Introduction

Influenza viruses cause significant health and economic harm through seasonal epidemics. Additionally, animal influenza viruses threaten food supplies and occasionally cross species barriers, causing disease and outbreaks in humans. The risk of emerging zoonotic influenza viruses with pandemic potential is exemplified by the extensive spread of clade 2.3.4.4b H5N1 avian influenza viruses in poultry and livestock, with sporadic cases of human illness1.

Current intramuscular influenza vaccines induce strain-specific systemic immune responses to the major surface glycoprotein, hemagglutinin (HA). These responses effectively prevent symptomatic illness when vaccines are well-matched to circulating strains but may be less effective at preventing infection[2](https://www.nature.com/articles/s41467-025-64686-3#ref-CR2 “White, E. B. et al. Influenza vaccine effectiveness against illness and asymptomatic infection in 2022-2023: a prospective cohort study. Clin. Infect. Dis. https://doi.org/10.1093/cid/ciae491

(2024).“),3. In contrast, mucosal vaccines, which stimulate immune responses at the site of infection, may provide superior protection against both viral shedding and transmission of influenza3. Recognizing this potential, public health organizations have advocated for improved mucosal vaccines, particularly those capable of broadening immunity against diverse influenza viruses[4](https://www.nature.com/articles/s41467-025-64686-3#ref-CR4 “Erbelding, E. J. et al. A universal influenza vaccine: the strategic plan for the National Institute of Allergy and Infectious Diseases. J. Infect. Dis. https://doi.org/10.1093/infdis/jiy103

(2018).“),5.

For traditional intramuscular influenza vaccines, a hemagglutination inhibition (HAI) titer ≥40 is considered an immune correlate of protection6 and serves as a standard for licensure of seasonal and pandemic influenza vaccines7,[8](https://www.nature.com/articles/s41467-025-64686-3#ref-CR8 “U.S. Food and Drug Administration. Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines, https://www.fda.gov/media/73706/download

(2007).“). In contrast, mucosal influenza vaccine development faces challenges due to the lack of established immune correlates of protection9. Only one mucosal influenza vaccine, a live-attenuated influenza vaccine (LAIV), is licensed in the United States10. This vaccine often does not meet the regulatory HAI standard[11](https://www.nature.com/articles/s41467-025-64686-3#ref-CR11 “Subbarao, K. Live attenuated cold-adapted influenza vaccines. Cold Spring Harb. Perspect. Med. 11, https://doi.org/10.1101/cshperspect.a038653

(2021).“), and its clinical development required extensive field trials to demonstrate efficacy[11](https://www.nature.com/articles/s41467-025-64686-3#ref-CR11 “Subbarao, K. Live attenuated cold-adapted influenza vaccines. Cold Spring Harb. Perspect. Med. 11, https://doi.org/10.1101/cshperspect.a038653

(2021).“). Developing mucosal vaccines for emerging pathogens like avian influenza faces even greater challenges. For instance, a 2007 clinical trial of an H5N1 LAIV failed to elicit HAI responses12; however, participants who later received an intramuscular H5N1 boost exhibited significant immune recall13, a pattern observed with other avian influenza LAIVs followed by intramuscular boosts14,15,16. For mucosal avian influenza vaccines, defining immune correlates of protection would be valuable in advancing products through clinical development.

To address these challenges, we conducted a phase I randomized controlled trial of an adjuvanted intranasal influenza A/H5 subtype vaccine. We assessed product safety and performed extensive immunologic analyses to explore markers of mucosal vaccine immune priming.

Results

Clinical trial design and participants

There were five vaccine groups. Three groups received clade 2.1 influenza A/H5 (A/Indonesia/05/2005) recombinant hemagglutinin glycoprotein (rH5)17 at one of three dose levels (25, 50, and 100 µg) combined with an oil-in-water nanoemulsion (NE) adjuvant (W805EC);18,19; one group received unadjuvanted rH5 (100 µg); and one group received placebo. Vaccines were administered intranasally on Days 1 and 29. Six months later (Day 197), all participants received a heterologous intramuscular boost with an unadjuvanted 90 µg dose of a licensed, inactivated clade 1 influenza A/H5N1 (A/Vietnam/1203/2004) vaccine (H5N1 IIV) (Sanofi Pasteur Inc, Swiftwater, PA, 2007).

