A Randomized, Double-Blind Clinical Trial of One vs Two Doses of High-Dose Influenza Vaccine in Solid Organ Transplant Recipients

Abstract

Background

Solid organ transplant recipients (SOTRs) exhibit suboptimal immune responses to standard-dose influenza vaccines. High-dose (HD) vaccines and booster strategies show promise, but the optimal vaccination approach for SOTRs remains undefined. We sought to evaluate a double vs a single HD influenza vaccine regimen, with the hypothesis that the double HD would yield superior immunogenicity.

Methods

This randomized clinical trial during the 2023–2024 and 2024–2025 influenza seasons compared the immunogenicity, clinical outcomes, and safety of 2 HD 1 month apart vs 1 HD influenza vaccines in 65 adult SOTRs at the University of Minnesota. The study comprised 3 visits: baseline, 1-month follow-up, and 4-month follow-up.

Results

The primary analysis showed greater but no statistically significant geometric mean fold rises (GMFRs) of ID₈₀ neutralizing and binding antibody titers from baseline to 4-month follow-up in the 2 HD group compared with the 1 HD group. However, exploratory analyses focusing on the period before to after the second injection (1–4 months) showed statistically significant differences in GMFRs for both ID₈₀ neutralizing and binding titers of most strains when comparing the 2 groups. Two participants from the 1 HD group developed laboratory-confirmed influenza. Adverse event frequency was balanced between groups, with reactogenicity symptoms within 7 days of injection being grade 1.

Conclusions

While a single HD vaccine provides an initial immune response, an HD booster appeared to improve overall immune responses to influenza strains contained in the vaccine formulation, although confirmation in larger, adequately powered studies is needed.

Clinical Trials Registration. NCT05663463

A randomized clinical trial of solid organ transplant recipients showed that a high-dose influenza vaccine booster generated greater immune responses compared with giving only 1 HD. This strategy may help overcome the lower immunogenicity associated with chronic immunosuppression in SOTRs.

Solid organ transplant recipients (SOTRs) are at an increased risk for severe influenza outcomes, including pneumonia, hospitalization, intensive care unit (ICU) admission, and death [1]. Vaccination is the cornerstone of prevention, yet SOTRs often mount suboptimal vaccine-induced immune responses compared with immunocompetent hosts [2]. Reported antibody responses to standard-dose (SD) influenza vaccination vary widely from 15% to 90% and are generally lower than in healthy controls [2]. Despite the importance of influenza prevention in this population, the optimal vaccination strategy for SOTRs remains undefined.

Strategies to improve immunogenicity include adjuvanted vaccine formulations, SD booster dosing within the influenza season, and the use of high-dose (HD) vaccines. In-season boosting with SD influenza vaccine in SOTRs has demonstrated increased seroconversion and seroprotection rates compared with a single SD dose [3, 4]. In phase 2 trials among pediatric and adult hematopoietic cell transplant (HCT) recipients, 2 doses of HD influenza vaccine elicited stronger and more durable antibody responses than SD vaccination without major safety concerns [5, 6]. The HD influenza vaccine, which contains 4-fold higher antigen content than SD formulations, improves immunogenicity and efficacy in older adults [7, 8] and has demonstrated improved immunogenicity in adult and pediatric transplant recipients compared with SD vaccination [9–12].

The relative benefit of single vs double HD influenza vaccination within a single season has not been evaluated in randomized trials among SOTRs [13]. To address this knowledge gap, we conducted a clinical trial comparing the immunogenicity and safety of 1 vs 2 doses of HD influenza vaccine (Fluzone®, Sanofi Pasteur) in adult SOTRs during the 2023–2024 and 2024–2025 seasons. We hypothesized that a 2-dose HD regimen would yield superior immunogenicity and more durable influenza-specific antibody responses throughout the influenza season in this vulnerable population.

METHODS

Study Design and Participants

A randomized, double-blind, placebo-controlled, single-center clinical trial was conducted during the 2023–2024 and 2024–2025 influenza seasons at the UMN to evaluate the immunogenicity and clinical outcomes of 1 vs 2 doses of HD influenza vaccine in adult SOTRs. Eligible participants were ≥18 years old and ≥1-year post-transplant (kidney, liver, heart, lung, or pancreas). Exclusion criteria included pregnancy, receipt of lymphocyte-depleting or B cell-directed therapy within 3 months, prednisone ≥20 mg/day, prior influenza vaccination during the influenza season of enrollment, or history of severe allergy to influenza vaccines.

