Safety and immunogenicity of an investigational mRNA-lipid nanoparticle-based monovalent influenza vaccine: Results from a phase 1, randomized, dose-escalation study

ABSTRACT

This first-in-human, randomized, controlled, phase 1 proof-of-principle study evaluated the safety, reactogenicity, and immunogenicity of an investigational mRNA-based monovalent influenza vaccine encoding influenza A/H1N1 hemagglutinin (FLUmHA). Younger adults (YA) aged 18–45 y received one dose of FLUmHA at one of 10 dose levels (0.5–100 µg, n = 24/25 per group) or licensed Flu Dresden-quadrivalent seasonal influenza vaccine (Flu D-QIV, n = 35) on day (D)1. Older adults (OA) aged 60–80 y received FLUmHA (18 µg, n = 32) or Flu D-QIV (n = 16). Reporting rates for solicited adverse events (AEs) occurring within 7 d post-vaccination generally increased with increasing FLUmHA dose levels, and were 62.5%-100% (severe: 0.0%-20.8%) in YA across FLUmHA dose levels versus 88.6% (severe: 2.9%) in Flu D-QIV-vaccinated YA, and 62.5% (severe: 0.0%) (FLUmHA) versus 56.3% (severe: 0.0%) (Flu D-QIV) in OA. Unsolicited AEs within 28 d post-vaccination were reported by 50.0%–70.8% of YA across FLUmHA dose levels versus 68.6% of Flu D-QIV-vaccinated YA, and by 43.8% (FLUmHA) versus 50.0% (Flu D-QIV) of OA. No safety concerns were identified. A/H1N1 hemagglutination inhibition titers increased from pre-vaccination to D22, with adjusted geometric mean increases (GMIs) of 6.2–36.7 across YA and OA groups; the observed response was dose-dependent and higher in FLUmHA (for doses > 1 µg) versus Flu D-QIV recipients. Titers decreased but remained above pre-vaccination levels at D183 (GMI: 2.9–14.0). Additionally, FLUmHA elicited a numerically higher hemagglutinin-specific CD4+ T-cell response (predominantly Th1 profile) than Flu D-QIV, both in YA and OA. These results support the progression to clinical development of a multivalent mRNA Flu vaccine candidate.

Trial registration: NCT05446740.

Plain Language Summary

What is the context?

• Seasonal flu is a common respiratory illness caused by influenza viruses. Most people only have mild symptoms, but flu can also be serious and lead to hospitalization and death.

• Flu vaccines are updated each year to match the most common influenza virus strains expected to be present that year. Because influenza viruses change easily, some years the vaccines do not work as well when strains chosen for the vaccine are a poor match with the circulating strains. Moreover, flu vaccines do not always work well in older adults.

• Investigational flu vaccines that use messenger RNA (mRNA) technology have been shown to cause a robust and durable immune response, also in older adults. In addition, the production of mRNA vaccines is flexible and scalable, and the vaccines contain the virus’s exact genetic code, avoiding the changes that can occur in traditional vaccines grown in eggs. This may lead to a better match between the vaccine and the influenza viruses circulating during the season.

What is new?

• We did a study to collect data on the safety and immune response of an investigational mRNA Flu vaccine targeting one flu strain.

• The study included 276 adults aged 18–45 y and 48 adults aged 60–80 y. Participants received either the mRNA Flu vaccine or a licensed flu vaccine as control. The younger participants receiving mRNA Flu were divided in 10 groups, each getting a different dose of the vaccine. The older participants received one specific dose.

• We found that side effects after mRNA Flu vaccination were mostly mild or moderate and were short-lived, even at the highest vaccine doses. Only a few serious adverse events occurred, and these were not related to the vaccine.

• The mRNA Flu vaccine induced an immune response against the flu strain targeted by the vaccine. The immune response was higher than with the control vaccine for most doses and increased with higher doses.

What is the impact?

• The safety data and robust and durable immune response of the tested vaccine support further clinical development of an mRNA Flu vaccine targeting multiple flu strains.

Introduction

Every year, seasonal influenza poses a significant burden on global health, causing an estimated 3.2–9.5 million hospitalizations^1,2 and 290,000– 650,000 respiratory deaths.³ Young children, older adults, and individuals with underlying health conditions are at increased risk of severe influenza, but a substantial clinical burden also exists in otherwise healthy younger adults.^1–5

The World Health Organization (WHO) recommends annual vaccination as the most effective way of preventing influenza and its complications.⁶ Two influenza types (A and B) are responsible for the yearly seasonal epidemics in humans.⁷ Because influenza viruses are prone to genetic mutations that change their antigenicity, vaccines must be reformulated and readministered each year.^4,8 Twice a year, the WHO provides recommendations for the influenza strains to be included in the Northern and Southern Hemisphere vaccines.⁹ Currently, the majority of influenza vaccines are manufactured using egg production platforms, with a smaller proportion produced in mammalian cell culture or using recombinant protein technology.^4,6,8 The effectiveness of the available influenza vaccines fluctuates from year to year and can be low when vaccine and circulating strains are mismatched.^10–13 In addition, host factors, such as immunosenescence at older age, can reduce vaccine immunogenicity and effectiveness.¹⁴

Vaccine platforms based on messenger RNA (mRNA) have the potential to overcome some of the limitations of current seasonal influenza vaccines.^12,14 Investigational vaccines containing mRNAs that encode the hemagglutinin proteins of seasonal influenza strains have been shown to elicit robust hemagglutination inhibition (HI) titers, particularly against influenza A strains, including in older adults.^15,16 The mRNA manufacturing process is flexible and scalable, and mRNA vaccines show a high antigen fidelity by encoding the exact viral sequences of the target antigens, thereby avoiding egg-adaptive mutations that can occur with egg-based production platforms and decreasing the risk of mismatch. Moreover, mRNA technology may allow for the inclusion of a greater number and variety of antigens, which may help broaden protection.^12,14 Additionally, the mRNA vaccine platform itself has the potential to induce strong cellular immune responses, which could improve effectiveness in older adults.^14,17

This first-in-human proof-of-principle study aimed to evaluate the safety, reactogenicity, and immunogenicity of an investigational lipid nanoparticle-encapsulated nucleoside-modified mRNA-based vaccine encoding hemagglutinin from a single influenza A/H1N1 strain (FLUmHA [monovalent HA]). The mRNA backbone was the same as that used for an mRNA COVID-19 vaccine (CV0501) that induced robust immune responses and had an acceptable safety profile over a dose range of 3 µg to 200 µg.¹⁸