The trial was conducted from July 7, 2022, through October 12, 2023. Forty healthy adults aged 18–45 years were enrolled and randomized, with eight participants each assigned to Group A (low-dose rH5-NE), Group B (medium-dose rH5-NE), Group C (high-dose rH5-NE), Group D (unadjuvanted high-dose rH5), and Group E (placebo) (Fig. 1).

Fig. 1: Consort diagram.

Forty participants were enrolled and received the first vaccination. The withdrawal of one participant (Group E) before the second vaccination was not related to any safety event. A second participant (Group D) left the study area after receiving the second vaccination and could not complete the trial. A third participant (Group E) did not receive the third vaccination and was lost to follow up. He did not respond to multiple inquiries from the study team, but his emergency contact informed the study team that the participant was well. Group A (maroon), Group B (orange), Group C (yellow), Group D (cyan), and Group E (gray) are represented by these standard colors in all figures.

Overall, 45% of participants were women, 18% were Black or African American, and 15% were of Hispanic ethnicity. Their mean age was 30.2 years. Participant demographic and baseline information by vaccine group are in Table 1.

Hemagglutination inhibition (HAI) responses

Baseline immunity by HAI Geometric Mean Titer (GMT) to rH5 (clade 2.1) and H5N1 IIV (clade 1) was low among all groups (GMT ≤ 5.5), and there were no significant increases on Days 57 or 197 after intranasal vaccinations (Fig. 2 panel A and Supplementary Table 1). Twenty-eight days after H5N1 IIV was administered (on Day 225), we observed significant GMT responses among Group A and Group C compared to baseline or to Day 57 (p < 0.05). Geometric mean fold rise (GMFR) to rH5 (clade 2.1) at Day 225 among the rH5-NE groups was 17.4 (95% CI 4.6, 66.0) for Group A, 3.7 (95% CI 0.82, 16.4) for Group B, and 14.7 (95% CI 10.1, 21.3) for Group C, while the GMFR at Day 225 remained low for comparator groups, 1.1 (95% CI 0.9, 1.4) for Group D and 1.6 (95% CI 0.5, 5.2) for Group E. The percentage of participants with seroconversion at Day 225 to rH5 (clade 2.1) was 87.5% (95% CI 47.3, 99.7) for Group A, 37.5% (95% CI 8.5, 75.5) for Group B, 100.0% (95% CI 63.1, 100.0) for Group C, 0.0% (95% CI 0.0, 41.0) for Group D, and 16.7% (95% CI 0.0, 64.1) for Group E.

Fig. 2: Hemagglutination inhibition, microneutralization, and surface plasmon resonance by vaccine strain and group.

Antibody responses to influenza A/H5N1 A/Indonesia/05/2005 (clade 2.1) and A/Vietnam/1203/2004 (clade 1) at baseline, Day 57 (28 days post-second intranasal vaccination), Day 197 (immediately before intramuscular H5N1 IIV boost), and Day 225 (28 days post intramuscular H5N1 IIV boost). Serum HAI (A) and microneutralization (B) titers are shown as individual results for each participant (n = 8 per group, except as per the consort diagram in Fig. 1), with lines connecting the geometric mean titers at each timepoint with 95% confidence intervals. C Individual Surface Plasmon Resonance (SPR) responses with connecting mean and standard deviations. HAI assays were also conducted with specimens from Day 204 (seven days post intramuscular H5N1 IIV boost, with samples available from n = 8 participants in group A and C, n = 7 in group B, and n = 6 in groups D, E). For the HAI assay, sera that were negative at the initial dilution were assigned a titer of 5. For the MN assay, sera that were negative at the initial dilution were assigned a titer of 10. Dotted lines in (A and B) show the 1:40 dilution. Low-dose (Group A), medium-dose (Group B), and high-dose (Group C) of rH5-NE are shown in maroon, orange, and yellow. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group E), are shown in cyan and gray.

Day 225 HAI GMTs responses were significantly higher than baseline or Day 57 (p < 0.05) for Group A and Group C against H5N1 IIV (clade 1), with the highest titers among the rH5-NE groups (Fig. 2 panel A and Supplementary Table 1). GMFR to H5N1 IIV (clade 1) at Day 225 was 20.7 (95% CI 10.4, 41.3) for Group A, 3.1 (95% CI 0.9, 11.0) for Group B, 10.4 (95% CI 3.9, 27.5) for Group C, 7.2 (95% CI 1.1, 48.6) for Group D, and 2.2 (95% CI 0.8, 6.5) for Group E. The percentage of participants with seroconversion at Day 225 to H5N1 IIV (clade 1) was 100.0% (95% CI 63.1, 100.0) for Group A, 37.5% (95% CI 8.5, 75.5) for Group B, 87.5% (95% CI 47.3, 99.7) for Group C, 57.1% (95% CI 18.4, 90.1) for Group D, and 33.3% (95% CI 4.3, 77.7) for Group E.

