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. 2025 Dec 23;11:27. doi: 10.1038/s41541-025-01350-3

Integrated analysis of humoral and memory B-cell responses reveals distinct immune landscapes shaped by influenza vaccine platforms

Laise Rodrigues Reis 1, Ted M Ross 1,2,3,4,✉
PMCID: PMC12848071  PMID: 41430048

Abstract

Influenza vaccination is central to reducing morbidity and mortality. However, vaccine-induced immune responses vary considerably across vaccine platforms. Hemagglutination inhibition (HAI) titers are widely used as correlates of protection, but do not fully capture the complexity of memory B-cell (MBC) responses. This study employed an integrated analysis of humoral and MBC responses elicited by three licensed influenza vaccines: inactivated Fluzone standard dose (FluZ), recombinant protein-based FluBlok (FluB), and live-attenuated intranasal FluMist (FluM). FluB-vaccinees had the most robust HAI and MBC responses, with increased frequencies of switched memory and IgG memory across all HA components (H1, H3, and IBV), along with increased IgA resting memory and IgG activated memory to H1 and H3, and IgG resting memory to H1 and IBV. FluZ-vaccinees had robust, but comparatively lower responses, including increased IgG memory to H1 and IBV, but reduced switched memory compared to FluB-vaccinees. FluM-vaccinees had the lowest HAI titers but increased unswitched memory and IgA memory to H1 and IBV, along with higher IgM memory to H3. Notably, FluM-vaccinees showed greater inter-correlation among multiple MBC subsets, particularly for H3. These findings uncover distinct platform-specific immune landscape and demonstrate that FluB induces superior MBC responses, providing a framework for designing next-generation vaccines.

Subject terms: Diseases, Immunology, Microbiology

Introduction

Seasonal influenza remains a major global public health challenge and is responsible for an estimated annual 3–5 million cases of severe illness and up to 650,000 respiratory-related deaths1,2. Vaccination is the most effective strategy to reduce the impact of influenza virus infection and is central to global prevention efforts3,4. However, vaccine effectiveness, as well as the magnitude and quality of the vaccine-induced immune responses, varies considerably depending on the vaccine platform, formulation, antigen content, route of administration, and host immune history, particularly prior influenza exposure through infection or vaccination5–7. Influenza vaccines are designed primarily to induce strain-specific neutralizing antibodies against the hemagglutinin (HA) protein, which is the major surface glycoprotein of the virus responsible for attachment and entry into host cells. Neutralizing antibodies targeting the HA head domain prevent infection by blocking viral binding to sialic acid receptors, and hemagglutination inhibition (HAI) titers have traditionally served as the major correlate of protection8–11. However, growing evidence indicates that HAI titers alone do not fully capture the complexity of vaccine-induced immune responses.

Memory B-cells (MBC) play a critical role in long-term immune protection, capable of mounting rapid and robust recall responses upon antigen re-exposure12,13. Even in the absence of detectable circulating antibodies, MBC can rapidly differentiate into antibody-secreting cells and contribute to the generation of high-affinity antibodies. This ability becomes particularly important as circulating antibody levels wane, positioning MBC as an essential component of the immune response. Importantly, MBC compartment is heterogeneous, comprising phenotypically and functionally distinct subsets with complementary roles. Circulating plasma cells provide an immediate source of antigen-specific antibodies following vaccination, whereas long-lived plasma cell and MBC populations are established to maintain durable protection14. Unswitched memory represents an early-response subset that rapidly produces IgM antibodies15,16. In contrast, class-switched memory has undergone germinal center maturation and is capable of a rapid proliferation and production of high-affinity IgA and IgG antibodies, providing a long-lasting immune response17,18. Additionally, activated and resting MBC reflect distinct stages of activation. Activated MBC function as professional antigen-presenting cells (APCs) that activate naïve T-cells19,20, whereas resting MBC, also known as classical MBC, can proliferate and rapidly differentiate into plasma cells21. Together, these subsets provide a multifaceted view of the memory compartment, and their evaluation offers mechanistic insight into the qualitative features of vaccine-elicited immune response that extend beyond serological measurements. Notably, different vaccine platforms may elicit distinct MBC phenotypes, with potential implications for the magnitude, quality, and durability of protection.

Despite the widespread use of licensed influenza vaccines, comprehensive comparative analyses of how different vaccine platforms shape MBC responses remain limited, particularly regarding cellular immune response. To address this critical knowledge gap, this study focused on a comprehensive and integrated analysis of humoral and MBC responses elicited by three licensed influenza vaccines: the standard dose inactivated vaccine (Fluzone), the recombinant protein-based vaccine (FluBlok), and the live-attenuated intranasal vaccine (FluMist). MBC responses were evaluated in peripheral blood mononuclear cells (PBMC), which reflect the circulating memory compartment and provide insight into systemic vaccine-induced immune response. By integrating traditional serological assessment through HAI titers with HA-specific MBC phenotyping, this study reveals a platform-specific immune landscape that reflects differences in antigen content, structure, and route of administration. Together, these findings advance our understanding of how distinct vaccine technologies shape both the magnitude and quality of the adaptive immune response and provide a mechanistic foundation for guiding the rational development of next-generation influenza vaccines.

Results

Influenza vaccines elicit distinct strain-specific HAI responses

To assess the humoral responses elicited by the three influenza vaccines (Fluzone standard dose inactivated vaccine—FluZ, FluBlok recombinant protein-based vaccine—FluB, and FluMist live-attenuated intranasal vaccine—FluM), HAI titers to H1N1 (A/Victoria/4897/2022), H3N2 (A/Massachusetts/18/2022), and B/Victoria (B/Austria/1359417/2021) components were measured in serum samples collected prior to (D0) and 28 days post-vaccination (D28) (Fig. 1).