Study Drug and Placebo

Fluzone® HD is an inactivated influenza vaccine (Sanofi Pasteur, Swiftwater, PA). Fluzone® HD for the 2024–2025 influenza season contained 60 μg hemagglutinin (HA) of each of 3 strains (trivalent): A/Victoria/4897/2022 IVR-238 (H1N1), A/California/122/2022 SAN-022 (an A/Thailand/8/2022-like virus) (H3N2), and B/Michigan/01/2021 (a B/Austria/1359417/2021-like virus, B Victoria lineage). Fluzone® HD for the 2023–2024 influenza season contained 60 μg HA of each of 4 strains (quadrivalent): A/Victoria/4897/2022 IVR-238 (H1N1), A/Darwin/9/2021 SAN-010 (H3N2), B/Phuket/3073/2013 (B Yamagata lineage), and B/Michigan/01/2021 (a B/Austria/1359417/2021-like virus, B Victoria lineage). The recommendation by the FDA's Vaccines and Related Biological Products Advisory Committee (VRBPAC) to transition from quadrivalent to trivalent influenza vaccines for 2024–2025 was made because influenza B/Yamagata viruses are no longer actively circulating since March 2020. Placebo injections contained 0.9% sodium chloride. The study drug was procured, and placebo injections were prepared in equal volume and appearance by the Investigational Drug Services (IDS) Pharmacy of M Health Fairview.

Study Activities and Procedures

Participants were recruited using institutional messages and physician referral. The study involved 3 in-person visits: baseline, 4–6 weeks after baseline, and 16–18 weeks after baseline. The final study visit at 16–18 weeks was selected to evaluate maintenance and durability of antibody responses later in the influenza season following the randomized second injection. Blood samples were obtained at all 3 timepoints. All participants received an HD influenza vaccine (Fluzone®) at the baseline visit after the blood draw. At the second visit, participants provided a repeat blood sample and then were randomized 1:1 to receive either a Fluzone® dose (booster group or “2 HD group”) or placebo (control group or “1 HD group”).

Participants completed a pre-vaccination screening questionnaire within 72 hours prior to each injection to confirm absence of acute illness and vaccine contraindications. Pregnancy testing was conducted in women of reproductive potential prior to study injections. Randomization was performed by the IDS Pharmacy using permuted blocks on the day of the second injection. Injections were administered intramuscularly in the deltoid. Participants, clinical staff, and research personnel remained blinded to treatment assignment until study completion.

At the end of the influenza season, medical records were reviewed, and participants were queried about influenza-like illness (ILI) symptoms and laboratory-confirmed respiratory infections during study participation. Demographics, medical history, clinical information, and adverse events (AEs) were recorded in a REDCap database.

Clinical Trial Oversight and Ethics

The study protocol was exempt from an FDA investigational new drug application per the criteria outlined in 21 CFR 312.2(b). The study underwent internal quality management and monitoring by the investigators. Safety was evaluated during trial conduct through ascertainment of AEs, including local and systemic solicited reactogenicity symptoms and signs, regardless of their clinical significance or relation to study drug. AEs were graded according to the 2007 FDA Guidance: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials with grades 1 to 5 (mild, moderate, severe, life-threatening, and fatal) [14]. The University of Minnesota Institutional Review Board approved the study (STUDY00017687). All participants provided informed consent.

Study Outcomes

The primary outcome was immunogenicity, assessed by fold change in geometric mean titers (GMTs) of ID₈₀ neutralizing and binding antibodies to influenza vaccine strains from baseline to the 4-month visit. Secondary outcomes included influenza-related clinical events (pneumonia, hospitalization, ICU admission, and death) during the influenza season and safety outcomes.

Laboratory Methods

Blood samples were processed at the Schacker Lab (UMN), stored at −80°C, and then shipped to the Vaccine Research Center at the U.S. National Institutes of Health for immunologic assays. Antibody titer measurements were conducted for 4 HA cell-based influenza strains: Austria (B Victoria lineage), Darwin (H3N2), Phuket (B Yamagata lineage), and Wisconsin (H1N1). As shown in Supplementary Table 1, these strains show 98%–100% amino acid homology relative to the Fluzone® HA egg-based component strains. For antibody neutralization assays, replication-restricted influenza reporter viruses (R3ΔPB1) expressing tdKatushka2 were generated as described previously [15]. Receptor-destroying enzyme–treated sera were serially diluted (8 2-fold dilutions, starting at 1:40) and incubated with virus for 1 hour at 37°C. MDCK-SIAT1-PB1 cells were added to each well in 384-well plates. Liquid handling was performed on a Beckman Biomek i7. Following incubation for 18–26 hours at 37°C, fluorescent foci were imaged using a Celigo image cytometer (Revvity). Neutralization was quantified as the reduction in fluorescent foci compared with virus control wells. The inhibitory dilution at 50% (ID₅₀) and 80% reductions (ID₈₀) were interpolated from a 5-parameter logistic regression curve using LabKey Server's neutralization analysis module. The binding antibody responses were measured using electrochemiluminescence immunoassays (ECLIA, Meso Scale Diagnostics). Streptavidin plates were blocked with MSD Blocker A, coated with biotinylated HA proteins, and incubated with serial dilutions of participant sera, controls, and reference standards in duplicate. Detection was performed with SULFO-TAG–labeled anti-human IgG/IgM/IgA (Thermo Fisher, cat# 31128). Plates were read on an MSD Sector Imager S600. Antibody concentrations were interpolated from standard curves generated from pooled human serum and reported as arbitrary units per mL (AU/mL). Samples with a coefficient of variation >30% between replicates were repeated.