In a post hoc analysis, we conducted HAI to rH5 (clade 2.1) and H5N1 IIV (clade 1) at Day 204, seven days after the intramuscular H5N1 IIV administration (Fig. 2 panel A and Supplementary Table 1). The GMFR results at Day 204 were similar to the Day 225 values for both H5N1 clades, indicating a recall response rather than a primary immune response to the intramuscular vaccination.

Microneutralization (MN) responses

Similar to HAI results, we measured no increases in MN titers on Days 57 or 197 to either A/Indonesia/05/2005 (clade 2.1) or H5N1 A/Vietnam/1203/2004 (clade 1). However, significant increases in MN titers were measured at Day 225 compared to baseline and to Day 57 among the rH5-NE groups to both strains (Fig. 2 panel B and Supplementary Table 2). MN GMFR to A/Indonesia/05/2005 (clade 2.1) at Day 225 was 20.7 (95% CI 1.0, 106.7) for Group A, 14.7 (95% CI 5.7, 38.0) for Group B, 7.3 (95% CI 3.6, 15.1) for Group C, 1.1 (95% CI 0.9, 1.4) for Group D, and 1.0 (95% CI 1.0, 1.0) for Group E. The percentage of participants with seroconversion at Day 225 to A/Indonesia/05/2005 (clade 2.1) was 75% (95% CI 34.9, 96.8) for Group A, 87.5% (95% CI 47.3, 99.7) for Group B, 87.5% (95% CI 47.3, 99.7) for Group C, 0.0% (95% CI 0.0, 41.0) for Group D, and 0.0% (95% CI 0.0, 45.9) for Group E.

MN GMFR to H5N1 A/Vietnam/1203/2004 (clade 1) at Day 225 was 17.5 (95% CI 3.7, 83.2) for Group A, 6.2 (95% CI 2.6, 14.8) for Group B, 4.8 (95% CI 2.3, 10.0) for Group C, 3.6 (95% CI 0.7, 17.8) for Group D, and 1.3 (95% CI 0.9, 1.8) for Group E (Fig. 2 panel B and Supplementary Table 2). The percentage of participants with seroconversion at Day 225 to H5N1 A/Vietnam/1203/2004 (clade 1) was 87.5% (95% CI 47.3, 99.7) for Group A, 75% (95% CI 34.9, 96.8) for Group B, 75% (95% CI 34.9, 96.8) for Group C, 42.9% (95% CI 9.9, 81.6) for Group D, and 0.0% (95% CI 0.0, 45.9) for Group E.

In a post hoc analysis, we assessed the breadth of serum MN at Day 225 against a panel of additional H5N1 viruses, clade 2.2, clade 2.2.1, clade 2.3.4, and clade 2.3.4.4b (Fig. 3 and Supplementary Table 2). We only performed the MN assays against the panel of heterologous H5N1 strains if participants had a measurable MN titer against clade 2.1 and clade 1 after unblinding. In earlier studies, no background responses against this panel of H5N1 viruses were found in healthy U.S. adults20. rH5-NE containing vaccines elicited seroconversion against clade 2.2 (50.0%–62.5%), clade 2.2.1 (50.0%–62.5%), clade 2.3.4 (75.0%–87.5%), and clade 2.3.4.4b (75.0%–87.5%) viruses, in the reverse dose response relationship observed with MN assays against clade 2.1 and clade 1 strains. Group D seroconversion against the panel of viruses was generally lower than the rH5-NE groups.

Fig. 3: Microneutralization on Day 225 by A/H5N1 virus panel and group.

