H1ssF + AddaVax generates a protective response in adult AGMs

While the primary goal in the current study was to determine whether a universal HA stem vaccine is effective in newborns, we first sought to establish the utility of this vaccine in adult AGMs, as previous studies with H1ssF were performed in adult macaques²⁴. This information was essential to appropriately interpret findings from newborn animals. H1ssF was delivered with the MF59-like adjuvant AddaVax, as previous studies have demonstrated that adjuvant is required for the generation of robust HA stem responses to H1ssF in IAV naïve animal models^22,24. Adult AGMs aged 5–7 years old (equivalent to ~20–28 year old humans, Supplementary Table S1) were vaccinated intramuscularly (i.m.) with H1ssF + AddaVax or with PBS (the control group (Ctrl)) (Fig. 1A). HA stem-specific Abs were measured by ELISA using a stabilised trimeric A/New Caledonia/20/1999 HA stem protein³¹. H1ssF + AddaVax vaccinated adults had a significant increase in HA stem-specific IgM (Fig. 1B) and IgG (Fig. 1C) compared to Ctrl animals following prime (p.v.) and boost (p.b). Modest but detectable levels of IgA were also generated (Fig. S1A). Interestingly, we observed significant generation of stem-specific IgG at d10 p.v. consistent with an early class switched plasmablast response. These data show that prime/boost vaccination with H1ssF + AddaVax in adult AGMs elicits high levels of circulating HA stem-specific Abs.

Adult AGM vaccination and sampling schedule (A). Circulating levels of NC99 stem-specific IgM (B) and IgG (C) were assessed by ELISA. HA-specific IgG cross-reactivity was measured against group 1 and group 2 HA molecules in the plasma at d45 p.b. The stem-reactive mAb FI6V3 (grey triangle) served as a positive control (D). Circulating levels of IgG binding to the full-length NC99 HA, H1-N27, H1-N45, or H1-N27/45 were assessed by ELISA at d45 p.b. Central and anchor Abs were quantified by subtracting AUC for H1-27/45 from the H1-27 (central) or H1-N45 (anchor) AUC values (E). NC99 stem-specific IgG avidity was measured by determining the NaSCN concentration that gave a 50% reduction in optical absorbance compared to the untreated sample (F). Adult AGMs were challenged with 5 × 10⁷ TCID₅₀ of Ca09 H1N1 at d45 p.b. Viral genomes were measured in BAL collected at d7 p.c. (G). NC99 HA stem-specific IgG (H) and IgA (I) were measured in the BAL at d7 p.c. The dotted line represents the limit of detection (LOD). Threshold titre (TT) is defined as the highest dilution resulting in an OD₄₅₀ greater than 3X the assay background. Ctrl (n = 3), H1ssF + AddaVax (n = 3). Symbols represent individual animals. The line in each column represents the median. Statistical significance: two-way ANOVA with Tukey’s post hoc analysis (B, C, H), a one-way ANOVA with either Tukey’s (D, F) post hoc analysis, or a paired (E), or unpaired two-tailed t test (G, I, J). Not significant p ≥ 0.05 (not indicated on graph). Figure 1A was created in BioRender. Alexander-Miller, M. (2025) https://BioRender.com/017wbod. Source data are provided as a Source Data file.

Previous studies with this vaccine have shown broad Ab reactivity across group 1 HA subtypes in adult animal models^22,24. Thus, we evaluated the breadth of HA recognition in our vaccinated adult AGMs at the latest time point prior to challenge (d45 p.b.) (Fig. 1D). FI6V3, a human HA stem-specific monoclonal Ab (mAb) that binds the central stem epitope on group 1 and group 2 HA subtypes³², was included as a positive control. Animals administered H1ssF + AddaVax displayed reactivity to multiple group 1 HA subtypes, with the highest recognition to H1 and H5 (Fig. 1D). Thus, the H1ssF + AddaVax prime/boost vaccination resulted in Abs that can react across group 1 HA subtypes in adult AGMs.