Statistical Analysis

We planned to enroll 47 participants per study arm to achieve 80% statistical power to detect a geometric mean ratio (GMR) of 3.0 between the 2 treatment groups at a 2-sided alpha of 0.05. Enrollment fell short of the planned sample size, reducing statistical power for the primary and secondary immunogenicity analyses.

Analyses were performed on the intention-to-treat population. Participant characteristics, clinical outcomes, and titer measurements were summarized descriptively by treatment group. Clinical AEs were tallied, and their type, severity, and relation to study drug/placebo were analyzed. Mean (SD) and median (IQR) were reported for continuous variables, while count and percentages were reported for categorical variables.

Analyses of the Phuket strain were restricted to participants enrolled during the 2023–2024 season as this strain was not included in the 2024–2025 Fluzone® HD formulation. For each strain and assay (ID₈₀ neutralizing and binding titers), the geometric mean fold rise (GMFR) was computed as the geometric mean of the fold change in the titer from baseline to the 4-month visit. The GMR, the ratio of the GMFRs between the 2 HD and 1 HD arms, was estimated under a generalized estimating equations framework by evaluating the difference between arms in the log-transformed titer and exponentiating. A Gaussian family model was used with an identity link and exchangeable working correlation structure. Robust variance estimation was used to compute the associated 95% confidence intervals (CIs) and P values.

Multiple imputation was used in the primary analysis to handle missing titers at the 4-month visit. Variables included in the imputation model were treatment arm, age, sex, receipt of influenza vaccine in the prior influenza season, time from most recent transplant (≤2 vs > 2 years), maintenance immunosuppression regimen (prednisone, mTOR/calcineurin inhibitors, and mycophenolate), and baseline titer. Complete-case analyses were also performed. Prespecified exploratory analyses included ID₅₀ neutralizing titers for each strain and evaluation of GMFRs from baseline to the second visit (∼1 month after baseline) and from the second visit to the final study visit (∼4 months after baseline). The latter analysis was performed to assess the incremental immunologic effect of the randomized second injection during the post-randomization interval. For immunogenicity analyses across influenza strains, Holm correction was applied to adjust for multiple comparisons and control the family-wise error rate. Two-sided P values <0.05 were considered statistically significant. Statistical analyses were performed using R version 4.5.1.

Further details about the laboratory methods are provided in the Supplementary Appendix.

RESULTS

Study Participants

We prescreened 228 participants, of whom 74 consented and were enrolled in the study (Figure 1). Seven participants withdrew because they were no longer interested or available (n = 4) or were lost to follow-up (n = 3). A total of 67 participants received an initial HD influenza vaccine dose at baseline, with 65 of them being randomized at their second visit to receive a second HD influenza vaccine (2 HD group, n = 32) or placebo (1 HD group, n = 33). Serologic data at all 3 study visits were available for 27 participants in the 2 HD group and 31 in the 1 HD group. Supplementary Figure 1 shows the study flow diagram by influenza season of enrollment.

Figure 1.

Study Flow Diagram. This diagram displays the progression of participants through screening, enrollment, intervention allocation, follow-up, and data analysis using the 2025 Consolidated Standards of Reporting Trials (CONSORT) template.

Demographics and Clinical Characteristics

Baseline participant characteristics were generally similar between groups (Table 1). Median age was 58.5 years (IQR, 55.5–69.5) for the 2 HD group and 64.0 years (IQR, 52.0–70.0) for the 1 HD group. Overall, 44/65 (64.6%) were male at birth, including 25/32 (78.1%) in the 2 HD group and 19/33 (57.6%) in the 1 HD group. Most study participants identified as white (60/65, 92.3%). The most common comorbidities were hypertension (78.5%), chronic kidney disease (76.9%), cancer (40%), dyslipidemia (38.5%), and diabetes mellitus (36.9%), with similar distributions across treatment arms.

Table 1.