Sera from Day 225 (28 days post intramuscular H5N1 IIV boost) were tested by MN assay using a panel of the following viruses: A/Indonesia/5/2005 (clade 2.1), A/Vietnam/1194/2004 (clade 1), A/Anhui/1/2000 (clade 2.3.4), A/Egypt/3072/2010 (clade 2.2.1), A/Turkey/15/2006 (clade 2.2), and A/Wigeon/sc/22/2021 (clade 2.3.4.4b). Sera that were negative at the initial dilution were assigned a titer of 10. Values shown as individual results for each participant, with geometric mean titers indicated by horizontal bar with 95% confidence intervals. Samples from the placebo group were not tested against the broader panel due to low MN titers against the vaccine strains (ND, not done). Low-dose (Group A), medium-dose (Group B), and high-dose (Group C) of rH5-NE are shown in maroon, orange, and yellow. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group E), are shown in cyan and gray.

Serum immunoglobulin responses

We measured rH5 (clade 2.1)-specific serum IgG and IgA and H5 stalk-specific serum IgG by ELISA (Fig. 4 and Supplementary Table 3). At baseline, IgA and IgG levels to rH5 were low, while participants had high titers of anti-stalk IgG. Unlike with MN or HAI assays, significant GMT increases (p < 0.05) from baseline were observed for most immunoglobulins measured at Days 57 and 197 in the rH5-NE groups. Significant GMT increases were measured from Day 197 to Day 225 in all groups (p < 0.05) for IgG and IgA, though significant boost responses at this timepoint for H5 stalk-specific serum IgG were only measured in Group C and Group E (p < 0.05).

Fig. 4: H5N1 clade 2.1 serum and nasal wash binding antibodies by group.

A shows the H5 A/Indonesia (Clade 2.1)-specific serum IgG and IgA responses, as well as H5 stalk-specific IgG responses (EU/mL); each dot represents one individual, with summary values shown as geometric mean concentration with 95% confidence intervals. B shows individual nasal wash responses (IgG and IgA) as well as the median and interquartile range of the ratio of H5-specific IgG or IgA (EU/µg) to total IgG or IgA at each timepoint per group. Inset shows nasal wash responses on an extended y-axis. Low-dose (Group A), medium-dose (Group B), and high-dose (Group C) of rH5-NE are shown in maroon, orange, and yellow. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group E), are shown in cyan and gray.

Surface plasmon resonance (SPR) binding

We assessed the quality of humoral immune response using SPR-based real-time kinetics assay against both H5N1 clade 2.1 and clade 1 (Fig. 2 panel C and Supplementary Table 4). Antibody binding was significantly higher in the rH5-NE groups than in controls on Day 57 (p = 0.0058). After intramuscular H5N1 IIV, antibody binding increased in each group, with values significantly higher in the rH5-NE groups than in controls at Day 225 (p = 0.0003). Similar patterns were seen with SPR against H5N1 clade 2.1 and against H5N1 clade 1 rHAs, though responses were consistently higher against the rH5 vaccine clade 2.1.

Antibody-dependent cell-mediated cytotoxicity (ADCC) responses

ADCC was assessed against rH5 clade 2.1. We defined ADCC seroconversion as ≥fourfold increase from baseline. Note that in this report, antibody seroconversion is referred to as seroconversion, while ADCC seroconversion is referred to as ADCC seroconversion. We measured ADCC seroconversion in all rH5-NE groups at Days 57 and 225 (p < 0.05), while comparator groups developed ADCC responses only at Day 225 (Fig. 5 panel A and Supplementary Table 5). The percentage of participants with ADCC seroconversion at Day 57 was 50% (95% CI 11.8, 88.2) for Group A, 57% (95% CI 18.4, 90.1) for Group B, and 75% (95% CI 34.9, 96.8) for Group C. ADCC seroconversion at Day 225 was 100% (95% CI 54.1, 100) for Group A, 86% (95% CI 42.1, 99.6) for Group B, 75% (95% CI 34.9, 96.8) for Group C, 33% (95% CI 4.3, 77.7) of Group D, and 50% (95% CI 11.8, 88.2) for Group E.

Fig. 5: Antibody-dependent cell-mediated cytotoxicity (ADCC), memory B cell, and memory T cell responses by group.

