Seasonal influenza causes significant global morbidity, mortality, and economic burden. Ongoing viral evolution can lead to vaccine mismatch and the emergence of antiviral resistance, highlighting the importance of genomic surveillance. The 2024-2025 influenza season was characterized by high incidence and increased hospitalizations. We analyzed influenza A virus (IAV) genomes and clinical characteristics from the 2024-2025 season. Whole-genome sequencing was performed on 648 influenza A-positive clinical specimens collected between October 2024 and April 2025. Hemagglutinin (HA) sequences were recovered from 74.23% (481/648) of samples and used for subtyping and phylogenetic analysis. A(H1N1)pdm09 and A(H3N2) viruses co-circulated, representing 55.5% and 44.5% of cases, respectively. Among A(H1N1)pdm09 viruses, the HA1 substitution T120A, located near the Sa antigenic site, increased more than twofold compared with the prior season. Circulating A(H3N2) viruses belonged to multiple HA subclades and exhibited distinct amino acid substitutions at key antigenic sites. Neutralization assays using sera from individuals vaccinated with the 2024-2025 seasonal influenza vaccine demonstrated reduced neutralization of three dominant A(H1N1)pdm09 isolates and two A(H3N2) isolates compared with vaccine strains, consistent with antigenic drift. In addition, the neuraminidase substitution S247N, previously associated with reduced oseltamivir susceptibility, was detected in 13.9% of A(H1N1)pdm09 samples. These findings demonstrate ongoing antigenic drift and the presence of antiviral resistance-associated mutations during the 2024-2025 influenza season, underscoring the need for continued genomic surveillance to guide vaccine and antiviral strategies.

Type 1 diabetes (T1D) is driven by autoreactive T cells, which destroy insulin-producing β cells. Antigen-specific immunotherapies (ASITs) aim to restore immune tolerance while avoiding the broad immunosuppression of current therapies. Soluble antigen arrays (SAgAs) are multivalent antigen constructs built on a low-molecular-weight hyaluronic acid (HA) backbone, previously validated in preclinical models. Here, we generated SAgAs displaying human T1D autoantigen epitopes, including Insulin B:9-23 (InsB:9-23) and a hybrid insulin peptide (HIP), alongside a hemagglutinin (HAg):306-318 control. We confirmed their structure, valency, and ability to engage human epitope-specific T cell clones. Both InsB:9-23-SAgA and HIP-SAgA demonstrated strong specificity, supporting their utility as T cell probes and potential candidates for ASIT in T1D.

Insights into antiviral activity of chlorpromazine against RNA viruses: Molecular docking, ADME profile, and semi-in vivo study.

Effective respiratory mucosal vaccines remain urgently needed to mitigate the rapid mutation and transmission of SARS-CoV-2. Here, we demonstrated that the spike protein (S-2P) of ancestral SARS-CoV-2 acted as a self-adjuvanted antigen for intranasal immunization, inducing robust systemic and mucosal immunity via integrin- and STING-dependent pathways. In contrast, H1N1 influenza hemagglutinin (HA) failed to generate measurable serum IgG or mucosal IgA following intranasal immunization. In mice, intranasal S-2P vaccination conferred complete protection against lethal ancestral SARS-CoV-2 challenge and partial cross-protection against heterologous Omicron variants, with both effects being IFN-γ- and CD8 + T cell-dependent. Co-administration of S-2P with the clinical immunomodulator lentinan (LNT) achieved complete protection against Omicron variants, mediated by IFN-γ but largely independent of CD8 + T cells. These findings establish S-2P + LNT as a safe, broad-spectrum mucosal vaccine candidate against emerging SARS-CoV-2 variants and reveal novel protection mechanisms beyond neutralizing antibodies and T cell immunity.