Fig. 1. HA-specific hemagglutination-inhibition assay (HAI) titers upon influenza vaccination in adults.

Fig. 1

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The heatmap represents the H1N1 (A/Victoria/4897/2022), H3N2 (A/Massachusetts/18/2022), and Influenza B (B/Austria/1359417/2021) HAI titers measured from the UGA9 cohort (2024–2025 Influenza season) at day 0 (D0) and 28 days (D28) post-vaccination. The rows represent the three vaccine groups: Fluzone standard dose inactivated vaccine (FluZ, median age = 34 y.o), FluBlok recombinant protein-based vaccine (FluB, median age = 33 y.o), and FluMist live-attenuated intranasal vaccine (FluM, median age = 35 y.o). Color gradient corresponds to HAI titer levels, with lighter pink shades indicating lower titers and darker blue shades representing higher titers. Asterisk represents participants who seroconverted (≥4-fold increase).

Participants vaccinated with any of the three influenza vaccines had, on average, increased HAI titers, however, the magnitude of the titers varied depending on the vaccine platform and vaccine strain (Fig. 1). Participants had highest post-vaccination HAI titers against the influenza B (IBV) component (B/Aus/21) compared to the two influenza A components (A/Vic/22 H1N1; A/Mass/22 H3N2). In comparison, participants vaccinated with FluB had the highest HAI titers, with several participants achieving seroprotective titers (≥1:40) and demonstrating seroconversion (≥4-fold increase) against all three components (H1N1, n = 11; H3N2, n = 11; and B/Victoria, n = 9). Participants vaccinated with FluZ had lower antibody titers compared to participants vaccinated with FluB, with the highest titers detected against the H3N2 (A/Mass/22) and B/Victoria (B/Aus/21) viruses. Conversely, participants vaccinated with FluM had the lowest serum HAI titers, with low fold increases in HAI titers and a lower proportion of individuals reaching the seroprotective threshold (titer ≥1:40) against all vaccine strains compared to participants vaccinated with FluZ or FluB. However, these findings reflect serum antibody response only, as mucosal immune response was not assessed in this study.

Influenza vaccine platforms differentially shape HA-specific memory B-cell responses

HA-specific memory B-cell (MBC) responses targeting the three vaccine strains (H1N1, H3N2, and B/Victoria) were evaluated using surface markers to define plasma cells (CD19+CD27+CD38+), total memory (CD19+CD24+CD38−), unswitched memory (CD19+IgD+IgM+), and switched memory (CD19+IgD−IgM−) B-cells (Supplementary Fig. 1 and Fig. 2).

Fig. 2. HA-specific memory B-cell response upon influenza vaccination in adults.

Fig. 2

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Analysis of HA-specific memory B-cell responses (Plasma Cells—CD19+CD27+CD38+; Total Memory—CD19+CD24+CD38−; Unswitched memory—CD19+IgD+IgM+; and Switched memory—CD19+IgD−IgM−) targeting the three strains of the vaccine (H1N1, H3N2, and B/Victoria) was carried out by Flow Cytometry, as described in Methods. The HA-specific B-cell response was evaluated in participants vaccinated with Fluzone standard dose (FluZ - blue bars), Flublok (FluB - pink bars), and Flumist (FluM - green bars). Data are displayed as a scattering distribution of individual values over bars expressed as fold change, determined by dividing post-vaccination (D28) results by corresponding baseline (D0) values for each participant. Comparative analyses between two groups were performed by Mann–Whitney test or Student t-test. For comparison across multiple groups, Kruskal–Wallis with Dunn’s post hoc correction or ANOVA followed by Tukey’s multiple comparison test was employed, as appropriate. Significant differences were considered at p ≤ 0.05 and represented by asterisk. Non-significant results are represented by ns.

For H1 HA-specific responses, participants vaccinated with FluB elicited significantly higher frequency of total MBC (CD24+CD38-) compared to participants vaccinated with FluM (*, p ≤ 0.05), along with significantly higher frequency of switched memory (IgD-IgM-) cells compared to participants vaccinated with FluZ and FluM (*, p ≤ 0.05). Additionally, participants vaccinated with FluM had a significantly higher frequency of unswitched memory (IgD+IgM+) cells compared to participants vaccinated with FluB (*, p ≤ 0.05). No statistical differences were observed in the frequency of plasma cells (CD27+CD38+) detected in participants vaccinated with any of the three vaccines.

For H3 HA-specific responses, participants vaccinated with FluB had significantly higher frequency of plasma cells (CD19+CD27+CD38+) compared to participants vaccinated with FluM (*, p ≤ 0.05). Total MBC (CD19+CD24+CD38−) were also significantly higher in participants vaccinated with either FluZ or FluB compared to FluM (*, p ≤ 0.05). Additionally, participants vaccinated with FluB had a significantly higher frequency of switched memory (CD19+IgD−IgM−) cells compared to participants vaccinated with either FluZ or FluM (*, p ≤ 0.05). There were no significant differences in the frequency of unswitched memory (CD19+IgD+IgM+) cells detected in participants vaccinated with any of the three vaccines. For IBV HA-specific responses, participants vaccinated with FluB had a significantly higher frequency of total MBC (CD19+CD24+CD38-) compared to participants vaccinated with FluZ (*, p ≤ 0.05). Conversely, participants vaccinated with FluM had a significantly greater frequency of unswitched memory (CD19+IgD+IgM+) cells compared to participants vaccinated with either FluZ or FluB (*, p ≤ 0.05). Switched memory (CD19+IgD-IgM-) levels were significantly higher in participants vaccinated with either FluZ or FluB compared to participants vaccinated with FluM (*, p ≤ 0.05). As with H1 HA-specific responses, there were no significant differences in the frequency of plasma cells (CD19+CD27+CD38+) identified in participants vaccinated with any of the three vaccines.

