VH)–variable light chains (VL) in peripheral B cells that provides a database for mass spectra interpretation and full-length antibody sequences, which can in turn be recombinantly produced for biochemical and functional characterisation . The VH-only or VH–VL paired high-throughput BCR-Seq was performed using bulk and single-cell day 7 circulating B-cell sequencing, respectively, as previously described ( pp 4–6). HA-binding antibodies were isolated from IgG plasma by affinity chromatography with immobilised A/Singapore/INFIMH-16–0019/2016 HA and analysed by LC-MS/MS, as described previously ( pp 6–7). , , The mass spectrometry search identified peptide spectra matches originating from heavy-chain complementarity determining region 3 (CDRH3) sequences, and the abundance of each clonotype was calculated by summing the extracted ion chromatogram (XIC) peak area of CDRH3 peptides mapped to a given clonotype ( pp 7–9). We selected monoclonal antibodies for which high-confidence CDRH3 peptides in serum were identified by LC-MS/MS at high abundance (as determined by XIC area), along with high peptide coverage of the VH, especially for the complementarity determining regions ( pp 26–27). The binding affinity of recombinant monoclonal antibodies was determined by ELISA against A/Texas/50/2012 and A/Singapore/INFIMH-16–0019/2016 HA ( pp 9–10). The high-throughput multiplex influenza antibody detection assay was conducted using multiplexed microsphere beads containing a broad panel of H3/HAs and nucleoprotein ( pp 4, 28). The binding kinetics of UT14 and its competition with known monoclonal antibodies were determined using biolayer interferometry ( pp 10–11). Cryo-electron microscopy structure of UT14 Fab in complex with A/Singapore/INFIMH-16–0019/2016 HA was determined using FEI Titan Krios G3 300kV cryo-EM (Thermo Fisher Scientific, Waltham, MA, USA) with a K3 direct detection camera ( pp 11, 31). Full details on sources and identifiers of reagents used in this study are in the (pp 3–11). Outcomes The primary exploratory outcome of this study was to compare the molecular composition of the HA-specific IgG antibody repertoire after vaccination by RIV4, eIIV4, or ccIIV4. As key secondary outcomes, the level of back-boosting, molecular features of serum clonotypes, binding affinity of representative monoclonal antibodies, HA serum-binding landscape against time-ordered H3 HA variants, correlation of antibody repertoire features with circulating T-follicular helper cell frequencies, and stereotypical B-cell receptor responses were evaluated. An additional exploratory outcome involved analysing the biochemical and structural features of an unusual near-stereotypical monoclonal antibody, which was detected at a high abundance in serum. Statistical analysis For multiple comparisons, ordinary one-way ANOVA tests followed by Tukey’s post-hoc tests, Welch’s ANOVA tests followed by Dunnett’s T3 post-hoc tests, or Kruskal–Wallis tests followed by Dunn’s post-hoc tests were used based on the assessment of normality and homogeneity of variance assumptions ( p 11). Unpaired or paired comparisons between two groups were conducted using the two-sided Mann–Whitney U or Wilcoxon matched-pairs signed rank tests, respectively. The Pearson correlation tests were conducted using Scipy python package version 1.9.1. The Tukey-style box-and-whisker plot was drawn using default geom_boxplot function by ggplot2 version 3.4.2. All raw data points are shown in the box-and-whisker or violin plot. Data are presented as median with 95% CI estimates or mean (SD). Statistical analyses were conducted using GraphPad Prism version 10.2.1 using a threshold for significance of p<0·05. Role of the funding source The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.   This comparative, prospective, observational cohort study is a preplanned exploratory analysis of the original randomised, open-label trial ( NCT03722589 ) involving 727 US health-care workers. We selected 15 female (mean age 47·6 years [SD 8]) trial participants who received either RIV4 (Flublok Quadrivalent by Sanofi Pasteur, Swiftwater, PA, USA; 45 μg of HA per strain), eIIV4 (Fluzone Quadrivalent by Sanofi Pasteur; 15 μg of HA per strain), or ccIIV4 (Flucelvax Quadrivalent by Seqirus, Holly Springs, NC, USA; 15 μg of HA per strain; n=5 per cohort) during the 2018–19 influenza season at Baylor Scott & White Health, Temple, TX, USA. Participants were excluded if they had experienced any previous hypersensitivity to influenza vaccines or received any vaccination within 4 weeks before and after the initial visit. Eligible individuals were selected based on comparable day 28 serum microneutralisation titres and similar vaccination history. This strategy for selecting individuals allows for a direct comparison of molecular features in anti-H3/HA serum repertoires induced by different vaccine platforms while mitigating confounding effects that could arise from comparing individuals with large variations in post-vaccination titres. Another consideration in the inclusion of individuals was the availability of sufficient amounts of sera and peripheral blood mononuclear cells, as required for B-cell receptor sequencing (BCR-Seq) and immunoglobulin sequencing (Ig-Seq). 