A(H2N2) and A(H3N2) influenza pandemics elicited durable cross-reactive and protective antibodies against avian N2 neuraminidases.

Human cases of avian influenza virus (AIV) infections are associated with an age-specific disease burden. As the influenza virus N2 neuraminidase (NA) gene was introduced from avian sources during the 1957 pandemic, we investigate the reactivity of N2 antibodies against A(H9N2) AIVs. Serosurvey of healthy individuals reveal the highest rates of AIV N2 antibodies in individuals aged ≥65 years. Exposure to the 1968 pandemic N2, but not recent N2, protected against A(H9N2) AIV challenge in female mice. In some older adults, infection with contemporary A(H3N2) virus could recall cross-reactive AIV NA antibodies, showing discernable human- or avian-NA type reactivity. Individuals born before 1957 have higher anti-AIV N2 titers compared to those born between 1957 and 1968. The anti-AIV N2 antibodies titers correlate with antibody titers to the 1957 N2, suggesting that exposure to the A(H2N2) virus contribute to this reactivity. These findings underscore the critical role of neuraminidase immunity in zoonotic and pandemic influenza risk assessment.

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 the effectiveness of the influenza vaccination in the current season. The mechanisms underlying these differences are unclear but might include "focusing" of the adaptive immune response to older strains. Methods: We established a 5-year randomized placebo-controlled trial of repeated influenza vaccination (Flublok, Sanofi Pasteur) in adults 18-45 years of age. Participants were randomized equally between five groups, with planned annual receipt of vaccination (V) or saline placebo (P) as follows: P-P-P-P-V, P-P-P-V-V, P-P-V-V-V, P-V-V-V-V, or V-V-V-VV. Serum samples were collected each year just before vaccination and after 30 and 182 days. A subset of sera were tested by hemagglutination inhibition assays, focus reduction neutralization tests and enzyme-linked immunosorbent assays against vaccine strains. Results: From 23 October 2020 through 11 March 2021 we enrolled and randomized 447 adults. We selected sera from 95 participants at five timepoints from the first two study years for testing. Among vaccinated individuals, antibody titers increased between days 0 and 30 against each of the vaccine strains, with substantial increases for first-time vaccinees and smaller increases for repeat vaccinees, who had higher pre-vaccination titers in year 2. There were statistically significant reductions in the proportion of participants achieving a four-fold greater rise in antibody titer for the repeat vaccinees for A(H1N1), B/Victoria and B/Yamagata, but not for influenza A(H3N2). There were no statistically significant differences between 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 two years, repeat vaccinees and first-time vaccinees had similar post-vaccination geometric mean titers to all four vaccine strains, indicative of similar levels of clinical protection. The vaccine strains of A(H1N1) and A(H3N2) were updated in year 2, providing an opportunity to explore antigenic distances between those strains in humans in subsequent years.

Hemagglutination inhibition antibody titers mediate influenza vaccine efficacy against symptomatic influenza A(H1N1), A(H3N2), and B/Victoria infections.

BACKGROUND: The hemagglutination inhibition antibody (HAI) titer mediates only a part of vaccine-induced protection against influenza virus infections. Using causal mediation analysis, we quantified the proportion of vaccine efficacy mediated by post-vaccination HAI titers. METHODS: Causal mediation analyses were conducted using data collected for a randomized, active-comparator controlled, phase 3 trial of a candidate inactivated, split-virion seasonal quadrivalent influenza vaccine (QIV) in children aged 3 to 8 years conducted from October 2010 to December 2011 in eight countries. Vaccine efficacy was estimated with a weighted Cox proportional hazards regression model. Estimates were decomposed into the direct and indirect effects mediated by post-vaccination HAI titers. RESULTS: The proportions of vaccine efficacy mediated by post-vaccination HAI titers were estimated to be 22% (95% CI: 18%, 47%) for influenza A(H1N1), 20% (95% CI: 16%, 39%) for influenza A(H3N2), and 37% (95% CI: 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 previous studies, possibly reflecting expected heterogeneity in antigenic similarity between vaccine and circulating viruses across seasons. Data from more influenza seasons are needed to understand better the mediating effects of HAI titers on vaccine efficacy.