Influenza vaccine platforms elicit distinct profiles of HA-specific memory B-cell isotypes

The isotype distribution of HA-specific MBC was assessed to evaluate the MBC features elicited by the three strains in the vaccine (H1N1, H3N2, and B/Victoria). Frequencies and fold changes of surface-expressing IgM Memory (CD19+IgD+IgM+), IgA Memory (CD19+IgD−IgA+), and IgG Memory (CD19+IgD−IgG+) were analyzed across FluZ, FluB, and FluM vaccine groups (Fig. 3).

Fig. 3. HA-specific memory B-cell isotypes upon influenza vaccination in adults.

Fig. 3

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Analysis of HA-specific memory B-cell isotypes (IgM Memory - IgD+IgM+, IgA Memory - IgD-IgA+, and IgG Memory - IgD-IgG+) was carried out by Flow Cytometry as described in Methods. The percentage of surface-expressing IgM (green bars), IgA (pink bars), and IgG (blue bars) memory was evaluated at day 0 (D0) and 28 (D28) days post vaccination with Fluzone standard dose (FluZ), FluBlok (FluB), or FluMist (FluM). Fold change analysis, determined by dividing post-vaccination (D28) results by corresponding baseline (D0) values for each participant, is displayed as a scattering distribution of individual values over bars (FluZ - blue bars, FluB - pink bars, and FluM - green bars). Comparative analyses between two groups were performed by Mann–Whitney test or Student t-test. For comparison across multiple groups, Kruskal–Wallis with Dunn’s post hoc correction or ANOVA followed by Tukey’s multiple comparison test was employed, as appropriate. Statistical significance is denoted as: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). Non-significant results are represented by ns. The percentage and fold change analysis were evaluated targeting the three strains of the vaccine: H1N1 (a), H3N2 (b), and B/Victoria (c).

For H1 HA-specific MBC responses, FluZ and FluB elicited significant increases in IgG Memory (CD19+IgD−IgG+) frequencies, while FluM induced a higher frequency of IgA Memory (CD19+IgD−IgA+) 28 days post-vaccination. There were significantly higher fold changes in the levels of IgA Memory (CD19+IgD−IgA+) in FluM vaccinated participants compared to FluZ and FluB participants (*, p ≤ 0.05; ***, p ≤ 0.001), whereas participants vaccinated with FluZ and FluB had significantly higher levels of IgG Memory (CD19+IgD−IgG+) compared to FluM vaccinated participants (**, p ≤ 0.01; ***, p ≤ 0.001). There were no significant differences in IgM Memory (CD19+IgD+IgM+) between vaccine groups.

For H3 HA-specific responses, participants vaccinated with FluZ or FluB had increased IgG Memory (CD19+IgD−IgG+) frequencies, while participants vaccinated with FluM had marked elevations in both IgM Memory (CD19+IgD+IgM+) and IgA Memory (CD19+IgD−IgA+) at 28 days after vaccination, with the most substantial increase in the IgA Memory (CD19+IgD−IgA+). Participants vaccinated with FluM had significantly higher IgA Memory (CD19+IgD-IgA+) and IgM Memory (CD19+IgD+IgM+) levels compared to participants vaccinated with FluZ or FluB (*, p ≤ 0.05; **, p ≤ 0.01), whereas FluB vaccinated participants had higher fold changes in IgG Memory (CD19+IgD−IgG+) compared to participants vaccinated with FluM (**, p ≤ 0.01).

For IBV HA-specific responses, participants vaccinated with FluZ or FluB had predominantly IgG Memory (CD19+IgD−IgG+) cells following vaccination, while participants vaccinated with FluM had higher IgA Memory (CD19+IgD−IgA+) frequencies than participants vaccinated with other influenza vaccines. Participants vaccinated with either FluZ and FluM had significantly higher IgA Memory (IgD-IgA+) compared to participants vaccinated with FluB (*, p ≤ 0.05), and IgG Memory (CD19+IgD−IgG+) was significantly greater in participants vaccinated with FluZ and FluB compared to participants vaccinated with FluM (*, p ≤ 0.05; **, p ≤ 0.01). There were no significant differences in IgM Memory (CD19+IgD+IgM+) were observed in participants vaccinated with any of the influenza vaccines.

High-dimensional analysis reveals vaccine-dependent differences in Memory B-cell landscapes

To further explore the HA-specific MBC response, a t-distributed Stochastic Neighbor Embedding (t-SNE) analysis was conducted to visualize the high-dimensional phenotypic landscape of antigen-specific (H1N1, H3N2, and B/Victoria) MBCs following vaccination with FluZ, FluB, and FluM. Fold change based on the number of events was calculated for surface-expressing IgM Memory (CD19+IgD+IgM+), IgA Memory (CD19+IgD−IgA+), and IgG Memory (CD19+IgD−IgG+) (Fig. 4).

Fig. 4. High-dimensional analysis of HA-specific memory B-cell subsets upon influenza vaccination in adults.

