Isolation of human antibodies against influenza B neuraminidase and mechanisms of protection at the airway interface.

Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo, and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates.

Human CD8+ T cell cross-reactivity across influenza A, B and C viruses.

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective.

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Triton X-100-treated virus-based ELLA demonstrates discordant antigenic evolution of influenza B virus hemagglutinin and neuraminidase.

Neuraminidase (NA)-specific antibodies have been associated with protection against influenza and thus NA is considered a promising target for next-generation vaccines against influenza A (IAV) and B viruses (IBV). NA inhibition (NI) by antibodies is typically assessed using an enzyme-linked lectin assay (ELLA). However, ELLA can be confounded by anti-hemagglutinin (anti-HA) antibodies that block NA by steric hindrance (termed HA interference). Although strategies have been employed to overcome HA interference for IAV, similar approaches have not been assessed for IBV. We found that HA interference is common in ELLA using IBV, rendering the technique unreliable. Anti-HA antibodies were not completely depleted from sera by HA-expressing cell lines, and this approach was of limited utility. In contrast, we find that treatment of virions with Triton X-100, but not Tween-20 or ether, efficiently separates the HA and NA components and overcomes interference caused by anti-HA antibodies. We also characterize a panel of recombinant IBV NA proteins that further validated the results from Triton X-100-treated virus-based ELLA. Using these reagents and assays, we demonstrate discordant antigenic evolution between IBV NA and HA over the last 80 years. This optimized ELLA protocol will facilitate further in-depth serological surveys of IBV immunity as well as antigenic characterization of the IBV NA on a larger scale.IMPORTANCEInfluenza B viruses (IBVs) contribute to annual epidemics and may cause severe disease, especially in children. Consequently, several approaches are being explored to improve vaccine efficacy, including the addition of neuraminidase (NA). Antigen selection and assessment of serological responses will require a reliable serological assay to specifically quantify NA inhibition (NI). Although such assays have been assessed for influenza A viruses (IAVs), this has not been done of influenza B viruses. Our study identifies a readily applicable strategy to measure the inhibitory activity of neuraminidase-specific antibodies against influenza B virus without interference from anti-hemagglutinin (anti-HA) antibodies. This will aid broader serological assessment of influenza B virus-specific antibodies and antigenic characterization of the influenza B virus neuraminidase.

Dissemination of influenza B virus to the lower respiratory tract of mice is restricted by the interferon response.

The global burden of disease caused by influenza B virus (IBV) is substantial; however, IBVs remain overlooked. Understanding host-pathogen interactions and establishing physiologically relevant models of infection are important for the development and assessment of therapeutics and vaccines against IBV. In this study, we assessed an upper respiratory tract (URT)-restricted model of mouse IBV infection, comparing it to the conventional administration of the virus to the total respiratory tract (TRT). We found that URT infections caused by different strains of IBV disseminate to the trachea but resulted in limited dissemination of IBV to the lungs. Infection of the URT did not result in weight loss or systemic inflammation even at high inoculum doses and despite robust viral replication in the nose. Dissemination of IBV to the lungs was enhanced in mice lacking functional type I IFN receptor (IFNAR2), but not IFNγ. Conversely, in mice expressing the IFN-inducible gene Mx1, we found reduced IBV replication in the lungs and reduced dissemination of IBV from the URT to the lungs. Inoculation of IBV in both the URT and TRT resulted in seroconversion against IBV. However, priming at the TRT conferred superior protection from a heterologous lethal IBV challenge compared to URT priming, as determined by improved survival rates and reduced viral replication throughout the respiratory tract. Overall, our study establishes a URT-restricted IBV infection model, highlights the critical role of IFNs in limiting dissemination of IBV to the lungs, and also demonstrates that the lack of viral replication in the lungs may impact protection from subsequent infections. IMPORTANCE: Our study investigated how influenza B virus (IBV) spreads from the nose to the lungs of mice and the impact this has on disease and protection from re-infection. We found that when applied to the nose only, IBV does not spread very efficiently to the lungs in a process controlled by the interferon response. Priming immunity at the nose only resulted in less protection from re-infection than priming immunity at both the nose and lungs. These insights can guide the development of potential therapies targeting the interferon response as well as of intranasal vaccines against IBV.

Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

A Multiseason Randomized Controlled Trial of Advax-Adjuvanted Seasonal Influenza Vaccine in Participants With Chronic Disease or Older Age.

BACKGROUND: The aim of the current study was to determine the safety and immunogenicity of trivalent inactivated influenza vaccine (TIV) alone or formulated with Advax delta inulin adjuvant in those who were older (aged >60 years) or had chronic disease. METHODS: Over 4 consecutive years from 2008 through 2011, adult participants with chronic disease or >60 years of age were recruited into a randomized controlled study to assess the safety, tolerability and immunogenicity of Advax-adjuvanted TIV (TIV + Adj) versus standard TIV. The per-protocol population with ≥1 postbaseline measurement of influenza antibodies comprised 1297 participants, 447 in the TIV and 850 in the TIV + Adj) group. RESULTS: No safety issues were identified. Variables negatively affecting vaccine responses included obesity and diabetes mellitus. Advax adjuvant had a positive impact on anti-influenza immunoglobulin M responses and on H3N2 and B strain seropositivity as assessed by hemagglutination inhibition. CONCLUSIONS: TIV + Adj was safe and well tolerated in individuals with chronic disease. There is an ongoing need for research into improved influenza vaccines for high-risk populations. CLINICAL TRIALS REGISTRATION: Australia New Zealand Clinical Trial Registry: ACTRN 12608000364370.

The Potential Benefits of Delaying Seasonal Influenza Vaccine Selections for the Northern Hemisphere: A Retrospective Modeling Study in the United States.

BACKGROUND: Antigenic similarity between vaccine viruses and circulating viruses is crucial for achieving high vaccine effectiveness against seasonal influenza. New non-egg-based vaccine production technologies could revise current vaccine formulation schedules. We aim to assess the potential benefit of delaying seasonal influenza vaccine virus selection decisions. METHODS: We identified seasons where season-dominant viruses presented increasing prevalence after vaccine formulation had been decided in February for the Northern Hemisphere, contributing to their antigenic discrepancy with vaccine viruses. Using a SEIR (susceptible-exposed-infectious-recovered) model of seasonal influenza in the United States, we evaluated the impact of updating vaccine decisions with more antigenically similar vaccine viruses on the influenza burden in the United States. RESULTS: In 2014-2015 and 2019-2020, the season-dominant A(H3N2) subclade and B/Victoria clade, respectively, presented increasing prevalence after vaccine decisions were already made for the Northern Hemisphere. Our model showed that the updated A(H3N2) vaccine could have averted 5000-65 000 influenza hospitalizations in the United States in 2014-2015, whereas updating the B/Victoria vaccine component did not substantially change influenza burden in the 2019-2020 season. CONCLUSIONS: With rapid vaccine production, revising current timelines for vaccine selection could result in substantial epidemiological benefits, particularly when additional data could help improve the antigenic match between vaccine and circulating viruses.

Hemagglutination Inhibition Antibody Titers as Mediators of Influenza Vaccine Efficacy Against Symptomatic Influenza A(H1N1), A(H3N2), and B/Victoria Virus Infections.

