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. Author manuscript; available in PMC: 2025 Dec 12.
Published in final edited form as: Vaccine. 2025 Jan 4;47:126689. doi: 10.1016/j.vaccine.2024.126689

Anti-Neuraminidase and Anti-Hemagglutinin Stalk Responses to Different Influenza A(H7N9) Vaccine Regimens

Hana M El Sahly 1, Evan J Anderson 2, Lisa A Jackson 3, Kathleen M Neuzil 4, Robert L Atmar 1, David I Bernstein 5, Wilbur H Chen 4, C Buddy Creech 6, Sharon E Frey 7, Paul Goepfert 8, Jeffery Meier 9, Varun Phadke 10, Nadine Rouphael 10, Richard Rupp 11, Jack T Stapleton 9, Paul Spearman 5, Emmanuel B Walter 12, Patricia L Winokur 9, Inci Yildirim 2, Tracie L Williams 13, Jennifer Oshinsky 4, Lynda Coughlan 4, Haye Nijhuis 4, Marcela F Pasetti 4, Florian Krammer 14, Daniel Stadlbauer 14, Raffael Nachbagauer 14, Rachel Tsong 15, Ashley Wegel 15, Paul C Roberts 16
PMCID: PMC12697238  NIHMSID: NIHMS2123204  PMID: 39756216

Abstract

Introduction:

Pandemic influenza vaccine development focuses on the hemagglutinin (HA) antigen for potency and immunogenicity. Antibody responses targeting the neuraminidase (NA) antigen, or the HA stalk domain have been implicated in protection against influenza. Responses to the NA and HA-stalk domain following pandemic inactivated influenza are not well characterized in humans.

Material and methods:

In a series of clinical trials, we determine the vaccines’ NA content and demonstrate that NA inhibition (NAI) antibody responses increase in a dose-dependent manner following a 2-dose priming series with AS03-adjuvanted influenza A(H7N9) inactivated vaccine (A(H7N9) IIV). NAI antibody responses also increase with interval extension of the 2-dose priming series or following a 5-year delayed boost with a heterologous adjuvanted A(H7N9) IIV. Neither concomitant seasonal influenza vaccination given simultaneously or sequentially, nor use of heterologous A(H7N9) IIVs in the 2-dose priming series had an appreciable effect on NAI antibody responses. Anti-HA stalk antibody responses were minimal and not durable.

Conclusions:

We provide evidence for strategies to improve anti-neuraminidase responses which can be further standardized for pandemic preparedness.

Clinical Trial Registry Numbers:

NCT03312231, NCT03318315, NCT03589807, NCT03738241.

Introduction

The current widespread outbreak of avian influenza disease in cattle, with several documented human infections, reminds us of the constant threat of novel influenza viruses and the importance of influenza vaccine development directed at potentially pandemic strains. The development of pandemic influenza vaccines has thus far emphasized targeting hemagglutinin (HA) as the principal antigen. The immunodominant but antigenically variable HA head results in largely strain-specific immunity. Neuraminidase (NA) is the second surface glycoprotein of the influenza virus, is subject to a lesser degree of immune pressure relative to HA, and anti-NA antibodies (Ab) have been demonstrated to have an independent protective role against influenza infection, illness, and disease severity1–3. Moreover, data suggest that anti-NA Abs have heterologous cross-reactive and cross protective properties that may prove beneficial in the face of drifting or shifting pandemic influenza strains4,5. Another influenza antigenic target of interest to pandemic preparedness is the HA stalk domain, which is more conserved than the HA head. Anti-stalk Abs have been shown to be broadly protective in animal models6. In a controlled human influenza infection model, pre-existing anti-stalk Ab levels were predictive of attenuating some aspects of disease severity7. The NA content in inactivated influenza vaccines (IIVs) is not routinely quantified, and as such can vary in quantity and quality. Therefore, a better understanding of anti-NA and anti-HA stalk Ab responses to various avian influenza vaccination strategies in humans is essential.

Soon after the emergence of 5th wave influenza A(H7N9) human infections in China in 2016, we conducted a series of pandemic influenza preparedness clinical trials utilizing the IIV platform targeting the 5th wave isolate influenza A/ Hong Kong/125/2017 (H7N9) (2017 A(H7N9) IIV) or the 1st wave isolate A/Shanghai/2/2013 (H7N9) (2013 A(H7N9) IIV). We previously reported on the hemagglutination inhibition (HAI) and neutralizing (Neut) Ab responses to increasing vaccine HA dosages, AS03 adjuvant inclusion, utilizing heterologous vs. homologous strains in the 2-dose priming regimen (2013 A(H7N9) IIV and 2017 A(H7N9) IIV), widening the interval between the first and second vaccination, concomitant seasonal influenza vaccination, and delayed heterologous prime boost approaches8–11. Pre-specified study objectives include quantifying the NA content of the vaccines and analyzing the anti-NA and anti-HA stalk Ab responses to the various vaccination strategies, the results of which are reported herein.

Material and methods

Study design and population

DMID 17–0075 (NCT03312231) was a randomized, double-blinded, phase 2 study that evaluated the immunogenicity, reactogenicity and safety of two intramuscular (IM) injections, administered 21 days apart, of A(H7N9) IIV given at three dose levels of HA (3.75, 7.5, and 15 mcg) combined with AS03A adjuvant and two dose levels (15 and 45 mcg) without adjuvant. After stratification by age group (19–64 and ≥ 65 years), participants were randomly assigned to one of the five study groups at a ratio of 2:2:2:1:1 (Table 1).

Table 1.

Influenza A(H7N9) vaccine strain, adjuvant, dosage per hemagglutinin content, interval between doses, and number of participants in the 4 clinical trials.