Fig. 4

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Dimensionality reduction data was performed using t-SNE (t-Distributed Stochastic Neighbor Embedding) to visualize high-dimensional phenotypic landscape of H1- (a), H3- (b), and IBV HA-specific (c) memory B-cells following vaccination with Fluzone standard dose (FluZ), Flublok (FluB), and Flumist (FluM). The color gradient represents the memory B-cell subsets (IgM Memory - CD19+IgD+IgM+ (blue), IgA Memory - CD19+IgD−IgA+ (green), and IgG Memory - CD19+IgD-IgG+ (peach pink). The left tables display the number of events and the percentage of memory B-cell subsets considering a total of 10,000 events for H1 and H3-specific, and 2500 for B-specific. The right tables show the fold change calculated based on the number of events.

For H1 HA-specific MBC responses, an increase in IgG Memory (CD19+IgD−IgG+) was observed in participants vaccinated with any of the influenza vaccines (FluZ, FluB, and FluM), while participants vaccinated with FluM or FluZ had elevated levels of IgA Memory (CD19+IgD−IgA+). Additionally, participants vaccinated with FluB had increased levels of IgM Memory (CD19+IgD+IgM+). For H3 HA-specific MBC responses, participants vaccinated with any of the three vaccines had IgG Memory (CD19+IgD−IgG+), while participants vaccinated with FluZ or FluM exhibited prominent increases in IgA Memory (CD19+IgD−IgA+). For IBV HA-specific MBC responses, participants vaccinated with FluZ or FluB had elevated levels of IgM Memory (CD19+IgD+IgM+), with increased IgG Memory (CD19+IgD-IgG+) in participants vaccinated with FluZ.

Influenza vaccine platforms differentially shape the activation phenotype of HA-specific memory B-cell subsets

HA-specific IgA and IgG memory subsets (activated memory - CD21-CD27+ and resting memory - CD21+CD27+) were evaluated in participants vaccinated with any of the three vaccines against the three vaccine strains (H1N1, H3N2, and B/Victoria) (Fig. 5).

Fig. 5. HA-specific memory B-cell response upon influenza vaccination in adults.

Fig. 5

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Analysis of HA-specific IgA and IgG memory subsets (activated memory - CD21−CD27+ and resting memory - CD21+CD27+) targeting the three strains of the vaccine (H1N1, H3N2, and B/Victoria) was carried out by Flow Cytometry as described in Methods. The HA-specific B-cell response was evaluated in participants immunized with Fluzone standard dose (FluZ - blue bars), Flublok (FluB - pink bars), and Flumist (FluM - green bars). Data are displayed as a scattering distribution of individual values over bars expressed as fold change, determined by dividing post-vaccination (D28) results by corresponding baseline (D0) values for each participant. Comparative analyses between two groups were performed by Mann–Whitney test or Student t-test. For comparison across multiple groups, Kruskal–Wallis with Dunn’s post hoc correction or ANOVA followed by Tukey’s multiple comparison test was employed, as appropriate. Statistical significance is denoted as: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). Non-significant results are represented by ns.

For H1 HA-specific response, participants vaccinated with either FluZ or FluB had significantly increased levels of IgA resting memory (CD19+IgA+CD21+CD27+) compared to participants vaccinated with FluM (**, p ≤ 0.01; ****, p ≤ 0.0001), and there were no differences in IgA activated memory (CD19+IgA+CD21−CD27+) in participants vaccinated with any of the three vaccines. Participants vaccinated with FluB also had significantly higher levels of IgG activated memory (CD19+IgG+CD21-CD27+) compared to participants vaccinated with FluM (*, p ≤ 0.05), and participants vaccinated with either FluZ or FluB had significantly higher levels of IgG resting memory (CD19+IgG+CD21+CD27+) cells compared to participants vaccinated with FluM (*, p ≤ 0.05).

For H3 HA-specific responses, participants vaccinated with FluZ or FluB had significantly higher levels of IgA resting memory (CD19+IgA+CD21+CD27+) compared to participants vaccinated with FluM (*, p ≤ 0.05; **, p ≤ 0.01), and there were no significant differences in IgA activated memory (CD19+IgA+CD21-CD27+). Participants vaccinated with FluB exhibited a robust increase in IgG activated memory (CD19+IgG+CD21−CD27+) that was significantly higher compared to participants vaccinated with FluZ or FluM (***, p ≤ 0.001; ****, p ≤ 0.0001). There were no significant differences in IgG resting memory (CD19+IgG+CD21+CD27+) participants vaccinated with any of the three vaccines.

In contrast, there were no significant differences in IgA activated memory (CD19+IgA+CD21−CD27+) or IgA resting memory (CD19+IgA+CD21+CD27+) in the IBV-specific responses among participants vaccinated with any of the three vaccines. However, the IgG resting memory (CD19+IgG+CD21+CD27+) was significantly higher in participants vaccinated with FluZ or FluB compared to participants vaccinated with FluM (*, p ≤ 0.05; **, p ≤ 0.01), while IgG activated memory (CD19+IgG+CD21-CD27+) remained in participants vaccinated with any of the three vaccines.

Influenza vaccine platforms elicited immune responses with distinct correlations between humoral and memory B-cell responses

Correlation analysis was performed to evaluate connections between humoral and MBC responses following vaccination with Fluzone standard dose (FluZ), Flublok (FluB), or Flumist (FluM) for each vaccine strain (H1N1, H3N2, and B/Victoria) (Fig. 6).

Fig. 6. Correlation matrices of HA-specific memory B-cell response upon influenza vaccination in adults.