To determine the fine specificity of HA stem-specific Abs, we utilised a mutant NC99 HA molecule wherein an N-linked glycosylation site was introduced (Ile545Asn and a Gly547Thr (H3 numbering)) that blocks Abs binding to the central epitope, hereafter referred to as H1-N45³³. We employed two additional NC99 HA mutants to better characterise epitope specificities, H1-N27 (Q27N and N29T) has a glycan in the anchor epitope that blocks recognition, and H1-N27/45 incorporates glycans in both the central and the anchor epitopes²⁶. We used this set of mutant HA antigens to profile stem-specific Abs in the vaccinated adult AGMs. To accurately determine Abs that bind either the central or anchor epitope only, we subtracted the area under the curve (AUC) obtained with the H1-N27/45 (non-central, non-anchor) from either the H1-N27 (central binding Abs) or the H1-N45 (anchor binding Abs) (Fig. 1E and Supplementary Fig. S2). We found readily detectable Abs recognising the central region of HA stem, with one animal making a much higher response (Fig. 1E). There was limited recognition of the anchor epitope. All animals made Abs that bound epitopes outside of the central or anchor regions (non-central/anchor epitopes) (Supplementary Fig. S2).

Based on a murine study that reported HA stem-specific Abs can increase the susceptibility to autoimmune diseases³⁴, we tested whether H1ssF+AddaVax elicited increases in autoreactive Abs through the measurement of IgM and IgG Abs specific to autologous nuclear antigens (ANA). There was no indication of vaccine-mediated induction of ANA Abs (Supplementary Fig. S3).

We next measured the avidity of HA stem-specific IgG over time in adults that received H1ssF + AddaVax. Increased avidity is associated with GC formation and somatic hypermutation³⁵. Boosting with H1ssF + AddaVax resulted in a rapid rise in HA stem-specific Ab avidity at d11 p.b.; this did not increase further at d45 p.b. (Fig. 1F).

To assess the ability of this vaccine to provide protection in the adult AGM model, we challenged animals with the clinically relevant H1N1 pandemic strain A/California/07/2009 (Ca09) (5 × 10⁷ TCID₅₀). We assessed HA stem-specific Ab and viral load in the lungs using bronchoalveolar lavage (BAL) samples collected at d7 p.c. Virus was significantly reduced in the lungs of H1ssF + AddaVax animals at d7 p.c., with 2 of 3 animals having cleared virus (Fig. 1G). We observed higher levels of HA stem-specific IgG in the BAL of H1ssF+AddaVax adults compared to Ctrl adults at d7p.c. (Fig. 1H). No HA stem-specific IgA was detected in the BAL (Fig. 1I). Thus, H1ssF + AddaVax vaccine elicited robust stem-specific, broadly reactive Ab responses and promoted enhanced viral clearance in the lungs of adult AGMs. These data establish the ability of this vaccine approach to generate protective responses in the AGM model.

HA stem-specific Abs have been shown to promote clearance through both neutralising activity^36,37 and Fc-mediated effector function^38,39. In adult naive animal models (3 doses)^22,24 and in human adults (2 doses)²⁵, the H1ssF vaccine elicits Abs that have both neutralising and Ab-dependent cellular cytotoxicity (ADCC) activity²⁵.

We evaluated the functional attributes, i.e., neutralising capacity, ADCC, Ab-dependent complement deposition (ADCD), and ADCP of the HA stem-specific Abs generated in adult AGMs following H1ssF+AddaVax prime/boost vaccination and after challenge with Ca09 H1N1. Neutralising capacity was assessed using a set of replication-restricted reporter influenza viruses (H1N1 A/New Caledonia/20/1999, H1N1 A/California/07/2009, and H5N1 A/Vietnam/1203/2004)⁴⁰. Interestingly, nAbs were not detected at d45 p.b. (Fig. 2A). However, neutralisation titres against the vaccine-matched NC99 were readily detected at d7 p.c. in animals administered H1ssF + AddaVax, but not in Ctrl animals (Fig. 2A). These data indicate a memory response was generated with vaccination that resulted in the rapid production of nAb after viral challenge. Neutralising Abs to the challenge virus (Ca09) were also detected, albeit at lower levels than to NC99 (Fig. 2B). Remarkably, all of the vaccinated adults also had nAb to H5N1 following challenge (Fig. 2C).