Methods

Study design and participants

This randomized, controlled, phase 1, dose-escalation study (https://www.clinicaltrials.gov/study/NCT05446740) with an adaptive design was conducted from 9 August 2022 to 26 March 2024 at seven study sites (clinical research sites and academic hospitals): two in Belgium, two in Canada, and three in Spain (see Supplementary methods for a list of study sites). The study enrolled participants aged 18–45 y (“younger adults”) or 60–80 y (“older adults”) who were medically stable, as judged by the investigator, and had a body mass index of 18–32 kg/m². Individuals were excluded if they had a close contact (i.e., being within ~1.8 m (6 feet) for a cumulative total of ≥15 minutes over 24 hours)¹⁹ with a person with influenza or laboratory-confirmed SARS-CoV-2 infection within 7 d before vaccination or if they had received a seasonal influenza vaccine within 180 d before enrollment or planned to receive one up to day 29 of the study. Detailed inclusion and exclusion criteria are provided in the Supplementary methods. The younger adults received a single dose of either the investigational FLUmHA at one of 10 different dose levels (0.5, 1, 2, 6, 9, 12, 18, 36, 54, or 100 µg) or the licensed Flu Dresden-quadrivalent seasonal influenza vaccine (Flu D-QIV, α-RIX-Tetra/Fluarix-Tetra, GSK) as active control on day 1 (Figure 1). Enrollment occurred before, during, or after the influenza seasons depending on the dose level (Supplementary methods) and was staggered within and between dose levels to allow safety monitoring of the participants by an internal safety review team (SRT). The SRT continuously assessed whether any protocol-defined pausing rules applied and determined whether, after the initial participants (referred to as sentinel participants), additional participants could be vaccinated at the same dose level, as well as whether the next dose level could be initiated. The 0.5 and 1 µg, and the 9 and 12 µg groups, which were added in amendments to the protocol after the 54 µg dose level was administered, were enrolled in parallel, without sentinel participants. The older adults received either a single 18 µg dose of FLUmHA or a single dose of Flu D-QIV as active control on day 1 (Figure 1). The 18 μg dose for older adults was selected following SRT review of the safety data obtained from younger adults after they had received the 2, 6, and 18 µg dose levels. Enrollment for older adults was staggered, starting with sentinel participants with a maximum age of 70 y. If a protocol-defined pausing rule was triggered, vaccine administration was paused for all study groups while the SRT and GSK’s Global Safety Board evaluated the cases. Further details on the oversight of the participants are included in the Supplementary methods.

Figure 1.

Study design.

The contact on D2 was for sentinel participants only (who were included for all groups except the 0.5, 1, 9, and 12 µg groups). Blood samples for humoral immunogenicity assessments were collected from all participants on D1, D22, D62, and D183, except on D62 and D183 for the 100 μg group and its control. Blood samples for cell-mediated immunity assessments were collected from a subset of participants from each group (except the 100 µg group and its control) on D1, D8, D62, and D183. FLUmHA, groups with participants who received the investigational mRNA-based monovalent H1 influenza vaccine at different dose levels; Flu D-QIV, control groups with participants who received the Flu Dresden-quadrivalent seasonal influenza vaccine; D, day; AE, adverse event; SAE, serious adverse event; AESI, adverse event of special interest. Detailed information on staggered enrollment and dose escalation is provided in the Supplementary methods.

Participants were followed up for 6 months after vaccination (day 1 to day 183), with study contacts or visits planned on days 1, 2 (for sentinel participants only), 3, 8, 22, 29, 62, and 183 (Figure 1). Handling of missing or unscheduled visits is described in the Supplementary methods.

The protocol, its amendments, and other relevant study-related documents were approved by the study sites’ independent ethics committees or institutional review boards (Supplementary methods). The study was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice guidelines, ethics guidelines by the Council for International Organizations of Medical Sciences, and all applicable laws and regulations. All participants signed an informed consent form before any study-specific procedure was performed.

Randomization and blinding

Younger adults were randomized to receive either one of the FLUmHA dose levels (2, 6, 18, 36, 54, and 100 µg) or Flu D-QIV active control. For the other dose levels, participants were randomized to receive either the 0.5 or 1 µg dose or either the 9 or 12 μg dose. Older adults were randomized to receive FLUmHA (18 µg) or Flu D-QIV. A justification for the selected dose levels is included in the Supplementary methods. A centralized, automated internet-based system was used for randomization and identification of the study intervention. The randomization algorithm accounted for sentinel, age, and dose level as stratification factors and study, country, site, and influenza vaccination history during the past 2 y as minimization factors.

The study was observer-blind with respect to the administered vaccine (i.e., participants and site personnel involved in the clinical evaluation of participants were blinded, but not the sponsor and study personnel that was involved in vaccine handling); it was open-label with respect to the dose level. For the 0.5, 1, 9, and 12 μg groups, the study was single-blind (i.e., only participants were blinded). The laboratories in charge of sample testing were blinded to the study interventions.

Vaccines

FLUmHA consisted of a sequence-optimized, capped, polyadenylated, and base-substituted mRNA encoding full-length influenza hemagglutinin antigen from the A/Wisconsin/588/2019 (H1N1)pdm09 virus (based on the WHO recommendations for recombinant vaccines for Northern Hemisphere season 2021–2022),⁹ encapsulated in a lipid nanoparticle delivery system (composed of a cationic lipid, PEGylated lipid, sterol lipid, and phospholipid).¹⁸ The vaccine uses a backbone with non-coding regions optimized to improve intracellular mRNA stability and translation, leading to increased protein expression and enhanced humoral, T-cell, and memory B-cell responses.^20,21 Flu D-QIV contained 15 µg hemagglutinin each of two influenza A-like viruses and two influenza B-like viruses. Viruses were those recommended by the WHO⁹ for the 2021–2022 Northern Hemisphere influenza season (A/Victoria/2570/2019 [H1N1]pdm09, A/Tasmania/503/2020 [H3N2], B/Washington/02/2019 [Victoria], and B/Phuket/3073/2013 [Yamagata]) in Flu D-QIV used as control for the 2, 6, 18, 36, and 54 μg FLUmHA groups, and for the 2022–2023 Northern Hemisphere season (A/Victoria/2570/2019 [H1N1], A/Darwin/06/2021 [H3N2], B/Austria/1359417/2021 [Victoria], B/Phuket/3073/2013 [Yamagata]) as control for the 100 μg FLUmHA group. Vaccines were administered by injection in the deltoid muscle.

Objectives

The primary objectives were to evaluate the safety and reactogenicity of the different dose levels (in terms of solicited and unsolicited adverse events [AEs], serious AEs [SAEs], AEs of special interest [AESIs], and laboratory parameters) and the humoral immune response (in terms of A/H1N1 HI titers at days 1 and 22). The secondary objective was to evaluate the humoral immune response in terms of A/H1N1 HI titers at days 62 and 183. Tertiary objectives included an evaluation of the cell-mediated immunity (CMI). Detailed endpoints are included in the Supplementary methods.

Safety assessments

Participants used electronic diaries to record information on solicited administration-site and systemic AEs occurring within 7 d post-vaccination. Unsolicited AEs occurring within 28 d post-vaccination, and SAEs and AESIs occurring within 6 months post-vaccination were reported (Figure 1); AESIs included potential immune-mediated diseases, severe hypersensitivity reactions within 24 hours post-vaccination, and myocarditis/pericarditis. Information on the AEs was collected during interviews with the participants and through review of their medical records, and was recorded in electronic case report forms. Hematology, clinical chemistry, coagulation, and urine analyses were performed on blood and urine samples collected on days 1, 8, and 29 (Figure 1). The intensity of solicited AEs was graded by the participants on a scale of 1 (mild) to 3 (severe). The intensity of other AEs, as well as their causal relationship to vaccination, was assessed by the investigators based on their clinical judgment.

Immunogenicity assessments

Blood samples for humoral immunogenicity assessments were collected from all participants on day 1 (pre-vaccination), and days 22, 62, and 183; no blood sampling was performed on days 62 and 183 for the 100 μg group and its control (Figure 1). Blood samples for CMI analyses were collected from the participants in the CMI subset (i.e., 50% of participants in each group at selected sites [Supplementary methods], except the 100 µg group and its control) on days 1, 8, 62, and 183 (Figure 1). Blood sampling on days 62 and 183 was not done in participants who had received a standard-of-care seasonal influenza vaccination prior to day 62 and did not report any AEs that required physical examination on site.