The widespread panzootic of clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 necessitates the development of vaccine platforms capable of rapid adaptation to emerging antigenic variants. Although commercial recombinant turkey herpesvirus (rHVT) vaccines are available, they often utilize heterologous inserts that may fail to optimally limit viral shedding of novel field strains. Here, we report the rapid construction of a homologous rHVT-H5 vaccine expressing the hemagglutinin (HA) gene of a representative clade 2.3.4.4b isolate via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). In vitro characterization confirmed stable HA surface expression and growth kinetics comparable to the parental virus. In specific-pathogen-free (SPF) chickens, rHVT-H5 elicited robust hemagglutination inhibition (HI) antibody titers. Following lethal challenge with a homologous clade 2.3.4.4b H5N1 virus, the vaccine conferred 100% protection against mortality and clinical signs while significantly reduced oropharyngeal sheddings and completely inhibited viral shedding in cloacal samples. These findings demonstrate that an antigenically matched rHVT-H5 constitutes a promising strategy for mitigating the ongoing global threat posed by clade 2.3.4.4b HPAI H5N1.

ABSTRACT Low pathogenic avian influenza viruses (LPAIVs) are typically associated with subclinical or mild disease in poultry. However, recent outbreaks involving atypical LPAIV strains, including H3N1 strains like A/chicken/Belgium/460/2019, have demonstrated severe clinical outcomes despite low intravenous pathogenicity index (IVPI) scores. These findings challenge current classification systems and raise questions about alternative markers of virulence, such as loss of a neuraminidase (NA) glycosylation site linked to plasminogen-binding and haemagglutinin (HA) cleavage. This study compared the pathogenicity of a wild-type H3N1 strain (wtH3N1), isolated from a disease outbreak in Belgium, with a genetically modified, loss-of function variant (mH3N1) carrying a single amino acid substitution (S122N) in NA that blocks plasminogen-dependent cleavage of HA in chickens. Four-week-old pullets and cockerels, and 30-week-old laying hens were inoculated with either wtH3N1 or mH3N1 and clinical signs, egg production, viral replication, post-mortem and tissue pathology were evaluated. Adult hens infected with wtH3N1 showed a complete cessation of egg production, systemic viral replication, and histopathological lesions in the reproductive tract, brain, and kidneys. In contrast, birds infected with mH3N1 displayed only mild, transient reductions in egg production and minimal viral detection. No mortality was observed in any group. All young chickens exhibited subclinical infections. Overall, the S122N mutation significantly attenuated viral virulence and tissue tropism. The study provides functional evidence that position 122 on NA contributes to increased virulence in H3N1 AIV. These findings support the role of molecular markers in risk assessment of non-H5/H7 LPAIVs and highlight the limitations of the current IVPI-based classification system.

Background/Objectives: While new vaccines are in development; one strategy to increase influenza vaccine coverage is to repurpose current influenza vaccines for intranasal delivery. Methods: To address this goal; mice were vaccinated intranasally with either a split inactivated virus vaccine (Fluzone) or a recombinant HA vaccine (Flublok) at one of two doses (1 μg high dose or 0.1 μg low dose). Both vaccines were adjuvanted with either a STING agonist; c-di-AMP (CDA); or a combination of a synthetic toll-like receptor (TLR) 4 and TLR7/8 agonist (TRAC478). Results: Mice vaccinated with either vaccine plus adjuvant had higher hemagglutination-inhibition titers than mice administered unadjuvanted vaccines. Mice vaccinated with either vaccine plus CDA had on average higher numbers of H3 and influenza B hemagglutinin (HA)-specific antibody-secreting cells (ASCs); whereas mice vaccinated with vaccine plus TRAC478 had on average higher number of H1 HA-specific ASCs. All vaccinated mice challenged with the H1N1 influenza virus were protected against both morbidity and mortality with no detectable virus in their lungs. Mice challenged with the H3N2 influenza virus all lost weight over the first 5 days of infection. Adding TRAC478 with either a high or low dose vaccine resulted in 80–100% survival following challenge. Almost all mice vaccinated with Flublok plus CDA died from H3N2 influenza virus challenged with ~2 logs higher viral lung titers than mice administered Flublok only or Flublok plus TRAC478. Conclusions: Overall; Fluzone and Flublok can effectively be used for intranasal vaccination.