Neutralising IC₈₀ titres against H1N1 A/New Caledonia/20/1999 (A), H1N1 A/California/07/2009 (B), and H5N1 A/Vietnam/1203/04 (C) were assessed by reporter-based microneutralization (MN) assays. The MN IC₈₀ titre was measured at the plasma dilution at which 80% of virus infection was inhibited. ADCC activity against HA Ca09 was measured using KHYG-1 cells expressing rhesus macaque CD16. ADCC activity was measured as the percentage of CD107a⁺ KHYG-1 cells present after sequential gating of cells by FSC/SSC and Zombie Violet negativity (D). ADCD scores were determined by calculating the percentage of HA FluoSpheres beads that were C3 + × GMFI/1000 at the highest dilution (1:5) (E). ADCP was measured by THP-1 uptake of Ab-opsonised HA FluoSpheres beads. THP-1 were gated through FSC/SSC and singlet gates prior to FITC bead analysis. Phagocytosis scores were determined by measuring the percentage of bead-positive THP-1 cells×the the geometric mean fluorescence/1000 at the 1:800 dilution. (F). The dotted line represents the limit of detection (LOD) for each assay. Ctrl (n = 3), H1ssF + AddaVax (n = 3). The line in each column represents the median. Statistical significance was determined using a one-way ANOVA with a Fisher’s LSD post hoc analysis. Not significant p = > 0.05 (not indicated on graph). Source data are provided as a Source Data file.

Next, we explored the ability of vaccine-elicited Abs to facilitate Fc-mediated effector function through ADCC, ADCD, and ADCP. Surprisingly, no ADCC, ADCD, or ADCP activity was detected in plasma from H1ssF + AddaVax vaccinated adults at d45 p.b. (Fig. 2D–F). However, there was a significant increase in all three Fc-mediated functions at d7 p.c. in vaccinated adults. Overall, these data support a rapid recall response in vaccinated adult animals that is associated with clearance and exhibits a broad array of Ab effector functions.

H1ssF + AddaVax promotes significant levels of HA stem-specific Abs in newborn AGMs that are broadly reactive

Having demonstrated the utility of H1ssF +AddaVax in adults, we sought to determine the capacity for newborns to respond to this vaccine. The AddaVax adjuvant used here is similar to the licensed MF59, which has a demonstrated safety profile in human newborns in the context of an HIV vaccine delivered at birth, 2w, 8w, and 20w⁴¹. AGMs aged 3–5 days old (equivalent to ~ 12–20 day old humans, Supplementary Table S2) received an H1ssF prime/boost regimen with or without AddaVax (Fig. 3A). As these studies are the first to explore the response to H1ssF in newborn NHPs, we selected a dose (40 µg) previously administered to adult ferrets²², which are approximately the same weight as our newborn AGMs. As a non-vaccinated control, newborns received PBS or an mRNA lipid nanoparticle vaccine expressing luciferase (leveraged from another ongoing study). These animals are referred to as the control group (Ctrl) moving forward. All animals were challenged with H1N1 Ca09 (1 × 10⁷ TCID₅₀) at d41/45 p.b. to assess the ability of this vaccine to promote responses that can reduce viral load.