The humoral immune response to FLUmHA and Flu D-QIV was measured on serum samples using an HI assay with the influenza A/Victoria/2570/2019 (H1N1) strain (Supplementary methods).²² The lower limit of quantification (LLOQ) was 10 1/dilution (dil). An HI titer of ≥40 1/dil has been considered as a surrogate marker for protection against influenza.^23,24

CMI was measured on peripheral blood mononuclear cells after in vitro stimulation with hemagglutinin peptides. Hemagglutinin-specific CD4+ and CD8+ T cells expressing different combinations of activation markers were measured using flow cytometry after intracellular cytokine staining. Frequencies of polypositive CD4+ and CD8+ T cells (i.e., expressing at least two activation markers, including at least one cytokine, among CD40 ligand [CD154], 4-1BB [CD137], interleukin [IL]-2, tumor necrosis factor-α, interferon-γ [IFN-γ], IL-13, or IL-17) were calculated. To characterize the CD4+ T-cell profile, the frequencies of CD4+ T cells expressing at least IFN-γ (indicating a Th1 profile), IL-13 (Th2 profile), or IL-17 (Th17 profile) were calculated. Hemagglutinin-specific immunoglobulin G-producing memory B cells were measured using an enzyme-linked immunospot assay (Supplementary methods).

Statistical analyses

The planned sample size was 24 participants per FLUmHA group (Supplementary methods). All analyses were descriptive and were performed using SAS software version 9.4 or later (SAS Institute, Cary, NC, USA). No adjustment for type 1 error was done. Data from the Flu D-QIV control groups for the different dose levels in younger adults were pooled.

Safety endpoints were analyzed in the exposed set (i.e., all participants who received FLUmHA or Flu D-QIV). The percentages of participants with solicited AEs, unsolicited AEs, SAEs, and AESIs occurring within the aforementioned follow-up periods were calculated with exact 95% confidence intervals (CIs). The duration of each solicited AE was summarized. Percentages of participants who showed a shift from a normal/missing laboratory value (i.e., a laboratory value within the normal range or a laboratory value missing) or from a non-clinically significant abnormal laboratory value to a clinically significant abnormal laboratory value between day 1 and day 8 or 29 for hematology, clinical chemistry, coagulation, and urine analysis were calculated.

Immunogenicity endpoints were evaluated in the per-protocol set (i.e., all participants who received FLUmHA or Flu D-QIV per protocol, had HI immunogenicity results pre- and post-vaccination, had no influenza illness, and had not received any prohibited concomitant medications or vaccinations before day 22). Adjusted HI geometric mean titers (GMTs) and adjusted geometric mean increases (GMIs, i.e., geometric means of the ratios of the post-vaccination over the pre-vaccination HI titers) with 95% CIs were derived from an analysis of covariance model on log₁₀-transformed titers. The model included study group, country, influenza vaccination history, and log₁₀-transformed pre-vaccination titer as fixed effects. To compare HI titers between the FLUmHA groups and Flu D-QIV control, the two-sided CIs for the adjusted GMT ratios (FLUmHA group over Flu D-QIV group [pooled for analysis in younger adults]) were obtained from the same model. Titers below the LLOQ were replaced by half of the LLOQ. Seroprotection rates (i.e., the percentage of participants with an HI titer ≥40 1/dil) and seroconversion rates (i.e., the percentage of participants with at least a fourfold increase in HI titer post- versus pre-vaccination if the pre-vaccination titer was ≥10 1/dil or with a post-vaccination titer ≥40 1/dil if the pre-vaccination titer was < 10 1/dil) were calculated with exact 95% CIs. The frequency (geometric mean and median with range and interquartile range) of hemagglutinin-specific CD4+/CD8+ T cells per 10⁶ CD4+/CD8+ T cells and the frequency of hemagglutinin-specific memory B cells per 10⁶ memory B cells were calculated in the per-protocol set of the CMI subset.

Results

Study participants

A total of 276 younger adults and 48 older adults were enrolled and vaccinated; 273 (98.9%) and 48 (100%), respectively, completed the 6 months of follow-up. The per-protocol set included 268 (97.1%) younger adults and 45 (93.8%) older adults (Figure 2).

Figure 2.

Enrollment, randomization, analysis sets, and follow-up.

In total, 396 younger adults were screened, of whom 120 were not randomized (65 were not eligible, 11 decided not to participate, 10 could not participate because of site termination, and 34 for other reasons); 89 older adults were screened, of whom 41 were not randomized (26 were not eligible, 2 decided not to participate, and 13 did not participate for other reasons). N, number of participants per group; FLUmHA, groups with participants who received the investigational mRNA-based monovalent H1 influenza vaccine at different dose levels; Flu D-QIV, control groups with participants who received the Flu Dresden-quadrivalent seasonal influenza vaccine; for younger adults, the different Flu D-QIV groups (randomized as controls for the FLUmHA 2, 6, 18, 36, 54, and 100 µg dose level groups) are represented as a single group on the figure because data from these groups were pooled for all analyses.

Baseline characteristics were generally balanced between groups. The mean age ranged between 27.8 and 35.8 y across the 11 younger adult groups and was 66.8 and 67.9 y for the two older adult groups. Across groups, 84.0%–100% of younger adults and 93.8%–100% of older adults were white, and 45.8%–80.0% and 31.3%–62.5%, respectively, were female (Table 1).

Table 1.

Baseline demographic characteristics of the study participants (exposed set).

Characteristic	Younger adults (18–45 y)											Older adults (60–80 y)
	FLUmHA										Flu D-QIV	FLUmHA	Flu D-QIV
	0.5 µg N = 24	1 µg N = 24	2 µg N = 24	6 µg N = 24	9 µg N = 24	12 µg N = 24	18 µg N = 24	36 µg N = 24	54 µg N = 25	100 µg N = 24	N = 35	18 µg N = 32	N = 16
Mean (SD) age, years	30.2 (8.5)	32.5 (7.6)	34.6 (7.3)	35.7 (7.8)	30.5 (9.5)	30.7 (8.1)	29.8 (7.6)	35.8 (7.1)	27.8 (5.7)	29.1 (5.8)	32.0 (7.8)	66.8 (4.1)	67.9 (4.6)
Female sex, n (%)	17 (70.8)	16 (66.7)	11 (45.8)	15 (62.5)	15 (62.5)	11 (45.8)	15 (62.5)	14 (58.3)	20 (80.0)	11 (45.8)	22 (62.9)	20 (62.5)	5 (31.3)
Race, n (%)
White	24 (100)	24 (100)	23 (95.8)	24 (100)	24 (100)	22 (91.7)	24 (100)	24 (100)	21 (84.0)	24 (100)	35 (100)	32 (100)	15 (93.8)
Asian	0 (0.0)	0 (0.0)	1 (4.2)	0 (0.0)	0 (0.0)	2 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Black	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Othera	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (12.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (6.3)
Country, n (%)
Belgium	24 (100)	24 (100)	16 (66.7)	17 (70.8)	22 (91.7)	23 (95.8)	14 (58.3)	6 (25.0)	0 (0.0)	0 (0.0)	18 (51.4)	26 (81.3)	10 (62.5)
Canada	0 (0.0)	0 (0.0)	8 (33.3)	7 (29.2)	2 (8.3)	1 (4.2)	10 (41.7)	10 (41.7)	7 (28.0)	0 (0.0)	7 (20.0)	0 (0.0)	0 (0.0)
Spain	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	8 (33.3)	18 (72.0)	24 (100)	10 (28.6)	6 (18.8)	6 (37.5)

FLUmHA, groups with participants who received the investigational mRNA-based monovalent H1 influenza vaccine at different dose levels; Flu D-QIV, control groups with participants who received the Flu Dresden-quadrivalent seasonal influenza vaccine; N, number of participants per group; SD, standard deviation; n (%), number (percentage) of participants with the indicated characteristic. ^aAmerican Indian or Alaska native.