A shows ADCC as fold-changes over Day 1. Blue text shows the percentage of volunteers that ADCC seroconverted (SC; ≥ fourfold rise; dotted line threshold). RLU data and stats reported in Supplementary Table 5. B shows the frequency of memory B cells producing anti-H5 IgG antibodies (SFU per 1 × 10e6 cells). Bars indicate median ± 95% CI. In (C and D) box (25th to 75th percentiles) and whiskers (minimum and maximum data points) plots display the frequency (net %) of memory CD4 T cells producing IL-2 (C) and IFN-γ (D) upon stimulation with an H5 peptide pool (A/Vietnam/1203/2004 (clade 1)). In plots A–D volunteers vaccinated with low-dose (Group A), medium-dose (Group B), and high-dose (Group C) of rH5-NE are shown in maroon, orange, and yellow. Unadjuvanted rH5 (Group D) and placebo (Group E) are shown in cyan and gray colors, respectively. E shows multifunctional (MF; IL-2+ & IFN-γ+) memory CD4 T cells (pooled Groups A–C) (white circles). IFN-γ-only and IL-2-only producing cells (Single Functional -SF- cells) are shown by the blue and grey circles, respectively. Bars indicate the median ± 95% CI. In (A–E), each dot represents one individual. In (B–E), the number of volunteers assessed at each timepoint (n) is shown in red font. F displays the frequency of MF and SF cells by Groups A–C at days 57 and 255. The data shown as percentage of the mean of MF and SF cells. Blue, gray, and white areas of the pie show IFN-γ-only, IL-2-only, and MF cells, respectively. Yellow semicircle shows the added percentage of SF cells (IL-2-only plus IFN-γ-only). Statistics from (B–E) derived from Wilcoxon signed-rank tests (2-sided) *p < 0.05, **p < 0.01, ***p < 0.005, **** p < 0.0001.

Mucosal immune responses

Nasal wash rH5-specific IgG normalized by total IgG was low at baseline for all groups (Fig. 4 panel B and Supplementary Table 6). Significant increases within groups were noted at Days 43, 57, and 197 compared to baseline for all rH5-NE groups (p < 0.05). At Day 225, significantly increased nasal IgG responses were measured for all groups compared to Day 197 (p < 0.05), with the highest responses in the rH5-NE groups.

Baseline levels of nasal rH5-specific IgA, normalized by total IgA, were low across all groups. However, significant increases were seen in Groups A and B on Days 43, 57, 197, and 225 compared to baseline (p < 0.05), but not for Groups C, D, or E (Fig. 4 panel B and Supplementary Table 6). A significant boost response in nasal IgA levels was seen on Day 225 compared to Day 197 in Groups A and B (*p *< 0.05).

Memory B cells

We assessed memory B cells with the potential to produce antibodies against rH5 (clade 2.1) in polyclonally expanded PBMC. Analysis of rH5-NE groups showed significant increases in vaccine-specific antibody-secreting cells (ASC) on Day 197 compared to baseline. Only Group C (high-dose rH5-NE) had a significant increase on Day 57 compared to baseline. rH5-NE groups B and C had significant increases in vaccine-specific ASCs after intramuscular H5N1 IIV on Days 57 and 197 compared to baseline (Fig. 5 panel B). The unadjuvanted rH5 group and the placebo group did not exhibit significant increases in H5-specific ASC from baseline at any subsequent timepoint.

T cell immunity

Activated (CD69+) memory CD4 T cells (CD4+, excluding CD45RA + CD62L+ cells) were assessed for their ability to produce cytokines or upregulate additional activation markers (e.g., CD154, CD137) after ex-vivo stimulation with rH5 (clade 2.1) (Supplementary Fig. 1panel A) and a H5 peptide pool (clade 1) (Fig. 5).

Peptide stimulations increased IL-2 expression significantly at Days 57, 197, and 225 compared to baseline among Groups A and B. Group C increased expression of this cytokine only after the intramuscular H5N1 IIV (Day 225) (Fig. 5panel C). IFN-γ was upregulated by all rH5-NE groups at Day 225. Only Group A had increased expression of this cytokine prior to intramuscular H5N1 IIV (Day 197) (Fig. 5 panel D). Memory CD4 T cells stimulated with the rH5 (clade 2.1) had IL-2 and IFN-γ responses of lower magnitude than those identified in cells stimulated with the H5 peptide pool (clade 1), but the trends were similar (Supplementary Fig. 1panels C-D). The comparator groups did not exhibit significant increases from baseline to Day 225 in memory CD4 T cells producing cytokines (Fig. 5 panels C and D), or in activation or degranulation markers.