15 was the maximum number of individuals we could analyse, given sample availability and the cost of immunoglobulin sequencing experiments. Participants provided written informed consent before enrolment and trial participation. Baseline characteristics for all individuals were collected from electronic medical records. Investigators were blinded to vaccine groups until the completion of the study. Participants had serum (day 0 and day 28) and peripheral blood mononuclear cells (day 0 and day 7) collected before and after vaccination ( p 25).   Serum microneutralisation assays were performed using cell-grown A/Singapore/INFIMH-16–0019/2016 viruses propagated in MDCK-SIAT1 cells (MilliporeSigma, Burlington, MA, USA; p 3). The microneutralisation titres were measured and reported by Dawood and colleagues as primary endpoints, which in turn guided donor selection in our preplanned exploratory analysis. Serum IgG binding titres were determined using ELISA to recombinant A/Singapore/INFIMH-16–0019/2016 HA ( p 3). Circulating T-follicular helper cells (CD4 + CXCR5 + PD1 + CD25 − ) were identified by multiparametric fluorescence-activated cell sorting using fluorescent-labelled antibodies ( p 3). We used the serum proteomics workflow, Ig-Seq, which capitalises on liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based serum proteomics combined with subject-specific, natively paired sequencing of variable heavy chains (VH)–variable light chains (VL) in peripheral B cells that provides a database for mass spectra interpretation and full-length antibody sequences, which can in turn be recombinantly produced for biochemical and functional characterisation . The VH-only or VH–VL paired high-throughput BCR-Seq was performed using bulk and single-cell day 7 circulating B-cell sequencing, respectively, as previously described ( pp 4–6). HA-binding antibodies were isolated from IgG plasma by affinity chromatography with immobilised A/Singapore/INFIMH-16–0019/2016 HA and analysed by LC-MS/MS, as described previously ( pp 6–7). , , The mass spectrometry search identified peptide spectra matches originating from heavy-chain complementarity determining region 3 (CDRH3) sequences, and the abundance of each clonotype was calculated by summing the extracted ion chromatogram (XIC) peak area of CDRH3 peptides mapped to a given clonotype ( pp 7–9). We selected monoclonal antibodies for which high-confidence CDRH3 peptides in serum were identified by LC-MS/MS at high abundance (as determined by XIC area), along with high peptide coverage of the VH, especially for the complementarity determining regions ( pp 26–27). The binding affinity of recombinant monoclonal antibodies was determined by ELISA against A/Texas/50/2012 and A/Singapore/INFIMH-16–0019/2016 HA ( pp 9–10). The high-throughput multiplex influenza antibody detection assay was conducted using multiplexed microsphere beads containing a broad panel of H3/HAs and nucleoprotein ( pp 4, 28). The binding kinetics of UT14 and its competition with known monoclonal antibodies were determined using biolayer interferometry ( pp 10–11). Cryo-electron microscopy structure of UT14 Fab in complex with A/Singapore/INFIMH-16–0019/2016 HA was determined using FEI Titan Krios G3 300kV cryo-EM (Thermo Fisher Scientific, Waltham, MA, USA) with a K3 direct detection camera ( pp 11, 31). Full details on sources and identifiers of reagents used in this study are in the (pp 3–11).   The primary exploratory outcome of this study was to compare the molecular composition of the HA-specific IgG antibody repertoire after vaccination by RIV4, eIIV4, or ccIIV4. As key secondary outcomes, the level of back-boosting, molecular features of serum clonotypes, binding affinity of representative monoclonal antibodies, HA serum-binding landscape against time-ordered H3 HA variants, correlation of antibody repertoire features with circulating T-follicular helper cell frequencies, and stereotypical B-cell receptor responses were evaluated. An additional exploratory outcome involved analysing the biochemical and structural features of an unusual near-stereotypical monoclonal antibody, which was detected at a high abundance in serum.   