BACKGROUND: The hemagglutination inhibition antibody (HAI) titer contributes only a part of vaccine-induced protection against influenza virus infections. Using causal mediation analysis, we quantified the proportion of vaccine efficacy mediated by postvaccination HAI titers. METHODS: We conducted causal mediation analyses using data from a randomized, active-comparator controlled, phase III, trial of an inactivated, split-virion seasonal quadrivalent influenza vaccine in children conducted from October 2010 to December 2011 in 8 countries. Vaccine efficacy was estimated using a weighted Cox proportional hazards model. Estimates were decomposed into the direct and indirect effects mediated by postvaccination HAI titers. RESULTS: The proportions of vaccine efficacy mediated by postvaccination HAI titers were estimated to be 22% (95% confidence interval, 18%--47%) for influenza A(H1N1), 20% (16%-39%) for influenza A(H3N2), and 37% (26%-85%) for influenza B/Victoria. CONCLUSIONS: HAI titers partially mediate influenza vaccine efficacy against influenza A(H1N1), A(H3N2), and B/Victoria. Our estimates were lower than in previous studies, possibly reflecting expected heterogeneity in antigenic similarity between vaccine and circulating viruses across seasons.

Preliminary Findings From the Dynamics of the Immune Responses to Repeat Influenza Vaccination Exposures (DRIVE I) Study: A Randomized Controlled Trial.

BACKGROUND: Studies have reported that repeated annual vaccination may influence influenza vaccination effectiveness in the current season. METHODS: We established a 5-year randomized placebo-controlled trial of repeated influenza vaccination (Flublok; Sanofi Pasteur) in adults 18-45 years of age. In the first 2 years, participants were randomized to receive vaccine or saline placebo as follows: placebo-placebo (P-P), placebo-vaccine (P-V), or vaccine-vaccine (V-V). Serum samples were collected each year just before vaccination and after 30 and 182 days. A subset of serum samples collected at 5 time points from 95 participants were tested for antibodies against vaccine strains. RESULTS: From 23 October 2020 through 11 March 2021 we enrolled and randomized 447 adults. Among vaccinated individuals, antibody titers increased between days 0 and 30 against each of the vaccine strains, with smaller increases for repeat vaccinees who on average had higher prevaccination titers in year 2. There were statistically significant differences in the proportions of participants achieving ≥4-fold rises in antibody titer for the repeat vaccinees for influenza A(H1N1), B/Victoria, and B/Yamagata, but not for A(H3N2). Among participants who received vaccination in year 2, there were no significant differences between the P-V and V-V groups in geometric mean titers at day 30 or the proportions of participants with antibody titers ≥40 at day 30 for any of the vaccine strains. CONCLUSIONS: In the first 2 years, during which influenza did not circulate, repeat and first-time vaccinees had similar postvaccination geometric mean titers to all 4 vaccine strains, indicative of similar levels of clinical protection. Clinical Trials Registration. NCT04576377.

Safety and Immunogenicity of Respiratory Syncytial Virus Prefusion F Protein Vaccine when Co-administered with Adjuvanted Seasonal Quadrivalent Influenza Vaccine in Older Adults: A Phase 3 Randomized Trial.

BACKGROUND: We evaluated co-administration of adjuvanted seasonal quadrivalent influenza vaccine (FLU-aQIV) and respiratory syncytial virus (RSV) prefusion F protein-based vaccine (RSVPreF3 OA) in ≥65-year-olds. METHODS: This phase 3, open-label trial randomized ≥65-year-olds to receive FLU-aQIV and RSVPreF3 OA concomitantly (Co-Ad) or sequentially, 1 month apart (Control). Primary objectives were to demonstrate the non-inferiority of FLU-aQIV and RSVPreF3 OA co-administration versus sequential administration in terms of hemagglutination inhibition (HI) titers for each FLU-aQIV strain and RSV-A and RSV-B neutralization titers, 1 month post-vaccination. Reactogenicity and safety were also assessed. RESULTS: Overall, 1045 participants were vaccinated (Co-Ad: 523; Control: 522). Non-inferiority of FLU-aQIV and RSVPreF3 OA co-administration versus sequential administration was demonstrated in terms of HI titers for the A/Victoria(H1N1), B/Victoria, and B/Yamagata influenza strains and RSV-A neutralization titers (upper limits [ULs] of 95% confidence intervals [CIs] for adjusted geometric mean titer [GMT] ratios [Control/Co-Ad] ≤1.50) but not for A/Darwin(H3N2) HI titers (95% CI UL = 1.53). The immune response to A/Darwin(H3N2) was further assessed post-hoc using a microneutralization assay; the post-vaccination adjusted GMT ratio (Control/Co-Ad) was 1.23 (95% CI: 1.06-1.42, ie, UL ≤1.50), suggesting an adequate immune response to A/Darwin(H3N2) following co-administration. RSV-B neutralization titers were comparable between groups (95% CI UL for adjusted GMT ratio ≤1.50). Solicited adverse events were mostly mild or moderate and transient; unsolicited and serious adverse event rates were balanced between groups. CONCLUSIONS: Adjuvanted FLU-aQIV and RSVPreF3 OA had acceptable reactogenicity/safety profiles when co-administered in ≥65-year-olds, without clinically relevant interference with the immune responses to either vaccine. CLINICAL TRIALS REGISTRATION: NCT05568797.