Safety

No safety signals were identified in the study. Among younger adults, the reporting rates of solicited AEs occurring within 7 d post-vaccination generally increased with increasing FLUmHA dose levels, ranging from 62.5% (0.5 and 6 µg) to 100% (54 and 100 µg). This compared to a rate of 88.6% after administration of the Flu D-QIV control (Figure 3). No grade 3 solicited AEs were reported after FLUmHA administration for the six groups up to a dose of 12 µg. The reporting rates for grade 3 solicited AEs for the higher dose levels were 12.5% (18 µg), 4.2% (36 µg), 0.0% (54 µg), and 20.8% (100 µg), compared to 2.9% for Flu D-QIV (Figure 3). Pain was the most common solicited administration-site AE across younger adult groups, reported by 20.8% (0.5 µg) to 100% (54 and 100 µg) of participants in the FLUmHA groups and by 71.4% in the Flu D-QIV group. Fatigue was the most frequent solicited systemic AE, with reporting rates ranging from 33.3% (0.5 and 1 µg) to 91.7% (100 µg) among FLUmHA groups and 54.3% in the Flu D-QIV control (Supplementary Table S1). The median duration of solicited AEs ranged from 1 to 3 d across all younger adult groups except for fatigue in the FLUmHA 12 µg group, for which the median duration was 5 d.

Figure 3.

Solicited adverse events with onset within 7 d after FLUmHA or Flu D-QIV administration (exposed set).

Error bars depict 95% confidence intervals for solicited AEs of any intensity (gray error bars) and grade 3 intensity (black error bars). Solicited administration-site AEs were pain, redness, swelling, and lymphadenopathy; solicited systemic AEs were fever (body temperature ≥ 38.0°C [100.4°F]), headache, fatigue, myalgia, arthralgia, and chills. AEs with grade 3 intensity were defined as: significant pain at rest or pain preventing normal everyday activities for administration-site pain, a diameter of >100 mm for administration-site redness and swelling, a body temperature ≥ 39.0°C (102.1°F) for fever, and an AE preventing normal everyday activities for all other AEs. The exposed set included 24 participants per younger adult FLUmHA group (except for the 54 µg group with 25 participants), 35 in the pooled younger adult Flu D-QIV, 32 in the older adult FLUmHA, and 16 in the older adult Flu D-QIV group. FLUmHA, investigational mRNA-based monovalent H1 influenza vaccine administered at different dose levels; Flu D-QIV, Flu Dresden-quadrivalent seasonal influenza vaccine; AE, adverse event; YA, younger adults (18–45 y of age); OA, older adults (60–80 y of age).

Among older adults, 62.5% in the FLUmHA group and 56.3% in the Flu D-QIV group reported solicited AEs (Figure 3), with pain (43.8% in both groups) and headache (37.5% in the FLUmHA versus 25.0% in the Flu D-QIV group) as the most commonly reported administration-site and systemic AEs (Supplementary Table S1). The median duration of the different solicited AEs among older adults was 1–2 d, and no grade 3 solicited AEs were reported (Figure 3).

Unsolicited AEs occurring within 28 d post-vaccination were reported without apparent dose-related trend by 50.0% (18 µg) to 70.8% (6 µg) of younger adult participants in the FLUmHA groups and by 68.6% in the Flu D-QIV control group; 8.3% (6, 9 µg) to 50.0% (100 µg) versus 31.4% (Flu D-QIV) reported vaccine-related unsolicited AEs (Table 2). The most common vaccine-related unsolicited AE in the FLUmHA groups was malaise. Overall, 0.0% (0.5, 1, 2, 12, 36, 54 µg) to 12.5% (100 µg) of younger adults in the FLUmHA groups versus 2.9% of Flu D-QIV controls reported grade 3 unsolicited AEs (Table 2). Cervical dysplasia (n = 1; Flu D-QIV control), upper respiratory tract infection (n = 1; 18 µg), COVID-19 (n = 2; 6 µg and 9 µg), and malaise (n = 3; 100 µg) were the grade 3 unsolicited AEs reported, with all events except malaise deemed unrelated to the vaccine. The study pausing rules were triggered twice due to the vaccine-related grade 3 malaise cases (Supplementary results). After review, the SRT and the Global Safety Board concluded that these events were self-limited, resolved within the predefined reactogenicity reporting window, and that the study could continue as planned.

Table 2.

Summary of safety outcomes (exposed set).

	Younger adults (18–45 y)
	FLUmHA
Type of AE	0.5 µg N = 24		1 µg N = 24		2 µg N = 24		6 µg N = 24		9 µg N = 24		12 µg N = 24		18 µg N = 24
	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)
Unsolicited AEs within 28 d post-vaccination
Any	13	54.2 (32.8–74.4)	13	54.2 (32.8–74.4)	14	58.3 (36.6–77.9)	17	70.8 (48.9–87.4)	13	54.2 (32.8–74.4)	16	66.7 (44.7–84.4)	12	50.0 (29.1–70.9)
Grade 3	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	1	4.2 (0.1–21.1)	1	4.2 (0.1–21.1)	0	0.0 (0.0–14.2)	1	4.2 (0.1–21.1)
Related	3	12.5 (2.7–32.4)	4	16.7 (4.7–37.4)	4	16.7 (4.7–37.4)	2	8.3 (1.0–27.0)	2	8.3 (1.0–27.0)	3	12.5 (2.7–32.4)	5	20.8 (7.1–42.2)
Grade 3 relateda	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)
Medically attendedb	3	12.5 (2.7–32.4)	1	4.2 (0.1–21.1)	4	16.7 (4.7–37.4)	0	0.0 (0.0–14.2)	2	8.3 (1.0–27.0)	4	16.7 (4.7–37.4)	3	12.5 (2.7–32.4)
SAEs and AESIs within 6 months post-vaccinationc
Any SAE	1	4.2 (0.1–21.1)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)	2	8.3 (1.0–27.0)	0	0.0 (0.0–14.2)	0	0.0 (0.0–14.2)
	Younger adults (18–45 y)										Older adults (60–80 y)
	FLUmHA				Flu D-QIV						FLUmHA		Flu D-QIV
	36 µg N = 24		54 µg N = 25		100 µg N = 24		N = 35				18 µg N = 32		N = 16
	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)			n	% (95% CI)	n	% (95% CI)
Unsolicited AEs within 28 d post-vaccination
Any	16	66.7 (44.7–84.4)	15	60.0 (38.7–78.9)	13	54.2 (32.8–74.4)	24	68.6 (50.7–83.1)			14	43.8 (26.4–62.3)	8	50.0 (24.7–75.3)
Grade 3	0	0.0 (0.0–14.2)	0	0.0 (0.0–13.7)	3	12.5 (2.7–32.4)	1	2.9 (0.1–14.9)			0	0.0 (0.0–10.9)	0	0.0 (0.0–20.6)
Related	5	20.8 (7.1–42.2)	3	12.0 (2.5–31.2)	12	50.0 (29.1–70.9)	11	31.4 (16.9–49.3)			7	21.9 (9.3–40.0)	2	12.5 (1.6–38.3)
Grade 3 relateda	0	0.0 (0.0–14.2)	0	0.0 (0.0–13.7)	3	12.5 (2.7–32.4)	0	0.0 (0.0–10.0)			0	0.0 (0.0–10.9)	0	0.0 (0.0–20.6)
Medically attendedb	3	12.5 (2.7–32.4)	3	12.0 (2.5–31.2)	1	4.2 (0.1–21.1)	2	5.7 (0.7–19.2)			2	6.3 (0.8–20.8)	2	12.5 (1.6–38.3)
SAEs and AESIs within 6 months post-vaccinationc
Any SAE	0	0.0 (0.0–14.2)	0	0.0 (0.0–13.7)	0	0.0 (0.0–14.2)	1	2.9 (0.1–14.9)			1	3.1 (0.1–16.2)	0	0.0 (0.0–20.6)