Avian influenza, caused by the avian influenza A virus (IAV), threatens poultry and public health. H6 subtype avian IAV is a low-pathogenic virus with hosts ranging from poultry and wild birds to mammals. H6 persists latently in poultry, which enables silent transmission and cross-species risk. The few fluorescence quantification assays that exist for H6 are mostly multiplexed. We developed a rapid, sensitive, efficient, monoplex fluorescence reverse-transcription qPCR (RT-qPCR) assay for H6 IAV. Specific primers and a TaqMan-MGB probe were designed based on the conserved hemagglutinin (HA) gene region of H6 IAVs from the GISAID database. The reaction components and conditions were optimized, and the assay was evaluated for specificity, sensitivity, and reproducibility. The optimized assay had excellent specificity, with no cross-reactivity with other avian viruses, including IAV subtypes H1-5, H7, H9, and H10, Newcastle disease virus, infectious bronchitis virus, fowl adenovirus, infectious laryngotracheitis virus, chicken anemia virus, Mycoplasmopsis (Mycoplasma) gallisepticum, and M. synoviae. Our method had a detection limit of 8.2 × 100 copies/μL, which is 1,000 times more sensitive than conventional RT-PCR. The intra- and inter-assay CVs for all tested concentrations were both <1.5%, indicating good reproducibility. When applied to clinical swab samples, the sensitivity of our fluorescence RT-qPCR assay was 98.8% and specificity was 96.2% compared with traditional virus isolation. Our method could provide strong technical support for the early detection, monitoring, and prevention of H6 subtype IAV infection.

Molecular basis of 60 years of antigenic evolution of human influenza A(H3N2) virus neuraminidase.

Influenza causes substantial morbidity and mortality worldwide. This randomized, open-label, phase 1 trial (ClinicalTrials.gov, NCT05397223, date of registration: May 31, 2022) compared the immunogenicity of an mRNA-based quadrivalent influenza hemagglutinin (HA) vaccine (mRNA-1010) with a licensed comparator (FLUAD) in adults aged 18-75 years. We evaluated humoral and cellular immune responses using hemagglutination inhibition assays, flow cytometry-based memory B cell (MBC) profiling, and intracellular cytokine staining for T-cell characterization. Both vaccines elicited durable hemagglutination inhibition titers and increased HA-specific MBC responses across four vaccine strains. Compared with FLUAD, mRNA-1010 induced higher frequencies of classical and activated MBCs specific to the H3 HA included in the vaccine, while inducing similar MBC responses to the other strains. mRNA-1010 and FLUAD generated strong HA-specific CD4+ T-cell responses; a trend toward higher CD8+ T-cell responses was observed in mRNA-1010 recipients compared with FLUAD recipients for two of the four strains. These findings support the potential of the mRNA platform for seasonal influenza vaccination.