Amino acids in the polymerase complex of shorebird-isolated H1N1 influenza virus impact replication and host-virus interactions in mammalian models.

A diverse population of avian influenza A viruses (AIVs) are maintained in wild birds and ducks yet the zoonotic potential of AIVs in these environmental reservoirs and the host-virus interactions involved in mammalian infection are not well understood. In studies of a group of subtype H1N1 AIVs isolated from migratory wild birds during surveillance in North America, we previously identified eight amino acids in the polymerase genes PB2 and PB1 that were important for the transmissibility of these AIVs in a ferret model of human influenza virus transmission. In this current study we found that PB2 containing amino acids associated with transmissibility at 67, 152, 199, 508, and 649 and PB1 at 298, 642, and 667 were associated with more rapid viral replication kinetics, greater infectivity, more active polymerase complexes and greater kinetics of viral genome replication and transcription. Pathogenicity in the mouse model was also impacted, evident as greater weight loss and lung pathology associated with greater inflammatory lung cytokine expression. Further, these AIVs all contained the avian-type amino acids of PB2-E627, D701, G590, Q591 and T271. Therefore, our study provides novel insights into the role of the AIV polymerase complex in the zoonotic transmission of AIVs in mammals.

High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains.

The high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we have developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes via a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify infectivity of all of them simultaneously using next-generation sequencing. With this approach, a single researcher performed the equivalent of 2,880 traditional neutralization assays (80 serum samples against 36 viral strains) in approximately one month. We applied the sequencing-based assay to quantify the impact of influenza vaccination on neutralization titers against recent human H1N1 strains for individuals who had or had not also received a vaccine in the previous year. We found that the viral strain specificities of the neutralizing antibodies elicited by vaccination vary among individuals, and that vaccination induced a smaller increase in titers for individuals who had also received a vaccine the previous year-although the titers six months after vaccination were similar in individuals with and without the previous-year vaccination. We also identified a subset of individuals with low titers to a subclade of recent H1N1 even after vaccination. This study demonstrates the utility of high-throughput sequencing-based neutralization assays that enable titers to be simultaneously measured against many different viral strains. We provide a detailed experimental protocol (DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3xdmpg25/v1) and a computational pipeline (https://github.com/jbloomlab/seqneut-pipeline) for the sequencing-based neutralization assays to facilitate the use of this method by others.

Potential for transmission of naturally mutated H10N1 avian influenza virus to mammalian hosts and causing severe pulmonary disease.

Subtype H10 avian influenza viruses (AIV) are distributed worldwide in wild aquatic birds, and can infect humans and several other mammalian species. In the present study, we investigated the naturally mutated PB2 gene in A/aquatic bird/South Korea/SW1/2018 (A/SW1/18, H10N1), isolated from wild birds during the 2018-2019 winter season. This virus was originally found in South Korea, and is similar to isolates from mainland China and Mongolia. It had low pathogenicity, lacked a multi-basic cleavage site, and showed a binding preference for α2,3-linked sialic acids. However, it can infect mice, causing severe disease and lung pathology. SW1 was also transmitted by direct contact in ferrets, and replicated in the respiratory tract tissue, with no evidence of extrapulmonary spread. The pathogenicity and transmissibility of SW1 in mouse and ferret models were similar to those of the pandemic strain A/California/04/2009 (A/CA/04, H1N1). These factors suggest that subtype H10 AIVs have zoonotic potential and may transmit from human to human, thereby posing a potential threat to public health. Therefore, the study highlights the urgent need for closer monitoring of subtype H10 AIVs through continued surveillance of wild aquatic birds.

The episodic resurgence of highly pathogenic avian influenza H5 virus.