Protocol Stratum Group (n) Dose 1 Dose 2 Dose 2 Day Dose 3 Dose 3 Day
17-0075 Age 19–64, n=106
Age ≥ 65, n=77		 1 (n=184) 2017 A(H7N9) 3.75 mcg+AS03 2017 A(H7N9) 3.75 mcg+AS03 22 NA NA
Age 19–64, n=102
Age ≥ 65, n=74		 2 (n=176) 2017 A(H7N9) 7.5 mcg+AS03 2017 A(H7N9) 7.5 mcg+AS03 22 NA NA
Age 19–64, n=105
Age ≥ 65, n=76		 3 (n=181) 2017 A(H7N9) 15 mcg+AS03 2017 A(H7N9) 15 mcg+AS03 22 NA NA
Age 19–64, n=53
Age ≥ 65, n=37		 4 (n=90) 2017 A(H7N9) 15 mcg 2017 A(H7N9) 15 mcg 22 NA NA
Age 19–64, n=53
Age ≥ 65, n=36		 5 (n=89) 2017 A(H7N9) 45 mcg 2017 A(H7N9) 45 mcg 22 NA NA
17-0077 1 (n=61) IIV4
2017 A(H7N9) 3.75mcg+AS03		 2017 A(H7N9) 3.75 mcg+AS03 22 NA NA
2 (n=58) IIV4 2017 A(H7N9) 3.75 mcg+AS03 22 2017 A(H7N9) 3.75 mcg+AS03 43
3 (n=30) IIV4 NA 22 NA NA
17-0078 1 (n=27) 2013 A(H7N9) 3.75 mcg+AS03 2017 A(H7N9) 3.75 mcg+AS03 22 NA NA
2 (n=30) 2013 A(H7N9) 3.75 mcg +AS03 2017 A(H7N9) 3.75 mcg+AS03 121 NA NA
3 (n=33) 2013 A(H7N9) 3.75 mcg +AS03 2017 A(H7N9) 15 mcg 121 NA NA
4 (n=32) 2017 A(H7N9) 3.75 mcg +AS03 2017 A(H7N9) 3.75 mcg+AS03 22 NA NA
5 (n=28) 2017 A(H7N9) 3.75 mcg +AS03 2017 A(H7N9) 3.75 mcg+AS03 121 NA NA
6 (n=30) 2017 A(H7N9) 3.75 mcg +AS03 2017 A(H7N9) 15 mcg 121 NA NA
17-0090 * 2013 A(H7N9) + MF59 1 (n=40) 2017 A(H7N9) 3.75 mcg+AS03 NA NA NA NA
2 (n=38) 2017 A(H7N9) 3.75 mcg
2013 A(H7N9) + AS03 3 (n=18) 2017 A(H7N9) 3.75 mcg+AS03 NA NA NA NA
4 (n=24) 2017 A(H7N9) 3.75 mcg
2013 A(H7N9) 5 (n=44) 2017 A(H7N9) 3.75 mcg+AS03 NA NA NA NA
6 (n=40) 2017 A(H7N9) 3.75 mcg
2013 A9(H7N9) +MF59/AS03 then 2013 A(H7N9) 15 mcg 7 (n=16) 2017 A(H7N9) 3.75 mcg +AS03 NA NA NA NA
8 (n=20) 2017 A(H7N9) 3.75 mcg
A/H7 IIV-Naïve 9 (n=35) 2017 A(H7N9) 3.75 mcg +AS03 NA NA NA NA
10 (n=29) 2017 A(H7N9) 3.75 mcg
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*

The first two doses of 2013 A(H7N9) IIV in the 17–0090 study strata were given in 2013–2014; and Dose 1 of 2017 A(H7N9) IIV was administered in 2018–2019

DMID 17–0077 (NCT03318315) was a randomized, unblinded, phase 2 study in adults aged 19 through 64 years that evaluated the safety, reactogenicity, and immunogenicity of an AS03-adjuvanted A(H7N9) IIV when two doses were administered 21 days apart, either sequentially or concomitantly with 2017–18 licensed IIV4. Participants were randomized 2:2:1 to receive two vaccine doses concomitantly in opposite arms, A(H7N9) IIV and IIV4, followed with a second dose of A(H7N9) IIV 21 days later, or one dose of IIV4 followed by the two-dose series of H7N9 IIV, with each dose 21 days after the preceding dose, or one dose of IIV4 as an open label comparison group.

DMID 17–0078 (NCT03589807) was a partially blinded, randomized, phase 2 study in adults aged 19–50 years that evaluated the safety, reactogenicity, and immunogenicity of different prime-boost vaccination schedules of the 2013 and 2017 A(H7N9) IIV. Participants were randomized with equal probability to 1 of 6 groups stratified by site and prior receipt of licensed, seasonal influenza vaccine in at least one of the 2017–2018 and/or 2018–2019 seasons. Participants in study groups 1 and 4 received vaccination on Days 1 and 22, while study groups 2, 3, 5, and 6 received vaccination on Days 1 and 121.

DMID 17–0090 (NCT03738241) was a randomized, blinded phase 2 study in healthy adults who were influenza A(H7N9) naive or previously primed. Participants were stratified by the priming regimen: 1 or 2 doses of 2013 A(H7N9) IIV with MF59, 1 or 2 doses of 2013 A(H7N9) IIV with AS03, 1 or 2 doses of unadjuvanted 2013 A(H7N9) IIV at 15 mcg or 45 mcg HA dosage, 1 dose of 2013 A(H7N9) IIV with MF59 or AS03 followed by 1 dose of unadjuvanted 2013 A(H7N9) IIV 15 mcg, and A(H7N9) naive (unprimed). Participants were randomized in a 1:1 ratio to receive 1 injection of 2017 A(H7N9) IIV at the 3.75 mcg HA dosage with or without AS03.

Vaccines

The study vaccines were a monovalent 2017 A(H7N9) IIV manufactured from a reverse genetics-derived reassortant CVV IDCDC RG56B (H7N9), containing the HA and NA gene segments from the low pathogenic avian influenza (LPAI) influenza A/ Hong Kong/125/2017 (H7N9) and the PB2, PB1, PA, NP, M, and NS gene segments from A/Puerto Rico/8/1934 (H1N1), or monovalent 2013 A(H7N9) IIV from CVV IDCDC RG32A (H7N9) with the HA and NA gene segments from A/Shanghai/2/2013 (H7N9). Vaccines were manufactured by Sanofi using a process similar to that used to produce the licensed seasonal IIV Fluzone® vaccine. AS03, manufactured by GSK, is an adjuvant system based on an oil-in-water emulsion that includes squalene and 11.86 mg α-tocopherol per 0.5 mL dose. MF59, manufactured by CSL Seqirus, is an adjuvant system based on oil-in-water emulsion containing squalene (4.3%) in citric acid buffer with stabilizing nonionic surfactants Tween 80 (0.5%) and Span 85 (0.5%).