Performance evaluation of the Panbio COVID-19/Flu A&B Panel for detection of SARS-CoV-2, influenza A, and influenza B antigens using mid-turbinate nasal swabs.

The Panbio COVID-19/Flu A&B Panel (Abbott) is an in vitro diagnostic rapid test designed for the qualitative detection of nucleocapsid proteins SARS-CoV-2 and nucleoprotein influenza A and B antigens in nasal mid-turbinate (NMT) swab specimens from symptomatic individuals meeting COVID-19 and influenza clinical and/or epidemiological criteria. This study, the largest global one to date using fresh samples, aimed to assess the diagnostic sensitivity and specificity of the Panbio COVID-19/Flu A&B Panel in freshly collected NMT swab specimens from individuals suspected of respiratory viral infection consistent with COVID-19 and/or influenza within the first 5 days of symptom onset compared with results obtained with the cobas SARS-CoV-2 and influenza A/B qualitative assay (cobas 6800/8800 systems), which were tested using nasopharyngeal swab samples. A total of 512 evaluable subjects were enrolled in the COVID-19 cohort across 18 sites, and 1,148 evaluable subjects were enrolled in the influenza cohort across 22 sites in the Asia-Pacific, Europe, and the USA. The Panbio COVID-19/Flu A&B Panel demonstrated a sensitivity of 80.4% and a specificity of 99.7% for COVID-19. For influenza A, the sensitivity and specificity rates were 80.6% and 99.3%, respectively. Likewise, for influenza B, the sensitivity and specificity rates were 80.8% and 99.4%, respectively. In conclusion, the Panbio COVID-19/Flu A&B Panel emerges as a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.4% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B. IMPORTANCE: The Panbio COVID-19/Flu A&B Panel is a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.0% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B.

Unlocking influenza B: exploring molecular biology and reverse genetics for epidemic control and vaccine innovation.

Influenza is a highly contagious acute viral illness that affects the respiratory system, posing a significant global public health concern. Influenza B virus (IBV) causes annual seasonal epidemics. The exploration of molecular biology and reverse genetics of IBV is pivotal for understanding its replication, pathogenesis, and evolution. Reverse genetics empowers us to purposefully alter the viral genome, engineer precise genetic modifications, and unveil the secrets of virulence and resistance mechanisms. It helps us in quickly analyzing new virus strains by viral genome manipulation and the development of innovative influenza vaccines. Reverse genetics has been employed to create mutant or reassortant influenza viruses for evaluating their virulence, pathogenicity, host range, and transmissibility. Without this technique, these tasks would be difficult or impossible, making it crucial for preparing for epidemics and protecting public health. Here, we bring together the latest information on how we can manipulate the genes of the influenza B virus using reverse genetics methods, most importantly helper virus-independent techniques.

Evaluation of CLINITEST® Rapid Covid-19 + Influenza antigen test in a cohort of symptomatic patients in an emergency department.