Highly pathogenic avian influenza (HPAI) H5N1 activity has intensified globally since 2021, increasingly causing mass mortality in wild birds and poultry and incidental infections in mammals1-3. However, the ecological and virological properties that underscore future mitigation strategies still remain unclear. Using epidemiological, spatial and genomic approaches, we demonstrate changes in the origins of resurgent HPAI H5 and reveal significant shifts in virus ecology and evolution. Outbreak data show key resurgent events in 2016-2017 and 2020-2021, contributing to the emergence and panzootic spread of H5N1 in 2021-2022. Genomic analysis reveals that the 2016-2017 epizootics originated in Asia, where HPAI H5 reservoirs are endemic. In 2020-2021, 2.3.4.4b H5N8 viruses emerged in African poultry, featuring mutations altering HA structure and receptor binding. In 2021-2022, a new H5N1 virus evolved through reassortment in wild birds in Europe, undergoing further reassortment with low-pathogenic avian influenza in wild and domestic birds during global dissemination. These results highlight a shift in the HPAI H5 epicentre beyond Asia and indicate that increasing persistence of HPAI H5 in wild birds is facilitating geographic and host range expansion, accelerating dispersion velocity and increasing reassortment potential. As earlier outbreaks of H5N1 and H5N8 were caused by more stable genomic constellations, these recent changes reflect adaptation across the domestic-bird-wild-bird interface. Elimination strategies in domestic birds therefore remain a high priority to limit future epizootics.

The impact of pre-existing influenza antibodies and inflammatory status on the influenza vaccine responses in older adults.

Age-associated immune changes and pre-existing influenza immunity are hypothesized to reduce influenza vaccine effectiveness in older adults, although the contribution of each factor is unknown. Here, we constructed influenza-specific IgG landscapes and determined baseline concentrations of cytokines typically associated with chronic inflammation in older adults (TNF-α, IL-10, IL-6, and IFN-γ) in 30 high and 29 low influenza vaccine responders (HR and LR, respectively). In a background of high H3 antibody titers, vaccine-specific H3, but not H1, antibody titers were boosted in LRs to titers comparable to HRs. Pre-vaccination concentrations of IL-10 were higher in LRs compared with HRs and inversely correlated with titers of pre-existing influenza antibodies. Baseline TNF-α concentrations were positively correlated with fold-increases in antibody titers in HRs. Our findings indicate that baseline inflammatory status is an important determinant for generating post-vaccination hemagglutinin-inhibition antibodies in older adults, and IgG responses can be boosted in the context of high pre-existing immunity.

Integrated Drivers of Basal and Acute Immunity in Diverse Human Populations.

Prior studies have identified genetic, infectious, and biological associations with immune competence and disease severity; however, there have been few integrative analyses of these factors and study populations are often limited in demographic diversity. Utilizing samples from 1,705 individuals in 5 countries, we examined putative determinants of immunity, including: single nucleotide polymorphisms, ancestry informative markers, herpesvirus status, age, and sex. In healthy subjects, we found significant differences in cytokine levels, leukocyte phenotypes, and gene expression. Transcriptional responses also varied by cohort, and the most significant determinant was ancestry. In influenza infected subjects, we found two disease severity immunophenotypes, largely driven by age. Additionally, cytokine regression models show each determinant differentially contributes to acute immune variation, with unique and interactive, location-specific herpesvirus effects. These results provide novel insight into the scope of immune heterogeneity across diverse populations, the integrative effects of factors which drive it, and the consequences for illness outcomes.

Global dissemination of influenza A virus is driven by wild bird migration through arctic and subarctic zones.