BackgroundInfluenza, particularly novel zoonotic strains, causes severe disease in immunocompromised persons (ISPs). Global outbreaks of highly pathogenic avian influenza (HPAI, e.g., H5N1, H7N9) may pose similar risk to ISPs, yet anti-HPAI immune landscape and impact of vaccination are unknown.Figure 1.HA Binding Titers Before and After Seasonal Influenza Vaccination.Antibodies to H1, H3, and H7 were measured using the Meso Scale Discovery Respiratory Panel 4 before (baseline) and 4 weeks (4W) after seasonal influenza vaccination in immunosuppressed persons. Results are reported on the y-axis in arbitrary units (AU)/mL. Vaccine type is indicated by the color of the dot.MethodsIn an ongoing national prospective cohort of ISPs reporting respiratory virus vaccination and infection, plasma from participants was analyzed for binding antibody (Ab) against seasonal (H1, H3) and HPAI (H7) hemagglutinin (HA) using a multiplex electrochemiluminescence assay (Meso Scale Discovery) and microneutralization using live contemporary vaccine strains of H1N1 and H3N2 before and after seasonal influenza vaccination. Changes in HA binding titer and neutralization were evaluated using Wilcoxon Signed Rank testing. Interstrain HA binding Pearson correlations were computed along with intrastrain HA binding and neutralization.Figure 2.Neutralizing Titers Before and After Seasonal Influenza Vaccination.Live-virus neutralization was measured using microneutralization assay against indicated seasonal influenza strains. Top. Reciprocal of the highest plasma dilution resulting in 50% protection (NT50) at baseline and 4 weeks post vaccination. Bottom. Area under the neutralization curve (AUC) log10 transformed at baseline and 4 weeks post vaccination for vaccine strains of H1N1 and H3N2.Figure 3.Interstrain HA Antibody Correlations.HA antibodies are plotted for each strain against one another and Pearson correlation coefficients are reported. ** and *** indicate p < 0.01 and 0.001, respectively, while no marking above the correlation coefficient indicates p > 0.05.ResultsAmong 11 ISPs, median [IQR] anti H1, H3, H7 titers did not change significantly from baseline (4.9 [4.7 – 5.0], 4.4 [4.3 – 4.6], and 3.7 [3.5 – 4] arbitrary units (AU)/mL, respectively) to 4 weeks after vaccination (4.9 [4.8 – 5.3], 4.6 [ 4.4 – 4.8], and 3.9 [3.7 - 4], respectively; p >0.05 for all) (Fig1). H7 titers were ∼5-fold and ∼11-fold lower than vs H3 and H1, respectively. Median area under the curve (AUC) neutralization was similar between baseline and 4 weeks for H1N1 (1.5 [1.1 – 1.8] to 1.5 [1.1 – 2.4], p = 0.23) but significantly increased for H3N2 (0.03 [0 – 0.6] to 0.06 [0 – 0.5], p < 0.05) (Fig2). H3 binding was positively correlated with H1 (r = 0.75, p< 0.05) and H7 (r = 0.58, p< 0.05), but H1 and H7 titers did not correlate (r = 0.21, p >0.05) (Fig3). H1 binding and neutralization were positively correlated (r = 0.65, p< 0.05), but H3 binding and neutralization were not significantly correlated (r = 0.04, p >0.05).ConclusionAnti-HPAI humoral immunity appears low in ISPs without substantial boosting by vaccination, raising concern for vulnerability to infection and disease. H3 binding may be associated with betters cross-reactivity with H7 than H1. Anti-H5 Ab evaluation is ongoing and of high importance given widespread H5N1 circulation and potential threat to ISPs.DisclosuresWilliam Werbel, MD PhD, AstraZeneca: Advisor/Consultant|Novavax: Advisor/Consultant Andrew H. Karaba, MD PhD, GSK: Advisor/Consultant|Hologic: Advisor/Consultant

BackgroundAnalgesics are sometimes taken prior to vaccination to prevent adverse reactions. Although several studies have examined the immunogenicity impact of post-vaccination analgesics, few have assessed the effect of analgesics taken prior to vaccination. This study compares antibody responses to hemagglutination (HA) among adults with and without analgesic use prior to influenza vaccine receipt. It further analyzes differences in immunogenicity according to analgesic type.MethodsPragmatic Assessment of Influenza Vaccine Effectiveness in the DoD (PAIVED) was an open-label, randomized clinical trial comparing the effectiveness of FDA-licensed influenza vaccines on adult military healthcare beneficiaries. A subset of participants provided venous blood samples collected pre-vaccination and 30 days post-vaccination. Participants included in this analysis provided venous blood samples and reported on analgesic use within 24 hours prior to vaccine receipt.ResultsOf 1,173 PAIVED participants with pre- and post-vaccination anti-HA antibody titers, 684 (58%) answered the analgesic question. Among those, 71 (10%) reported taking an analgesic within 24 hours prior to vaccine receipt. The most prevalent analgesics were nonsteroidal anti-inflammatory drugs (NSAIDs) including ibuprofen (total NSAIDs, n=37; ibuprofen, n=19) and acetaminophen (n=28). Those who took a pre-vaccination analgesic were significantly older and more often female (Table 1). There were no significant differences in the average change in geometric mean titer (GMT) among subjects with a pre-vaccination analgesic compared to those without, including when stratified by analgesic type (Figure 1). While a higher percentage of people who took acetaminophen seroconverted (4-fold rise in antibody titer) compared to those who took ibuprofen or other NSAIDs, this finding was not statistically significant (Figure 2).ConclusionOverall, pre-vaccination analgesic use in PAIVED did not have a significant impact on influenza vaccine immunogenicity, although there was a suggestion that acetaminophen may have a more favorable effect on anti-HA seroconversion than NSAIDs for some vaccine strains. Assessment of the interplay among factors potentially influencing these findings may provide useful insight.DisclosuresAll Authors: No reported disclosures