Participant Characteristics at Enrollment (n = 65)

Characteristica	Overall (n = 65)	2 HD Group (n = 32)	1 HD Group (n = 33)
Age, median (IQR), years	61 (55.0, 70.0)	58.5 (55.5, 69.5)	64 (52.0, 70.0)
Male sex at birth	44 (67.7%)	25 (78.1%)	19 (57.6%)
Raceb
White	60 (92.3%)	31 (96.9%)	29 (87.9%)
Black	2 (3.1%)	0 (0%)	2 (6.1%)
Other	3 (4.6%)	1 (3.1%)	2 (6.1%)
Hispanic/Latino ethnicity	1 (1.5%)	0 (0%)	1 (3.0%)
Influenza season (enrollment)
2023–2024	35 (53.8%)	17 (53.1%)	18 (54.5%)
2024–2025	30 (46.2%)	15 (46.9%)	15 (45.5%)
Medical history of comorbiditiesc,d
Hypertension	51 (78.5%)	23 (71.9%)	28 (84.8%)
Chronic kidney disease	50 (76.9%)	22 (68.8%)	28 (84.8%)
Cancer	26 (40.0%)	12 (37.5%)	14 (42.4%)
Dyslipidemia	25 (38.5%)	12 (37.5%)	13 (39.4%)
Diabetes mellitus	24 (36.9%)	11 (34.4%)	13 (39.4%)
Obesity	14 (21.5%)	9 (28.1%)	5 (15.2%)
Coronary artery disease	14 (21.5%)	7 (21.9%)	7 (21.2%)
Liver disease	11 (16.9%)	7 (21.9%)	4 (12.1%)
Cardiomyopathy	3 (4.6%)	2 (6.3%)	1 (3.0%)
COPD	2 (3.1%)	2 (6.3%)	0 (0%)
Cystic fibrosis	2 (3.1%)	1 (3.1%)	1 (3.0%)
Other lung diseases	5 (7.7%)	4 (12.5%)	1 (3.0%)
HIV infection	1 (1.5%)	0 (0%)	1 (3.0%)
Most recent SOT
Kidney	33 (50.8%)	13 (40.6%)	20 (60.6%)
Liver	16 (24.6%)	9 (28.1%)	7 (21.2%)
Heart	6 (9.2%)	5 (15.6%)	1 (3.0%)
Lung	4 (6.2%)	3 (9.4%)	1 (3.0%)
Kidney and pancreas	4 (6.2%)	1 (3.1%)	3 (9.1%)
Pancreas	1 (1.5%)	0 (0%)	1 (3.0%)
Liver and kidney	1 (1.5%)	1 (3.1%)	0 (0%)
History of SOT
Any kidney	39 (60.0%)	15 (46.9%)	24 (72.7%)
Any liver	17 (26.2%)	10 (31.3%)	7 (21.2%)
Any heart	8 (12.3%)	5 (15.6%)	3 (9.1%)
Any lung	4 (6.2%)	3 (9.4%)	1 (3.0%)
Any kidney and pancreas	5 (7.7%)	2 (6.3%)	3 (9.1%)
≥2 SOTs	9 (13.8%)	4 (12.5%)	5 (15.2%)
Time from most recent SOT, median (IQR), months	95.9 (42.8, 191.6)	74.3 (36.0, 178.3)	106.7 (54.3, 191.9)
Time since most recent SOT
1–2 y	8 (12.3%)	4 (12.5%)	4 (12.1%)
2.1–5 y	15 (23.1%)	9 (28.1%)	6 (18.2%)
>5 y	42 (64.6%)	19 (59.4%)	23 (69.7%)
Induction immunosuppressionc
Steroids	44 (67.7%)	23 (71.9%)	21 (63.6%)
ATG	25 (38.5%)	12 (37.5%)	13 (39.4%)
Mycophenolate	18 (27.7%)	10 (31.3%)	8 (24.2%)
Basiliximab	18 (27.7%)	11 (34.4%)	7 (21.2%)
Tacrolimus	5 (7.7%)	2 (6.3%)	3 (9.1%)
Rituximab	1 (1.5%)	0 (0%)	1 (3.0%)
Maintenance immunosuppressionc
Tacrolimus	44 (67.7%)	20 (62.5%)	24 (72.7%)
Mycophenolate	44 (67.7%)	22 (68.8%)	22 (66.7%)
Prednisone	27 (41.5%)	15 (46.9%)	12 (36.4%)
Cyclosporine	9 (13.8%)	4 (12.5%)	5 (15.2%)
Sirolimus	5 (7.7%)	4 (12.5%)	1 (3.0%)
Everolimus	4 (6.2%)	2 (6.3%)	2 (6.1%)
Azathioprine	4 (6.2%)	1 (3.1%)	3 (9.1%)
Prior influenza immunityc
Laboratory-confirmed influenza in the previous season	3 (4.6%)e	3 (9.4%)e	0 (0%)
Seasonal influenza vaccine in the previous season	56 (86.2%)	27 (84.4%)	29 (87.9%)
Participants enrolled in the  2023–2024 season (n = 35)	31/35 (88.6%)	15/17 (88.2%)	16/18 (88.9%)
Participants enrolled in the 2024–2025 season (n = 30)	25/30 (83.3%)	12/15 (80%)	13/15 (86.7%)

Abbreviations: ATG, anti-thymocyte globulin; COPD, chronic obstructive pulmonary disease; HD, high-dose; IQR, interquartile range; SOT, solid organ transplant.