Influenza A viruses (IAV) circulate endemically among many wild aquatic bird populations that seasonally migrate between wintering grounds in southern latitudes to breeding ranges along the perimeter of the circumpolar arctic. Arctic and subarctic zones are hypothesized to serve as ecologic drivers of the intercontinental movement and reassortment of IAVs due to high densities of disparate populations of long distance migratory and native bird species present during breeding seasons. Iceland is a staging ground that connects the East Atlantic and North Atlantic American flyways, providing a unique study system for characterizing viral flow between eastern and western hemispheres. Using Bayesian phylodynamic analyses, we sought to evaluate the viral connectivity of Iceland to proximal regions and how inter-species transmission and reassortment dynamics in this region influence the geographic spread of low and highly pathogenic IAVs. Findings demonstrate that IAV movement in the arctic and subarctic reflects wild bird migration around the perimeter of the circumpolar north, favouring short-distance flights between proximal regions rather than long distance flights over the polar interior. Iceland connects virus movement between mainland Europe and North America, consistent with the westward migration of wild birds from mainland Europe to Northeastern Canada and Greenland. Though virus diffusion rates were similar among avian taxonomic groups in Iceland, gulls play an outsized role as sinks of IAVs from other avian hosts prior to onward migration. These data identify patterns of virus movement in northern latitudes and inform future surveillance strategies related to seasonal and emergent IAVs with potential public health concern.

Using discrete event simulation to optimize nucleic acid testing process for coronavirus disease 2019 (COVID-19).

Background: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus-2 (SARS-CoV-2) has placed enormous diagnostic burden on hospitals and testing laboratories. It is thus critical for such facilities to optimize the diagnostic process to enable maximum testing on minimum resources. The current standard of diagnosis is the detection of the viral nucleic acid in clinical specimens. Methods: In order to optimize the laboratory's nucleic acid testing system for COVID-19, we performed a Discrete-Event-Simulation using the Arena Simulation Software to model the detection process based on the data obtained from the First Affiliated Hospital of Guangzhou Medical University (FAHGMU). The maximum of total time that specimens spent and the equipment consumption was compared under different scenarios in the model. Results: Seven scenarios were performed to simulate actual situation and improved situations. We analyzed conditions that adding a new nucleic acid extraction system (NAES), shifting a member from night duty to morning duty, using specimen tubes containing guanidine isothiocyanate (GITC), then tested the maximum testing capacity in the current number of technicians. In addition, the costs including personal protective equipment (PPE) and testing kits was calculated. Conclusions: A work schedule based on specimen-load improves efficiency without incurring additional costs, while using the specimen tubes containing GITC could reduce testing time by 30 min. In contrast, adding new NAESs or polymerase chain reaction (PCR) instruments has minimal impact on testing efficiency.

Human adenovirus (HAdV) infection in children with acute respiratory tract infections in Guangzhou, China, 2010-2021: a molecular epidemiology study.

BACKGROUND: Human adenovirus (HAdV) infection can cause a variety of diseases. It is a major pathogen of pediatric acute respiratory tract infections (ARIs) and can be life-threatening in younger children. We described the epidemiology and subtypes shifting of HAdV among children with ARI in Guangzhou, China. METHODS: We conducted a retrospective study of 161,079 children diagnosed with acute respiratory illness at the Guangzhou Women and Children's Medical Center between 2010 and 2021. HAdV specimens were detected by real-time PCR and the hexon gene was used for phylogenetic analysis. RESULTS: Before the COVID-19 outbreak in Guangzhou, the annual frequency of adenovirus infection detected during this period ranged from 3.92% to 13.58%, with an epidemic peak every four to five years. HAdV demonstrated a clear seasonal distribution, with the lowest positivity in March and peaking during summer (July or August) every year. A significant increase in HAdV cases was recorded for 2018 and 2019, which coincided with a shift in the dominant HAdV subtype from HAdV-3 to HAdV-7. The latter was associated with a more severe disease compared to HAdV-3. The average mortality proportion for children infected with HAdV from 2016 to 2019 was 0.38% but increased to 20% in severe cases. After COVID-19 emerged, HAdV cases dropped to 2.68%, suggesting that non-pharmaceutical interventions probably reduced the transmission of HAdV in the community. CONCLUSION: Our study provides the foundation for the understanding of the epidemiology of HAdV and its associated risks in children in Southern China.

