Enhanced isolation of influenza viruses in qualified cells improves the probability of well-matched vaccines.

Influenza vaccines are utilised to combat seasonal and pandemic influenza. The key to influenza vaccination currently is the availability of candidate vaccine viruses (CVVs). Ideally, CVVs reflect the antigenic characteristics of the circulating virus, which may vary depending upon the isolation method. For traditional inactivated egg-based vaccines, CVVs are isolated in embryonated chicken eggs, while for cell-culture production, CVV's are isolated in either embryonated eggs or qualified cell lines. We compared isolation rates, growth characteristics, genetic stability and antigenicity of cell and egg CVV's derived from the same influenza-positive human clinical respiratory samples collected from 2008-2020. Influenza virus isolation rates in MDCK33016PF cells were twice that of eggs and mutations in the HA protein were common in egg CVVs but rare in cell CVVs. These results indicate that fully cell-based influenza vaccines will improve the choice, match and potentially the effectiveness, of seasonal influenza vaccines compared to egg-based vaccines.

Which influenza viruses will emerge following the SARS-CoV-2 pandemic?

The world has experienced five pandemics in just over one hundred years, four due to influenza and one due to coronavirus (SARS-CoV-2). In each case of pandemic influenza, the pandemic influenza strain has replaced the previous seasonal influenza virus. Notably, throughout the SARS-CoV-2 pandemic, there has been a 99% reduction in influenza isolation globally. It is anticipated that influenza will re-emerge following the SARS-CoV-2 pandemic and circulate again. The potential for which influenza viruses will emerge is examined.

Immune cellular networks underlying recovery from influenza virus infection in acute hospitalized patients.

How innate and adaptive immune responses work in concert to resolve influenza disease is yet to be fully investigated in one single study. Here, we utilize longitudinal samples from patients hospitalized with acute influenza to understand these immune responses. We report the dynamics of 18 important immune parameters, related to clinical, genetic and virological factors, in influenza patients across different severity levels. Influenza disease correlates with increases in IL-6/IL-8/MIP-1α/ß cytokines and lower antibody responses. Robust activation of circulating T follicular helper cells correlates with peak antibody-secreting cells and influenza heamaglutinin-specific memory B-cell numbers, which phenotypically differs from vaccination-induced B-cell responses. Numbers of influenza-specific CD8+ or CD4+ T cells increase early in disease and retain an activated phenotype during patient recovery. We report the characterisation of immune cellular networks underlying recovery from influenza infection which are highly relevant to other infectious diseases.

Impact of prior vaccination on antibody response and influenza-like illness among Australian healthcare workers after influenza vaccination in 2016.

BACKGROUND: Epidemiological studies suggest that influenza vaccine effectiveness decreases with repeated administration. We examined antibody responses to influenza vaccination among healthcare workers (HCWs) by prior vaccination history and determined the incidence of influenza infection. METHODS: HCWs were vaccinated with the 2016 Southern Hemisphere quadrivalent influenza vaccine. Serum samples were collected pre-vaccination, 21-28 days and 7 months post-vaccination. Influenza antibody titres were measured at each time-point using the haemagglutination inhibition (HI) assay. Immunogenicity was compared by prior vaccination history. RESULTS: A total of 157 HCWs completed the study. The majority were frequently vaccinated, with only 5 reporting no prior vaccinations since 2011. Rises in titres for all vaccine strains among vaccine-naïve HCWs were significantly greater than rises observed for HCWs who received between 1 and 5 prior vaccinations (p < 0.001, respectively). Post-vaccination GMTs against influenza A but not B strains decreased as the number of prior vaccinations increased from 1 to 5. There was a significant decline in GMTs post-season for both B lineages. Sixty five (41%) HCWs reported at least one influenza-like illness episode, with 6 (4%) identified as influenza positive. CONCLUSIONS: Varying serological responses to influenza vaccination were observed among HCWs by prior vaccination history, with vaccine-naïve HCWs demonstrating greater post-vaccination responses against A(H3N2).

New Technologies for Influenza Vaccines.