An AE with grade 3 intensity is an AE that prevented normal, everyday activities. A related AE is an AE that was considered related to vaccination based on the investigator’s clinical judgment. FLUmHA, groups with participants who received the investigational mRNA-based monovalent H1 influenza vaccine at different dose levels; Flu D-QIV, control groups with participants who received the Flu Dresden-quadrivalent seasonal influenza vaccine; AE, adverse event; N, number of participants per group; n/%, number/percentage of participants reporting at least one of the specified AEs; CI, confidence interval; SAE, serious AE; AESI, AE of special interest, i.e., potential immune-mediated disease, severe hypersensitivity reaction within 24 hours post-vaccination, or myocarditis/pericarditis. ^aAll grade 3 related unsolicited AEs reported in the 100 µg group were malaise. ^bNone of the medically attended AEs were considered related to vaccination. ^cNo SAEs considered related to vaccination, no fatal SAEs, no AESIs, and no AEs leading to withdrawal from the study were reported throughout the study.

Among older adults, 43.8% of FLUmHA and 50.0% of Flu D-QIV recipients reported unsolicited AEs; and 21.9% versus 12.5% had a vaccine-related unsolicited AE. None were of grade 3 intensity (Table 2).

During the 6-month follow-up period, five participants reported SAEs: one younger adult in the FLUmHA 0.5 µg group (craniofacial fracture), two younger adults in the 9 µg group (deliberate poisoning in one participant, malignant melanoma and ovarian benign germ cell teratoma in a second participant), one younger adult in the Flu D-QIV group (depression), and one older adult in the FLUmHA group (abdominal infection). None of these SAEs were considered by the investigator as vaccine-related, and all were resolving or had resolved by study end. No AESIs or deaths were reported during the study, and no participants withdrew due to an AE (Table 2).

A shift from a non-clinically significant to a clinically significant abnormal laboratory value (for hematology, biochemistry, coagulation, or urine analysis) between day 1 and day 8 or 29 was observed in three younger adults across the FLUmHA groups (two in the 18 µg, one in the 36 µg group), and in one younger and one older adult in the Flu D-QIV groups. A shift from a normal/missing laboratory value to a clinically significant abnormal laboratory value was observed in eight younger adults (two in the 0.5 µg, one each in the 1, 6, and 12 µg, three in the 18 µg group) and one older adult across the FLUmHA groups, and in one older adult in the Flu D-QIV group. These abnormal values were transient and/or were associated with AEs assessed by the investigator as not related to the study intervention. There was no evidence of a dose effect nor of a specific pattern of abnormal laboratory values (Supplementary results).

Immunogenicity

Humoral immune response

In younger adults, A/H1N1 HI titers increased from pre-vaccination to day 22 in all groups, with adjusted GMIs at day 22 versus day 1 ranging from 8.6 (0.5 µg) to 36.7 (18 µg) in the FLUmHA groups and 10.0 in the Flu D-QIV control. Adjusted GMTs at day 22 were numerically higher in the FLUmHA groups than in the control group for all dose levels above 1 µg and were highest for the 18 and 54 µg doses (Figure 4A). Adjusted GMT ratios (FLUmHA over Flu D-QIV) at day 22 ranged from 0.86 (0.5 µg) to 3.66 (18 µg) across the different FLUmHA groups, with lower 95% confidence limits >1.00 for all dose levels of 18 µg and higher (Figure 4B). Seroprotection rates at day 22 were 100% in all younger adult groups except for the 0.5 µg group (95.7%). Seroconversion rates ranged from 47.8% (0.5 µg) to 95.8% (12, 54, and 100 µg) in the FLUmHA groups compared to 59.4% in the Flu D-QIV group (Table 3).

Figure 4.

Adjusted geometric mean hemagglutination inhibition titers, geometric mean increases post-vaccination (A), and adjusted geometric mean titer ratios (B) for the influenza A/Victoria/2570/2019 (H1N1) strain (per-protocol set).

Error bars depict 95% CIs. In panel A, the horizontal dashed line marks the assay’s LLOQ (10 1/dil). Titers <LLOQ were replaced by half the LLOQ. No blood sampling was performed on days 62 and 183 for the 100 μg group and its control. For days indicated underneath the graph, day 1 is the pre-vaccination timepoint; days 22, 62, and 183 are post-vaccination timepoints. In panel B, the vertical dashed line marks an adjusted GMT ratio of 1.00. FLUmHA, groups with participants who received the investigational mRNA-based monovalent H1 influenza vaccine at different dose levels; (Flu) D-QIV, control groups with participants who received the Flu Dresden-quadrivalent seasonal influenza vaccine; N, number of participants per group with available results; as N varied by timepoint, the range is given across timepoints in panel A; GMT, geometric mean A/H1N1 hemagglutination inhibition titer; dil, dilution; LLOQ, lower limit of quantification; GMI, geometric mean increase in A/H1N1 hemagglutination inhibition titer from day 1 (pre-vaccination) until the indicated timepoints post-vaccination; CI, confidence interval; NA, not applicable.

Table 3.

Seroprotection and seroconversion rates for hemagglutination inhibition titers for the influenza A/Victoria/2570/2019 (H1N1) strain (per-protocol set).