Neuraminidase inhibition antibody assay

NAI Ab titers (IC50) were determined using a fit-for-purpose neuraminidase inhibition enzyme-linked lectin assay (ELLA) adapted by Battelle Biomedical Research Center (BBRC; West Jefferson, OH) based on published procedures and optimized at Battelle for detection of N1 and N9 specific responses using H6N9 and H6N1 beta- propiolactone, inactivated, reassortant viruses12. The inactivated, reassortant H6N1 virus strain (Lot No. 138) was kindly provided by Laura Couzens at the FDA; H6 HA derived from influenza A(H6N2) virus strain A/turkey/Massachusetts/3740/1975, N1 NA from influenza A(H1N1) virus strain A/California/04/2009, and internal proteins derived from influenza A (H1N1) virus strain A/Puerto Rico/8/1934. The inactivated, reassortant A(H6N9) virus was established by Battelle; H6 HA derived from influenza A(H6N1) virus strain A/mallard/Sweden/81/2002, N9 NA derived from influenza A(H7N9) virus strain A/Hong Kong/125/2017 with internal proteins derived from influenza A(H1N1) virus strain A/Puerto Rico/8/1934.

Stalk antibody assay

Anti-stalk serum IgG was measured by enzyme-linked immunosorbent assay (ELISA) using optimized/qualified procedures13,14. Briefly, Immulon 2HB microtiter plates were coated overnight at 4°C with 1.0mcg of group 1 rH1 stalk from A/California/07/2009 and 0.5 mcg/ml group 2 rH3 from A/Texas/71/2017 expressed in Expi293F cells. Phosphate buffered saline (PBS) with 0.05% Tween (PBS-T) was used as washing buffer. PBS-T containing 5% non-fat dry milk (NFDM), and PBS containing 3% goat serum and 0.5% NFDM were used as diluent/blocking buffers for the H1 and H3 stalk ELISAs respectively. The stalk proteins were validated using a panel of well-characterized mAbs which bind conformational epitopes on the HA stalk domain to ensure that the proteins are authentically folded. Bound antibodies were detected with anti-human Fcγ peroxidase-labeled goat antisera (Jackson ImmunoResearch, West Grove, PA) diluted 1:5,000. Tetramethylbenzidine (TMB) 2-component (SeraCare Life Sciences, Gaithersburg. MD) was used was used as substrate followed by TMB stop solution. An in-house standard with assigned ELISA Units (EU) corresponding to the inverse of the dilution that produced an OD450 value of 0.5, as well as negative and positive controls were included in all assays. Sera were tested in a single dilution. Ab titers were calculated by interpolation of OD450 values in the 4PL curve of the in-house standard and reported in EU/ml.

Neuraminidase content assessments

The concentration of HA and NA in vaccine lots were measured by Isotope-Dilution Mass Spectrometry (ID-MSand VaxArray (InDevR Inc., Bolder, CO) using published methods15,16. In addition, NA content was measured by standard capture ELISA using an N9 recombinant protein derived from H7N9 A/Hong Kong/125/2017 reference antigen (lot #88) as a calibrator. The NA content of the reference antigen was determined by Isotope-Dilution Mass Spectrometry, and Anti influenza N9 antibody 10F4 was used for antigen capture and HRP-labelled anti influenza N9 mAb 2E6 for antigen detection17,18.

Statistics

N1 and N9 NAI Ab GMTs and anti-HA stalk Ab GMCs along with GMFR from baseline were calculated and presented with 95% confidence intervals based on the Student’s t-distribution for each protocol and study group within protocol. Proportion with detectable antibody and with four-fold rise from baseline or greater are presented with 95% exact (Clopper-Pearson) confidence intervals. Generalized estimating equations were used to estimate the model parameters for a longitudinal model exploring the relationship between vaccine NA content and difference from baseline of log-2 transformed NAI response. Covariates with known effects on NAI responses were included in the model to account for the impact of AS03 adjuvant, prior A(H7N9) IIV vaccination, and IIV4. For the purposes of creating linear contrasts, compared how different dosages of NA content affect NAI responses. Using the NA content of all study groups, we determined the highest dose, the median dose, and the lowest dose and set up contrasts to compare them. Contrasts were used to compare the effect of differing NA dosages on estimated mean NAI response.

Study approval

The clinical trials were approved by the Institutional Review Boards at all participating sites, and all participants provided written informed consent prior to the performance of study procedures.

Data availability

Data will be made available as widely as possible while safeguarding the privacy of participants and protecting confidential and proprietary data.

Results

Hemagglutinin and Neuraminidase vaccine content

All vaccinations were prepared from one of three lots: 2017 A(H7N9) IIV labeled at 15 mcg/mL HA, 2017 A(H7N9) IIV labeled at 30 mcg/mL HA, and 2013 A(H7N9) IIV labeled at 15 mcg/mL HA. The HA content as measured by single radial immunodiffusion (SRID), isotope dilution mass spectrometry (IDMS), and VaxArray® is presented in Figure S1. The HA content was within specifications, approximately 1.5–2 times the labeled content measured by SRID, which was known at the time of study enrollment, and adjustments were made during vaccine preparation to ensure receipt of the correct target HA dosage. The HA content measured by VaxArray® was comparable to SRID for the 2013 vaccine but tended to over-estimate the HA content for the 2017 vaccines when compared to SRID. The NA content of the vaccines as measured by IDMS, VaxArray®, and enzyme-linked immunosorbent assay (ELISA) is presented in Figure S1. The NA content measurements of the 2017 A(H7N9) IIV 15mcg/ml lot were below the limits of quantification by IDMS. NA content measurements by ELISA were higher than IDMS and VaxArray. The rest of the analysis below utilized the VaxArray® method of NA content measurement (Table S1).