^aValues are shown as number (%) or as otherwise noted.

^bRace and ethnicity categories were self-reported by trial participants.

^cFrequencies do not add up to 100% as multiple values/options are possible.

^dPast medical history of comorbidities includes remote and current events.

^eThe 3 participants with previous laboratory-confirmed influenza had also received a seasonal influenza vaccine in that prior season.

In this study, kidney (33/65, 50.8%) and liver (16/65, 24.6%) recipients were the most frequent SOTRs. A total of 39 individuals (60%) had a history of kidney transplant, and 9 individuals (13.8%) had a history of 2 or more SOTs. Most participants (42/65, 64.6%) were >5 years from their most recent transplant at enrollment. Induction and maintenance immunosuppression medications were similar between the 2 groups. The maintenance immunosuppression regimens most frequently included tacrolimus (67.7%), mycophenolate (67.7%), and prednisone (41.5%). Most participants were considered to have prior influenza immunity (56/65, 86.2%), defined as having received an influenza vaccine and/or having a history of laboratory-confirmed influenza in the prior influenza season. The 56 participants had received an influenza vaccine in the previous influenza season, of whom 3 had laboratory-confirmed influenza during that prior season. No participants reported laboratory-confirmed influenza during the influenza season prior to enrollment.

ID₈₀ Neutralizing and Binding Antibody Titers From Baseline to 1-Month Follow-up

Following the initial HD influenza vaccine at baseline, we observed GMFRs >1.0 for both ID₈₀ neutralizing and binding antibody titers across all strains at the 1-month visit, consistent with an expected immune response (Supplementary Table 2, Supplementary Figure 2). Responses were similar among those with and without prior immunity (Supplementary Tables 3 and 4).

ID₈₀ Neutralizing and Binding Antibody Titers From Baseline to 4-Month Follow-up (2 HD vs 1 HD)

In the prespecified primary immunogenicity analysis from baseline to the final 4-month follow-up visit incorporating multiple imputation for missing 4-month titers, GMFRs in both ID₈₀ neutralizing and binding titers were consistently higher in the 2 HD group compared with the 1 HD group across all strains (Table 2, Figure 2). However, between-group differences did not reach statistical significance for any strain based on the GMRs and corresponding 95% CIs, and the study may have been underpowered to detect modest between-group differences. The largest differences were observed for the Phuket strain, particularly for binding titers. Complete-case analyses restricted to participants with observed titers during the study yielded similar results (Supplementary Table 5).

Table 2.

Geometric Mean Fold Rises (GMFRs) in ID₈₀ Neutralizing and Binding Antibody Titers From Baseline to 4-Month Follow-up After Multiple Imputation (n = 65). Multiple Imputation was Used to Fill in the Titer Values for 5 Individuals of the 2 HD Group and 2 Individuals of the 1 HD Group Who Missed Visit 3 and Therefore did not Have a 4-Month Follow-up Titer Measurement. Treatment Arm, Age, Sex, Receipt of Influenza Vaccine in the Prior Influenza Season, Time From Most Recent SOT, Maintenance Immunosuppression Regimen, and Baseline Titer Value Were Included in the Imputation Model

	GMFR (95% CI)
Assay—Influenza strain	2 HD group (n = 32)	1 HD group (n = 33)	GMRa (95% CI)	P value	Adjusted P value b
ID80—Austria	1.51 (1.31, 1.74)	1.41 (1.24, 1.60)	1.07 (.89, 1.28)	.484	1.000
ID80—Darwin	1.55 (1.31, 1.84)	1.34 (1.12, 1.62)	1.15 (.90, 1.49)	.267	1.000
ID80—Phuketc	1.58 (1.28, 1.96)	1.30 (1.15, 1.46)	1.22 (.96, 1.56)	.108	.756
ID80—Wisconsin	1.65 (1.37, 1.99)	1.54 (1.33, 1.79)	1.07 (.84, 1.36)	.587	1.000
Binding—Austria	1.58 (1.34, 1.87)	1.45 (1.28, 1.65)	1.09 (.88, 1.34)	.441	1.000
Binding—Darwin	2.03 (1.65, 2.50)	1.94 (1.64, 2.28)	1.05 (.81, 1.37)	.724	1.000
Binding—Phuketc	1.83 (1.48, 2.25)	1.40 (1.18, 1.67)	1.30 (.99, 1.71)	.057	.456
Binding—Wisconsin	2.02 (1.64, 2.47)	1.85 (1.59, 2.15)	1.09 (.84, 1.41)	.507	1.000

^aGeometric mean ratio (GMR) is the ratio of the GMFRs between the 2 HD group and the 1 HD group.