Fig. 6

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Correlation analysis between humoral and memory B-cell response was performed using fold-change values (D28/D0). Pearson’s or Spearman’s rank correlation coefficient was applied according to data distribution. The color gradient represents the strength of positive (blue) or negative (red) correlation between two ranked variables. Only significant data are displayed.

The correlation matrix revealed distinct interactions between humoral and MBC response in participants vaccinated with any of the three vaccine platforms and vaccine strains. Participants vaccinated with FluB and FluM had a significantly higher number of MBC subsets, with participants vaccinated with FluM having the highest network complexity of H3 HA-specific responses, particularly involving IgA memory compartments. In contrast, participants vaccinated with FluZ had fewer interactions across MBC subpopulations for all strains. When comparing the responses for all three vaccine strains, influenza B-specific responses had the most frequent correlations between HAI titers and MBC subsets.

Discussion

Seasonal influenza vaccination remains a cornerstone of public health efforts for reducing morbidity and mortality associated with influenza virus infection3,22,23. However, the magnitude and quality of vaccine-induced immune response can vary considerably depending on the vaccine platform, the formulation, as well as host factors24–26. Although hemagglutination inhibition (HAI) titers are widely used as correlates of protection, they do not fully capture the complexity of vaccine-elicited immune responses. MBC, which include diverse phenotypes and activation states, play a central role in long-term immune protection and rapid recall responses upon antigen re-exposure27. Despite the widespread use of various influenza vaccine technologies, comparative data detailing how their platforms differentially shape the magnitude and phenotypic features of the immune response remain limited, particularly regarding cellular responses. To address this gap, the present study employed an integrated analysis of the humoral and MBC responses elicited by three licensed influenza vaccine platforms in healthy adults: the standard dose inactivated vaccine (Fluzone), the recombinant protein-based vaccine (FluBlok), and the live-attenuated intranasal vaccine (FluMist).

Although participants vaccinated with any of the three influenza vaccines had, on average, increased HAI antibody titers following vaccination, participants vaccinated with FluB had the most robust HAI titers against all three vaccine HA components (H1, H3, and IBV). These results aligned with previous reports showing that recombinant HA vaccines outperform egg-based inactivated vaccines25,28,29, likely due its higher HA content. FluB contains approximately three times more HA than standard dose formulations30. In addition, the absence of egg-adapted mutations commonly introduced during production that can alter antigenic HA epitopes and reduce antigenic match that ultimately compromising immunogenicity31–33. In contrast, participants vaccinated with FluM had lower serum HAI titers compared to participants vaccinated with FluZ or FluB. This observation is consistent with prior studies, suggesting that intranasal live attenuated vaccines may elicit lower antibody responses due to pre-existing mucosal immunity, which can constrain viral replication and limit systemic antigen exposure34,35.

To more comprehensively assess adaptive immune responses, HA-specific MBC phenotypes were also evaluated. Participants vaccinated with FluB had the most robust expansion of total memory, switched memory, and IgG MBC against all three vaccine HA components (H1, H3, and IBV). This enhanced response not only reflects the higher antigen content in FluB but also its composition of intact, soluble, and non-particulate HA proteins that preserve native conformations, facilitating more effective B-cell receptor engagement. Such antigen presentation promotes robust germinal center formation, leading to class-switch recombination and affinity maturation, generating MBC primed for rapid and potent antibody-mediated recall responses upon antigen re-exposure, contributing to an effective and long-lasting immune response25,36. In addition, differences in antigen presentation pathways may further contribute to the superior MBC response elicited by FluB. The soluble, non-particulate HA protein in FluB can be directly captured and internalized by B-cells through BCR-mediated uptake and rapidly trafficked to germinal center follicles, promoting efficient T follicular helper (Tfh) cells engagement and germinal center activity37–39. In contrast, particulate HA protein in FluZ and FluM requires additional processing via APCs, such as dendritic cells, and may preferentially engage distinct innate immunity pathways40–42. These differences in antigen form and processing can influence the B-cell response, providing a mechanistic explanation for the platform-specific differences observed in this study.

In comparison, participants vaccinated with FluZ had robust, but lower MBC responses compared to FluB. Although participants vaccinated with FluZ had a notable expansion of IgG memory, particularly for H1 and IBV HA components. Participants vaccinated with FluZ had a lower frequency of switched memory compared to participants vaccinated with the FluB recombinant protein-based platform. This may reflect limited germinal center engagement, potentially driven by the lower HA content and the presence of egg-adapted mutations that can alter critical antigenic sites33,43–45.

In contrast, participants vaccinated with FluM had higher frequencies of unswitched memory and IgA memory, particularly for H1 and IBV components, with higher IgM memory observed against the H3 HA. Unlike FluZ and FluB, which primarily stimulate systemic immunity, FluM is designed to replicate in the upper respiratory tract, triggering mucosal immune responses. This local stimulation favors the generation of IgA and IgM memory cells, which are important for early frontline defense at mucosal sites46–49. However, in adults who often have pre-existing immunity to influenza, the replication of the attenuated virus may be limited and result in lower antigen exposure and reduced germinal center formation needed for class-switch recombination50,51. Together, these findings correlate with previous studies showing that FluM elicits strong mucosal immunity, but lower antibody titers and systemic MBC24,46,52.