Newborn AGM vaccination and sampling schedule (A). Circulating levels of NC99 stem-specific IgM (B) and IgG (C) were assessed by ELISA. IgG reactivity was measured against group 1 and group 2 HA molecules in the plasma at d41/45 p.b. The stem-reactive mAb FI6V3 was included as a positive control (D). The ratio of the TT for each HA divided by that for NC99 HA was calculated as a measure of the efficiency of recognition of each HA molecule (E). Circulating levels of IgG that binds to full-length NC99 HA, H1-N27, H1-N45, or H1-N27/45 were assessed by ELISA at d41/45 p.b. The quantity of central and anchor region Abs was determined by subtracting the AUC for H1-27/45 from that of H1-27 (central) and H1-N45 (anchor) (F). NC99 stem-specific IgG avidity was measured by determining the concentration of NaSCN that resulted in a 50% reduction in optical absorbance compared to the untreated sample (G). The dotted line represents the LOD. TT was defined as the highest dilution resulting in an OD₄₅₀ greater than 3X assay background. Ctrl (n = 5), H1ssF (n = 4), H1ssF + AddaVax (n = 8). Symbols represent individual animals and are maintained throughout the datasets. The line in each column represents the median (F, G). Statistical analysis: two-way ANOVA with Tukey’s post hoc analysis (B–D, G), a one-way ANOVA with a Dunnet’s (H1 NC99 is the control) (E), or a two-sided paired t test (F). Statistics represent H1ssF + AddaVax vs. Ctrl. (Fig. 3B, C, and D). Ctrl vs. H1ssF was not significant. Statistical analysis in Fig. 3E is for each HA vs. WT NC99 HA. Not significant p = >0.05 (not included on graph). Figure 3A was created in BioRender. Alexander-Miller, M. (2025) https://BioRender.com/vqpx526. Source data are provided as a Source Data file.

Infants were monitored for 15 minutes following vaccination; no sign of adverse reaction was noted during this period. At 24 h p.v., infants were assessed for changes at the vaccination site as well as general health characteristics (e.g., alert, latching on to mothers). No vaccine-induced changes were observed. As a measure of systemic inflammation, we assessed C-reactive protein (CRP) on d1 p.v. A CRP level lower than 10 mg/L is considered low, with levels greater than 10 mg/L indicating an overly robust inflammatory reaction⁴². All animals were below 10 mg/L (median = 0.82 mg/L, Supplementary Fig. S4A). There was no statistically significant difference in the CRP levels between the vaccine groups. We also measured rectal temperatures on d1 p.v. All were within the normal range (Supplementary Fig. S4B). There was also no difference in weight gain across the groups over the course of the experiment (Supplementary Fig. S4C). These data suggest that the vaccine does not induce adverse systemic reactions.

Newborns receiving H1ssF + AddaVax exhibited a significant increase in the quantity of HA stem-specific IgM (Fig. 3B) and IgG (Fig. 3C) at all-time points following vaccination compared to Ctrl animals. Newborns administered H1ssF in the absence of adjuvant were not statistically significant from Ctrl newborns. Interestingly, in 4 of 8 H1ssF + AddaVax vaccinated newborns, we found detectable stem-specific IgG responses as early as d10 p.v. Interestingly, as was observed in adults, we found increased levels of stem-specific IgA in the plasma in the H1ssF + AddaVax vaccinated animals compared to Ctrl and H1ssF vaccinated animals (Supplementary Fig. S1B).

Next, we determined the capacity of HA stem-specific Abs to recognise divergent subtypes of HA, assessing the time point at which the response was most mature in our experimental regimen, d41/45 p.b. (Fig. 3D). The H1ssF + AddaVax elicited Abs with the capacity for broad recognition against group 1 HAs (H1, H2, H5, and H9) when compared to Ctrl and H1ssF vaccinated newborns. Recognition of Ca09 H1 HA was strongest, with a titre similar to that for NC99, the stem used in the vaccine. Recognition of H1 from PR8, H5, and H9 (group 1) was robust, although reduced compared to NC99. The weakest recognition among the group 1 HAs was H2. Recognition of group 2 HA by Abs from the H1ssF+AddaVax newborns was not above Ctrl animals (Fig. 3D). Although not statistically significant as a group, there were select H1ssF + AddaVax animals that exhibited higher levels of H7 binding, suggesting their repertoire may allow recognition of this group 2 HA.

