Longitudinal nasopharyngeal swabs sample and reveal upper airway humoral memory generated after intranasal FluMist vaccination.

Licensure of influenza vaccines relies on serum hemagglutination inhibition (HAI) titers, a correlate of protection (CoP) that was developed more than 50 years ago and which is only poorly predictive of protection. This is especially true of immunity induced by intranasal live attenuated influenza vaccines (LAIVs). Unlike intramuscular inactivated influenza vaccines (IIVs), LAIV and natural infection selectively stimulate mucosal immunity. Developments in mucosal immunology now enable measurement of diverse aspects of mucosal immunity, including the frequencies and functions of nasal antibodies, T cells, and B cells. This review assesses the potential of new sampling and assay approaches that may overcome inconsistent findings from previous methodologies. Standardization of the collection and assessment of mucosal samples is essential in developing new CoPs for vaccine development and licensure of novel vaccines that induce nasal protection and are more effective in prevention of viral transmission.

Seasonal influenza vaccines are most frequently delivered as intramuscular inactivated vaccines, which elicit systemic responses against the immunodominant hemagglutinin (HA) head domain. An intranasally administered, live-attenuated influenza vaccine designed to stimulate mucosal immunity, FluMist, is the sole intranasal vaccine approved in the United States. However, FluMist has lower systemic immunogenicity and efficacy in adults compared with intramuscular formulations. In this study, human mucosal and systemic immunity were examined after seasonal intramuscular or intranasal vaccination. Nasopharyngeal swabs of adenoid tissue were used to longitudinally sample the upper airway. FluMist induced substantial increases in upper respiratory tract IgG+ and IgA+ HA-specific memory B cells, which displayed an activated CD27+CD21- phenotype. H1, H3, and influenza B virus HA-specific memory B cells were all detected in the upper airway after intranasal immunization, remained elevated at 6 months postvaccination, and were associated with the presence of circulating T follicular helper (cTFH) cells. Recently activated upper airway memory B cells were not readily detected in intramuscular vaccinees, despite the elevation of systemic antibodies and circulating HA-specific memory B cells. Thus, despite minimal immune responses detected in circulation, adults vaccinated with the live-attenuated influenza vaccine generate substantial local antigen-specific memory B cell responses. These findings have implications for improving influenza vaccines and for mucosal vaccination against other respiratory pathogens.

Seasonal influenza vaccines are available in multiple formulations including egg-based inactivated standard-dose, cell-based inactivated, recombinant, and live-attenuated. However, vaccine effectiveness and antibody responses vary by vaccine type, recipient age, and prior influenza exposures. Both age and repeated annual vaccination can modulate immune outcomes through antigenic differences between historical and updated vaccine formulations. In this three-season (2022-2023-2024-2025) study, 1,328 participants aged 9-89 years received one of six vaccine types (Fluzone standard dose (SD) or high dose (HD), Fluad, Flucelvax, Flublok, Flumist). Collected serum samples were tested for hemagglutination inhibition (HAI) activity against vaccine strains and historical circulating strains before vaccination and day 28 post-vaccination. Seroprotection and seroconversion rates were analyzed by age group and vaccine platform. Repeated vaccination across consecutive seasons was associated with reduced HAI boosting to current vaccine strains in some participants, although vaccination broadly increased antibody titers to prior strains (back-boosting) and maintained high seroprotection rates from year to year. Participants ≥65 years had lower post-vaccination HAI titers compared to younger adults and had rapid waning of HAI titers than vaccinated younger adults, yet elderly participants with HAI titers <1:40 had higher seroconversion rates following vaccination, which may in part reflect lower baseline titers in this group allowing a ≥ 4-fold rise to meet the seroconversion definition with Fluzone HD or Fluad adjuvanted vaccines compared to younger vaccinated participants. Over the three seasons, younger adult participants vaccinated with Flublok had higher HAI titers than participants vaccinated with Fluzone SD against H1N1 and H3N2 strains. In contrast, post-vaccination HAI titers in elderly participants were comparable between Fluzone HD and Fluad recipients for each vaccine component, including influenza A and B strains. Participants vaccinated with Flumist had low or no measurable serum HAI activity. Overall, the elicitation of vaccine induced immune responses is strongly influenced by vaccine formulation, age and immune history of the participant.