BackgroundInfluenza A viruses pose a persistent pandemic threat due to their zoonotic potential and capability of antigenic shift, enabling the emergence of novel strains with pandemic potential.MethodsThis parallel design, dose-ranging, blinded study, assessed the safety and immunogenicity of three dose-levels (12.5, 25 and 50 µg) of mRNA-1018. This pre-pandemic vaccine, which encodes for the hemagglutinin (HA) of H5-A/chicken/Ghana/2021, was given as two doses 21 days apart to 304 healthy adults (> 18 to < 65 and > 65 years of age). Immune responses were assessed by hemagglutination inhibition (HAI) and microneutralization (MN) at multiple time points during the 6-month study.ResultsMost local and systemic solicited adverse reactions (AR) were Grade 1–2, and the most common AR were injection site pain, fatigue, and headache. Grade 3 AR were infrequent (< 5%), and no Grade 4 events occurred. The rates of solicited AR following the second dose were comparable to the first, with no notable increase in severity. One related SAE (syncope) was reported.Three weeks after the second dose, the percentage of participants in all dose groups with HAI titers ≥ 1:40 (defined as seroprotection or SP) were 97.8% (95% confidence interval [95%CI]: 95.4, 99.2). The percentage with seroconversion (SC) by HAI (defined as post-vaccination titer ≥ 1:40 if baseline is < 1:10 or a 4-fold or greater rise if baseline is ≥ 1:10) was 97.1% (95%CI: 94.4–98.7). Immune responses were detected early across all dose groups and remained detectable at study end. Three weeks after the first dose, 79.5% (95%CI: 74.3, 84.1) of participants had achieved SP and 78.1% (95%CI: 72.8, 82.8) met criteria for SC. Six months after the second dose, 70.6 % (95%CI: 64.8, 76.0) maintained SP levels. Titers measured by both HAI and microneutralization (MN) assays increased with increasing dose of mRNA-1018.ConclusionAcross all dose levels, mRNA-1018 was safe, well-tolerated, and demonstrated rapid and persistent immune response which are considered key attributes of a pandemic vaccine.DisclosuresAll Authors: No reported disclosures

A rare intramuscular hydatid cyst of the gluteus: a case report

A novel calcium influx inducer and a TLR7 agonist are synergistic co-adjuvants that enhance cross-reactive immunity against influenza in young and aged mice.

The high mutation rate of the influenza virus poses a significant challenge to global health, highlighting the urgent need for broad-spectrum vaccines. Here, we engineered Ad-Hex, a pan-influenza vaccine comprising three chimpanzee adenoviral vectors (Ad-H1H3, Ad-BYBV, and Ad-H5H7), each encoding the hemagglutinin (HA) genes from two distinct influenza virus subtypes/lineages in the △E1 region. A single intranasal dose of Ad-Hex induced robust humoral, cellular, and mucosal immunity, conferring complete protection against lethal challenge with six vaccine-matched strains in female C57BL/6 or Balb/c mice. Notably, the vaccine achieved 60% survival rates against mismatched strains. Mechanistic studies revealed that Ad-Hex activated germinal center B-cell responses not only against the cognate HAs but also against the conserved HA stalks. This drove the production of cross-reactive antibodies, which contributed to heterologous protection along with the CD8+ T cell response. Our study confirms the feasibility of this multivalent strategy by mixing multiple recombinant adenoviruses and provides insights into the development of multivalent vaccines against other respiratory pathogens.

Extracellular vesicles are key mediators for direct antigen transport to draining lymph nodes.