For multiple comparisons, ordinary one-way ANOVA tests followed by Tukey’s post-hoc tests, Welch’s ANOVA tests followed by Dunnett’s T3 post-hoc tests, or Kruskal–Wallis tests followed by Dunn’s post-hoc tests were used based on the assessment of normality and homogeneity of variance assumptions ( p 11). Unpaired or paired comparisons between two groups were conducted using the two-sided Mann–Whitney U or Wilcoxon matched-pairs signed rank tests, respectively. The Pearson correlation tests were conducted using Scipy python package version 1.9.1. The Tukey-style box-and-whisker plot was drawn using default geom_boxplot function by ggplot2 version 3.4.2. All raw data points are shown in the box-and-whisker or violin plot. Data are presented as median with 95% CI estimates or mean (SD). Statistical analyses were conducted using GraphPad Prism version 10.2.1 using a threshold for significance of p<0·05.   The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.   We selected 15 female health-care personnel (n=5 per vaccine cohort) who were enrolled in a large clinical trial for cohorts RIV4 (participants A1–5; mean age 47·8 years [SD 4·7]), eIIV4 (participants B1–5; 46·8 years [9·5]), and ccIIV4 (participants C1–5; 48·2 years [11·3]). Participants received a single dose of RIV4, standard-dose eIIV4, or ccIIV4 during the 2018 September-to-October period of the 2018–19 influenza season . We chose our study cohorts to have statistically similar serum micro-neutralisation and ELISA binding titres to A/Singapore/INFIMH-16–0019/2016 (H3N2) virus on day 28 after vaccination . Serum IgG ELISA binding titres significantly correlated with the serum microneutralisation titres to the vaccine H3 strain (p=0·0001, Pearson r =0·64 [95% CI 0·21-0·86]) . Ig-Seq serum proteomics analysis of A/Singapore/INFIMH-16–0019/2016 H3/HA affinity-purified IgGs showed that all three vaccines elicited a highly polarised serological repertoire, dominated by back-boosted antibodies that were also detectable at day 0 (median percentage preexisting: 98% [95% CI 23–100] for RIV4, 98% [89–99] for eIIV4, 92% [23–100] for ccIIV4; p=1·0 for all multiple comparisons; , pp 12–13). The serological repertoires comprised a few highly abundant clonotypes, with the top three most abundant clonotypes accounting for a median 58% (95% CI 46–69) of the post-vaccination repertoire by abundance. The back-boosted (pre-existing) antibodies constituted a median 98% (95% CI 88–99) of the anti-H3/HA serum response, with no significant differences observed among the three vaccine cohorts . Interestingly, two individuals (participant identifiers A5 in the RIV4 cohort and C4 in the ccIIV4 cohort) had an unexpectedly 3·8-times lower fraction of pre-existing antibodies at day 28 (28% and 23% by abundance, respectively) compared with the other 13 individuals who had a median 98% (95% CI 89–99) of back-boosted antibodies in their sera. We noticed that these two individuals, compared with the rest of the cohort, had a significantly higher increase in serum nucleoprotein (NP) titre by a median 3·2 times (95% CI 3·0–3·4) on day 28 comparedwith day 0, whereas the other individuals did not have appreciable changes in NP titre between the two timepoints (differences in NP titre change, p=0·02; ; p 14). This finding suggests that the two donors with atypical fractions of pre-existing antibodies and NP titres might have had subdinical influenza infection around the time of vaccination, given that they received recombinant HA-based and inactivated subunit vaccines. We analysed the molecular features of the serological IgG repertoire associated with different vaccine platforms, specifically among the 13 donors who showed no sign of infection. For these individuals, the clonal composition and the extent of repertoire polarisation on day 28 versus day 0 were not influenced by the type of vaccination received, as measured by D80 diversity index (p>0·05; ). Furthermore, the IgG antibodies comprising the anti-H3/HA serum response had the same molecular features in terms of VH somatic hypermutation, CDRH3 hydrophobicity, and the CDRH3 amino acid length across all three vaccine cohorts (p>0·05; – ). In the case of the typical donors, pre-existing antibodies had a higher level of VH somatic hypermutation