Given the outbred nature of the newborn AGMs, we were curious whether the binding pattern across the HA molecules differed among individual animals. To address this, we calculated the efficiency of recognition of each HA type vs. NC99 (threshold titre of HA X:threshold titre of HA NC99). This ratio allowed us to normalise for any differences in total HA stem-specific Abs among animals. We observed diverse binding patterns (Fig. 3E), with some infants binding H5 with high efficiency (e.g., 2661 (○)), while others more efficiently bound H9 (e.g., 2698 (□)). Together, these data show that the vaccine-elicited stem-specific Abs bind diverse group 1 HA subtypes; however, the efficiency of binding across the HA molecules in relation to the parent strain differed among animals. The divergent binding patterns suggest there may be differences among newborns in the quality of Abs generated, i.e., avidity, or differences in epitope specificity represented within the polyclonal response.

To test the latter, we used the glycan mutant HA molecules described above to quantify the relative response to the central and anchor epitopes at d41/45 p.b. Vaccinated newborns generated a strong Ab response to the central epitope compared to anchor or non-central/anchor epitopes (Fig. 3F, primary data Supplementary Fig. S5). We noted that the quantity of central stem Ab varied considerably among the H1ssF + AddaVax animals (Fig. 3F). In contrast to adult animals, the response to regions outside of the central or anchor was modest.

One mechanism that has been postulated to contribute to the subdominance of the stem response is steric hindrance in accessing this region of HA on the surface of the virion²¹. To understand the capacity of the Abs generated in response to vaccination with the stem region to bind HA on an intact virus, we assessed recognition of the purified PR8 virus (Supplementary Fig. S6A). Recognition of the PR8 virus correlated with that of PR8 HA (r = 0.86, r² = 0.74, p = 0.006) (Supplementary Fig. S6B). This finding supports the ability of Abs elicited by H1ssF to recognise HA on the influenza virion.

We also assessed the capacity of stem-specific Abs elicited by H1ssF to recognise drifted H1 variants using the 2023-2024 Fluzone quadrivalent vaccine containing A/Victoria/4897/2022 (H1N1), A/Darwin/9/2021 (H3N2), B/Phuket/3073/2013 (B Yamagata lineage), and B/Michigan/01/2021 (B Victoria lineage). We found significant binding to the Fluzone vaccine in plasma from H1ssF + AddaVax vaccinated newborns compared to the H1ssF and Ctrl animals (Supplementary Fig. S6C). A strong correlation was observed between Fluzone-specific IgG and NC99 stem-specific IgG in H1ssF + AddaVax newborns (Supplementary Fig. S6D). These data show the vaccine-elicited stem-specific IgG is capable of recognising modern HA molecules. We would predict this is via binding to H1 in the vaccine, based on our finding of limited group 2 recognition by these Abs. As in adults, there was no indication of a vaccine-mediated induction of ANA-specific IgM or IgG in newborn AGM (Supplementary Fig. S7).

Finally, we measured the avidity of HA stem-specific IgG over time in newborns that received H1ssF+AddaVax (Fig. 3G). Boosting resulted in increased avidity, with a rapid rise in HA stem-specific Ab avidity by d10/11 p.b. Overall, these data demonstrate vaccination of newborn AGMs with H1ssF + AddaVax results in uniformly high levels of HA stem Ab. These Abs are broadly reactive, including recognition of modern strains, and can bind the stem region of HA on a virion.

Vaccination with H1ssF + AddaVax results in reduced virus following Ca09 challenge in a subset of newborns

To evaluate the capacity for H1ssF + AddaVax to control infection in the newborn AGMs, we challenged animals with H1N1 Ca09 (1 × 10⁷ TCID₅₀) at d41/45 p.b. Viral load was measured by RT-PCR in the lung at d7 p.c. A significantly lower level of virus was found in the lungs of animals vaccinated with H1ssF + AddaVax compared to H1ssF alone, with two animals exhibiting markedly superior clearance, 2661 (○) and 2698 (□) (Fig. 4A). No significant difference was observed between Ctrl and H1ssF animals.