Severe acute respiratory syndrome coronavirus 2 and influenza A virus co-infection alters viral tropism and haematological composition in Syrian hamsters.

The ongoing coronavirus disease 2019 pandemic and its overlap with the influenza season lead to concerns over severe disease caused by the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infections. Using a Syrian hamster co-infection model with SARS-CoV-2 and the pandemic influenza virus A/California/04/2009 (H1N1), we found (a) more severe disease in co-infected animals, compared to those infected with influenza virus alone but not SARS-CoV-2 infection alone; (b) altered haematological changes in only co-infected animals and (c) altered influenza virus tropism in the respiratory tracts of co-infected animals. Overall, our study revealed that co-infection with SARS-CoV-2 and influenza virus is associated with altered disease severity and tissue tropism, as well as haematological changes, compared to infection with either virus alone.

Molecular alterations in human milk in simulated maternal nasal mucosal infection with live attenuated influenza vaccination.

Breastfeeding protects against mucosal infections in infants. The underlying mechanisms through which immunity develops in human milk following maternal infection with mucosal pathogens are not well understood. We simulated nasal mucosal influenza infection through live attenuated influenza vaccination (LAIV) and compared immune responses in milk to inactivated influenza vaccination (IIV). Transcriptomic analysis was performed on RNA extracted from human milk cells to evaluate differentially expressed genes and pathways on days 1 and 7 post-vaccination. Both LAIV and IIV vaccines induced influenza-specific IgA that persisted for at least 6 months. Regulation of type I interferon production, toll-like receptor, and pattern recognition receptor signaling pathways were highly upregulated in milk on day 1 following LAIV but not IIV at any time point. Upregulation of innate immunity in human milk may provide timely protection against mucosal infections until antigen-specific immunity develops in the human milk-fed infant.

Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity.

Infection and vaccination repeatedly expose individuals to antigens that are conserved between influenza virus subtypes. Nevertheless, antibodies recognizing variable influenza epitopes greatly outnumber antibodies reactive against conserved epitopes. Elucidating factors contributing to the paucity of broadly reactive influenza antibodies remains a major obstacle for developing a universal influenza vaccine. Here, we report that inducing broadly reactive influenza antibodies increases autoreactive antibodies in humans and mice and exacerbates disease in four distinct models of autoimmune disease. Importantly, transferring broadly reactive influenza antibodies augments disease in the presence of inflammation or autoimmune susceptibility. Further, broadly reactive influenza antibodies spontaneously arise in mice with defects in B cell tolerance. Together, these data suggest that self-tolerance mechanisms limit the prevalence of broadly reactive influenza antibodies, which can exacerbate disease in the context of additional risk factors.

Diagnostic performance and clinical feasibility of a novel one-step RT-qPCR assay for simultaneous detection of multiple severe acute respiratory syndrome coronaviruses.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID50/mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin.

Subtype H3N2 Influenza A Viruses: An Unmet Challenge in the Western Pacific.

Subtype H3N2 influenza A viruses (A(H3N2)) have been the dominant strain in some countries in the Western Pacific region since the 2009 influenza A(H1N1) pandemic. Vaccination is the most effective way to prevent influenza; however, low vaccine effectiveness has been reported in some influenza seasons, especially for A(H3N2). Antigenic mismatch introduced by egg-adaptation during vaccine production between the vaccine and circulating viral stains is one of the reasons for low vaccine effectiveness. Here we review the extent of this phenomenon, the underlying molecular mechanisms and discuss recent strategies to ameliorate this, including new vaccine platforms that may provide better protection and should be considered to reduce the impact of A(H3N2) in the Western Pacific region.

Clinical impact of nosocomial infection with pandemic influenza A (H1N1) 2009 in a respiratory ward in Guangzhou.