OBJECTIVES: Rapid management of patients with respiratory tract infections in hospital emergency departments is one of the main objectives since the concurrent circulation of respiratory viruses following the SARS-CoV-2 pandemic. The use of new combined point-of-care antigen tests for detecting influenza A/B and SARS-CoV-2 represents an advantage in response time over the molecular tests. The objective was to evaluate the suitability of the CLINITEST® Rapid Covid-19 + Influenza Antigen test (Siemens Healthineers, Germany) (RCIA test) by measuring the sensitivity, specificity, Cohen's kappa, and cut-off values. METHODS: Nasopharyngeal samples were collected from a randomised group of symptomatic patients of all ages at emergency department during January-February 2023. In parallel, these patients were screened for influenza A/B, and SARS-CoV-2 using RT-PCR. The Ct (cycle threshold) values were collected for positive [RT-PCR (+) /RCIA test (+)] and false negative [(RT-PCR (+) /RCIA test (-)] samples. A subanalysis was performed in the paediatric population (< 16 years-old). RESULTS: We included 545 patients (55.8% females) with a median age of 7 years-old (IQR: 1-66.5). The RCIA test showed a sensitivity of 59.7% [95%CI: 46.9-67.33] for influenza A, 65.6% [95%CI: 49.5-80.3] for influenza B, and 76.9% [95%CI: 45.8-84.8] for SARS-CoV-2. The specificity was between 90.7%-99.7% with a moderate/high level of agreement with RT-PCR (kappa score: 0.6-0.8) for the three respiratory viruses included in the RCIA test. CONCLUSIONS: The sensitivity of the RCIA test is insufficient for screening of patients, including patients with low Ct values (Ct > 20). Despite its good specificity and Cohen's kappa value, its use as a screening test is not comparable to RT-PCR systems in the ED environment with a high number of false negative results.

Establishment of a Raman nanosphere based immunochromatographic system for the combined detection of influenza A and B viruses' antigens on a single T-line.

A simple and rapid system based on Raman nanosphere (R-Sphere) immunochromatography was developed in this study for the simultaneous detection of Influenza A, B virus antigens on a single test line (T-line). Two types of R-Sphere with different characteristic Raman spectrum were used as the signal source, which were labeled with monoclonal antibodies against FluA, FluB (tracer antibodies), respectively. A mixture of antibodies containing anti-FluA monoclonal antibody and anti-FluB monoclonal antibody (capture antibody) was sprayed on a single T-line and goat anti-chicken IgY antibody was coated as a C-line, and the antigen solution with known concentration was detected by the strip of lateral flow immunochromatography based on surface-enhanced Raman spectroscopy (SERS). The T-line was scanned with a Raman spectrometer and SERS signals were collected. Simultaneous specific recognition and detection of FluA and FluB were achieved on a single T-line by analyzing the SERS signals. The findings indicated that the test system could identify FluA and FluB in a qualitative manner in just 15 minutes, with a minimum detection threshold of 0.25 ng ml-1, excellent consistency, and specificity. There was no interference with the other four respiratory pathogens, and it exhibited 8 times greater sensitivity compared to the colloidal gold test strip method. The assay system is rapid, sensitive, and does not require repetitive sample pretreatment steps and two viruses can be detected simultaneously on a single T-line by titrating one sample, which improves detection efficiency, and provide a reference for developing multiplexed detection techniques for other respiratory viruses.

Effectiveness of influenza vaccines in children aged 6 to 59 months: a test-negative case-control study at primary care and hospital level, Spain 2023/24.

During 2023/24, all children aged 6 to 59 months were targeted for seasonal influenza vaccination in Spain nationally. Using a test-negative case-control design with sentinel surveillance data, we estimated adjusted influenza vaccine effectiveness (IVE) against any influenza type to be 70% (95% confidence interval (CI): 51 to 81%) for primary care patients with acute respiratory illness (ARI) and 77% (95% CI: 21 to 93%) for hospitalised patients with severe ARI. In primary care, where most subtyped viruses (61%; 145/237) were A(H1N1), adjusted IVE was 77% (95% CI: 56 to 88%) against A(H1N1)pdm09.

