[Show abstract][Hide abstract] ABSTRACT: Importance:
Both influenza A and B viruses co-circulate in the human population and annual influenza seasons are typically dominated by an influenza A subtype or an influenza B lineage. Surveillance data indicates that the burden of disease is higher in some seasons, yet it is unclear whether this is due to specific virus strains or to other factors, such as cross reactive immunity or clinical definitions of influenza. We directly compared disease and the localised inflammatory response to different seasonal influenza strains, including the 2009 pandemic strain, in healthy naïve ferrets. We have found that disease, as well as the cytokine and chemokine responses, were similar irrespective of the seasonal strain or the location of the infection in the respiratory tract. This suggests that factors other than the immune response to a particular virus (sub)type contribute to the variable impact of influenza infection in a population.
Full-text · Article · Dec 2015 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: H1N1 influenza viruses mutate rapidly, rendering vaccines developed in any given year relatively ineffective in subsequent years. Thus it is necessary to generate new vaccines every year, but this is time-consuming and resource-intensive. Should a highly virulent influenza strain capable of human-to-human transmission emerge, these factors will severely limit the number of people that can be effectively immunised against that strain in time to prevent a pandemic. An adjuvant and mode of administration capable of rendering ordinarily unprotective vaccine doses protective would thus be highly advantageous.
The carbohydrate mannan was conjugated to whole inactivated H1N1 influenza virus at a range of ratios, and mixed with it at a range of ratios, and various doses of the resulting preparations were administered to mice via the intranasal (IN) route. Serum immunity was assessed via antigen-specific IgG ELISA and the haemagglutination-inhibition (HI) assay, and mucosal immunity was assessed via IgA ELISA of bronchio-alveolar lavages.
IN-administered inactivated H1N1 mixed with mannan induced higher serum IgG and respiratory-tract IgA than inactivated H1N1 conjugated to mannan, and HIN1 alone. Adjuvantation was mannan-dose-dependent, with 100 μg of mannan adjuvanting 1 μg of H1N1 more effectively than 10 or 50 μg of mannan. Serum samples from mice immunised with 1 μg H1N1 adjuvanted with 10 μg mannan did not inhibit agglutination of red blood cells (RBCs) at a dilution factor of 10 in the HI assay, but samples resulting from adjuvantation with 50 and 100 μg mannan inhibited agglutination at dilution factors of ≥ 40. Both serum IgG1 and IgG2a were induced by IN mannan-adjuvanted H1N1 vaccination, suggesting the induction of humoral and cellular immunity.
Mixing 100 μg of mannan with 1 μg of inactivated H1N1 adjuvanted the vaccine in mice, such that IN immunisation induced higher serum IgG and respiratory tract IgA than immunisation with virus alone. The serum from mice thus immunised inhibited H1N1-mediated RBC agglutination strongly in vitro. If mannan similarly adjuvants low doses of influenza vaccine in humans, it could potentially be used for vaccine 'dose-sparing' in the event that a vaccine shortage arises from an epidemic involving a highly virulent human-to-human transmissable influenza strain.
Full-text · Article · Dec 2015 · BMC Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Neuraminidase-inhibition (NI) antibody titers can be used to evaluate the immunogenicity of inactivated influenza vaccines and have provided evidence of serologic cross-reactivity between seasonal and pandemic H1N1 viruses. The traditional thiobarbituric acid assay is impractical for large serologic analyses, and therefore many laboratories use an enzyme-linked lectin assay (ELLA) to determine serum NI antibody titers. The comparability of ELLA NI antibody titers when measured in different laboratories was unknown. Here we report a study conducted through the Consortium for the Standardisation of Influenza SeroEpidemiology (CONSISE) to evaluate the variability of the ELLA. NI antibody titers of a set of 12 samples were measured against both N1 and N2 neuraminidase antigens in 3 independent assays by each of 23 laboratories. For a sample repeated in the same assay, ≥96% of N1 and N2 assays had less than a 4-fold difference in titer. Comparison of the titers measured in assays conducted on 3 different days in the same laboratory showed that a four-fold difference in titer was uncommon. Titers of the same sera measured in different laboratories spanned 3 to 6 two-fold dilutions (i.e., 8-64 fold difference in titer), with an average percent geometric coefficient of variation (%GCV) of 112 and 82% against N1 and N2 antigens, respectively. The difference in titer as indicated by fold range and %GCV was improved by normalizing the NI titers to a standard that was included in each assay. This study identified background signal and the amount of antigen in the assay as critical factors that influence titer, providing important information toward development of a consensus ELLA protocol.
[Show abstract][Hide abstract] ABSTRACT: 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 Å by 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.
No preview · Article · Oct 2015 · Developmental and comparative immunology
[Show abstract][Hide abstract] ABSTRACT: Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus-innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.
Full-text · Article · Aug 2015 · PLoS Computational Biology
[Show abstract][Hide abstract] ABSTRACT: Background:
Epidemiological studies suggest that, following infection with influenza virus, there is a short period during which a host experiences a lower susceptibility to infection with other influenza viruses. This viral interference appears to be independent of any antigenic similarities between the viruses. We used the ferret model of human influenza to systematically investigate viral interference.
Ferrets were first infected then challenged 1-14 days later with pairs of influenza A(H1N1)pdm09, influenza A(H3N2), and influenza B viruses circulating in 2009 and 2010.
Viral interference was observed when the interval between initiation of primary infection and subsequent challenge was <1 week. This effect was virus specific and occurred between antigenically related and unrelated viruses. Coinfections occurred when 1 or 3 days separated infections. Ongoing shedding from the primary virus infection was associated with viral interference after the secondary challenge.