Bioengineered tissues offer vital platforms capable of fundamental research, screening interventions and reducing the use of animals in scientific research. However, their predictive accuracy is dependent upon how closely their structure and function resemble their native counterpart. In this study, we present a novel in vitro engineered construct representative of the human nasal mucosa, consisting of both stromal and epithelial compartments. Communication between the stroma and the overlying epithelium is an essential factor involved in tissue development and homeostasis yet is often lacking in the majority of published tissue constructs. Described is the construction of a pseudostratified epithelium consisting of an organised heterogeneous cell population consistent with the respiratory region of the nasal mucosa that forms on a stromal foundation populated with tissue-specific fibroblasts and endogenous extracellular matrix. In addition, for the first time, we provide an extensive ultrastructural analysis of a bioengineered nasal tissue using both scanning and transmission electron microscopic techniques. This in-depth characterisation revealed microanatomical hallmarks consistent with the native tissue, including motile cilia, mucin secretions, intercellular junctions, dynamic basement membrane, microvilli and glycocalyx. Given each of these features play pivotal physiological roles in the specialised functions of the respiratory epithelium, their presence in vitro lends itself to tissue equivalents with enhanced physiological relevance and greater predictive accuracy. In summary, we present a highly characterised in vitro nasal mucosal construct that accurately reflects native microanatomy. Such technology will be of value to a wide range of applications, most notably, undertaking basic research and in vitro pharmaceutical screening, of which nasal mucosal models have become increasingly applicable due to the popularity of intranasal drug and vaccine delivery methods.

Annual influenza vaccination is the cornerstone for seasonal protection, yet antibody responses are highly variable across individuals and over time. To systematically assess the determinants of this heterogeneity, we compiled 20,449 hemagglutination inhibition and neutralization titers from 4,540 participants enrolled in 14 new vaccine studies we conducted and 50 prior studies that collectively span 2010-2023. Seasonal effects dominated, with pre- and post-vaccination titers declining steadily from 2017 onwards, outweighing the influence of age, sex, or repeated vaccination. Titers to B Yamagata remained steady throughout all years examined, suggesting unique durability and offering a reason for lineage extinction. Vaccine timing emerged as a strong and previously underappreciated determinant of immunity, with individuals vaccinated later in the season exhibiting larger post-vaccination titers. Not being vaccinated or receiving the live-attenuated FluMist vaccine in one year significantly enhanced the response to inactivated vaccines in 45% or 68% of cohorts, respectively, whereas antigen dose and adjuvants had modest impact. These findings identify vaccine timing and seasonal context as underrecognized drivers of immunogenicity and provide actionable insights for optimizing influenza vaccination strategies.

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.

For FluMist®/Fluenz live attenuated influenza vaccine (LAIV), efficient virus replication in human nasal epithelial cells (hNEC) is an important factor in vaccine effectiveness (VE). The necessity of antigenic similarity of LAIV viruses to the WHO-recommended strains for VE is less clear. Here, we aimed to describe the relative importance of optimal hNEC replication and antigenic match in protection from wild-type (wt) challenge in vivo. The efficacy of an A/H1N1pdm09 LAIV strain (LAIVH1.opt) with optimised hNEC replication but reduced antigenicity relative to its parent (LAIVH1.par) was assessed. Delivered at a low dose, animals vaccinated with antigenically matched LAIVH1.par shed wt challenge virus and exhibited influenza-like illness post-challenge. Conversely, antigenically divergent LAIVH1.opt provided protection from both endpoints. LAIVH1.opt was also found to be more immunogenic in ferrets than LAIVH1.par, generating superior anti-HA and anti-NA antibody responses. We conclude that efficient virus replication can supersede antigenic match for high LAIV efficacy.

Vaccination against seasonal influenza is considered an effective means of reducing morbidity and mortality. Live-attenuated vaccines offer more protection against influenza than inactivated vaccines as they efficiently induce cellular immunity and provide cross-immunogenicity against various antigenic subtypes. For the production of safer live-attenuated vaccines, it is important to develop a common master donor vaccine strain in which pathogenic revertants are much less likely to appear. In this study, we introduced a single amino acid substitution of Lys471 into the PB1 polymerase subunit of influenza A virus and succeeded in isolating an attenuated mutant virus that exhibits a temperature-sensitive phenotype. The Lys471 residue is located in the polymerase motif D on PB1 and is positioned near the entrance tunnel domain for incoming nucleotide triphosphate. Although 10 viable PB1-Lys471 mutants did not proliferate at 37°C, their variants could replicate at 31°C and 34°C. Moreover, we found that PB1-Lys471Pro variant induces a genetically stable influenza virus phenotype; this mutant virus did not revert to wild-type phenotype from the temperature-sensitive phenotype by serial virus passages. Animal experiments have demonstrated that these PB1 mutant strains work effectively as live-attenuated vaccines. Application of the PB1-Lys471 substitution to a master donor strain is expected to lead to the development of a safer, high-performance, and widely used live-attenuated vaccine with the antigen of circulating influenza viruses.

