Hemagglutinin and Neuraminidase Antibodies Are Induced in an Age- and Subtype-Dependent Manner after Influenza Virus Infection.

Despite evidence that antibodies targeting the influenza virus neuraminidase (NA) protein can be protective and are broadly cross-reactive, the immune response to NA during infection is poorly understood compared to the response to hemagglutinin (HA) protein. As such, we compared the antibody profile to HA and NA in two naturally infected human cohorts in Auckland, New Zealand: (i) a serosurvey cohort, consisting of pre- and post-influenza season sera from PCR-confirmed influenza cases (n = 50), and (ii) an immunology cohort, consisting of paired sera collected after PCR-confirmation of infection (n = 94). The induction of both HA and NA antibodies in these cohorts was influenced by age and subtype. Seroconversion to HA was more frequent in those <20 years old (yo) for influenza A (serosurvey, P = 0.01; immunology, P = 0.02) but not influenza B virus infection. Seroconversion to NA was not influenced by age or virus type. Adults ≥20 yo infected with influenza A viruses were more likely to show NA-only seroconversion compared to children (56% versus 14% [5 to 19 yo] and 0% [0 to 4 yo], respectively). Conversely, children infected with influenza B viruses were more likely than adults to show NA-only seroconversion (88% [0 to 4 yo] and 75% [5 to 19 yo] versus 40% [≥20 yo]). These data indicate a potential role for immunological memory in the dynamics of HA and NA antibody responses. A better mechanistic understanding of this phenomenon will be critical for any future vaccines aimed at eliciting NA immunity.IMPORTANCE Data on the immunologic responses to neuraminidase (NA) is lacking compared to what is available on hemagglutinin (HA) responses, despite growing evidence that NA immunity can be protective and broadly cross-reactive. Understanding these NA responses during natural infection is key to exploiting these properties for improving influenza vaccines. Using two community-acquired influenza cohorts, we showed that the induction of both HA and NA antibodies after infection is influenced by age and subtypes. Such response dynamics suggest the influence of immunological memory, and understanding how this process is regulated will be critical to any vaccine effort targeting NA immunity.

Risk Factors and Attack Rates of Seasonal Influenza Infection: Results of the Southern Hemisphere Influenza and Vaccine Effectiveness Research and Surveillance (SHIVERS) Seroepidemiologic Cohort Study.

Background: Understanding the attack rate of influenza infection and the proportion who become ill by risk group is key to implementing prevention measures. While population-based studies of antihemagglutinin antibody responses have been described previously, studies examining both antihemagglutinin and antineuraminidase antibodies are lacking. Methods: In 2015, we conducted a seroepidemiologic cohort study of individuals randomly selected from a population in New Zealand. We tested paired sera for hemagglutination inhibition (HAI) or neuraminidase inhibition (NAI) titers for seroconversion. We followed participants weekly and performed influenza polymerase chain reaction (PCR) for those reporting influenza-like illness (ILI). Results: Influenza infection (either HAI or NAI seroconversion) was found in 321 (35% [95% confidence interval, 32%-38%]) of 911 unvaccinated participants, of whom 100 (31%) seroconverted to NAI alone. Young children and Pacific peoples experienced the highest influenza infection attack rates, but overall only a quarter of all infected reported influenza PCR-confirmed ILI, and one-quarter of these sought medical attention. Seroconversion to NAI alone was higher among children aged <5 years vs those aged ≥5 years (14% vs 4%; P < .001) and among those with influenza B vs A(H3N2) virus infections (7% vs 0.3%; P < .001). Conclusions: Measurement of antineuraminidase antibodies in addition to antihemagglutinin antibodies may be important in capturing the true influenza infection rates.

Severe Influenza Is Characterized by Prolonged Immune Activation: Results From the SHIVERS Cohort Study.

Background: The immunologic factors underlying severe influenza are poorly understood. To address this, we compared the immune responses of influenza-confirmed hospitalized individuals with severe acute respiratory illness (SARI) to those of nonhospitalized individuals with influenza-like illness (ILI). Methods: Peripheral blood lymphocytes were collected from 27 patients with ILI and 27 with SARI, at time of enrollment and then 2 weeks later. Innate and adaptive cellular immune responses were assessed by flow cytometry, and serum cytokine levels were assessed by a bead-based assay. Results: During the acute phase, SARI was associated with significantly reduced numbers of circulating myeloid dendritic cells, CD192+ monocytes, and influenza virus-specific CD8+ and CD4+ T cells as compared to ILI. By the convalescent phase, however, most SARI cases displayed continued immune activation characterized by increased numbers of CD16+ monocytes and proliferating, and influenza virus-specific, CD8+ T cells as compared to ILI cases. SARI was also associated with reduced amounts of cytokines that regulate T-cell responses (ie, interleukin 4, interleukin 13, interleukin 12, interleukin 10, and tumor necrosis factor ß) and hematopoiesis (interleukin 3 and granulocyte-macrophage colony-stimulating factor) but increased amounts of a proinflammatory cytokine (tumor necrosis factor α), chemotactic cytokines (MDC, MCP-1, GRO, and fractalkine), and growth-promoting cytokines (PDGFBB/AA, VEGF, and EGF) as compared to ILI. Conclusions: Severe influenza cases showed a delay in the peripheral immune activation that likely led prolonged inflammation, compared with mild influenza cases.