Effect of vaccine dosage and adjuvant use on NAI Ab responses (DMID 17–0075; NCT03312231)

This study evaluated the effects of vaccine dosage, co-administration of the AS03 adjuvant, and age on immune responses to A (H7N9) IIV (Table 1). Almost all participants in the trial had detectable N9 NAI Abs at baseline, with higher geometric mean titers (GMTs) observed in those ≥65 years of age compared to those between 19 and 64 years of age (GMTs range 252.8–295.5 vs 124.4–155.5, respectively). Non-overlapping confidence intervals for the GMTs within study arm indicate a statistical difference in GMTs between age groups. Titers rose modestly after the first dose, and after a second dose of the vaccine, N9 NAI GMTs increased further in a vaccine dosage dependent fashion and with the inclusion of an AS03 adjuvant (Fig 1, Table S2). By 21 days post dose 2 (Day 43), the N9 NAI GMTs were comparable between the older and younger age groups.

Figure 1.

Figure 1.

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Geometric Mean Titers of Neuraminidase Inhibition Antibody Against A/Hong Kong/125/2017 (H7N9) by Study Day, Study Group, and Age Stratum, DMID 17–0075 Exploratory Immunogenicity Population. Day 1 N=702, Day 22 N=672, Day 43 N=640

Effect of simultaneous or sequential seasonal influenza vaccination on NAI Ab responses (DMID 17–0077; NCT03318315)

This study evaluated the effects of co- or sequential administration of quadrivalent seasonal influenza vaccine (IIV4) on immune responses to A (H7N9) IIV (Table 1). Concomitant receipt of IIV4 with AS03-adjuvanted A (H7N9) IIV resulted in N9 and N1 NAI GMTs that were comparable at 21 and 180 days after the first dose to those observed following sequential administration of the vaccines. A single dose of IIV4 resulted in a 4.5-fold rise in N9 NAI GMTs at 21 days after vaccination, and by day 181 N9 and N1 NAI GMTs were lower than the GMTs observed in the groups that received the concomitant and sequential doses. (Fig 2, Tables S3, S4).

Figure 2.

Figure 2.

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Geometric Mean Titers of Neuraminidase Inhibition Antibody by Strain, Study Day, and Study Group, DMID 17–0077 Exploratory Immunogenicity Population. Day 1 N=138, Day 22 N=136, Day 181 N=125

Effect of varying the strain and the interval between the first and second dose (DMID 17–0078; NCT03589807)

This study evaluated the effects of administering a homologous vs heterologous second vaccine dose, increasing the interval between first and second dose from 21 days to 120 days, and including the AS03 adjuvant as part of the second dose of vaccine (all initial doses were AS03 adjuvanted) (Table 1). At 21 days post-second vaccination, N9 NAI GMTs were similar following a heterologous prime-boost regimen with AS03-adjuvanted 2013/2017 A(H7N9) IIVs compared to the two-dose regimen with homologous 2017 A(H7N9) IIV (Figure 3; Groups 1 and 4 and Groups 2 and 5, respectively, Table S5). However, prolonging the interval between the two administered doses from 21 to 120 days resulted in increases in N9 NAI GMTs for both prime-boost regimens and with GMTs also qualitatively higher following the homologous 2 dose regimen (Group 5). The lowest N9 NAI responses were observed when the second dose was unadjuvanted (adjuvant-sparing) with no appreciable effect of prolonging the interval between the 2 doses in the absence of an adjuvant (Fig 3; Groups 3 and 6, Table S5).

Figure 3.

Figure 3.

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Geometric Mean Titers of Neuraminidase Inhibition Antibody Against A/Hong Kong/125/2017 (H7N9) by Study Day and Study Group, DMID 17–0078 Exploratory Immunogenicity Population. Day 1 N=132, Day 22 N=128, Day 43 N=48 (only Groups 1, 4), Day 121 N=97 (only Groups 2, 3, 5, 6), Day 142 N=94, Day 202 N=50(only Groups 1, 4), Day 301 N=84 (only Groups 2, 3, 5, 6),

Responses to a delayed heterologous boost vaccination (DMID 17–0090; NCT03738241)

This study evaluated the immune response to a single heterologous boost vaccination with a 2017 A(H7N9) IIV with or without AS03 in participants who had been primed ~5 years earlier with two doses of 2013 A(H7N9) IIV with or without an adjuvant (MF59 or AS03) (Table 1). At baseline, N9 NAI GMTs were higher in the groups that previously received 2013 A(H7N9) IIV compared to the unprimed group. All groups that received AS03 adjuvant in the single delayed boost of 2017 A(H7N9) IIV had higher N9 NAI GMTs at 8- and 21-days post boost compared to those similarly primed who received an unadjuvanted vaccine. Unprimed participants had the lowest responses overall, while participants who were primed with a regimen that contained an oil-in-water adjuvant (MF59 or AS03) in at least one of the doses had the highest and most sustained responses through D181, especially if the delayed boost also included AS03 (Fig 4, Table S6).

Figure 4.

Figure 4.

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Geometric Mean Titers of Neuraminidase Inhibition Antibody Against A/Hong Kong/125/2017 (H7N9) by Study Day and Study Group, DMID 17–0090 Exploratory Immunogenicity Population. Day 1 N=301, Day 8 N=301, Day 22 N=300, Day 181 N=298

Correlation of HAI and NAI responses

At baseline, HAI and NAI Ab GMTs correlated weakly (Spearman’s rho=0.212, p<0.001), and after two vaccinations at any dosage level, the correlation between the two parameters was moderate (Spearman’s rho=0.552, p<0.001) (Fig S2). A similar correlation dynamic was observed between Neut and NAI Ab titers (Fig S3), and when examining the effect of concomitant vs sequential vaccination with seasonal IIV4, variable intervals between prime and boost, and delayed heterologous boosting on the correlation between HAI and NAI Ab titers (Fig S4–S6).