^bAdjusted P values were calculated using the Holm method to account for multiple comparisons across influenza strains and assay types.

^cAnalysis of the Phuket titer levels is restricted to participants in the 2023–2024 influenza season.

Figure 2.

ID₈₀ Neutralizing and Binding Antibody Titer Fold Changes for Each Influenza Strain from Baseline (Visit 1) to 4-Month Follow-up (Visit 3). GMFRs with their 95% CIs are shown for each study arm.

Exploratory Analyses of ID₈₀ Neutralizing and Binding Antibody Titers From 1-Month Follow-up to 4-Month Follow-up (2 HD vs 1 HD)

To explore the incremental immunologic effect of the randomized second injection during the post-randomization interval, we compared antibody responses from the 1-month visit (immediately prior to the second injection) to the 4-month follow-up visit. During this post-randomization interval, GMFRs in both ID₈₀ neutralizing and binding titers were higher in the 2 HD group than the 1 HD group across all strains (Table 3, Figure 3). In exploratory analyses, binding antibody responses were consistently higher in the 2 HD group across all strains, and differences for Austria, Darwin, and Phuket strains remained statistically significant after adjustment for multiple comparisons. No ID₈₀ neutralizing antibody comparisons remained statistically significant after adjustment. The largest increase was observed for Darwin binding titers (GMR 1.38, 95% CI 1.13–1.69). These results were similar in the complete-case analysis (Supplementary Table 6). Antibody response trajectories across all study visits are shown in Supplementary Figure 3 to facilitate visualization of longitudinal changes in GMFRs over time in each study group.

Table 3.

Exploratory Analysis of Geometric Mean Fold Rises (GMFRs) in ID₈₀ Neutralizing and Binding Antibody Titers From 1-Month Follow-up to 4-Month Follow-up After Multiple Imputation (n = 65). Multiple Imputation was Used to Fill in the Titer Values for 5 Individuals of the 2 HD Group and 2 Individuals of the 1 HD Group Who Missed Visit 3 and Therefore did not Have a 4-Month Follow-up Titer Measurement. Treatment Arm, Age, Sex, Receipt of Influenza Vaccine in the Prior Influenza Season, Time From Most Recent SOT, Maintenance Immunosuppression Regimen, and Baseline Titer Value Were Included in the Imputation Model

	GMFR (95% CI)
Assay—Influenza strain	2 HD group (n = 32)	1 HD group (n = 33)	GMRa (95% CI)	P value	Adjusted P valueb
ID80—Austria	1.00 (0.89, 1.13)	0.84 (0.75, 0.94)	1.19 (1.02, 1.38)	.028	.095
ID80—Darwin	0.96 (0.82, 1.13)	0.82 (0.73, 0.93)	1.17 (.95, 1.43)	.133	.266
ID80—Phuketc	1.06 (0.93, 1.22)	0.89 (0.82, 0.96)	1.20 (1.03, 1.40)	.023	.095
ID80—Wisconsin	1.00 (0.88, 1.14)	0.88 (0.79, 0.98)	1.13 (.95, 1.34)	.163	.266
Binding—Austria	1.05 (0.94, 1.18)	0.81 (0.73, 0.90)	1.30 (1.11, 1.52)	.001	.008
Binding—Darwin	1.09 (0.94, 1.27)	0.79 (0.69, 0.90)	1.38 (1.13, 1.69)	.001	.008
Binding—Phuketc	1.10 (0.94, 1.29)	0.81 (0.73, 0.90)	1.36 (1.12, 1.65)	.002	.012
Binding—Wisconsin	1.09 (0.91, 1.29)	0.84 (0.75, 0.94)	1.29 (1.04, 1.60)	.019	.095

^aGeometric mean ratio (GMR) is the ratio of the GMFRs between the 2 HD group and the 1 HD group.

^bAdjusted P values were calculated using the Holm method to account for multiple comparisons across influenza strains and assay types.

^cAnalysis of the Phuket titer levels is restricted to participants in the 2023–2024 influenza season.

Figure 3.

ID₈₀ Neutralizing and Binding Antibody Titer Fold Changes for Each Influenza Strain from 1-Month Follow-up (Visit 2) to 4-Month Follow-up (Visit 3). GMFRs with their 95% CIs are shown for each study arm.

ID₅₀ Neutralizing Antibody Titers

The GMFRs in ID₅₀ neutralizing titers for all strains were greater in the 2 HD group compared with the 1 HD group, yet no statistically significant differences were observed in both the complete-case and imputed data analyses (Supplementary Tables 7 and 8).