Activated CD4+ T cells and CD14hiCD16+ monocytes correlate with antibody response following influenza virus infection in humans.

The failure to mount an antibody response following viral infection or seroconversion failure is a largely underappreciated and poorly understood phenomenon. Here, we identified immunologic markers associated with robust antibody responses after influenza virus infection in two independent human cohorts, SHIVERS and FLU09, based in Auckland, New Zealand and Memphis, Tennessee, USA, respectively. In the SHIVERS cohort, seroconversion significantly associates with (1) hospitalization, (2) greater numbers of proliferating, activated CD4+ T cells, but not CD8+ T cells, in the periphery during the acute phase of illness, and (3) fewer inflammatory monocytes (CD14hiCD16+) by convalescence. In the FLU09 cohort, fewer CD14hiCD16+ monocytes during early illness in the nasal mucosa were also associated with the generation of influenza-specific mucosal immunoglobulin A (IgA) and IgG antibodies. Our study demonstrates that seroconversion failure after infection is a definable immunological phenomenon, associated with quantifiable cellular markers that can be used to improve diagnostics, vaccine efficacy, and epidemiologic efforts.

Potential Association Between Dietary Fibre and Humoral Response to the Seasonal Influenza Vaccine.

Influenza vaccination is an effective public health measure to reduce the risk of influenza illness, particularly when the vaccine is well matched to circulating strains. Notwithstanding, the efficacy of influenza vaccination varies greatly among vaccinees due to largely unknown immunological determinants, thereby dampening population-wide protection. Here, we report that dietary fibre may play a significant role in humoral vaccine responses. We found dietary fibre intake and the abundance of fibre-fermenting intestinal bacteria to be positively correlated with humoral influenza vaccine-specific immune responses in human vaccinees, albeit without reaching statistical significance. Importantly, this correlation was largely driven by first-time vaccinees; prior influenza vaccination negatively correlated with vaccine immunogenicity. In support of these observations, dietary fibre consumption significantly enhanced humoral influenza vaccine responses in mice, where the effect was mechanistically linked to short-chain fatty acids, the bacterial fermentation product of dietary fibre. Overall, these findings may bear significant importance for emerging infectious agents, such as COVID-19, and associated de novo vaccinations.

Impact of the COVID-19 nonpharmaceutical interventions on influenza and other respiratory viral infections in New Zealand.

Stringent nonpharmaceutical interventions (NPIs) such as lockdowns and border closures are not currently recommended for pandemic influenza control. New Zealand used these NPIs to eliminate coronavirus disease 2019 during its first wave. Using multiple surveillance systems, we observed a parallel and unprecedented reduction of influenza and other respiratory viral infections in 2020. This finding supports the use of these NPIs for controlling pandemic influenza and other severe respiratory viral threats.

Pandemic (H1N1) 2009 and seasonal influenza A (H1N1) co-infection, New Zealand, 2009.

Co-infection with seasonal influenza A (H1N1) and pandemic (H1N1) 2009 could result in reassortant viruses that may acquire new characteristics of transmission, virulence, and oseltamivir susceptibility. Results from oseltamivir-sensitivity testing on viral culture suggested the possibility of co-infections with oseltamivir-resistant (seasonal A [H1N1]) and -susceptible (pandemic [H1N1] 2009) viruses.

Impact of the COVID-19 nonpharmaceutical interventions on influenza and other respiratory viral infections in New Zealand.

Stringent nonpharmaceutical interventions (NPIs) such as lockdowns and border closures are not currently recommended for pandemic influenza control. New Zealand used these NPIs to eliminate coronavirus disease 2019 during its first wave. Using multiple surveillance systems, we observed a parallel and unprecedented reduction of influenza and other respiratory viral infections in 2020. This finding supports the use of these NPIs for controlling pandemic influenza and other severe respiratory viral threats.

A Modular Cytokine Analysis Method Reveals Novel Associations With Clinical Phenotypes and Identifies Sets of Co-signaling Cytokines Across Influenza Natural Infection Cohorts and Healthy Controls.