To further characterize the B-cell compartment, the analysis was extended to assess the activation status of HA-specific MBC. By distinguishing between activated and resting memory subsets within IgA and IgG memory populations, these data provide a more refined understanding of the immune landscape shaped by each vaccine. Participants vaccinated with FluB had robust activation of antigen-specific MBC profile with significantly higher frequencies of IgA resting memory and IgG activated memory against H1 and H3 HA proteins, as well as IgG resting memory against H1 and IBV HA components. Notably, the FluB HA content and structure may result in more sustained antigen availability, supporting extended germinal center activity and class-switch recombination53. Moreover, FluB may more effectively recruit and engage Tfh cells, which are essential for germinal center reactions, high-affinity antibody production, and the development of long-lived MBC25,54. The presence of both activated and resting subsets in FluB vaccinated individuals indicates that FluB effectively primes long-term MBC while preserving a pool of antigen-experienced B-cells ready to respond upon re-exposure.

In comparison to FluB, the MBC responses in participants vaccinated with FluZ, although robust, had generally a lower magnitude and breadth of B-cells. While the lower HA content and egg-based production may explain this outcome, other factors likely also influence it. Notably, FluZ contains not only the HA antigen, but also other influenza proteins such as matrix protein 1 (M1), neuraminidase (NA), and nucleoprotein (NP)55,56. Given the prior influenza exposure from infection virus or vaccination, most adults have MBC and antibodies against these proteins. This pre-existing immunity may redirect or compete for immune recognition, thereby reducing the response to HA. Moreover, antibodies against non-HA components could impair antigen uptake or processing by APCs, further diminishing HA-specific germinal center responses. Therefore, the MBC response following split inactivated standard dose FluZ vaccination may be particularly limiting in individuals with pre-existing immunity, who require stronger stimulation to generate a diverse and more robust MBC response57,58. These factors may collectively contribute to the comparatively lower HA-specific MBC responses observed in participants vaccinated with FluZ compared to FluB and support continued optimization of vaccine formulations for long-lasting immune response.

The correlation analysis between anti-HA humoral and MBC responses provided additional insights into the immune responses elicited by each vaccine platform. Although participants vaccinated with FluM had the lowest HAI titers among the three vaccine groups, there was a strong correlation within the MBC compartment, particularly for H3 HA-specific responses. This complex B-cell network suggests that, while these MBC subsets may not directly contribute to circulating antibodies, they may play a critical role in frontline protection by mediating rapid recall responses at mucosal sites upon re-exposure. Tissue-resident MBC, in particular, can act as sentinels in the respiratory tract, enabling localized antibody production even in the absence of high antibody titers59. Following re-exposure to the antigen, these MBC rapidly differentiate into plasma cells, capable of producing antibodies. Mucosal IgA responses, for example, contribute to immune protection through both neutralizing and non-neutralizing mechanisms. Polymeric IgA antibodies exhibit enhanced antigen-binding avidity due to their multimeric structure and have cross-reactive binding and neutralization across homologous and heterologous influenza strains60–62. In addition to classical neutralization, IgA antibodies also had cross-protective immunity to multiple HA subtypes, protecting against a broad range of influenza viruses63. Moreover, IgA can restrict viral release from infected cells and mediate antibody-dependent cellular cytotoxicity (ADCC), further contributing to viral control64–66. These findings reinforce the need for multifaceted immune assessments to fully evaluate vaccine-induced immunity, highlighting that serological assays alone may underestimate the true protective capacity of mucosal vaccines.

There are limitations to this study that should be considered. The sample size, while sufficient for detecting large effect sizes, may underpower analyses of rare MBC subsets. Additionally, the study population consisted only of healthy adults within a specific age range, and the results may not be generalizable to other populations, such as children, older adults, or immunocompromised individuals. Mucosal antibody and mucosal cellular immune responses, especially Tfh cells, were not assessed in this study but represent key components of FluM-elicited immune response. Additional studies evaluating these mucosal mechanisms are essential for fully capturing the protective potential of this vaccine platform. Although FluB elicited higher frequencies of MBC, the longevity and functional quality of these cells were not evaluated. Additional studies are essential to determine whether FluB-generated MBC are qualitatively superior or just more abundant. Moreover, although MBCs are long-lived, longitudinal studies are essential to evaluate the longevity of vaccine-induced responses and whether platform-specific features confer lasting protection against drifted or novel strains.

In summary, this study provides a comprehensive comparison of the humoral and memory B-cell responses elicited by three licensed influenza vaccine platforms, demonstrating that the recombinant protein-based FluB vaccine induces the most robust MBC response. These findings have important implications for optimizing vaccine strategies in individuals with pre-existing immunity, such as older adults or those with repeated influenza exposures. In such populations, platforms that minimize antigenic imprinting may offer improved immunogenicity. Recombinant protein-based vaccines, by delivering high-purity HA antigens without additional viral components, may reduce recall of non-protective memory and better prime novel responses. Importantly, the differences observed across vaccine platforms cannot be attributed solely to antigen content or manufacturing method. Antigen structure, protein purity, and route of administration are equally critical in shaping the quality and magnitude of vaccine-induced memory. The distinct immune response elicited by FluB, FluZ, and FluM reflects the complex interplay of these factors and offers a framework for rational design of next-generation influenza vaccines aimed at achieving broad, long-lasting, and population-wide protection.

Methods

Study design and population

Participants were recruited during the 2024–2025 Influenza season (UGA9 - September 2024 to March 2025) in Athens, Georgia, USA. All participants were part of a larger longitudinal cohort with approximately 10 years of follow-up, in which individuals are consistently vaccinated with the same vaccine platform each season. Written informed consent was obtained from all participants prior to enrollment, and the study protocol was approved by the Institutional Review Board of the University of Georgia (IRB #20224877). An overview of the study population, design, and methods is presented in Fig. 7.