This report updates the 2024-25 recommendations of the Advisory Committee on Immunization Practices (ACIP) concerning the use of seasonal influenza vaccines in the United States. Routine annual influenza vaccination is recommended for all persons aged ≥6 months who do not have a contraindication to vaccination. Multiple formulations of the trivalent inactivated influenza vaccines (IIV3s), trivalent recombinant influenza vaccine (RIV3), and trivalent live attenuated influenza vaccine (LAIV3) are expected to be available for the 2025-26 influenza season. Updates for the 2025-26 season include 1) antigenic composition of 2025-26 U.S. seasonal influenza vaccines, 2) Food and Drug Administration (FDA) approval of FluMist (LAIV3) for self-administration or caregiver administration, 3) FDA approval of a change in age indication for Flublok (RIV3) from ≥18 years to ≥9 years, and 4) a new ACIP recommendation that children aged ≤18 years, pregnant women, and all adults receive seasonal influenza vaccines only in single-dose formulations that are free of thimerosal as a preservative. A comprehensive summary of recommendations, including those discussed in this report, as well as previous recommendations concerning topics not addressed in this report and that remain unchanged for the 2025-26 season, is available at Influenza | ACIP Recommendations for Vaccination. Additional background information also is available at Prevention and Control of Seasonal Influenza with Vaccines.

Seasonal influenza vaccines are most frequently delivered as intramuscular inactivated vaccines which elicit systemic responses against the immunodominant hemagglutinin (HA) head domain. An intranasally administered, live-attenuated influenza vaccine designed to stimulate mucosal immunity, FluMist, is the sole intranasal vaccine approved in the United States. However, FluMist has lower systemic immunogenicity and efficacy in adults compared to intramuscular formulations. In this study, human mucosal and systemic immunity were examined following seasonal intramuscular or intranasal vaccination. Nasopharyngeal swabs of adenoid tissue were used to longitudinally sample the upper airway. Notably, FluMist induced substantial increases in upper respiratory tract IgG [+] and IgA [+] HA-specific memory B cells, which displayed an activated CD27 [+] CD21 [-] phenotype. H1, H3, and influenza B virus HA-specific memory B cells were all detected in the upper airway after intranasal immunization and remained elevated at 6-months post-vaccination. Recently activated upper airway memory B cells were not readily detected in intramuscular vaccinees, despite marked elevation of systemic antibody and memory B cells. Thus, despite minimal immune response detected in circulation, live-attenuated influenza vaccine can generate substantial local antigen-specific memory B cell responses in adults. These findings have implications for improving influenza vaccines and for mucosal vaccination against other respiratory pathogens.

Alpha-gal syndrome (AGS) is an allergic reaction caused by sensitization to alpha-gal, a sugar molecule in gelatin-containing vaccines. Tick bites can induce alpha-gal IgE sensitization, as ticks inject alpha-gal through their saliva. However, there is no real-world data on the anaphylaxis rates associated with gelatin-containing vaccines in patients with tick bite history. We utilized TriNetX, a research network database, to analyze data in November 2024. The history of tick bites (i.e., the presence of a tick-bite history) was categorized into three groups: ehrlichiosis, tularemia, and bites from nonvenomous arthropods. Vaccines were classified into two categories: (1) gelatin-containing vaccines (live intranasal influenza [FluMist], varicella, measles/mumps/rubella [MMR], oral typhoid, rabies [RabAvert], and yellow fever vaccines [YF-VAX]) and (2) gelatin-free vaccines. Anaphylactic episodes were identified in patients with a history of tick bites who developed anaphylaxis within one day following vaccination. The anaphylaxis rates after receiving gelatin-containing vaccines were 0.06 %, 0.7 %, and 0.2 % in patients with ehrlichiosis, tularemia, and nonvenomous arthropod bites, respectively. In contrast, the anaphylaxis rates after receiving gelatin-free vaccines were 0.04 %, 0.0 %, and 0.1 % in patients with ehrlichiosis, tularemia, and nonvenomous arthropod bites, respectively. Among the gelatin-containing vaccines, live intranasal influenza and varicella vaccines posed a higher rate of anaphylaxis compared to other gelatin-containing vaccines. While tick bites are associated with AGS, a history of tick bites is not a contraindication for most gelatin-containing vaccines, including MMR, oral typhoid, rabies, and yellow fever vaccines. Further investigations are warranted to evaluate the safety of intranasal influenza and varicella vaccines in this population.