Cytokines and chemokines are key signaling molecules of the immune system. Recent technological advances enable measurement of multiplexed cytokine profiles in biological samples. These profiles can then be used to identify potential biomarkers of a variety of clinical phenotypes. However, testing for such associations for each cytokine separately ignores the highly context-dependent covariation in cytokine secretion and decreases statistical power to detect associations due to multiple hypothesis testing. Here we present CytoMod-a novel data-driven approach for analysis of cytokine profiles that uses unsupervised clustering and regression to identify putative functional modules of co-signaling cytokines. Each module represents a biosignature of co-signaling cytokines. We applied this approach to three independent clinical cohorts of subjects naturally infected with influenza in which cytokine profiles and clinical phenotypes were collected. We found that in two out of three cohorts, cytokine modules were significantly associated with clinical phenotypes, and in many cases these associations were stronger than the associations of the individual cytokines within them. By comparing cytokine modules across datasets, we identified cytokine "cores"-specific subsets of co-expressed cytokines that clustered together across the three cohorts. Cytokine cores were also associated with clinical phenotypes. Interestingly, most of these cores were also co-expressed in a cohort of healthy controls, suggesting that in part, patterns of cytokine co-signaling may be generalizable. CytoMod can be readily applied to any cytokine profile dataset regardless of measurement technology, increases the statistical power to detect associations with clinical phenotypes and may help shed light on the complex co-signaling networks of cytokines in both health and infection.

A feasibility study: association between gut microbiota enterotype and antibody response to seasonal trivalent influenza vaccine in adults.

Objective: We investigated the potential feasibility of a randomized controlled trial of a nutritional intervention that may alter human gut microbiota and support immune defence against respiratory tract infection in adults (Proposed Study). Methods: In total, 125 healthy adults aged 18-64 participated in a 6-month study that measured antibody response to the seasonal trivalent influenza vaccine. We assessed completion rates, procedure adherence rates and the influence of possible exclusion criteria on potential recruitment into the Proposed Study. We examined whether the gut microbiota could be categorised into enterotypes, and whether there was an association between enterotypes and the antibody response to the influenza vaccine. Results: The participant completion rate was 97.6% (95% CI 93.1-99.5%). The proportions (95% CI) of participants who may be excluded for antibiotic or corticosteroid use in the 30 days prior to the study, or due to receiving the influenza vaccine in the previous two years were 9.6% (5.1-16.2), 8.0% (3.9-14.2) and 61.6% (52.5-70.2), respectively. All participants were stratified into four gut microbiota enterotypes. There was no association between these enterotypes and the antibody response to the influenza vaccine, although the study was not powered for this outcome. Conclusion: This study design is suitable for the Proposed Study. The completion rate is likely to be high, although exclusion criteria should be selected with care. Further analyses of gut microbiota composition or function in association with antibody and immune responses are warranted to explore the role of host-microbiota interactions on protective immunity.

Tracking oseltamivir-resistance in New Zealand influenza viruses during a medicine reclassification in 2007, a resistant-virus importation in 2008 and the 2009 pandemic.

INTRODUCTION: Oseltamivir (Tamiflu®) is an important pharmaceutical intervention against the influenza virus. The importance of surveillance for resistance to oseltamivir has been highlighted by two global events: the emergence of an oseltamivir-resistant seasonal influenza A(H1N1) virus in 2008, and emergence of the influenza A(H1N1)pdm09 virus in 2009. Oseltamivir is a prescription medicine in New Zealand, but more timely access has been provided since 2007 by allowing pharmacies to directly dispense oseltamivir to patients with influenza-like illness. OBJECTIVE: To determine the frequency of oseltamivir-resistance in the context of a medicine reclassification in 2007, the importation of an oseltamivir-resistant seasonal influenza virus in 2008, and the emergence of a pandemic in 2009. METHODS: A total of 1795 influenza viruses were tested for oseltamivir-resistance using a fluorometric neuraminidase inhibition assay. Viruses were collected as part of a sentinel influenza surveillance programme between the years 2006 and 2010. RESULTS: All influenza B, influenza A(H3N2) and influenza A(H1N1)pdm09 viruses tested between 2006 and 2010 were shown to be sensitive to oseltamivir. Seasonal influenza A(H1N1) viruses from 2008 and 2009 were resistant to oseltamivir. Sequencing of the neuraminidase gene showed that the resistant viruses contained an H275Y mutation, and S247N was also identified in the neuraminidase gene of one seasonal influenza A(H1N1) virus that exhibited enhanced resistance. DISCUSSION: No evidence was found to suggest that increased access to oseltamivir has promoted resistance. A probable importation event was documented for the global 2008 oseltamivir-resistant seasonal A(H1N1) virus nine months after it was first reported in Europe in January 2008.