Clinical Outcomes

Of the 65 randomized participants, one participant from the 2 HD group died of influenza-unrelated causes, leaving a total of 64 participants for clinical outcome analyses. Among the 64 study participants, 16 participants (25%) reported ILI symptoms; 7 were in the 2 HD group, and 9 were in the 1 HD group (Supplementary Table 9). Of these 16 participants, 6 (3 in each group) underwent respiratory viral testing. Two participants (3.1% of 64 participants) developed laboratory-confirmed influenza: one influenza A case in the 2024–2025 season and one influenza B case in the 2023–2024 season. Both participants were in the 1 HD group, and the influenza infections occurred 5–6 months after the baseline study visit where they received a single HD influenza vaccine dose. The remaining 4 participants who underwent testing included 2 with COVID-19 and 1 with RSV in the 2 HD group and 1 with COVID-19 in the 1 HD group. There were no influenza-related hospitalizations, ICU admissions, needs for noninvasive or mechanical ventilation, or deaths.

Adverse Events

The proportion of participants experiencing any clinical AE was similar between groups: 22/32 (69%) in the 2 HD group and 22/33 (67%) in the 1 HD group (Table 4). The vast majority of AEs were grade 1 or 2 (95/97, 98%). Solicited reactogenicity symptoms within 7 days of injection were reported in 19/65 participants (29%), were all grade 1, and were less frequent in the 2 HD group (6/32, 19%) than in the 1 HD group (13/33, 39%). Reactogenicity symptoms after the first HD influenza vaccine dose, in particular pain at the injection site, were more common in the 1 HD group. Reactogenicity after the second HD influenza vaccine dose was similar regardless of administration of HD vaccine or placebo. Most other AEs recorded during the study were deemed unrelated to the study injections.

Table 4.

Clinical Adverse Events in Study Participants (n = 65). Data are Presented as No. or No. (%) of Participants Experiencing AEs. Where Two Numbers are Shown Separated by a Hyphen, the First Number Refers to AEs After the First Injection (HD Influenza Vaccine) Visit While the Second Number Refers to AEs After the Second Injection (Either HD Influenza Vaccine or Placebo)

Clinical AEs	No. (any Grade, N = 65a)	2 HD Group (n = 32)				1 HD Group (n = 33)
		Any grade	G1	G2	G5b	Any grade	G1	G2	G3
Any AE	44 (68%)	22 (69%)	15 (47%)	7 (22%)	1 (3%)	22 (67%)	18 (55%)	8 (24%)	1 (3%)
SAEs	1	1	…	…	1	…	…	…	…
Death	1	1	…	…	1c	…	…	…	…
Any solicited AEd	19 (29%)	6 (19%)	5–3	…	…	13 (39%)	8–6	…	…
Solicited local AEs	…	…	…	…	…	…	…	…	…
Pain or tenderness	12	4	2–3	…	…	8	6–3	…	…
Bruising	2	…	…	…	…	2	0–2	…	…
Solicited systemic AEs	…	…	…	…	…	…	…	…	…
Fatigue	5	3	3–0	…	…	2	2–0	…	…
Malaise	2	1	1–0	…	…	1	0–1	…	…
Other AEse	32 (49%)	17 (53%)	4–7	1–6	…	15 (45%)	3–7	2–6	0–1

Abbreviations: AE, adverse event; HD, high-dose; SAE, serious AE.

^aOf the 74 enrolled individuals, 7 withdrew before the first HD influenza vaccine visit and did not report any study AEs. Of the 67 recipients who received an HD influenza vaccine dose at baseline, 2 withdrew shortly after vaccine administration and did not report any study AEs.

^bGrade 5 indicates death.

^cThe cause of death was subarachnoid hemorrhage with the contributory cause of acute myeloid leukemia.

^dSolicited reactogenicity symptoms and signs, either systemic or local, that started within 7 days after study injections.

^eOther AEs include solicited-type AEs starting after 7 days of either injection visit or unsolicited AEs documented throughout study conduct. All these AEs were deemed unrelated to the study injections. Further details are provided in the Supplementary Table 10.

^fBiopsy-proven acute cellular liver rejection.

Two grade ≥3 AEs occurred during the 2023–2024 season: one participant in the 1 HD group experienced grade 3 biopsy-proven acute cellular liver rejection approximately 3 months after the HD vaccination, and one participant in the 2 HD group died from subarachnoid hemorrhage and acute myeloid leukemia approximately 2 months after the second injection; this event was deemed unrelated to study injections. A detailed assessment of clinical AEs is provided in Supplementary Table 10.

DISCUSSION

In the prespecified primary analysis of this randomized trial, GMFRs from baseline to 4-month follow-up were consistently higher in the 2 HD group than in the 1 HD group across strains, although between-group differences did not reach statistical significance. In the analysis focusing on the post-randomization interval from 1-month to 4-month follow-up, which may better reflect the incremental effect of the second HD dose than analyses anchored at baseline, antibody responses were higher in the 2 HD group and reached statistical significance for multiple strains. Together, these findings suggest the possibility that an in-season HD booster may enhance or better maintain influenza-specific antibody responses later in the season among immunosuppressed SOTRs, although confirmation in larger, adequately powered cohorts is needed to substantiate these findings given the limited sample size.