compared with newly elicited antibodies (p=0·030; p 15) for all vaccine cohorts. Given the indistinguishable features of the anti-H3/HA serum repertoires, we examined whether molecular characteristics of the day 28 repertoire correlate with immunological parameters regardless of the vaccine received. Across all 15 individuals, serum antibodies encoded by IGHV4–59, IGHV3–30, IGHV1–69, IGHV4–31, and IGHV4–39 were used with higher frequency than other VH gene families . IGHV1–2, IGHV5–51, and IGHV1–18 showed higher serum abundance when calculated by LC-MS/MS XIC peak area, albeit less frequently ( p 16). Additionally, we found that a reduction in the repertoire diversity (in other words, an increase in polarisation) correlated with an increase in circulating follicular helper T-cell frequency at day 7 versus day 0, although this observation did not reach statistical significance (p=0·068; p 17). To compare the quality of monoclonal antibodies identified in the serum repertoire among different vaccine cohorts, we recombinantly expressed antibodies representative of dominant serum clonotypes ( p 18). Although monoclonal antibodies from all three vaccine cohorts had similar levels of VH somatic hypermutation (p>0·05; p 18), we found that the monoclonal antibodies induced by RIV4 had a substantially higher affinity to the current vaccine A/Singapore/INFIMH-16–0019/2016 HA and A/Texas/50/2012 HA used in the preceding 2014–15 season than did those induced by the other two vaccines ( – , p 18). For the RIV4 cohort, the median half-maximal effective concentration (EC50) of monoclonal antibodies was 0·037 μg/mL (95% CI 0·012–0·12) and 0·037 μg/mL (0·017–0·32) for A/Singapore/INFIMH-16–0019/2016 and A/Texas/50/2012 HAs, respectively, which is approximately two orders of magnitude (30 to 500 times) lower than the median EC50 of monoclonal antibodies induced by either eIIV4 or ccIIV4 (H3 Singapore, 4·43 μg/mL [95% CI 0·030–100] for eIIV4, 18·50 μg/mL [0·99–100] for ccIIV4; H3 Texas, 1·10 μg/mL [0·045–100] for eIIV4, and 12·63 μg/mL [1·83–100] for ccIIV4; – ). Notably, we found that higher affinity monoclonal antibodies boosted by RIV4 contributed a significantly larger fraction of the serum response than those elicited by eIIV4 or ccIIV4 . There was no significant difference in the quality of monoclonal antibodies constituting the serum response in the eIIV4 and ccIIV4 cohort . We analysed the binding landscapes of bulk serum and also of top-dominant monoclonal antibodies (ie, detected at high concentrations in the serum) against a time-ordered panel of H3/HAs via multiplexed Luminex assay. In two individuals (A4 and B5) for whom we detected dominant clonotypes that accounted for more than 50% of the anti-H3/HA serum response, the binding landscape for these two dominant clonotypes, M81 and M91, closely mirrored the binding landscape observed with whole serum. The concordance in the binding pattern of sera and the dominant antibodies identified by Ig-Seq suggest that a single antibody lineage can largely dictate the functional properties of the polyclonal serum response . Deep scanning saturation mutagenesis could, in principle, further assist in delineating the role of non-dominant serum antibodies in shaping binding breadth and possibly viral escape. Additionally, consistent with the larger fraction of high affinity monoclonal antibodies boosted by RIV4, complete sera from RIV4 vaccine recipients had a substantially higher and broader increase in H3/HA binding response to contemporary strains that have been circulating after the year 2000 ( ; pp 19, 29), compared with eIIV4 and ccIIV4 recipients. Given that we generated a very large set (> 10 6 ) of antibody VH–VL paired sequences from day 7 total B cells, a population in which antigen-specific plasmablasts are highly enriched after influenza vaccination, we examined B-cell receptor clonotypes with known stereotypical HA binding sequence features within this dataset ( pp 20, 30). Although no significant differences were detected in the stereotypical B-cell responses targeting the central stalk, trimer interface, and group 1 and group 2 broadly neutralising stem epitopes, eIIV4 elicited a significantly higher frequency of canonical egg-glycan binding antibodies than did ccIIV4 (median 0·196% [95% CI 0·067–0·372] for eIIV4, 0·035% [0·000–0·062] for ccIIV4, p=0·0071; p 20). Furthermore, stereotyped B-cell receptors associated with binding to membrane-proximal anchor epitope were three times less frequent in the RIV4 cohort than in the ccIIV4 cohort, although the difference was not