Since memory CD4 T cells from rH5-NE groups stimulated with the clade 1 peptide pool had significant expression of IL-2 and IFN-γ, we assessed whether these cells had multifunctional activity, defined as production of more than one cytokine by the same cell. First, we performed these assessments in peptide-stimulated cells pooled from all rH5-NE groups (Fig. 5 panel E). We identified a significant increase in the frequency of multifunctional cells (IL-2+, IFN-γ+) on Days 57 and 197 compared to baseline. After intramuscular H5N1 IIV vaccination, the frequency of these cells increased significantly. Notably, IL-2+ single functional cells had the highest frequency at every timepoint after intranasal and intramuscular vaccination (Fig. 5 panel E). Intramuscular H5N1 IIV increased the frequency of multifunctional memory CD4 T cells in all the rH5-NE groups (shown as percentage of mean multifunctional and single function cells), but the change was highest in Group A and Group B (Fig. 5 panel F). For example, in Group A, the frequencies of multifunctional cells changed from 8.4% (95% CI 0, 27.1) at Day 57 to 15.5% (95% CI 5.8, 90) by Day 225. We identified no significant production of cytokines or upregulation of CD107a in memory CD8 T cells upon stimulation with H5N1 clade 2.1 or clade 1.

Safety

The rH5-NE vaccines were well tolerated. Among participants receiving the first intranasal vaccination, immediate (within 60 min) reactogenicity symptoms were common and mostly mild (Grade 1) (Fig. 6 and Supplementary Table 7). Solicited reactogenicity symptoms occurring in >5% of participants included runny nose (55.0%), postnasal drip (52.5%), stuffy nose (32.5%), sore throat (35.0%), and itchy nose (10.0%). Participants receiving rH5-NE containing vaccines experienced more local symptoms and three of four moderate-severity events (Grade 2). Immediate reactogenicity symptoms after the second intranasal dose in >5% of participants included runny nose (61.5%), postnasal drip (48.7%), sore throat (48.7%), stuffy nose (41.0%), itchy nose (5.1%), feverishness (5.1%), headache (5.1%), watery eyes (5.1%). Most symptoms occurred in rH5-NE groups, including one moderate-severity event (Grade 2). Solicited 7-day post-vaccination reactogenicity symptoms were mostly mild (Grade 1), remained more common in rH5-NE groups, and were similar to the immediate reactogenicity profiles.

Fig. 6: Solicited symptoms through 7 days after each intranasal vaccination.

All solicited events were mild or moderate. No reports of nosebleed, hearing difficulty, double vision, joint pain, ear ringing, slurred speech, eye swelling, chest tightness, or wheezing. Figure colors: immediate mild (light gray), immediate moderate (black), 7-day mild (pink), 7-day moderate (red).

Five related unsolicited adverse events within 1 h of vaccination were mild, including elevated diastolic blood pressure, toothache, sinus pain, and nasal discomfort (2 participants) (Supplementary Tables 8 and 9). Two solicited events (mild cough and postnasal drip) began during the 7-day post-vaccination period and extended beyond it, qualifying as unsolicited events. All adverse events were self-limited. There were two other related unsolicited adverse events (cough and upper-airway cough) within the 28-day post-vaccination period, and none of the related unsolicited adverse events was medically attended. No adverse events after either intranasal vaccination were of severe or higher severity.

Laboratory abnormalities within seven days post-dose 1- and 14-days post-dose 2 were mostly mild (Grade 1) (Supplementary Tables 8 and 10). Two moderate abnormalities (Grade 2) occurred after the first intranasal vaccination with no severe or higher severity events. There were no moderate or greater severity adverse events after the second intranasal vaccination.

The licensed H5N1 IIV was also well tolerated with few solicited adverse events and no related unsolicited events. Two participants had unrelated Grade 3 low hemoglobin after H5N1 IIV vaccination, assessed as due to study phlebotomy. No other moderate or greater severity events occurred after H5N1 IIV vaccination (Supplementary Tables 10 and 11).

Throughout the trial there were no potentially immune-mediated medical conditions, new onset chronic medical conditions, or serious adverse events (Supplementary Table 12).

Discussion

In this study, we evaluated the safety and immunogenicity of an intranasal adjuvanted recombinant influenza A/H5 vaccine. Aware of the absence of an immune correlate of protection for mucosal influenza vaccines, we performed extensive assessments of cell-mediated, humoral, and mucosal immune responses to evaluate vaccine immunogenicity. As shown in prior studies of avian influenza LAIV, vaccine-induced immune responses were evident only after a subsequent intramuscular inactivated vaccine boost13,14,16,21.