Relationship between NA content and NAI antibody responses

To explore the relationship between NA content and NAI responses, we constructed a logistic linear regression model that included NA content in the first dose, inclusion of the AS03 adjuvant, prior A(H7N9) IIV vaccination, and IIV4 receipt as covariates, and we evaluated the difference from baseline of log-2 transformed NAI titer (Table S7). We found that the NA content, adjuvant use, and previous receipt of A(H7N9) IIV correlated with increased NAI Ab responses. In a second model that also included the NA content in first and second dose and day from vaccination we found that a single unadjuvanted dose with a median NA content was not associated with increased NAI Ab responses compared to a single unadjuvanted dose with a low NA content. However, NAI Ab responses correlated with increased NA content if a second vaccine dose was given or if an adjuvant was included (Table S8).

Anti-HA stalk responses

Anti-HA stalk Abs were assessed in two of the four clinical trials to evaluate the levels observed in naïve subjects and in response to homologous or heterologous prime-boost regimens with different dosing intervals (DMID 17–0078 and 17–0090). There are two HA phylogenetic groups: 1 and 2, and the H7 HA belongs to Group 2. In DMID 17–0078, baseline geometric mean concentrations (GMCs) of Abs against the phylogenetic Group 2 HA stalk antigen from A/Texas/71/2017 (H3N2) were lower than those against the Group 1 HA stalk, and at 21 days post dose 2 there was a geometric mean fold rise (GMFR) of 2.0–3.1, which declined but remained elevated above baseline (GMFR 1.4–2.0) through D180. Using a Phylogenetic Group 1 HA stalk from A/California/07/2009 (H1N1) as the antigen, the GMCs of anti-HA stalk Abs were found to be elevated at baseline, with minimal rises observed at Day 21 post dose 2 with GMFR 1.2–1.6, followed by a return to baseline levels by Day 180. There was no discernible effect of varying the interval or the use of heterologous strains between the first and second dose on the magnitude of the anti-HA stalk Ab responses (Fig 5A).

Figure 5.

Figure 5.

Figure 5.

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Geometric Mean Concentration of Anti-Hemagglutinin-Stalk Antibody by Study Day and Study Group, DMID 17–0078 (Panel A) and DMID 17–0090 (Panel B) Exploratory Immunogenicity Populations. DMID 17–0078: Day 1 N=132, Day 22 N=128, Day 43 N=48 (only Groups 1, 4), Day 121 N=97 (only Groups 2, 3, 5, 6), Day 142 N=94, Day 202 N=50(only Groups 1, 4), Day 301 N=84 (only Groups 2, 3, 5, 6). DMID 17–0090 Day 1 N=299, Day 8 N=300, Day 22 N=299, Day 181 N=297.

A single heterologous 2017 A(H7N9) IIV boost injection at approximately five years after a 2 dose priming series with various 2013 A(H7N9) IIV adjuvanted regimens, and regardless of the priming regimen, led to only minimal increases in anti-H3 stalk responses, with a GMFR ranging from 1.1–2.4 at 21 days post boosting (DMID 17–0090). The largest increases were seen in participants who were unprimed and who received the adjuvanted 2017 A(H7N9) IIV, and in those who were primed and boosted with AS03-adjuvanted regimens. By Day 181, the GMCs of anti-HA stalk Abs were comparable between all groups and close to baseline levels except in the two aforementioned groups in whom a GMFR 1.6 compared to baseline was observed (Fig 5B).

Overall, we found no to minimal correlation between anti- HA stalk Ab responses and HAI or Neut Ab responses.

Discussion

In a series of randomized clinical trials, we found that almost all adults had low but detectable N9 NAI antibody levels prior to vaccination. Following vaccination there was a dose-dependent increase in N9 NAI activity that was associated with increased NA content, use of adjuvanted vaccines, and longer interval between the first and second dose of the vaccine regimen. At 5 years post-priming with 2 doses of adjuvanted or unadjuvanted 2013 A(H7N9) IIV, a single booster dose of adjuvanted 2017 A(H7N9) IIV led to a rapid increase in N9 specific NAI responses. Notably, seasonal influenza vaccination resulted in a modest, albeit transient increase in N9 NAI levels.

In certain aspects, these findings mirror the HAI responses to the same vaccination strategies: improved antibody responses with the AS03 adjuvant use and increasing the interval between the first and second dose of the regimen, but not with the use of heterologous strains in doses 1 and 2. However, there are important distinctions in the dynamics of A(H7N9) HAI and NAI Ab responses. First, while most adults have low levels of pre-existing N9 NAI Abs, most adults do not have detectable H7 HAI Abs before vaccination, owing to the cross-reactive nature of NAI Ab compared to the specificity of HAI Ab elicited by seasonal influenza infections and vaccination19–21. This is further demonstrated in our data: vaccination with seasonal influenza vaccine did boost N9 NAI Ab levels. While detecting N9 NAI Ab in the majority of adult participants may not be surprising, the frequency (>90%) observed in our study was high. For example, Jiang et al found that 53% of blood donors had detectable N9 NAI Ab in Thailand22. These differences in frequencies could be due to variability between the assays, the populations under evaluation, and variation in influenza exposure histories.

NAI Abs’ role in protection against seasonal influenza viral infection and disease is well documented, but their contribution to protection against a novel avian virus remains to be verified in humans1–3. The data in animal models suggest a role in protection: N9 NA-specific monoclonal antibodies protected mice from lethal influenza infection, and chickens vaccinated with recombinant NA replicon particles had decreased or no shedding following infection with low pathogenicity influenza A(H7N7)23,24. Of note, we observed higher pre-existing N9 NAI Ab GMTs in older persons, likely due to repeated immunologic exposures to influenza A(H3N2) vaccines and infections, which raises the hypothesis that children and adolescents may have lower N9 NAI Ab GMTs, and thus could represent a subpopulation of particular vulnerability to avian influenza.