	Younger adults (18–45 y)
	FLUmHA
Timepoint	0.5 µg N = 17–23		1 µg N = 17–22		2 µg N = 16–23		6 µg N = 17–24		9 µg N = 23–24		12 µg N = 24		18 µg N = 8–23
	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)
Seroprotection rates
Day 1	13	56.5 (34.5–76.8)	15	68.2 (45.1–86.1)	12	52.2 (30.6–73.2)	10	41.7 (22.1–63.4)	8	33.3 (15.6–55.3)	9	37.5 (18.8–59.4)	14	60.9 (38.5–80.3)
Day 22	22	95.7 (78.1–99.9)	22	100 (84.6–100)	23	100 (85.2–100)	23	100 (85.2–100)	23	100 (85.2–100)	24	100 (85.8–100)	23	100 (85.2–100)
Day 62	20	95.2 (76.2–99.9)	19	100 (82.4–100)	23	100 (85.2–100)	21	100 (83.9–100)	23	95.8 (78.9–99.9)	24	100 (85.8–100)	14	100 (76.8–100)
Day 183	15	88.2 (63.6–98.5)	16	94.1 (71.3–99.9)	16	100 (79.4–100)	17	100 (80.5–100)	21	91.3 (72.0–98.9)	22	91.7 (73.0–99.0)	8	100 (63.1–100)
Seroconversion rates
Day 22	11	47.8 (26.8–69.4)	11	50.0 (28.2–71.8)	19	82.6 (61.2–95.0)	18	78.3 (56.3–92.5)	22	95.7 (78.1–99.9)	23	95.8 (78.9–99.9)	20	87.0 (66.4–97.2)
Day 62	10	47.6 (25.7–70.2)	7	36.8 (16.3–61.6)	15	65.2 (42.7–83.6)	16	76.2 (52.8–91.8)	21	87.5 (67.6–97.3)	19	79.2 (57.8–92.9)	11	78.6 (49.2–95.3)
Day 183	8	47.1 (23.0–72.2)	6	35.3 (14.2–61.7)	13	81.3 (54.4–96.0)	13	76.5 (50.1–93.2)	18	78.3 (56.3–92.5)	18	75.0 (53.3–90.2)	5	62.5 (24.5–91.5)
	Younger adults (18–45 y)										Older adults (60–80 y)
	FLUmHA						Flu D-QIV				FLUmHA		Flu D-QIV
	36 µg N = 15–23		54 µg N = 20–24		100 µg N = 24		N = 19–34				18 µg N = 12–29		N = 6–16
	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)			n	% (95% CI)	n	% (95% CI)
Seroprotection rates
Day 1	10	43.5 (23.2–65.5)	16	66.7 (44.7–84.4)	16	66.7 (44.7–84.4)	17	50.0 (32.4–67.6)			18	62.1 (42.3–79.3)	7	43.8 (19.8–70.1)
Day 22	23	100 (85.2–100)	24	100 (85.8–100)	24	100 (85.8–100)	32	100 (89.1–100)			28	96.6 (82.2–99.9)	15	93.8 (69.8–99.8)
Day 62	17	100 (80.5–100)	20	100 (83.2–100)	NA	NA	23	100 (85.2–100)			14	93.3 (68.1–99.8)	9	90.0 (55.5–99.7)
Day 183	15	100 (78.2–100)	20	100 (83.2–100)	NA	NA	18	94.7 (74.0–99.9)			10	83.3 (51.6–97.9)	6	100 (54.1–100)
Seroconversion rates
Day 22	22	95.7 (78.1–99.9)	23	95.8 (78.9–99.9)	23	95.8 (78.9–99.9)	19	59.4 (40.6–76.3)			26	89.7 (72.6–97.8)	9	56.3 (29.9–80.2)
Day 62	16	94.1 (71.3–99.9)	17	85.0 (62.1–96.8)	NA	NA	14	60.9 (38.5–80.3)			10	66.7 (38.4–88.2)	6	60.0 (26.2–87.8)
Day 183	13	86.7 (59.5–98.3)	12	60.0 (36.1–80.9)	NA	NA	12	63.2 (38.4–83.7)			4	33.3 (9.9–65.1)	2	33.3 (4.3–77.7)

FLUmHA, groups with participants who received the investigational mRNA-based monovalent H1 influenza vaccine at different dose levels; Flu D-QIV, control groups with participants who received the Flu Dresden-quadrivalent seasonal influenza vaccine; N, number of participants with available results per group; as N varied by timepoint, the range is given across timepoints; n/%, number/percentage of participants who were seroprotected (i.e., hemagglutination inhibition titer ≥40 1/dilution [dil]) or who seroconverted (i.e., had at least a fourfold increase in hemagglutination inhibition titer post- versus pre-vaccination if the pre-vaccination titer was ≥10 1/dil or had a post-vaccination titer ≥40 1/dil if the pre-vaccination titer was <10 1/dil); CI, confidence interval; NA, not applicable (no blood samples for immunogenicity analysis taken at these time points for this study group).

A/H1N1 HI titers declined between day 22 and day 183 but remained above baseline levels, with adjusted GMIs at day 183 versus day 1 ranging from 5.4 (1 µg) to 14.0 (54 µg) in the FLUmHA groups and 8.7 in the Flu D-QIV control (Figure 4A). Seroprotection rates at days 62 and 183 remained similar to those at day 22 in all younger adult groups, ranging from 88.2% (0.5 µg) to 100% (2, 6, 18–54 µg) in the FLUmHA groups versus 94.7% in the Flu D-QIV group at day 183. Seroconversion rates at day 183 ranged from 35.3% (1 µg) to 86.7% (36 µg) in the FLUmHA groups compared to 63.2% in the Flu D-QIV group (Table 3).

In older adults, FLUmHA tended to induce a stronger A/H1N1 HI response than Flu D-QIV; adjusted GMIs at day 22 versus day 1 were 14.5 in the FLUmHA group and 6.2 in the Flu D-QIV group. Likewise, adjusted GMTs, seroprotection rates, and seroconversion rates at day 22 were numerically higher in FLUmHA than in Flu D-QIV recipients (Figure 4A, Table 3). The adjusted GMT ratio (FLUmHA over Flu D-QIV) was 2.33, with a lower 95% confidence limit > 1.00 (Figure 4B), and the seroconversion rate reached 89.7% in the FLUmHA group, compared to 56.3% in the Flu D-QIV group (Table 3). Adjusted HI GMTs declined between day 22 and day 183 but remained above baseline levels in both groups (Figure 4A).

Cell-mediated immune response

The median frequency of hemagglutinin-specific polypositive CD4+ T cells increased from pre-vaccination to day 8 in all evaluated FLUmHA groups (i.e., receiving 0.5 to 36 µg) among both younger and older adults (Supplementary Tables S2 and S3). This was driven by an increase in CD4+ T cells expressing at least IFN-γ (indicative of Th1 profile). Observed median frequencies of CD4+ Th1 cells at day 8 were higher in the FLUmHA groups compared to the Flu D-QIV groups and increased with increasing dose levels (Figure 5A). The median frequency of hemagglutinin-specific CD4+ Th1 cells declined at the later timepoints but remained above baseline at day 183 in all FLUmHA groups. No increase was observed in CD4+ T cells expressing at least IL-13 (Th2 profile) or IL-17 (Th17 profile) (Figure 5B, Supplementary Tables S2 and S3). A hemagglutinin-specific CD8+ T-cell response was low to undetectable across the groups (Supplementary Tables S2 and S3).

Figure 5.

Frequencies of hemagglutinin-specific CD4+ T cells expressing at least IFN-γ (A) or IL-13 (B) in the FLUmHA and Flu D-QIV groups (per-protocol set of cell-mediated immunity subset).

Analyses not done for the 54 µg and 100 µg dose levels (no blood sampling for cell-mediated immunity analysis was performed for the 100 μg group and its control, and only one participant could be assessed for the 54 μg dose level). For days indicated underneath the graph, day 1 is the pre-vaccination timepoint; days 8, 62, and 183 are post-vaccination timepoints. IFN-γ, interferon-γ; IL-13, interleukin-13; FLUmHA, groups with participants who received the investigational mRNA-based monovalent H1 influenza vaccine at different dose levels; Flu D-QIV, control group with participants who received the Flu Dresden-quadrivalent seasonal influenza vaccine; HA, hemagglutinin; N, number of participants per group with available results (as N varied by timepoint, the range is given across timepoints); max, maximum; Q3, third quartile; Q1, first quartile; min, minimum.