Newborn AGMs were challenged with 1×10⁷ TCID₅₀ of Ca09 H1N1 at d41/45 p.b. Viral genomes were measured in the BAL at d7p.c. (A). IL-6 levels were measured in the BAL at d7 p.c. (B). NC99 stem-specific IgG (C) and IgA (D) were assessed in the BAL at d7 p.c. in newborn AGM. The dotted line represents the LOD for the assay. The line in each column represents the median. Statistical significance was determined by using a one-way ANOVA with pairwise comparisons between groups (A), a non-parametric Wilcoxon rank scores with Kruskal-Wallis analysis (B), or a one-way ANOVA with Tukey’s post hoc analysis (C, D). Not significant p ≥ 0.05 (not indicated on graph). Source data are provided as a Source Data file.

A strong inflammatory environment in the lung following influenza virus infection is associated with increased disease⁴³. In children with influenza, a higher level of circulating IL-6 is correlated with greater disease severity⁴⁴. In our study, newborns that received H1ssF+AddaVax had significantly lower IL-6 compared to both H1ssF and Ctrl animals (Fig. 4B). Overall, these data show that H1ssF+AddaVax vaccination resulted in lower viral load and lower levels of IL-6 in newborn AGM. Further, they suggest that, although the vaccine induced high levels of Ab in all newborns, there were differences in the ability of the vaccine response to promote viral clearance in the lung.

The level of HA stem-specific Ab in the lung does not predict viral load in newborn AGMs

One possibility to explain the differences in clearance was the level of stem-specific IgG present in the respiratory tract. Although vaccination resulted in higher levels of stem-specific IgG in the BAL, this was not the determinant of viral clearance, as not all animals that had higher levels of Ab had lower viral loads (Fig. 4C). IgA was not detectable in the BAL at this time (Fig. 4D). While it is possible that IgA is present at a level that is below the limit of detection in our assay, it seems unlikely that such a level would be sufficient for the clearance observed in the subset of newborns.

Reduced viral load in the lower respiratory tract of newborn AGMs is correlated with Ab function

Given the lack of correlation between Ab quantity and viral clearance in the newborns, we evaluated the functional attributes of the HA stem-specific Abs generated in response to H1ssF + AddaVax. This has not, to our knowledge, been explored in newborns. Neutralising Abs against the vaccine matched NC99 virus were present at d41/45 p.b in 5 of 8 newborns that received H1ssF + AddaVax (Fig. 5A). Three newborns had detectable titres to Ca09 (Fig. 5B) and 2 to H5N1 (Fig. 5C). Importantly, the 2 newborns with the highest neutralising titres across all three viruses (2661 (○) and 2698 (□)) also had the lowest viral titres in the BAL at d7 p.c. (Fig. 4A).

Neutralising Ab was measured using a reporter-based microneutralization (MN) assay at d1 p.v., d41/45 p.b., and d7 p.c. Neutralising IC₈₀ titres against H1N1 A/New Caledonia/20/1999 (A), H1N1 A/California/07/2009 (B), and H5N1 A/Vietnam/1203/04 (C) were quantified. A Spearman’s correlation analysis was performed on A/New Caledonia/20/1999 nAb IC₈₀ titre from d41/45 p.b and the NC99 central epitope IgG AUC from d41/45 p.b in H1ssF + AddaVax newborns (D). A Spearman’s correlation analysis was performed on A/New Caledonia/20/1999 nAb IC₈₀ titre and viral genomes/mL in the BAL at d7 p.c. (E). Ctrl (n = 5), H1ssF (n = 4), H1ssF+AddaVax (n = 8). The dotted line represents the limit of detection (LOD) for the assay. The line in each column represents the median. Statistical significance was determined using a one-way ANOVA with a Fisher’s LSD post hoc analysis. Not significant p ≥ 0.05 (not indicated on graph). Source data are provided as a Source Data file.