The other distinction we observed was that when using AS03-adjuvanted vaccines, increasing the vaccine’s NA content is associated with increased N9 NAI Ab responses. This is in contrast to our previous finding that increasing an AS03- or MF59-adjuvanted vaccine’s A/H7 content is not associated with increased anti-HAI levels8,25,26. The reason for this observation is unclear. Pre-pandemic vaccine manufacturing and regulatory reviews require standardization of the HA content and assessment of the HAI responses. To capitalize on the potentially protective NAI Ab responses, our data suggest that alternate manufacturing strategies that specifically enrich for the NA content or NA antigen supplementation approaches should be considered and evaluated in persons of all ages to confirm improved immunogenicity, given the theoretical potential for competition between HA and NA for B cells27. The finding of improved NAI Ab responses with increasing the interval between vaccine doses as seen with other antigens, likely due to improved somatic hypermutation of antigen-specific B cells and improved CD4+ helper responses, might have salutary effects by allowing more persons to get a first dose of the vaccine which needs to be weighed at a population level against the risk of unknown reduction in efficacy28–30.31

Generating anti-HA stalk Abs is one approach to advancing influenza vaccines that are potentially less impacted by viral shifts and drifts. Natural infection with influenza A(H7N9) virus was shown to elicit broadly cross-reactive anti-stalk antibodies that reacted with HA from groups 1 and 2 influenza viruses, and this cross reactivity is stalk-specific32. Priming with an adjuvanted avian inactivated vaccine or live attenuated vaccines did result in improved anti-stalk responses, although repeated dosing re-focused the responses to the HA head33,34. Our study shows that A(H7N9) vaccination results only in small anti-stalk Ab responses against a Group 2 HA and transient minimal rises against a Group 1 HA. The differences in the responses to the two antigens is likely due to the fact that influenza A(H7N9) viruses belong to Group 2 viruses, and possibly to differences in immunological imprinting by previous exposures35. While not measured in our study, it is possible that anti-H7 stalk Ab levels may be higher than the anti-H1 and anti-H3 stalk Ab levels. Responses in our studies to the H3 and H1 stalk antigens are comparable to the 1.5–2-fold responses elicited to the stalk’s vaccine antigen following inactivated seasonal influenza virus vaccine36,37. Strategies proposed to boost the production of stalk Ab include optimizing the antigen, the adjuvant, the platform, and using sequential HA chimeric constructs36,38,39. While anti-HA stalk Ab have been shown in animal models to be protective against homologous and heterologous influenza infections, data on an independent role in protecting humans against infection and disease are conflicting, including a recent clinical trial of an anti-HA stalk monoclonal Ab that failed to prevent virologically confirmed influenza disease7,37,40–42.

The study findings have to be taken in the context of their limitations. We could not make cross-study comparisons because these were separately conducted clinical trials. Other limitations include a healthy adult study population that excluded children, pregnant women, and immunocompromised participants. In addition, our study investigated NAI and anti-HA stalk Ab responses elicited by inactivated influenza A(H7N9) vaccines and our results may not apply to other pre-pandemic vaccine platforms under development, such as mRNA-, vector-, and recombinant protein-based technologies. We evaluated the responses to various pre-pandemic vaccine strategies using an influenza A(H7N9) strain, which may not be the strain causing a pandemic. The antigens used to measure anti-HA stalk Abs belonged to phylogenetic Groups 1 and 2; and vaccine-matched stalk antigens might result in possibly higher anti-HA stalk Ab levels. While higher NA antibody levels are associated with protection from disease in humans, a putatively protective NAI Ab level has not been established. Moreover, there are variabilities in the performance of NAI Ab assays and NA content. However, given the dearth of data on NAI and anti-HA stalk responses to avian influenza IIV, the findings of our study provide insight into the general patterns of NAI and anti-HA stalk Ab responses to IIV, against which responses to other platforms and other strains can be compared. From a mechanistic standpoint, there is evidence in the literature that anti-NA and anti-HA stalk Abs have significant Fc-mediated antiviral functions43–47. Our study assessed NAI and anti-HA stalk Ab levels as a function of avian influenza vaccine dose, dosing interval and inclusion of adjuvants but not the effects these variables had on the Fc-mediated functions such as antibody dependent cellular cytotoxicity and complement activation.

Vaccine preparedness to mitigate the morbidity and mortality of a potential avian influenza pandemic has thus far relied on standardizing the HA content and improving HAI antibody responses. Our studies provide evidence on actionable strategies that increase NAI antibody responses to avian influenza vaccines in humans and point to the need for additional research to better characterize anti-stalk Ab responses and their contribution to protection.

Supplementary Material

Supp material

Highlights.

  • Neuraminidase and hemagglutinin’s stalk antibodies are not well characterized post pandemic influenza vaccines.

  • Neuraminidase inhibition antibody responses increase in a dose dependent fashion after 2 doses of influenza A(H7N9) vaccine.

  • Increasing the interval of the 2-dose series and a delayed heterologous boost increased neuraminidase responses.

  • Concomitant seasonal influenza vaccination or use of heterologous A(H7N9) IIVs in the 2- series had no effect on neuraminidase responses.

  • Anti-hemagglutinin stalk responses were of small magnitude and transient.

Acknowledgements

We are thankful for study design, protocol implementation and regulatory support provided by Wendy Buchanan RN, Mohamed Elsafy, MD, Francisco J. Leyva MD, PhD, ScM, Rhonda Pikaart-Tautges, BSBA, BS, Diane Post, PhD, Chelsea Lane, PhD, Sonnie Kim, MSc., Nancy Ulbrandt, PhD, Jae Arega, MSc., and Hyung Koo, RN, BSN at the Division of Microbiology and Infectious Diseases at the National Institute of Allergy and Infectious Diseases. We are also thankful for the study participants, the Data and Safety Monitoring Board members (Jeanne S. Sheffield, MD, Bryan Murray MBBS, John Bruce McClain, MD, David Carlin, PhD)