Median frequencies of hemagglutinin-specific memory B cells increased after vaccination in all FLUmHA groups and in the Flu D-QIV groups in both younger and older adults and remained elevated at all assessed timepoints. Among younger adults, a stronger response was observed at the higher dose levels. Median frequencies in the Flu D-QIV control groups were numerically lower than those in the younger adult FLUmHA 18 and 36 µg groups and in the older adult FLUmHA group but higher than those in the lower dose level groups (Supplementary Figure S1).

Discussion

This phase 1 proof-of-principle study showed that an investigational mRNA-lipid nanoparticle-based monovalent influenza vaccine encoding influenza A/H1N1 hemagglutinin was well tolerated, had an acceptable safety profile, and induced a durable humoral and cellular immune response in adults 18–45 y as well as in those 60–80 y of age.

The study evaluated 10 dose levels (from 0.5 µg to 100 µg) of hemagglutinin mRNA in younger adults and a single dose level (18 µg) in older adults. No safety signals were identified during the study period for any of the doses. No deaths, AESIs, vaccine-related SAEs, or withdrawals due to AEs were reported in any of the study groups. Reactogenicity among younger adults appeared to be dose-dependent. The proportions of participants reporting solicited AEs tended to be higher for the highest FLUmHA dose levels (12–100 µg), lower for the lower dose levels (0.5–6 µg), and similar for the 9 µg dose level compared to the licensed Flu D-QIV control. In general, solicited AEs were transient and had a mild-to-moderate intensity. Unsolicited AEs with grade 3 intensity were reported infrequently, with none considered related to the vaccine, except for three cases of grade 3 malaise that occurred within 7 d post-vaccination in the 100 µg group. These events were transient and self-limited. FLUmHA vaccination tended to be less reactogenic in older adults than in younger adults receiving the same dose (18 µg). This is consistent with previous studies across various targeted pathogens and vaccine platforms (including mRNA vaccines), showing fewer and milder AEs as age increases.^25–29

The investigational FLUmHA vaccine induced a humoral response against the hemagglutinin component of the A/H1N1 strain targeted by the vaccine both in younger adults and older adults. A response was observed with all dose levels tested in younger adults, including the lowest doses of 0.5 and 1 µg. In general, A/H1N1 HI titers at day 22 increased with increasing dose levels and were numerically higher than those measured after Flu D-QIV vaccination for all FLUmHA doses above 1 µg in younger adults, and in older adults. HI GMTs declined after day 22, but they remained above baseline levels, with high seroprotection rates (≥83.3%) at 6 months post-vaccination regardless of dose level and age group. The magnitude of the humoral response in older adults is encouraging and raises the possibility that FLUmHA may partially mitigate the effects of immunosenescence, as previously observed with enhanced influenza vaccines,^30,31 but this will need to be confirmed in larger studies using a multivalent vaccine.

Most licensed seasonal influenza vaccines elicit poor T-cell responses.³² Consistent with these findings, in our study, only a limited increase in hemagglutinin-specific CD4+ T-cell frequencies was observed after vaccination with the licensed Flu D-QIV. By contrast, FLUmHA vaccination in both younger and older adults elicited a strong CD4+ T-cell response at day 8. The CD4+ T-cell response was skewed toward a Th1 profile, which is consistent with observations for other mRNA-based influenza and COVID-19 vaccines^15,18,33 and after influenza infection.³² The induction of a strong CD4+ T-cell response may play a significant role in enhancing protection against influenza.^34,35 The limited CD8+ T-cell response directed to the H1 antigen is in line with observations with another mRNA-based influenza vaccine.¹⁵ A dose-dependent memory B-cell response was observed after FLUmHA vaccination, which was evident at all timepoints. Both in older adults and at the 18 µg and 36 µg dose levels in younger adults, the median frequencies of hemagglutinin-specific memory B cells exceeded those in the Flu D-QIV control groups.

Strengths of our study include the use of a standard-of-care seasonal influenza vaccine as an active control, the extensive dose ranging, and the broad panel of immunological and safety endpoints evaluated at multiple timepoints in both younger and older adults. Our study has several limitations. As this was a phase 1, descriptive study with no adjustment for multiplicity, analyses were descriptive rather than inferential, and therefore conclusions should be drawn with caution. Group sizes by dose level were small, limiting precision and the ability to detect rare safety signals. CMI responses were only analyzed in 50% of participants enrolled at selected sites; as such, these analyses are exploratory, and their generalizability is limited. Due to the study’s dose-escalation design with staggered enrollment, dose groups were enrolled at different times throughout the year, over a period spanning more than one influenza season, with vaccines administered before, after, or during the influenza seasons, depending on the dose group. The participants in the different groups were therefore not all exposed to the same levels of circulating virus, which might have influenced the immunogenicity results. In addition, some dose groups were enrolled in only one country, limiting the generalizability of the results. Finally, we used a standard seasonal influenza vaccine as control in older adults, whereas enhanced influenza vaccines (e.g., adjuvanted or high-dose vaccines) are typically recommended in this population to overcome immunosenescence.^30,31 Enhanced influenza vaccines may need to be considered as comparator in future studies with multivalent influenza mRNA vaccines in older adults.

In conclusion, this investigational mRNA-lipid nanoparticle-based monovalent influenza vaccine encoding A/H1N1 hemagglutinin had an acceptable safety profile and elicited durable humoral and cellular immune responses. These results support the progression to clinical development of a multivalent seasonal influenza mRNA vaccine candidate targeting all WHO-recommended strains using this platform.

Biographies

Isabel Leroux-Roels is a medical doctor and clinical researcher with over 20 y of experience in vaccinology and infectious disease prevention. She is Head of the Center for Vaccinology (CEVAC) at Ghent University and Ghent University Hospital, where she leads early- and late-phase clinical vaccine trials in collaboration with academic partners, biotech companies, and global health organizations. Her research focuses on innovative vaccine technologies, immune response profiling, and infection prevention strategies in both general and vulnerable populations. Dr. Leroux-Roels has played a pivotal role in the development and evaluation of vaccines against emerging and re-emerging pathogens, including during the COVID-19 pandemic. She is committed to translating scientific advances into public health impact and is actively involved in national and international networks dedicated to vaccine research, clinical trial innovation, and pandemic preparedness.

Ilse De Coster is heading the Ambulatory Clinical Trial Unit of the Center for the Evaluation of Vaccination, a research center specialized in the conduct of clinical vaccine trials at Vaccinopolis, University of Antwerp in Belgium. She obtained her MD as general practitioner in 1993. Following completion of the post-graduate course Public Health and Tropical Medicine at the Institute for Tropical Medicine, Antwerp, she joined the University of Antwerp in 2010. Since then, she has been committed to a large number of clinical vaccine trials as principal and co-investigator and participated in the coordination of vaccine trials within EU projects. She is associate professor at the University of Antwerp and teaches infectious diseases and vaccinology. Since 2024, she is chair of the National Certification Committee for the Eradication of Poliomyelitis and co-chair of the Antwerp Pediatric Clinical Trial Network, with a focus on vaccine trials.

Alberto M. Borobia is a medical doctor, specialized in clinical pharmacology. He is the head of the Clinical Trial Unit of La Paz University Hospital and deputy scientific director of IdiPAZ (Research Institute of La Paz University Hospital). Dr. Borobia is a professor of pharmacology at Universidad Autónoma de Madrid.

Joanne M. Langley is a Professor of Pediatrics and Community Health and Epidemiology at Dalhousie University, and Head, Infectious Diseases at IWK Health. https://www.linkedin.com/in/joanne-langley-00181771/.