We therefore administered the intramuscular heterologous H5N1 IIV (A/Vietnam/1203/2004, clade 1) vaccine boost dose six months after the two intranasal rH5-NE doses (A/Indonesia/05/2005, clade 2.1) as an immune probe to potentially uncover vaccine priming. Like those previous studies, we observed low HAI and MN responses after the primary intranasal vaccination series but rapid, robust responses after intramuscular boost13,14,15,16,21. Additionally, the rH5-NE groups elicited high neutralizing antibody titers against diverse H5N1 virus clades.

The immune responses observed after the intramuscular H5N1 IIV boost indicate a strong recall response in groups that received the intranasal rH5-NE vaccines. Seroconversion rates following H5N1 IIV in the rH5-NE groups ranged from 38 to 100% for the H5N1 clade 1 virus, surpassing the 19–26% seroconversion rates after a single-dose of H5N1 IIV in a previous study[22](https://www.nature.com/articles/s41467-025-64686-3#ref-CR22 “Oshansky, C. M. et al. Safety and immunogenicity of influenza A(H5N1) vaccine stored up to twelve years in the national pre-pandemic influenza vaccine stockpile (NPIVS). Vaccine https://doi.org/10.1016/j.vaccine.2018.11.069

(2018).“). The rapid HAI responses detected seven days after the H5N1 IIV boost, along with differential immune responses at 28 days post-boost in the intranasal rH5-NE groups, support a recall response of cross-reactive memory B cells rather than a primary response to the intramuscular vaccination.

We observed a reverse dose response to the rH5-NE vaccine in several immune assays, a phenomenon previously noted in adjuvanted intramuscular vaccines for avian influenza23,24. This finding may suggest that increased antigen doses may activate extrafollicular B cells with lower affinity, which are not targeted to the germinal centers for further affinity maturation. Such B cells are more likely to produce non-neutralizing antibodies upon boosting25,26. An alternative possibility is that larger antigen doses with the NE adjuvant favor the activation of CD4 regulatory T cells (Tregs) over Effector Memory CD4 T cells, with activated Tregs suppressing immune responses27. Some of the CD4 T cell responses that we assessed showed a similar reverse dose response pattern, though it is unclear whether the frequencies of T follicular helper cells (essential for germinal center seeding and somatic hypermutation) would also follow this pattern. Of note, ADCC-mediating antibodies followed an increasing dose response, suggesting a disassociated evolution of antibodies with different functions.

The rH5-NE vaccine was well-tolerated at all doses. Mild local nasal symptoms were common, occurred more frequently in the adjuvanted vaccines than the comparators, and were often reported within 60 min of intranasal vaccination. While rH5-NE reactogenicity was minimal in preclinical studies and our trial, we remain cautious given the history of Bell’s palsy associated with an intranasal split-virus influenza vaccine adjuvanted with E. coliheat-labile toxin28. While the cause was likely related to the specific adjuvant29, which was more immunostimulatory than the nanoemulsion used in our trial, future studies of adjuvanted intranasal vaccines should monitor for this adverse event closely.

Our study suggests that the absence of HAI or MN responses following the primary intranasal series may reflect immunological compartmentalization, with priming potentially focused on the upper respiratory tract. We saw increases in both nasal IgG and IgA levels after the intranasal vaccination series, but low magnitude nasal IgA boost after H5N1 IIV, further supporting this notion. It remains unclear whether the nasal wash immunoglobulins originated from local production or were due, at least in part, to extravasation from the vasculature. Attempts to investigate this by isolating mononuclear cells from nasal wash samples for flow cytometry were unsuccessful due to insufficient cell yields.

Despite these limitations, immunogenicity signals elicited by the primary intranasal rH5-NE series, including significant serum and mucosal IgG and IgA responses, SPR binding antibodies, and ADCC activity, warrant further investigation. ADCC, in particular, has been historically underappreciated; however, recent studies have demonstrated that antibodies with ADCC capacity that target conserved regions of HA are cross-reactive and can be protective in adoptive transfer models30. Additionally, within the framework of the European Union’s Innovative Medicines Initiative-funded project FLUCOP, ADCC assays have been shown to be standardized, cost-effective, and viable as an alternative immune correlate of influenza protection31. In our study, robust ADCC activity was detected among all rH5-NE groups following intranasal vaccination, suggesting that ADCC assays could serve as a valuable tool for evaluating mucosal vaccine priming. However, this hypothesis requires confirmation in larger, future studies.