Neutralising Abs against all three strains increased following challenge in H1ssF + AddaVax vaccinated infants (Fig. 5A–C). Although there may be some HA head directed nAbs in the post challenge response, we expect the majority of the nAb in the H1ssF + AddaVax animals is a stem induced vaccine response, as 7 out of 8 animals are capable of neutralising heterosubtypic H5N1 (Fig. 5C). Interestingly, when we compared the level of central binding IgG Abs at d41/45 p.b. against NC99 nAb d41/45 p.b. (Fig. 5D), we observed a positive correlation (r = 0.94, p = 0.0012), consistent with a model where higher levels of central binding stem specific Abs contribute to higher nAb titres. We performed another correlation analysis plotting nAb to NC99 present at d7 p.c. against viral load in the BAL for all newborns, finding higher nAb against NC99 was also associated with lower viral load (r = − 0.68, p = 0.0035) (Fig. 5E). These data are consistent with a role for nAbs as a component of viral clearance in newborn AGMs. Newborns vaccinated with H1ssF in the absence of adjuvant and Ctrl animals had lower nAb titres against H1 Ca09 at d7 p.c., while the presence of nAb was variable for H1 NC99 and absent for H5 Viet04 (Fig. 5A–C).

Lastly, we explored the ability of these Abs to promote ADCC, ADCD, and ADCP. Neither ADCC nor ADCD activity was detected in H1ssF+AddaVax newborns prior to challenge (d41/45 p.b.) (Fig. 6A and B). In contrast, ADCP activity was readily detected (Fig. 6C). Challenge resulted in a statistically significant increase in all three activities in newborns who had received H1ssF+AddaVax. Interestingly, ADCP was also robustly induced in naive newborns following challenge (Fig. 6C). Given the correlation between nAb and central epitope Abs, we probed the relationship between binding to the central epitope and ADCP, finding no significant correlation between these Abs and ADCP (Supplementary Fig. S8). We speculate that this is due to the ability of Ab binding to any site on the HA molecule to facilitate ADCP.

Plasma samples were assessed for Ab function at d41/45 p.b. and d7 p.c. in newborn AGM. ADCC activity was quantified using the percent of CD107a + KHYG-1 cells. Cells were sequentially gated as follows: FSC/SSC and viability staining, followed by CD107a positivity (A). ADCD scores were determined as follows: percent C3 + beads \(\times\) GMFI/1000 at a 1:5 dilution of plasma (B). ADCP scores were determined using the percent of bead-positive THP-1 cells X the geometric mean/1000 at a 1:800 dilution of plasma. THP-1 were gated through FSC/SSC and singlet gates prior to FITC bead+ analysis (C). A Pearson correlation analysis was performed on viral genomes/mL in the BAL at d7 p.c. versus d41/45 p.b. ADCP scores (D). Ctrl (n = 4), H1ssF (n = 4), H1ssF + AddaVax (n = 8)). The dotted line represents the limit of detection (LOD) for the assay. The line in each column represents the median. Statistical significance was determined using a one-way ANOVA with a Fisher’s LSD post hoc analysis. Not significant p ≥ 0.05 (not indicated on graph). Source data are provided as a Source Data file.

To evaluate the relationship between ADCP activity and viral load, we plotted the vaccine-induced ADCP scores at d41/45 p.b. against viral load in the BAL at d7 p.c. for each newborn. We found these two readouts were negatively correlated (r = − 0.76, r² = 0.58, p = 0.0006), with higher ADCP scores associated with lower viral load (Fig. 6D). Interestingly, the newborn that had the highest ADCP score pre-challenge (2661 (○)) also had the lowest viral load in the lower respiratory tract (Fig. 4C). Overall, these data support a model wherein neutralising capacity and ADCP activity can mediate influenza viral clearance in newborn AGMs.