To our knowledge, this study is the first randomized trial to compare the immunologic response and clinical outcomes in SOTRs receiving 2 HD influenza vaccine doses 1 month apart vs 1 HD influenza vaccine during an influenza season. Our findings are consistent with prior studies demonstrating enhanced immunogenicity of HD vaccine formulations [5, 6, 9–11, 16] and align with evidence supporting in-season vaccine booster strategies for SOTRs and HCT recipients [4–6]. Although these studies present differences in the specific vaccine intervention and design, they build on the principle of using a greater amount of antigen in immunocompromised individuals to stimulate a more robust and protective antibody response. Cordero et al. reported higher rates of seroconversion among SOTRs receiving two doses of SD of influenza vaccine compared to one dose [4]. In a phase 2 randomized trial in HCT recipients, Thomas et al. found that 2 doses of the trivalent HD influenza vaccine produced higher post-vaccination GMTs compared with SD vaccination when given 4 weeks apart, although differences were not uniformly statistically significant for influenza A(H1N1) [6]. Interestingly, a systematic review of 4 studies found that 2 doses of HD influenza vaccine led to higher hemagglutination inhibition assay (HAI) GMTs across all strains, but no difference was observed in seroprotection or seroconversion compared with 2 SD influenza vaccines [17].

Our results suggest that repeat HD vaccination within a single influenza season may augment influenza-specific antibody responses in immunocompromised persons. While the study was not powered to assess clinical effectiveness, laboratory-confirmed influenza occurred only in the placebo group. However, the small number of events precludes any conclusions regarding clinical protection. Most participants had evidence of prior influenza exposure, either by influenza infection or vaccination. Due to the small sample size, we could not reliably determine whether prior exposure to vaccination/infection modified the titer response from baseline through the 4-month follow-up.

In our study, the safety profile of a 2 HD influenza vaccination regimen was reassuring, with AEs being mild and not more frequent in the 2 HD group. Similarly to previous influenza vaccine studies, our findings confirm that in-season boosting is safe [4, 17]. The redemonstration of both the safety and efficacy in influenza vaccine studies over the past few years has informed the 2025 influenza vaccine recommendations from the Centers for Disease Control and Prevention, which state that the HD influenza vaccine is an acceptable option for seasonal influenza vaccination for people aged 18–64 years who have undergone a SOT or are receiving immunosuppressants [18]. Although current recommendations do not include routine in-season boosting, this approach warrants further evaluation and should be considered in future guideline recommendations for immunocompromised persons.

This study has limitations. Enrollment did not reach the target sample size. Recruitment was limited by the relatively short seasonal vaccination window, competing opportunities for influenza vaccination outside the study, and the logistical challenges of completing study procedures during a concentrated period early in each influenza season. In addition, several participants missed the 4-month visit, further reducing statistical power for the primary and secondary analyses. Consequently, the study may have been unable to detect modest but clinically meaningful differences between groups, and the exploratory findings should be interpreted cautiously. Although several exploratory post-randomization comparisons remained statistically significant after adjustment for multiple comparisons, these findings should be interpreted cautiously because the analyses were exploratory and not primary efficacy analyses. Additionally, the cohort was predominantly kidney and liver transplant recipients, limiting generalizability to other transplant types, including lung transplant recipients, who are at particularly high risk for severe respiratory viral infections. We also excluded participants within 1 year of SOT to reduce the confounding effects of heightened immunosuppression early post-SOT. While individuals with less than 1-year post-SOT might also benefit from a 2 HD vaccination regimen, our findings cannot be generalized to that population. The study did not include a dedicated visit 4–6 weeks after the second injection and therefore could not directly assess peak post-booster antibody responses. Also, ILI was documented at the end of the season, which—in the absence of active surveillance—may have been underestimated if participants sought care locally or outside the primary service area. Lastly, the influenza assays did not include HAI titers to determine seroprotection and seroconversion correlates, and the Fluzone® vaccine formulation differed between study seasons. Due to difficulties in rescuing a matched virus to the Fluzone® egg-based strains, we used cell-based influenza strains with 98%–100% amino acid homology. However, we acknowledge this strain mismatch and the use of a surrogate assay instead of HAI as limitations. Future multicenter studies powered for clinical and laboratory outcomes are warranted to confirm our findings; such studies would also allow sub-analyses by transplant type or prior immunity profile.

In conclusion, our findings suggest that an in-season second dose of HD influenza vaccine may improve influenza-specific antibody responses compared with a single HD dose and may optimize protection against influenza illness in adult SOTRs, although these observations require confirmation in larger, adequately powered studies.

Supplementary Data

Supplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.