Funding Source

The work was supported by the National Institutes of Allergy and Infectious contract HHSN272201300015I (Baylor College of Medicine), 75N93021C00012 (The Emmes Company, LLC), HHSN2722013000017I (Duke University School of Medicine), HHSN272201300018I (Emory University), HHSN272201300022I (University of Maryland), HHSN272201300019I (Kaiser Washington), HHSN272201300020I (University of Iowa), HHSN272201300023I (Vanderbilt University Medical Center), HHSN27220130021I (Saint Louis University) HHSN272201300016I (Cincinnati Children’s Hospital Medical Center). The candidate vaccines evaluated under this study have been funded in whole or in part with Federal funds from the Department of Health and Human Services; Administration for Strategic Preparedness and Response; Biomedical Advanced Research and Development Authority, under Contracts HHSO100201600006I, HHSO1002012000131I, HHSO100201600004I. The contracts and federal funding are not an endorsement of the study results, product, or company. The vaccine antigen was manufactured by Sanofi (H7N9 vaccine). AS03 is a trademark owned by or licensed to the GSK group of companies. Both manufacturers were provided the opportunity to review a preliminary version of this manuscript for factual accuracy, but the authors are solely responsible for final content and interpretation. The NAI assessments were funded with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Preclinical Services Contract No. HHSN272201800003I/75N93020F00001. HA stalk antigens were designed and produced with support from R01AI148369 (LC) and NIAID contract and 75N93019C00055. The HA stalk antibody assessments were funded by NIAID contract HHSN272201300022I. Work in the Krammer laboratory was supported via the NIAID Centers of Excellence for Influenza Research and Response (CEIRR, 75N93021C00014).

Conflict of interest statement

Drs. El Sahly, Roberts, Bernstein, Pasetti, Jackson, Frey, Rupp, Atmar, Williams, Nijhuis, Coughlan, Neuzil, Stapleton, and Phadke have no conflict of interest. Rachel Tsong and Ashley Wegel have no conflicts of interest.

EBW received support from Pfizer, Moderna, Sequiris, Najit Technologies and Clinetic as an investigator for clinical trials or studies. He has served as an advisor to Vaxcyte and Pfizer, a consultant to Iliad Biotechnologies, and as a DSMB member for Shionogi.

Emory received funds for NR to conduct research from Sanofi, Lilly, Merck, Quidel, Immorna, Vaccine Company and Pfizer. NR served on selected advisory boards for Sanofi, Seqirus, Pfizer and Moderna and was a paid clinical trials safety consultant for ICON, CyanVac, Imunon and EMMES.

WHC served as a safety consultant for FluGen, ICON, Rho, Emmes, and PATH.

IY received funding to her institution to conduct clinical research from Merck, Moderna, Pfizer outside the submitted work; and received honorarium for advisory board from Merck and Sanofi Pasteur.

EJA is currently an employee of Moderna.

PW received research funding from Pfizer, and Sanofi.

CBC received grant funding from Vedanta, and Moderna; he serves as a consultant to GSK, Sanofi Pasteur, Moderna, Debiopharma, CommenseBio, TDCowen, and Guidepoint Global; and received royalties from UpToDate.

The Icahn School of Medicine at Mount Sinai filed patent applications relating to SARS-CoV-2 serological assays, New Castle virus-based SARS-CoV-2 vaccines influenza virus vaccines and influenza virus therapeutics which list FK as co-inventor. Mount Sinai spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, CastleVax, to develop SARS-CoV-2 vaccines. FK is co-founder and scientific advisory board member of CastleVax. FK has consulted for Merck, Curevac, Seqirus, GSK and Pfizer and is currently consulting for 3rd Rock Ventures, Gritstone and Avimex. The Krammer laboratory is collaborating with Dynavax on influenza vaccine development and with VIR regarding influenza therapeutics.

PG is on a patent for a COVID-19 monoclonal antibody made by Aridis Pharmaceuticals.

DS and RN are employees and shareholders of Moderna.