Vaccine development has been hampered by the long lead times and the high cost required to reach the market. The 2020 pandemic, caused by a new coronavirus (SARS-CoV-2) that was first reported in late 2019, has seen unprecedented rapid activity to generate a vaccine, which belies the traditional vaccine development cycle. Critically, much of this progress has been leveraged off existing technologies, many of which had their beginnings in influenza vaccine development. This commentary outlines the most promising of the next generation of non-egg-based influenza vaccines including new manufacturing platforms, structure-based antigen design/computational biology, protein-based vaccines including recombinant technologies, nanoparticles, gene- and vector-based technologies, as well as an update on activities around a universal influenza vaccine.

Immunogenicity of the inactivated influenza vaccine in children who have undergone allogeneic haematopoietic stem cell transplant.

Influenza vaccination is recommended for children following allogeneic haematopoietic stem cell transplant (HSCT), however there is limited evidence regarding its benefit. A prospective multicentre study was conducted to evaluate the immunogenicity of the inactivated influenza vaccine in children who have undergone HSCT compared with healthy age-matched controls. Participants were vaccinated between 2013 and 2016 according to Australian guidelines. Influenza-specific hemagglutinin inhibition antibody titres were performed prior to each vaccination and 4 weeks following the final vaccination. A nasopharyngeal aspirate for influenza was performed on participants that developed influenza-like illness. There were 86 children recruited; 43 who had undergone HSCT and 43 controls. For the HSCT group, seroprotection and seroconversion rates were 81.4% and 60.5% for H3N2, 41.9% and 32.6% for H1N1, and 44.2% and 39.5% for B strain respectively. There was a significant geometric mean fold increase to the H3N2 (GMFI 5.80, 95% CI 3.68-9.14, p < 0.001) and B (GMFI 3.44, 95% CI 2.36-5.00, p = 0.048) strains. Serological response was superior in age-matched controls to all vaccine strains. There were no serious adverse events following vaccination. For children who underwent HSCT, incidence of laboratory-proven influenza infection was 2.3%. Overall, this study provides evidence to support annual inactivated influenza vaccine administration to children following HSCT.

Influenza A Virus Infection Induces Viral and Cellular Defective Ribosomal Products Encoded by Alternative Reading Frames.

The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.

Safety, immunogenicity and protection of A(H3N2) live attenuated influenza vaccines containing wild-type nucleoprotein in a ferret model.

Live attenuated influenza vaccines (LAIVs) are promising tools for the induction of broad protection from influenza due to their ability to stimulate cross-reactive T cells against influenza pathogens. One of the major targets for cytotoxic T-cell immunity is viral nucleoprotein (NP), which is relatively conserved among antigenically distant influenza viruses. Nevertheless, a diversity of epitope composition has been found in the NP protein of different lineages of influenza A viruses. The H2N2 master donor virus which is currently used as a backbone for the LAIV and donor of the six genomic segments encoding the internal proteins, A/Leningrad/134/17/57 (MDV Len/17), was isolated 60 years ago. As such, NP-specific T-cell immunity induced upon vaccination with classical LAIVs with a 6:2 genome composition containing this older NP might be suboptimal against currently circulating influenza viruses. In this study, a panel of H3N2 LAIV candidates with wild-type NP genes derived from circulating viruses were generated by reverse genetics (5:3 genome composition). These viruses displayed the cold adaptation and temperature sensitivity phenotypes of MDV Len/17 in vitro. LAIVs with both 6:2 and 5:3 genome compositions were attenuated and replicated to a similar extent in the upper respiratory tract of ferrets. LAIVs were immunogenic as high neutralizing and hemagglutination inhibition serum antibody titers were detected 21 days after infection. All vaccinated animals were protected against infection with heterologous H3N2 influenza A viruses. Thus, LAIV with a 5:3 genome composition is safe, immunogenic and can induce cross-protective immunity.

Investigating Viral Interference Between Influenza A Virus and Human Respiratory Syncytial Virus in a Ferret Model of Infection.

Epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hRSV) infection, with the peak incidence of hRSV infection delayed. This is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. We investigated viral interference between hRSV and 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) in the ferret model. Infection with A(H1N1)pdm09 prevented subsequent infection with hRSV. Infection with hRSV reduced morbidity attributed to infection with A(H1N1)pdm09 but not infection, even when an increased inoculum dose of hRSV was used. Notably, infection with A(H1N1)pdm09 induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hRSV. Minimal cross-reactive serological responses or interferon γ-expressing cells were induced by either virus ≥14 days after infection. These data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease.