Fig. 7. Compendium of study population and methods.

Fig. 7

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The study comprises 33 healthy adults vaccinated with one of the three trivalent seasonal influenza vaccines: Fluzone standard dose inactivated vaccine (n = 11, median age = 34 y.o.), FluBlok recombinant protein-based vaccine (n = 11, median age = 33 y.o.), or FluMist live-attenuated intranasal vaccine (n = 11, median age = 35 y.o.). The humoral response was assessed by HAI at day 0 (D0) and 28 days (D28) after vaccination. Flow Cytometry was performed to determine HA-specific memory B-cell response prior to (D0) and 28 days (D28) post-vaccination. Created with BioRender.com.

A total of 33 healthy adults, aged 18–49 years (Table 1), who had not received seasonal influenza vaccination during the current season, were enrolled. Participants were randomized to receive one of the three trivalent seasonal influenza vaccines: Fluzone standard-dose inactivated vaccine, or FluBlok recombinant protein-based vaccine (Sanofi Pasteur, Swiftwater, PA, USA), or FluMist live-attenuated intranasal vaccine (AstraZeneca, Cambridge, UK). Vaccine-induced humoral and cellular immune responses were assessed to the H1N1, H3N2, and Influenza B components of the vaccine.

Table 1.

Demographic data of the study population

FluZ (N, %) FluB (N, %) FluM (N, %)
No. of individuals, N (%) 11 (33.3%) 11 (33.3%) 11 (33.3%)
Age, y Median = 34 Median = 33 Median = 35
Range = 18–48 Range = 21–48 Range = 21–49
Male 4 (36.4%) 1 (9.1%) 1 (9.1%)
Female 7 (63.6%) 10 (90.9%) 10 (90.9%)
White: non-Hispanic/Latino 9 (81.8%) 10 (90.9%) 10 (90.9%)
White: Hispanic/Latino 0 (0.0%) 0 (0.0%) 1 (9.1%)
Black: Hispanic/Latino 1 (9.1%) 0 (0.0%) 0 (0.0%)
Asian 1 (9.1%) 1 (9.1%) 0 (0.0%)
Comorbidities 0 (0.0%) 0 (0.0%) 0 (0.0%)
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Biological samples

Peripheral blood samples were collected from all participants before vaccination (day 0; D0) and 28 days (D28) post-vaccination following immunization with one of the three trivalent seasonal influenza vaccines: Fluzone standard dose inactivated vaccine (FluZ), FluBlok recombinant protein-based vaccine (FluB), or FluMist live-attenuated intranasal vaccine (FluM). Each vaccine formulation included three viral strains recommended for the 2024–2025 Northern Hemisphere Influenza season: A/Victoria/4897/2022 or A/Wisconsin/67/2022 (H1N1), A/Thailand/8/2022 or A/Massachusetts/18/2022 (H3N2), and B/Austria/1359417/2021 (B/Victoria lineage).

For humoral response assessment, samples were collected into serum separation tubes (SST) (BD Biosciences, Franklin Lakes, NJ, USA) and centrifuged at 1,000 x g for 10 min at RT. After centrifugation, the serum was carefully isolated from the gel layer and red blood cell pellet and stored at −80 °C until use.

For cellular immune profiling, peripheral blood samples were collected into K2 EDTA tubes (BD Biosciences, Franklin Lakes, NJ, USA), homogenized, and transferred to cell preparation tubes (CPT) (BD Biosciences, Franklin Lakes, NJ, USA) for PBMC isolation. CPT tubes were centrifuged at 1800 × g for 20 min at RT. The mononuclear cell layer was collected, washed with 1x Phosphate-buffered saline (PBS), and subjected to red blood cell (RBC) lysis using ACK lysis buffer (Life Technologies, Carlsbad, CA, USA). After additional PBS washes, PBMC were adjusted to a final concentration of 1 × 10⁷ cells/mL, resuspended in cold cryopreservation medium (90% fetal bovine serum with 10% dimethyl sulfoxide), and cryopreserved in liquid nitrogen until further analysis by flow cytometry.

Influenza Hemagglutination-inhibition (HAI) Assay

Influenza hemagglutination-inhibition (HAI) assays were performed to quantify HA-specific antibodies by assessing the ability to inhibit the binding of influenza virus to sialic acid receptors on erythrocytes. The assay was conducted in accordance with the World Health Organization (WHO) Manual for Laboratory Diagnosis and Virological Surveillance of Influenza67. Briefly, serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Co., Japan) at a 1:3 ratio and incubated overnight at 37 °C to eliminate non-specific inhibitors. The enzyme was subsequently inactivated by incubation at 56 °C for 45 min. Following inactivation, samples were diluted with PBS at a 1:6 ratio and serially two-fold diluted in V-bottom 96-well microtiter plates. The H1N1 (A/Victoria/4897/2022), H3N2 (A/Massachusetts/18/2022) and Influenza B (B/Austria/1359417/2021) virus was adjusted to contain 8 hemagglutination units (HAU) per 50 μL, and turkey red blood cells (TRBC) (Lampire Biologicals, Pipersville, PA, USA) were prepared at a final concentration of 0.8% in PBS. Equal volumes of virus and diluted serum were mixed and incubated for 20 min at RT. Subsequently, 0.8% TRBC was added to each well, and the plates were gently mixed and incubated for 30 min at RT. HAI titers were defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. Seroprotection was defined as an HAI titer ≥1:40, and seroconversion as a ≥4-fold increase in titer.