statistically significant (median 0·062% [95% CI 0·000–0·084] for RIV4, 0·181% [0·016–0·195] for ccIIV4, p=0·064; p 20). Interestingly, even though we detected multiple antibodies with stereotypical HA binding features in day 7 peripheral B cells, only two near stereotypical monoclonal antibodies, UT14 and M47 (a stereotypical trimer interface monoclonal antibody), were detected in the serum of two of the 15 individuals analysed. UT14 is a heterosubtypic monoclonal antibody that was found to be abundant in the serum of a ccIIV4 recipient and that possessed conserved anchor binding sequence features, such as the use of VH3–30, IGKV3–11, and IGKJ5 genes, along with Asn-Trp-Pro amino acid motif in the CDR3 of the kappa light chain (CDRK3; , ). However, UT14 has a nine amino acid-long CDRK3, as opposed to a ten amino acid-long CDRK3 seen in other antibodies of this class ( p 21). In addition to detecting the UT14 CDRH3 peptide that defines the lineage in the serum, we also detected by LC-MS/MS unique tryptic peptides that contain the Asn-Trp-Pro region from the CDRK3 region, further confirming its serological relevance . Biolayer interferometry competition assays revealed that UT14 does not compete with anchor nor stem monoclonal antibodies; however, it competes with known trimer interface monoclonal antibodies ( ; p 21). , – The higher binding affinity of UT14 Fab towards monomeric HA than trimeric HA indicates that the UT14 epitope could be less readily accessible in trimeric HA . We further obtained a 3·8Å resolution cryo-electron microscopy structure of UT14 with HA, revealing that this antibody buries 808Å 2 lateral surface on the H3 head at the interface between two H3 protomers of the trimer ( ; pp 22–24). Similar to the human monoclonal antibodies, FluA-20, H2214, and S5V2–29, and the murine monoclonal antibody, FL-1066, the UT14 Fab interacts with the HA 220-loop and 90-loop via both its heavy (220 loop) and light chains (220 and 90 loop) while demonstrating a distinct angle of approach relative to the aforementioned trimer interface antibodies . UT14 utilises its light chain extensively for HA recognition, a feature that distinguishes UT14 and the murine FL-1066 from the human FluA-20, H2214, and S5V2–29-monoclonal antibodies, and faces the HA in a similar orientation with FL-1066. Unlike the Asn-Trp-Pro motif critical for binding to the anchor epitope, P95 in the Asn-Trp-Pro residues of UT14 CDRK3 does not interact with the HA. Instead, CDRK3 residues, including R91 and Y92, along with the N93 and W94, engage in epitope contact ( p 24).   Our findings suggest that for all three influenza A vaccines—RIV4, eIIV4, and ccIIV4—the serological repertoire was heavily shaped by back-boosting, with more than 80% of the antigen-specific clonal lineages found in serum having been elicited by previous exposures and thus detected at day 0. Due to this high degree of serological imprinting, vaccination with RIV4, eIIV4, or ccIIV4 results in serological anti-H3/HA clonotypes having similar repertoire diversity, VH somatic hypermutation, and CDRH3 features. These results are in line with the fact that A/Singapore/INFIMH-16–0019/2016 is antigenically similar to A/Hong Kong/4801/2014, which had been used as H3 vaccine strains in the two preceding 2016–17 and 2017–18 seasons, and that all individuals had extensive influenza vaccination records in the past 5 consecutive years. Furthermore, irrespective of the vaccine received, our findings support the notion that the serological response to H3/HA is driven by antibodies derived from a small set of select VH gene families. , The trend in correlation between circulating T-follicular helper cell responses and the degree of polarisation suggests that highly polarised serum response, likely to be derived from a few dominant expanded B cells, might require more robust help from circulating T-follicular helper cells. Importantly, from a clinical standpoint, we present data showing that the RIV4 vaccine preferentially boosts H3/HA-specific clonotypes that have much higher affinity for the vaccine HA as well as greater binding breadth to contemporary H3N2 strains than do eIIV4 and ccIIV4. Since we saw no statistical difference in the amount of HA-specific IgG that could be isolated from sera by affinity purification with immobilised H3/HA, we conclude that it is the preferential back-boosting of high-affinity monoclonal antibodies by RIV4 that is probably responsible for the greater increase in H3 serum landscape observed with RIV4 vaccination. Clinical serological testing for