References

  • 1.Couch RB, et al. Antibody correlates and predictors of immunity to naturally occurring influenza in humans and the importance of antibody to the neuraminidase. J Infect Dis 207, 974–981 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Monto AS, et al. Antibody to Influenza Virus Neuraminidase: An Independent Correlate of Protection. J Infect Dis 212, 1191–1199 (2015). [DOI] [PubMed] [Google Scholar]
  • 3.Memoli MJ, et al. Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy Human Challenge Model. mBio 7, e00417–00416 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wan H, et al. Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses. J Virol 87, 9290–9300 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Walz L, Kays SK, Zimmer G & von Messling V Neuraminidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype. J Virol 92(2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Impagliazzo A, et al. A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen. Science 349, 1301–1306 (2015). [DOI] [PubMed] [Google Scholar]
  • 7.Park JK, et al. Evaluation of Preexisting Anti-Hemagglutinin Stalk Antibody as a Correlate of Protection in a Healthy Volunteer Challenge with Influenza A/H1N1pdm Virus. mBio 9(2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Jackson LA, et al. Immunogenicity and safety of varying dosages of a fifth-wave influenza A/H7N9 inactivated vaccine given with and without AS03 adjuvant in healthy adults. Vaccine 42, 295–309 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Ortiz JR, et al. Safety and immunogenicity of monovalent H7N9 influenza vaccine with AS03 adjuvant given sequentially or simultaneously with a seasonal influenza vaccine: A randomized clinical trial. Vaccine 40, 3253–3262 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Rostad CA, et al. A Phase 2 Clinical Trial to Evaluate the Safety, Reactogenicity, and Immunogenicity of Different Prime-Boost Vaccination Schedules of 2013 and 2017 A(H7N9) Inactivated Influenza Virus Vaccines Administered with and without AS03 Adjuvant in Healthy US Adults. Clin Infect Dis (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.El Sahly HM, et al. Safety and Immunogenicity of a Delayed Heterologous Avian Influenza A(H7N9) Vaccine Boost Following Different Priming Regimens: A Randomized Clinical Trial. J Infect Dis 229, 327–340 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Couzens L, et al. An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. J Virol Methods 210, 7–14 (2014). [DOI] [PubMed] [Google Scholar]
  • 13.Yassine HM, et al. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nat Med 21, 1065–1070 (2015). [DOI] [PubMed] [Google Scholar]
  • 14.Corbett KS, 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). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Santana WI, Williams TL, Winne EK, Pirkle JL & Barr JR Quantification of viral proteins of the avian H7 subtype of influenza virus: an isotope dilution mass spectrometry method applicable for producing more rapid vaccines in the case of an influenza pandemic. Anal Chem 86, 4088–4095 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kuck LR, et al. VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines. Vaccine 36, 2937–2945 (2018). [DOI] [PubMed] [Google Scholar]
  • 17.Wan H, et al. Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection. J Virol 92(2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Rajendran M, et al. An immuno-assay to quantify influenza virus hemagglutinin with correctly folded stalk domains in vaccine preparations. PLoS One 13, e0194830 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Levine MZ, et al. Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity. Vaccines (Basel) 10(2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chen YQ, et al. Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies. Cell 173, 417–429 e410 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Pichyangkul S, et al. Pre-existing cross-reactive antibodies to avian influenza H5N1 and 2009 pandemic H1N1 in US military personnel. Am J Trop Med Hyg 90, 149–152 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Jiang L, et al. Cross-reactive antibodies against H7N9 and H5N1 avian influenza viruses in Thai population. Asian Pac J Allergy Immunol 35, 20–26 (2017). [DOI] [PubMed] [Google Scholar]
  • 23.Gilchuk IM, et al. Influenza H7N9 Virus Neuraminidase-Specific Human Monoclonal Antibodies Inhibit Viral Egress and Protect from Lethal Influenza Infection in Mice. Cell Host Microbe 26, 715–728 e718 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Halbherr SJ, et al. Biological and protective properties of immune sera directed to the influenza virus neuraminidase. J Virol 89, 1550–1563 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Jackson LA, et al. Effect of Varying Doses of a Monovalent H7N9 Influenza Vaccine With and Without AS03 and MF59 Adjuvants on Immune Response: A Randomized Clinical Trial. JAMA 314, 237–246 (2015). [DOI] [PubMed] [Google Scholar]
  • 26.Mulligan MJ, et al. Serological responses to an avian influenza A/H7N9 vaccine mixed at the point-of-use with MF59 adjuvant: a randomized clinical trial. JAMA 312, 1409–1419 (2014). [DOI] [PubMed] [Google Scholar]
  • 27.Johansson BE & Kilbourne ED Dissociation of influenza virus hemagglutinin and neuraminidase eliminates their intravirionic antigenic competition. J Virol 67, 5721–5723 (1993). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Rodrigues CMC & Plotkin SA The influence of interval between doses on response to vaccines. Vaccine 39, 7123–7127 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ledgerwood JE, et al. DNA priming and influenza vaccine immunogenicity: two phase 1 open label randomised clinical trials. Lancet Infect Dis 11, 916–924 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Payne RP, et al. Immunogenicity of standard and extended dosing intervals of BNT162b2 mRNA vaccine. Cell 184, 5699–5714 e5611 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kim W, et al. Germinal centre-driven maturation of B cell response to mRNA vaccination. Nature 604, 141–145 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Liu L, et al. Induction of Broadly Cross-Reactive Stalk-Specific Antibody Responses to Influenza Group 1 and Group 2 Hemagglutinins by Natural H7N9 Virus Infection in Humans. J Infect Dis 215, 518–528 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Halliley JL, et al. High-Affinity H7 Head and Stalk Domain-Specific Antibody Responses to an Inactivated Influenza H7N7 Vaccine After Priming With Live Attenuated Influenza Vaccine. J Infect Dis 212, 1270–1278 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ellebedy AH, et al. Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans. Proc Natl Acad Sci U S A 117, 17957–17964 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Chen Y, et al. Anamnestic broadly reactive antibodies induced by H7N9 virus more efficiently bind to seasonal H3N2 strains. Hum Vaccin Immunother 18, 2128014 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Folschweiller N, et al. Reactogenicity, safety, and immunogenicity of chimeric haemagglutinin influenza split-virion vaccines, adjuvanted with AS01 or AS03 or non-adjuvanted: a phase 1–2 randomised controlled trial. Lancet Infect Dis 22, 1062–1075 (2022). [DOI] [PubMed] [Google Scholar]
  • 37.Dhar N, et al. Hemagglutinin Stalk Antibody Responses Following Trivalent Inactivated Influenza Vaccine Immunization of Pregnant Women and Association With Protection From Influenza Virus Illness. Clin Infect Dis 71, 1072–1079 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Pardi N, et al. Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies. Nat Commun 9, 3361 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Moin SM, et al. Co-immunization with hemagglutinin stem immunogens elicits cross-group neutralizing antibodies and broad protection against influenza A viruses. Immunity 55, 2405–2418 e2407 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ng S, et al. Novel correlates of protection against pandemic H1N1 influenza A virus infection. Nat Med 25, 962–967 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Aydillo T, et al. Pre-existing Hemagglutinin Stalk Antibodies Correlate with Protection of Lower Respiratory Symptoms in Flu-Infected Transplant Patients. Cell Rep Med 1, 100130 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Tan SK, et al. A Randomized, Placebo-Controlled Trial to Evaluate the Safety and Efficacy of VIR-2482 in Healthy Adults for Prevention of Influenza A Illness (PENINSULA). Clin Infect Dis (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.DiLillo DJ, Tan GS, Palese P & Ravetch JV Broadly neutralizing hemagglutinin stalk-specific antibodies require FcgammaR interactions for protection against influenza virus in vivo. Nat Med 20, 143–151 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.He W, et al. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus. Proc Natl Acad Sci U S A 113, 11931–11936 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Chen X, et al. Functional antibody-dependent cell mediated cytotoxicity (ADCC) responses to vaccine and circulating influenza strains following vaccination. Virology 569, 44–55 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Stadlbauer D, et al. Broadly protective human antibodies that target the active site of influenza virus neuraminidase. Science 366, 499–504 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Wohlbold TJ, et al. Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat Microbiol 2, 1415–1424 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Supp material
NIHMS2123204-supplement-Supp_material.pdf (617.9KB, pdf)

Data Availability Statement

Data will be made available as widely as possible while safeguarding the privacy of participants and protecting confidential and proprietary data.

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