Comparison of Two High-Dose Versus Two Standard-Dose Influenza Vaccines in Adult Allogeneic Hematopoietic Cell Transplant Recipients.

BACKGROUND: Adult hematopoietic cell transplant (HCT) recipients are at high risk for influenza-related morbidity and mortality and have suboptimal influenza vaccine immune responses compared to healthy adults, particularly within 2 years of transplant. METHODS: This phase II, double-blind, multicenter randomized controlled trial compared 2 doses of high-dose trivalent (HD-TIV) to 2 doses of standard-dose quadrivalent (SD-QIV) influenza vaccine administered 1 month apart in adults 3-23 months post-allogeneic HCT. Hemagglutinin antibody inhibition (HAI) titers were measured at baseline, 4 weeks following each vaccine dose, and approximately 7 months post-second vaccination. Injection-site and systemic reactions were assessed for 7 days post-vaccination. The primary immunogenicity comparison was geometric mean HAI titer (GMT) at visit 3 (4 weeks after the second dose); we used linear mixed models to estimate adjusted GMT ratios (aGMRs) comparing HD-TIV/SD-QIV for each antigen. RESULTS: We randomized 124 adults; 64 received SD-QIV and 60 received HD-TIV. Following the second vaccination, HD-TIV was associated with higher GMTs compared to SD-QIV for A/H3N2 (aGMR = 2.09; 95% confidence interval [CI]: [1.19, 3.68]) and B/Victoria (aGMR = 1.61; 95% CI: [1.00, 2.58]). The increase was not statistically significant for A/H1N1 (aGMR = 1.16; 95% CI: [0.67, 2.02]). There was a trend to more injection-site reactions for HD-TIV after the second vaccination compared to SD-QIV (50% vs 33%; adjusted odds ratio [aOR] = 4.53; 95% CI: [0.71, 28.9]), whereas systemic reactions were similar between groups with both injections. CONCLUSIONS: Adult allogeneic HCT recipients who received 2 doses of HD-TIV produced higher HAI antibody responses for A/H3N2 and B/Victoria compared with 2 doses of SD-QIV, with comparable injection-site or systemic reactions.

Divergent evolutionary trajectories of influenza B viruses underlie their contemporaneous epidemic activity.

Influenza B viruses have circulated in humans for over 80 y, causing a significant disease burden. Two antigenically distinct lineages ("B/Victoria/2/87-like" and "B/Yamagata/16/88-like," termed Victoria and Yamagata) emerged in the 1970s and have cocirculated since 2001. Since 2015 both lineages have shown unusually high levels of epidemic activity, the reasons for which are unclear. By analyzing over 12,000 influenza B virus genomes, we describe the processes enabling the long-term success and recent resurgence of epidemics due to influenza B virus. We show that following prolonged diversification, both lineages underwent selective sweeps across the genome and have subsequently taken alternate evolutionary trajectories to exhibit epidemic dominance, with no reassortment between lineages. Hemagglutinin deletion variants emerged concomitantly in multiple Victoria virus clades and persisted through epistatic mutations and interclade reassortment-a phenomenon previously only observed in the 1970s when Victoria and Yamagata lineages emerged. For Yamagata viruses, antigenic drift of neuraminidase was a major driver of epidemic activity, indicating that neuraminidase-based vaccines and cross-reactivity assays should be employed to monitor and develop robust protection against influenza B morbidity and mortality. Overall, we show that long-term diversification and infrequent selective sweeps, coupled with the reemergence of hemagglutinin deletion variants and antigenic drift of neuraminidase, are factors that contributed to successful circulation of diverse influenza B clades. Further divergence of hemagglutinin variants with poor cross-reactivity could potentially lead to circulation of 3 or more distinct influenza B viruses, further complicating influenza vaccine formulation and highlighting the urgent need for universal influenza vaccines.