Memory B-cell phenotyping by flow cytometry

PBMC samples were used to assess the HA-specific MBC response, as previously described68,69 with modifications. In-house produced biotinylated HA proteins were used to generate antigen-specific probes by multimerization with fluorochrome-conjugated streptavidin (SA): A/Victoria/4897/2022 (H1N1) conjugated to SA-PE (#405245, Biolegend, San Diego, CA, USA), A/Thailand/8/2022 (H3N2) conjugated to SA-APC (#405243, Biolegend, San Diego, CA, USA), and B/Austria/1359417/2021 (B/Victoria lineage) conjugated to SA-BV421 (#405225, Biolegend, San Diego, CA, USA). Each biotinylated HA protein was multimerized with SA at a 1:2.6 mass ratio (60 ng HA with 156 ng SA for H1 and H3; 40 ng HA with 104 ng SA for B) for 1 h at 4 °C. To minimize non-specific binding, 5 µM free D-biotin (Avidity Biosciences, Inc., San Diego, CA, USA) was added to the final probe mix. A decoy probe consisting of SA-APC/Cy7 (#405208, BioLegend), lacking biotinylated HA, was also included to identify cells with non-specific streptavidin binding. Fluorescence-minus-one (FMO) controls were also performed for each HA probe (H1, H3, and IBV) to establish gating thresholds for antigen-specific B cells. FMOs included the complete antibody panel minus the respective HA probe, enabling precise definition of background fluorescence.

For MBC phenotyping, cryopreserved PBMC were thawed at 37 °C water bath and immediately resuspended in 10 mL of pre-warmed supplemented RPMI 1640 medium (10% heat-inactivated AB human serum, 1% penicillin/streptomycin, 2 mM L-glutamine) containing 25 U/mL benzonase (Millipore Sigma, Burlington, MA, USA). Cells were centrifuged at 500 × g for 7 min at 4 °C, washed twice with supplemented RPMI (without benzonase), and resuspended in 1 mL of medium. Cell concentration was adjusted to 2–5 × 10⁶ cells/mL using a LUNA-II™ Automated Cell Counter (Logos Biosystems, South Korea), then transferred to U-bottom 96-well plates (200 µL/well) and rested at 37 °C for 2 h. After resting, cells were washed and resuspended in human Fc-blocking solution (BD, Franklin Lakes, NJ, USA), followed by viability staining with LIVE-DEAD NIR (#L10119, Thermo Scientific, Waltham, Massachusetts, USA) for 20 min at 4 °C. After washing with FACS buffer, cells were incubated for 1 h on ice at 4 °C with a pre-prepared HA-probe mix (H1 HA-PE, H3 HA-APC, IBV HA-BV421, and the APC-Cy7 decoy probe in FACS buffer with 5 µM free D-biotin). Following incubation with the antigen probes, cells were washed 3 times with FACS buffer and stained with fluorochrome-conjugated monoclonal antibodies targeting B-cell surface markers (Supplementary Table 1) for 30 min at 4 °C, protected from light. Stained cells were washed 3 times and resuspended in FACS buffer for acquisition. All samples were acquired on an Aurora Spectral Flow Cytometer system with four lasers (Cytek, Fremont, CA, USA).

Data analysis

Flow cytometry data were analyzed in FlowJo 10.10.0 (BD Biosciences, Franklin Lakes, NJ, USA). Gating strategies for MBC subset identification are provided in Supplementary Fig. 1. Statistical analyses were performed using GraphPad Prism 10.5.0 (Dotmatics, Boston, MA, USA). The fold change was determined by dividing post-vaccination (D28) results by corresponding baseline (D0) values for each participant. Comparative analyses between two groups were performed by Mann–Whitney test or Student t-test. For comparison across multiple groups, Kruskal–Wallis with Dunn’s post hoc correction or ANOVA followed by Tukey’s multiple comparison test was employed, as appropriate. In all cases, significant differences were considered when p ≤ 0.05. High-dimensional analysis was carried out in Cytobank (Beckman Coulter, Brea, CA, USA) to generate t-distributed Stochastic Neighbor Embedding (t-SNE) plots. Correlation analyses between variables were performed using fold-change values (D28/D0). Pearson’s or Spearman’s rank correlation coefficient was applied according to data distribution. Heatmaps displaying the strength and direction of correlations were generated using R software (R Foundation for Statistical Computing, Vienna, Austria), and in all cases, significant data were considered when p ≤ 0.05.

Supplementary information

Supplementary information (612.6KB, pdf)

Acknowledgements

The authors acknowledge Dr. James Thomas from the FRIC Flow Cytometry Core for his support and assistance. We also extend our appreciation to Vanessa Moraes, Spencer Pierce, Julia Aguirre, and Wayne Grant for their valuable technical contributions. Special thanks to the University of Georgia (UGA) Flu Vaccine team for their efforts in sample collection and processing. Finally, the authors express their sincere gratitude to all study participants for their time, participation, and commitment to this research. This project has been funded by the National Institute of Allergy and Infectious Diseases, a component of the NIH, Department of Health and Human Services, under contract 75N93019C00052.

Author contributions

Funding acquisition: T.M.R. Designing of research protocol: L.R.R. and T.M.R. Conducting experiments: L.R.R. Data analysis: L.R.R. Writing and revising the manuscript: L.R.R. and T.M.R.

Data availability

The results included in the present study are available at [https://immport.niaid.nih.gov/home].

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at 10.1038/s41541-025-01350-3.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary information (612.6KB, pdf)

Data Availability Statement

The results included in the present study are available at [https://immport.niaid.nih.gov/home].

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