Circulating TFH cells, serological memory, and tissue compartmentalization shape human influenza-specific B cell immunity.

Immunization with the inactivated influenza vaccine (IIV) remains the most effective strategy to combat seasonal influenza infections. IIV activates B cells and T follicular helper (TFH) cells and thus engenders antibody-secreting cells and serum antibody titers. However, the cellular events preceding generation of protective immunity in humans are inadequately understood. We undertook an in-depth analysis of B cell and T cell immune responses to IIV in 35 healthy adults. Using recombinant hemagglutinin (rHA) probes to dissect the quantity, phenotype, and isotype of influenza-specific B cells against A/California09-H1N1, A/Switzerland-H3N2, and B/Phuket, we showed that vaccination induced a three-pronged B cell response comprising a transient CXCR5-CXCR3+ antibody-secreting B cell population, CD21hiCD27+ memory B cells, and CD21loCD27+ B cells. Activation of circulating TFH cells correlated with the development of both CD21lo and CD21hi memory B cells. However, preexisting antibodies could limit increases in serum antibody titers. IIV had no marked effect on CD8+, mucosal-associated invariant T, γδ T, and natural killer cell activation. In addition, vaccine-induced B cells were not maintained in peripheral blood at 1 year after vaccination. We provide a dissection of rHA-specific B cells across seven human tissue compartments, showing that influenza-specific memory (CD21hiCD27+) B cells primarily reside within secondary lymphoid tissues and the lungs. Our study suggests that a rational design of universal vaccines needs to consider circulating TFH cells, preexisting serological memory, and tissue compartmentalization for effective B cell immunity, as well as to improve targeting cellular T cell immunity.

Evidence for Viral Interference and Cross-reactive Protective Immunity Between Influenza B Virus Lineages.

Background: Two influenza B virus lineages, B/Victoria and B/Yamagata, cocirculate in the human population. While the lineages are serologically distinct, cross-reactive responses to both lineages have been detected. Viral interference describes the situation whereby infection with one virus limits infection and replication of a second virus. We investigated the potential for viral interference between the influenza B virus lineages. Methods: Ferrets were infected and then challenged 3, 10, or 28 days later with pairs of influenza B/Victoria and B/Yamagata viruses. Results: Viral interference occurred at challenge intervals of 3 and 10 days and occasionally at 28 days. At the longer interval, shedding of challenge virus was reduced, and this correlated with cross-reactive interferon γ responses from lymph nodes from virus-infected animals. Viruses from both lineages could prevent or significantly limit subsequent infection with a virus from the other lineage. Coinfections were rare, indicating the potential for reassortment between lineages is limited. Conclusions: These data suggest that innate and cross-reactive immunity mediate viral interference and that this may contribute to the dominance of a specific influenza B virus lineage in any given influenza season. Furthermore, infection with one influenza B virus lineage may be beneficial in protecting against subsequent infection with either influenza B virus lineage.

Pathogenesis, Humoral Immune Responses, and Transmission between Cohoused Animals in a Ferret Model of Human Respiratory Syncytial Virus Infection.

Small-animal models have been used to obtain many insights regarding the pathogenesis and immune responses induced following infection with human respiratory syncytial virus (hRSV). Among those described to date, infections in cotton rats, mice, guinea pigs, chinchillas, and Syrian hamsters with hRSV strains Long and/or A2 have been well characterized, although clinical isolates have also been examined. Ferrets are also susceptible to hRSV infection, but the pathogenesis and immune responses elicited following infection have not been well characterized. Here, we describe the infection of adult ferrets with hRSV Long or A2 via the intranasal route and characterized virus replication, as well as cytokine induction, in the upper and lower airways. Virus replication and cytokine induction during the acute phase of infection (days 0 to 15 postinfection) were similar between the two strains, and both elicited high levels of F glycoprotein-specific binding and neutralizing antibodies following virus clearance (days 16 to 22 postinfection). Importantly, we demonstrate transmission from experimentally infected donor ferrets to cohoused naive recipients and have characterized virus replication and cytokine induction in the upper airways of infected contact animals. Together, these studies provide a direct comparison of the pathogenesis of hRSV Long and A2 in ferrets and highlight the potential of this animal model to study serological responses and examine interventions that limit transmission of hRSV.IMPORTANCE Ferrets have been widely used to study pathogenesis, immunity, and transmission following human influenza virus infections; however, far less is known regarding the utility of the ferret model to study hRSV infections. Following intranasal infection of adult ferrets with the well-characterized Long or A2 strain of hRSV, we report virus replication and cytokine induction in the upper and lower airways, as well as the development of virus-specific humoral responses. Importantly, we demonstrate transmission of hRSV from experimentally infected donor ferrets to cohoused naive recipients. Together, these findings significantly enhance our understanding of the utility of the ferret as a small-animal model to investigate aspects of hRSV pathogenesis and immunity.