Background: Seasonal influenza remains a significant public health problem, and the constant antigenic drift of viruses requires regular vaccine updates. mRNA vaccines offer a promising platform for the development of new, effective influenza vaccines. Administration of the naked mRNA vaccine using a needle-free jet injection system further enhances its safety, reduces cost, and eliminates the need for lipid nanoparticles, which are traditionally used for mRNA delivery. Lyophilization of naked mRNA allows for long-term storage at +4 °C. Methods: We designed and produced an mRNA vaccine against seasonal influenza, designated mRNA-Vector-Flu, encoding the hemagglutinin (HA) of the A/Wisconsin/67/2022(H1N1)pdm09, A/Darwin/9/2021(H3N2), and B/Austria/1359417/2021 strains. The vaccine was lyophilized and stored for 1 month in a refrigerator (+4 °C). A comparative immunogenicity study was conducted between synthesized immediately before use prepared and lyophilized naked mRNA-Vector-Flu. The preparations were administered to BALB/c mice using a jet needleless injection twice, 3 weeks apart. Immunogenicity was assessed on day 35 of the study. Results: A comparative immunogenicity study of naked mRNA-Vector-Flu demonstrated that both the synthesized immediately before use prepared formulation and the lyophilized form, stored at +4 °C for a month, induced similar levels of virus-specific antibodies and generated a pronounced T-cell immune response. Conclusions: Delivery of the naked mRNA vaccine using a needle-free jet injection ensures a high-level immune response, which improves its safety, reduces its cost, and eliminates the need for lipid nanoparticles traditionally used for mRNA delivery. At the same time, lyophilization of the naked mRNA vaccine preserves its biological activity and ensures its storage for at least a month at +4 °C temperatures. Our results demonstrate that our proposed approach can be considered a promising direction for the development and improvement of the mRNA vaccine platform.

Influenza presents a global threat representing hundreds of thousands of deaths each year worldwide, yet influenza vaccines currently do not offer full protection against infection and must be updated each year to account for antigenic drift. Subunit vaccines using hemagglutinin (HA) are a promising approach to decrease reactogenicity while still providing protection, but these vaccines require large antigen doses or inclusion of reactogenic adjuvants. Here, we use multivalent presentation of HA on the self-assembling nanofiber platform Q11 coupled with a nanoscale adjuvant to augment responses to HA vaccines. We leverage polypeptides containing random combinations of lysine, glutamic acid, tyrosine, and alanine (KEYA) as a nanoscale, non-reactogenic adjuvant and demonstrate two methods of bioconjugation of HA to Q11 nanofibers, namely β-tail co-assembly and SortaseA (SrtA)-mediated ligation. Both conjugation methods demonstrated effective conjugation to Q11, with minimal perturbation of HA antigenicity with the inclusion of low concentrations of KEYA. β-tail co-assembly offered increased humoral responses, and the inclusion of KEYA-Q11 enabled modest increases in breadth of antibody binding and survival against influenza challenge. SrtA-mediated conjugation additionally augmented humoral immune responses and also elicited a significant increase in breadth of antibody binding, achieving significant binding to HA trimers with up to 30 years of antigenic drift from the immunized antigen, increased hemagglutinin inhibition to homologous and heterologous viruses, and survival in a lethal disease challenge. Together, these results demonstrate the utility of peptide nanofiber bioconjugation strategies to augment subunit vaccines, particularly in the context of influenza. STATEMENT OF SIGNIFICANCE: Influenza causes hundreds of thousands of deaths each year worldwide, yet current influenza vaccines do not offer full protection and must be updated each year. In this report, we describe self-assembled peptide nanofiber subunit vaccines comprised of the Q11 nanofiber platform, hemagglutinin antigens, and a nanoscale adjuvant consisting of randomized lysine, glutamic acid, tyrosine, and alanine (KEYA). Optimized formulations augmented humoral immune responses and elicited a significant increase in the breadth of antibody binding, achieving significant binding to HA trimers with up to 30 years of antigenic drift from the original antigen. These findings illustrate the utility of peptide nanofibers as platforms for subunit vaccines.

Replacement of the Loop 4 region in CPV VP2 with corresponding PPV segments affects N-terminal cleavage, VLP morphology, hemagglutination, and cell binding.