Belén Ruiz-Antorán is a specialist in Clinical Pharmacology at University Hospital Puerta de Hierro Majadahonda (Madrid, Spain), where she leads the Clinical Research Unit within the Department of Clinical Pharmacology. She combines her clinical work with teaching responsibilities in the Medicine and Nursing programs at Universidad Antonio de Nebrija. Her research focuses on pharmacoepidemiology, clinical toxicology, and clinical research methodology. She has been involved in numerous national and international multicenter studies, both observational and interventional, and has authored multiple publications in peer-reviewed journals in the field of clinical pharmacology.

Stefano Berrè is a clinical assay scientist at GSK since 2020. He is responsible for laboratory assays deployed as cell-mediated immunity readouts in vaccine clinical trials. https://www.linkedin.com/in/stefano-berr%C3%A8-6b88b88b/.

Christine Knauer is a medical doctor and drug safety expert. She has more than 15 y of experience in drug safety, pharmacovigilance, and clinical development, 2.5 y in public health and almost 5 y in biometry. Prior to her career in the pharmaceutical industry, she held academic positions in clinical research and as a clinician in internal medicine, oncology, and pediatrics. https://www.linkedin.com/in/dr-med-christine-knauer-5603b68a/.

Sven D. Koch is a Clinical R&D professional holding a Ph.D. (Dr. rer. Nat) from the University of Tübingen. He has over 15 y of experience in translational human immunology, infectious disease and immuno-oncology. Skills: Clinical research and science, biomarkers, xOMICS, flow cytometry, biopharmaceuticals, biosimilars, GCP, mRNA therapeutics, mRNA vaccines, immunology, infectious disease, cancer immunotherapy, immuno-oncology, project management, leadership. https://www.linkedin.com/in/sven-koch-6961a755/.

Philipp Mann is a board-certified physician in anesthesiology and tropical medicine with approximately 15  of experience in vaccine clinical trials. During the first decade of his career, he held academic positions focused on preventive HIV vaccine trials spanning phases 1 to 3, primarily in sub-Saharan Africa and Latin America. His roles included both site-level responsibilities and coordinating positions. Over the subsequent 5 y, he transitioned to the biotech and pharmaceutical industry, where he coordinated phase 1 to 3 trials predominantly for COVID-19 and influenza vaccines. In 2024, Dr. Mann joined the Coalition for Epidemic Preparedness Innovations (CEPI), further contributing to the advancement of vaccine research and development.

Doris Mesia Vela is a medical doctor specialized in pediatrics and tropical medicine. In 2003, she earned a master’s degree in public health. She has over 13 y of experience in clinical research, epidemiology, and medical affairs, with a strong focus on vaccine development within the pharmaceutical industry. Her background also includes 15 y of dedicated medical service in humanitarian settings around the world. Additionally, she has 2 y of experience practicing as a physician and pediatrician. Her experience in vaccines includes HIV, tuberculosis, malaria, COVID-19, and influenza vaccines. As a former CureVac employee, she was a medical lead for influenza and COVID-19 candidate vaccines. https://www.linkedin.com/in/dmv-08064242/.

Leroy Jide Ovbude is a project lead statistician at GSK with nearly 10 y of experience in clinical development. https://www.linkedin.com/in/leroyjide.

Iris Alessandra Pardo is a board-certified dermatologist and currently serves as a safety physician at GSK. She earned her medical degree from the University of the Philippines College of Medicine. With more than 10 y of experience in pharmacovigilance, she has contributed to the safety oversight of multiple clinical development programs. Her experience in pharmacovigilance spans from first-in-human studies to post-marketing surveillance, across various therapeutic areas within vaccines, oncology, and consumer health. https://www.linkedin.com/in/iaspardo/.

Barkha Srivastava is a senior clinical biomarker scientist at CureVac, with over 4 y of experience in the clinical team. She contributes to designing and executing biomarker strategies in various phase 1 and phase 2 clinical trials for infectious diseases and early-phase cancer vaccines. Dr. Srivastava holds a Ph.D. in Infection Biology from Hannover Medical School and completed her postdoctoral research at the Institute of Lung Biology, Helmholtz Zentrum, München. She has more than a decade of experience as a translational scientist specializing in lung diseases, infection biology, virology, and clinical immunology.

Brian Moldt has a Ph.D. in medicine from Aarhus University and currently works as a clinical lead at GSK. He has over 10 y of experience in drug development for respiratory and chronic infectious diseases. https://www.linkedin.com/in/brian-moldt-0aa86b2a.

Marcus Nordgren is a clinical R&D professional holding a Ph.D. in Biomedical Sciences from the University of KU Leuven. He has more than 10 y of experience within vaccines R&D. https://www.linkedin.com/in/marcus-nordgren-0010474/.

Funding Statement

This study was funded by GlaxoSmithKline. GlaxoSmithKline also funded all costs associated with the development and publication of this manuscript.

Disclosure statement

Christine Knauer, Sven D. Koch, Philipp Mann, Doris Mesia Vela, and Barkha Srivastava are or were employees of CureVac SE at the time of study design and/or conduct. Christine Knauer and Philipp Mann hold financial equities in CureVac SE. Sven D. Koch holds financial equities in CureVac SE and Novo Nordisk. Doris Mesia Vela holds financial equities in CureVac SE and GSK. Stefano Berrè, Sophie Germain, Leroy Jide Ovbude, Iris Alessandra Pardo, Brian Moldt, and Marcus Nordgren are employees of GSK. Stefano Berrè, Leroy Jide Ovbude, Iris Alessandra Pardo, Brian Moldt, and Marcus Nordgren hold financial equities in GSK as part of their employee remuneration. Brian Moldt declares patent applications filed in the name of GSK. Isabel Leroux-Roels reports institutional research grants from GSK, CSL Seqirus, Moderna, LiteVax, Osivax, Sumitomo Pharma, J&J, Virometix AG, AstriVax Therapeutics, Bavarian Nordic, Vaccitech, and MinervaX for the conduct of clinical trials; institutional lecture fees by MSD and CSL Seqirus; and institutional financial compensation for the participation in Data Safety Monitoring and Advisory Boards by JnJ, MSD, and CSL Seqirus. Ilse De Coster reports support from GSK for the current study through her institution and declares grants or contracts from GSK, Moderna, MSD, Astrivax Therapeutics, J&J, Bavarian Nordic, Icosavax Inc, Osivax, Abbott, and Merck paid to her institution; she declares participation in an Advisory Board for GSK paid to her institution. Joanne M. Langley reports institutional grants from GSK, Moderna, Merck, and Pfizer; institutional consulting fees from GSK, CSL Seqirus, and Sanofi; support from GSK for attending meetings; participation on the Advisory Board for the SNAP clinical trial; and unpaid roles in the Meningitis Foundation of Canada, the Council of Expert Advisors to Health Emergency Readiness Agency, Government of Canada, and the NACI Influenza Working Group. Alberto M. Borobia, Dolores Ochoa Mazarro, and Belén Ruiz-Antorán have no conflicts of interest. The authors declare no other financial or non-financial interests, relationships, or activities.

Data availability statement

The protocol and statistical analysis plan are available on https://www.gsk-studyregister.com/trials/217895. Please refer to the link https://www.gsk-studyregister.com/en/ to access GSK’s data sharing policies and to request anonymized participant-level data.

Trademark statement

α-RIX-Tetra/Fluarix-Tetra are trademarks owned by or licensed to the GSK group of companies.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21645515.2026.2631804.