Influenza vaccination responses: Evaluating impact of repeat vaccination among health care workers.

OBJECTIVE: To compare the antibody response to influenza between health care workers (HCWs) who have received multiple vaccinations (high vaccination group) and those who have received fewer vaccinations (low vaccination group). DESIGN: Prospective serosurvey. SETTING: Tertiary referral hospital. PARTICIPANTS: Healthcare workers. METHODS: Healthcare workers were vaccinated with the 2015 southern hemisphere trivalent influenza vaccine. Influenza antibody titres were measured pre-vaccination, 21-28days post-vaccination and 6months post-vaccination. Antibody titres were measured using the haemagglutination inhibition assay. Levels of seropositivity and estimated geometric mean titres were calculated. RESULTS: Of the 202 HCWs enrolled, 182 completed the study (143 high vaccination and 39 low vaccination). Both vaccination groups demonstrated increases in post-vaccination geometric mean titres, with greater gains in the low vaccination group. Seropositivity remained high in both high and low vaccination groups post-vaccination. The highest fold rise was observed among HCWs in the low vaccination group against the H3N2 component of the vaccine. CONCLUSIONS: Both high and low vaccination groups in our study demonstrated protective antibody titres post-vaccination. The findings from the current study are suggestive of decreased serological response among highly vaccinated HCWs. More studies with larger sample sizes and a greater number of people in the vaccine-naïve and once-vaccinated groups are required to confirm or refute these findings before making any policy changes.

Modelling cross-reactivity and memory in the cellular adaptive immune response to influenza infection in the host.

The cellular adaptive immune response plays a key role in resolving influenza infection. Experiments where individuals are successively infected with different strains within a short timeframe provide insight into the underlying viral dynamics and the role of a cross-reactive immune response in resolving an acute infection. We construct a mathematical model of within-host influenza viral dynamics including three possible factors which determine the strength of the cross-reactive cellular adaptive immune response: the initial naive T cell number, the avidity of the interaction between T cells and the epitopes presented by infected cells, and the epitope abundance per infected cell. Our model explains the experimentally observed shortening of a second infection when cross-reactivity is present, and shows that memory in the cellular adaptive immune response is necessary to protect against a second infection.

CONSISE statement on the reporting of Seroepidemiologic Studies for influenza (ROSES-I statement): an extension of the STROBE statement.

BACKGROUND: Population-based serologic studies are a vital tool for understanding the epidemiology of influenza and other respiratory viruses, including the early assessment of the transmissibility and severity of the 2009 influenza pandemic, and Middle East respiratory syndrome coronavirus. However, interpretation of the results of serologic studies has been hampered by the diversity of approaches and the lack of standardized methods and reporting. OBJECTIVE: The objective of the CONSISE ROSES-I statement was to improve the quality and transparency of reporting of influenza seroepidemiologic studies and facilitate the assessment of the validity and generalizability of published results. METHODS: The ROSES-I statement was developed as an expert consensus of the CONSISE epidemiology and laboratory working groups. The recommendations are presented in the familiar format of a reporting guideline. Because seroepidemiologic studies are a specific type of observational epidemiology study, the ROSES-I statement is built upon the STROBE guidelines. As such, the ROSES-I statement should be seen as an extension of the STROBE guidelines. RESULTS: The ROSES-I statement presents 42 items that can be used as a checklist of the information that should be included in the results of published seroepidemiologic studies, and which can also serve as a guide to the items that need to be considered during study design and implementation. CONCLUSIONS: We hope that the ROSES-I statement will contribute to improving the quality of reporting of seroepidemiologic studies.

Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine.

UNLABELLED: This study seeks to assess the ability of seasonal trivalent inactivated influenza vaccine (TIV) to induce nonneutralizing antibodies (Abs) with Fc-mediated functions in HIV-uninfected and HIV-infected subjects. Functional influenza-specific Ab responses were studied in 30 HIV-negative and 27 HIV-positive subjects immunized against seasonal influenza. All 57 subjects received the 2015 TIV. Fc-mediated antihemagglutinin (anti-HA) Ab activity was measured in plasma before and 4 weeks after vaccination using Fc-receptor-binding assays, NK cell activation assays, and phagocytosis assays. At baseline, the HIV-positive group had detectable but reduced functional Ab responses to both vaccine and nonvaccine influenza antigens. TIV enhanced Fc-mediated Ab responses in both HIV-positive and HIV-negative groups. A larger rise was generally observed in the HIV-positive group, such that there was no difference in functional Ab responses between the two groups after vaccination. The 2015 TIV enhanced functional influenza-specific Ab responses in both HIV-negative and HIV-positive subjects to a range of influenza HA proteins. The increase in functional Ab responses in the HIV-positive group supports recommendations to immunize this at-risk group. IMPORTANCE: Infection with HIV is associated with increasing disease severity following influenza infections, and annual influenza vaccinations are recommended for this target group. However, HIV-infected individuals respond relatively poorly to vaccination compared to healthy individuals, particularly if immunodeficient. There is therefore a need to increase our understanding of immunity to influenza in the context of underlying HIV infection. While antibodies can mediate direct virus neutralization, interactions with cellular Fc receptors may be important for anti-influenza immunity in vivo by facilitating antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent phagocytosis (ADP). The ability of seasonal influenza vaccines to induce antibody responses with potent Fc-mediated antiviral activity is currently unclear. Probing the ADCC and ADP responses to influenza vaccination has provided important new information in the quest to improve immunity to influenza.

Neutralizing inhibitors in the airways of naïve ferrets do not play a major role in modulating the virulence of H3 subtype influenza A viruses.

Many insights regarding the pathogenesis of human influenza A virus (IAV) infections have come from studies in mice and ferrets. Surfactant protein (SP)-D is the major neutralizing inhibitor of IAV in mouse airway fluids and SP-D-resistant IAV mutants show enhanced virus replication and virulence in mice. Herein, we demonstrate that sialylated glycoproteins, rather than SP-D, represent the major neutralizing inhibitors against H3 subtype viruses in airway fluids from naïve ferrets. Moreover, while resistance to neutralizing inhibitors is a critical factor in modulating virus replication and disease in the mouse model, it does not appear to be so in the ferret model, as H3 mutants resistant to either SP-D or sialylated glycoproteins in ferret airway fluids did not show enhanced virulence in ferrets. These data have important implications for our understanding of pathogenesis and immunity to human IAV infections in these two widely used animal models of infection.

Structural and functional characterisation of ferret interleukin-2.

While the ferret is a valuable animal model for a number of human viral infections, such as influenza, Hendra and Nipah, evaluating the cellular immune response following infection has been hampered by the lack of a number of species-specific immunological reagents. Interleukin 2 (IL-2) is one such key cytokine. Ferret recombinant IL-2 incorporating a C-terminal histidine tag was expressed and purified and the three-dimensional structure solved and refined at 1.89 Šby X-ray crystallography, which represents the highest resolution and first non-human IL-2 structure. While ferret IL-2 displays the classic cytokine fold of the four-helix bundle structure, conformational flexibility was observed at the second helix and its neighbouring region in the bundle, which may result in the disruption of the spatial arrangement of residues involved in receptor binding interactions, implicating subtle differences between ferret and human IL-2 when initiating biological functions. Ferret recombinant IL-2 stimulated the proliferation of ferret lymph node cells and induced the expression of mRNA for IFN-γ and Granzyme A.