eIIV4, ccIIV4, and RIV4 , – cannot address the question of how antigenic and structural differences among the three licensed vaccines affect the composition and functional features of the serological repertoire at the molecular level. One recent study analysing plasmablast-encoded monoclonal antibodies reported that RIV4 elicited a broader homo-subtypic breadth relative to ccIIV4. However, the singlecell cloning from peripheral B cells, although immensely valuable, does not provide information about the antigen-specific serological repertoire that constitutes the polyclonal serum response for multiple reasons, including the fact that less than 5% of plasmablast-encoded antibodies are detectable in circulation and thus have a role in protection against viral infection. Extensive earlier studies and more recent mathematical modelling of the germinal centre reaction indicate a non-linear correlation between antigen dosage and antibody affinity, in which an optimally moderate dose, one that is not too high or too low, can lead to the production of high-affinity antibodies. , A recent structural study revealed that the RIV4 vaccine exclusively contains starfish-like HA multivalent structures consisting of five to 12 copies of HA trimers clustered together to form a transmembrane core. By contrast, the ccIIV4 and eIIV4 vaccines contain comparable or higher fractions of individually isolated HA trimers than HA multimers. Given that the multivalent presentation of immunogens has been shown to enhance antibody responses and increase the affinity of bound monoclonal antibodies, we speculate that RIV4 immunogen structure probably affects the binding affinity of RIV4-boosted monoclonal antibodies observed in our study. Furthermore, the crowding of HA stem domains within the starfish-like HA structures of RIV4 was estimated to occlude about 28% of stem epitopes due to steric clashes. This structural feature of the RIV4 immunogen might affect the ability to activate B cells binding to less accessible epitopes in the HA stem region, which is consistent with our finding of a decrease in the frequency of day 7 B-cell clonotypes with membrane-proximal anchor selective stereotypical features. By analogy, stabilised HIV-1 envelop antigens displayed on nanoparticles reduced the accessibility of epitopes proximal to the base of the antigen due to steric crowding with neighboring antigens on the nanoparticle surface. Multivalent display of antigens has been further shown to enhance trafficking to follicular dendritic cells and accumulation in germinal centres. Lastly, we report on UT14, a highly abundant monoclonal antibody, that has nearly all stereotypical features of membrane-proximal anchor antibodies reported by Guthmiller and colleagues, although it differs by having a nine amino acid-long CDRK3 instead of a canonical ten amino acid-long CDRK3. A closer inspection revealed that one more conserved proline adjacent to Asn-Trp-Pro (ie, Asn-Trp-Pro-Pro) is likely an essential feature of the anchor stereotype (Guthmiller JJ, personal communication). This additional Pro amino acid is 100% conserved in all anchor-binding antibodies and is likely critical for stabilising the Asn-Trp-Pro loop for epitope binding. Our findings thus highlight how antibodies with highly similar sequence features might have evolved to recognise distinct epitopes on the same HA antigen. The discovery of diverse heavy and light chain gene rearrangements in antibodies targeting the recurrent HA epitopes will continue refining our understanding of canonical and non-canonical antibody responses. There are several limitations in our study, including the small sample size (n=5 individuals per cohort) and the fact that the cohorts comprise female health-care personnel (mean age 47·6 years [SD8]) and thus are not representative of diverse populations with different baseline characteristics, such as age, sex, ethnicity, or health status. Additionally, the anti-H1/HA serological repertoire could not be analysed due to limitations in the amount of serum and peripheral blood mononuclear cells that had been obtained under the institutional review board protocol. Further studies are needed to determine how the vaccine platform-specific repertoires are shaped in a diverse cohort of individuals and for different HA subtypes. Nevertheless, our finding of back-boosted antibodies dominant in all three vaccine cohorts, along with the prevalence of high-affinity monoclonal antibodies boosted by RIV4, points to strategies for designing more efficacious vaccines.   Supplementary Material   