The separate rH5-NE vaccine components have been evaluated in human trials previously. A 2010 randomized trial evaluated the nanoemulsion adjuvant combined with seasonal inactivated influenza vaccine at doses of 4–10 μg in a single intranasal dose32. Compared to the approved intramuscular vaccine and an intranasal placebo, 28-day HAI seroconversion ranged from 0 to 25% for intranasal groups and 60–80% for the intramuscular group. Vaccine-specific IgA levels were similar across all groups. A 2011 trial of intramuscular rH5 in a dose-ranging study (15–90 μg, with and without Alhydrogel adjuvant) found 10% seroconversion after the second dose in the best-performing group17.

Given prior clinical experience with the nanoemulsion adjuvant and rH5 antigen, the intranasal rH5-NE formulation’s performance in our study is noteworthy. In the 2010 trial, serum HAI titers were not expected to correlate with intranasal vaccine performance, and prior exposure to seasonal influenza viruses likely confounded immune response assessments. For the intramuscular rH5 study, inactivated avian influenza vaccines are known to have low immunogenicity, often requiring high antigen doses or potent adjuvants to induce measurable HAI responses33. These studies highlight challenges in influenza vaccine development, including unclear immunogenicity measures of success for mucosal vaccines, confounding by pre-existing immunity, and challenges advancing novel H5N1 prevention technologies.

The influenza A/H5N1 strains in the rH5 antigen (A/Indonesia/05/2005, clade 2.1) and H5N1 IIV (A/Vietnam/1203/2004, clade 1) were isolated nearly 20 years before our trial. Since then, H5N1 viruses have evolved substantially, including the emergence of clade 2.3.4.4b, which has caused widespread infections in poultry, livestock, and over 50 sporadic human cases in North America in 2024 and a recent death34,[35](https://www.nature.com/articles/s41467-025-64686-3#ref-CR35 “Centers for Disease Control and Prevention. H5 Bird Flu: Current Situation. https://www.cdc.gov/bird-flu/situation-summary/index.html

(2024).“). To assess cross-protective potential, we performed MN assays against a panel of H5N1 strains, including the vaccine clades and clade 2.3.4.4b. The rH5-NE groups attained the highest neutralization titers and elicited cross-protective MN responses. Similar cross-protection against H5N1 clade 2.3.4.4b has been shown with licensed adjuvanted H5N1vaccines20. These findings suggest the rH5-NE vaccine has potential as a preventive intervention.

Given the design of our trial, we could not directly determine whether the breadth of immunological memory induced by the two intranasal rH5-NE doses was attributable to the adjuvant or the mucosal priming. However, a 2024 study demonstrated that individuals who received two doses of an intramuscular unadjuvanted H5N1 (A/Vietnam) vaccine had significantly less cross-reactivity against heterologous H5N8 (clade 2.3.4.4b) viruses compared with those who received two doses of an adjuvanted H5N1 (A/Indonesia) vaccine20. In our trial, intranasal vaccination with the unadjuvanted rH5 failed to induce strong memory B cells that could be recalled by the intramuscular boost. Thus, in the absence of an adjuvanted intramuscular priming dose followed by an adjuvanted intramuscular boost, we can only hypothesize that mucosal vaccination may contribute to immune priming, but a strong adjuvant is likely critical for successful priming. The nanoemulsion adjuvant’s ability to elicit strong memory immune responses at low antigen doses may reduce the required vaccine dose and help expand the available supply of avian influenza vaccines.

Our trial has notable strengths, including extensive immunological assessments of cell-mediated, humoral, and mucosal immune responses, with consistent patterns across varied readouts and signs of mucosal immune priming that warrant further investigation. The intramuscular H5N1 IIV vaccine used to probe rH5-NE priming revealed immunological priming that otherwise would not have been detected. This heterologous prime-boost approach, previously used in H5N1 trials, elicits broader neutralizing and anti-stalk antibody responses compared to homologous regimens36,37,38,39,40. The rH5-NE vaccine provides an excellent model for advancing mucosal immune priming research. It was well-tolerated, and the absence of pre-existing H5N1 immunity in the general population, simplified the interpretation of the observed immune responses