BACKGROUND: Nosocomial outbreaks of pandemic influenza A (H1N1) 2009 virus [A(H1N1)pdm09] easily develop due to its high transmissibility. This study aimed to investigate the clinical impacts of a nosocomial outbreak of A(H1N1)pdm09 between 21 January and 17 February 2016. METHODS: Patients who developed influenza-like illness (ILI) more than 48 hours after hospitalization in the index ward were enrolled as suspected patients, defined as group A and quarantined. Patients in other wards were defined as group B. A phylogenetic tree was constructed to determine the origins of the hemagglutinin and neuraminidase genes. RESULTS: After the implementation of an infection control measure bundle, the outbreak was limited to eight patients with ILIs in group A. Nasal swabs from seven patients were positive for A(H1N1)pdm09. All the patients recovered after treatment. Prolonged viral shedding was observed in a patient with bronchiectasis and Penicillium marneffei infection. Compared to the expected duration of hospitalization in patients without fever, those with fever had a median 7-day delay in discharge and a mean excess cost of 3,358 RMB. The four influenza strains identified were genetically identical to the A/California/115/2015 strain. Six of the 54 patients in group B who underwent bronchoscopy developed transient fever. These patients were hospitalized in various wards of the hospital and recovered after a short-term course of empirical antibiotics. CONCLUSIONS: After the implementation of infection control measures, the nosocomial A(H1N1)pdm09 outbreak was rapidly contained; infected patients had a delay in discharge and excess costs, but no deaths occurred.

Seroprevalence of Antibodies to SARS-CoV-2 in Guangdong Province, China between March to June 2020.

Guangdong province, located in South China, is an important economic hub with a large domestic migrant population and was among the earliest areas to report COVID-19 cases outside of Wuhan. We conducted a cross-sectional, age-stratified serosurvey to determine the seroprevalence of antibodies against SARS-CoV-2 after the emergence of COVID-19 in Guangdong. We tested 14,629 residual serum samples that were submitted for clinical testing from 21 prefectures between March and June 2020 for SARS-CoV-2 antibodies using a magnetic particle based chemiluminescent enzyme immunoassay and validated the results using a pseudovirus neutralization assay. We found 21 samples positive for SARS-CoV-2 IgG, resulting in an estimated age- and sex-weighted seroprevalence of 0.15% (95% CI: 0.06-0.24%). The overall age-specific seroprevalence was 0.07% (95% CI: 0.01-0.24%) in persons up to 9 years old, 0.22% (95% CI: 0.03-0.79%) in persons aged 10-19, 0.16% (95% CI: 0.07-0.33%) in persons aged 20-39, 0.13% (95% CI: 0.03-0.33%) in persons aged 40-59 and 0.18% (95% CI: 0.07-0.40%) in persons ≥60 years old. Fourteen (67%) samples had pseudovirus neutralization titers to S-protein, suggesting most of the IgG-positive samples were true-positives. Seroprevalence of antibodies to SARS-CoV-2 was low, indicating that there were no hidden epidemics during this period. Vaccination is urgently needed to increase population immunity to SARS-CoV-2.

Antibody Responsiveness to Influenza: What Drives It?

The induction of a specific antibody response has long been accepted as a serological hallmark of recent infection or antigen exposure. Much of our understanding of the influenza antibody response has been derived from studying antibodies that target the hemagglutinin (HA) protein. However, growing evidence points to limitations associated with this approach. In this review, we aim to highlight the issue of antibody non-responsiveness after influenza virus infection and vaccination. We will then provide an overview of the major factors known to influence antibody responsiveness to influenza after infection and vaccination. We discuss the biological factors such as age, sex, influence of prior immunity, genetics, and some chronic infections that may affect the induction of influenza antibody responses. We also discuss the technical factors, such as assay choices, strain variations, and viral properties that may influence the sensitivity of the assays used to measure influenza antibodies. Understanding these factors will hopefully provide a more comprehensive picture of what influenza immunogenicity and protection means, which will be important in our effort to improve influenza vaccines.