The interval between infections and the sequential combination of viruses were important determinants of viral interference. The influenza viruses in this study appear to have an ordered hierarchy according to their ability to block or delay infection, which may contribute to the dominance of different viruses often seen in an influenza season.
Full-text · Article · May 2015 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Background:
Intravenous immunoglobulin (IVIG) is a purified pool of human antibodies from thousands of donors that is used to prevent or treat primary immune deficiency, several infectious diseases, and autoimmune diseases. The antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against heterologous influenza strains may be present in IVIG preparations.
We tested 8 IVIG preparations prior to the 2009 H1N1 swine-origin influenza pandemic and 10 IVIG preparations made after 2010 for their ability to mediate influenza-specific ADCC.
ADCC mediating antibodies to A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) were detected in IVIG preparations prior to the 2009-H1N1 pandemic. The HA-specific ADCC targeted both the HA1 and HA2 regions of A(H1N1)pdm09 HA and was capable of recognizing a broad range of HA proteins including those from recent avian influenza strains A(H5N1) and A(H7N9). The low but detectable ADCC recognition of A(H7N9) was likely due to rare individuals in the population contributing cross-reactive antibodies to IVIG.
IVIG preparations contain broadly cross-reactive ADCC mediating antibodies. IVIG may provide at least some level of protection for individuals at high risk of severe influenza disease, especially during influenza pandemics prior to the development of effective vaccines.
No preview · Article · Jun 2014 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.
Full-text · Article · May 2014 · Journal of virological methods
[Show abstract][Hide abstract] ABSTRACT: Historical records of influenza pandemics demonstrate variability in incidence and severity between waves. The influenza A(H1N1)pdm09 pandemic was the first in which many countries implemented strain-specific vaccination to mitigate subsequent seasons. Serosurveys provide opportunity to examine the constraining influence of antibody on population disease experience.
Changes in the proportion of adults seropositive to influenza A(H1N1)pdm09over the 2009/10 (summer) interepidemic period and 2010 (winter) influenza season were measured to determine whether there was a temporal relationship with vaccine distribution and influenza activity, respectively.
Plasma samples were collected from healthy blood donors from seven cities at the end of the first wave (November 2009), and before (March/April 2010) and after (November 2010) the subsequent influenza season.
Haemagglutination inhibition (HI) assays were performed to assess reactivity of plasma against A(H1N1)pdm09, and the proportion seropositive (HI titre ≥ 40) compared over time, by age group and location.
Between the 2009 and 2010 influenza seasons, the seropositive proportion rose from 22% to 43%, an increase observed across all ages and sites. Brisbane alone recorded a significant rise in seropositivity over the 2010 influenza season - from a baseline of 35% to 53%. The seropositive proportion elsewhere was ≥40% pre-season, and did not rise over winter.
A vaccine-associated increase in seropositive proportion preceding the influenza season correlated with low levels of disease activity in winter 2010. These observations support the role of immunisation in mitigating the 'second wave' of A(H1N1)pdm09, with timing critical to ensure sustained herd protection.
Full-text · Article · Dec 2013 · Influenza and Other Respiratory Viruses
[Show abstract][Hide abstract] ABSTRACT: For in vivo studies of influenza dynamics where within-host measurements are fit with a mathematical model, infectivity assays (e.g. 50% tissue culture infectious dose; TCID50) are often used to estimate the infectious virion concentration over time. Less frequently, measurements of the total (infectious and non-infectious) viral particle concentration (obtained using real-time reverse transcription-polymerase chain reaction; rRT-PCR) have been used as an alternative to infectivity assays. We investigated the degree to which measuring both infectious (via TCID50) and total (via rRT-PCR) viral load allows within-host model parameters to be estimated with greater consistency and reduced uncertainty, compared with fitting to TCID50 data alone. We applied our models to viral load data from an experimental ferret infection study. Best-fit parameter estimates for the "dual-measurement" model are similar to those from the TCID50-only model, with greater consistency in best-fit estimates across different experiments, as well as reduced uncertainty in some parameter estimates. Our results also highlight how variation in TCID50 assay sensitivity and calibration may hinder model interpretation, as some parameter estimates systematically vary with known uncontrolled variations in the assay. Our techniques may aid in drawing stronger quantitative inferences from in vivo studies of influenza virus dynamics.
[Show abstract][Hide abstract] ABSTRACT: Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) provide a degree of cross-strain protection that is potentially subverted by mutation. Here we describe the sequential emergence of such variants within CTL epitopes for a persistently infected, immunocompromised infant. Further analysis in immunodeficient and wild-type mice supports the view that CTL escape variants arise frequently in influenza, accumulate with time and revert in the absence of immune pressure under MHCI-mismatched conditions. Viral fitness, the abundance of endogenous CD8(+) T cell responses and T cell receptor repertoire diversity influence the nature of these de novo mutants. Structural characterization of dominant escape variants shows how the peptide-MHCI interaction is modified to affect variant-MHCI stability. The mechanism of influenza virus escape thus looks comparable to that recognized for chronic RNA viruses like HIV and HCV, suggesting that immunocompromised patients with prolonged viral infection could have an important part in the emergence of influenza quasispecies.
Full-text · Article · Oct 2013 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce ADCC responses that can cross-recognize divergent influenza strains is unknown. We immunized 6 influenza-naïve pigtail macaques twice with the 2011-2012 season TIV and then challenged macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza-specific CD8 T cell (CTL) responses using a sensitive MHC-tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by ELISA, did not induce detectable influenza-specific ADCC or CTLs responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins but not influenza HA proteins from different subtypes (H2-H7). There was no anamnestic influenza-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or divergent H3 proteins but did boost CTL responses. ADCC or CTLs responses are not induced by TIV vaccination in influenza-naïve macaques. There was a marked difference in the ability of infection compared to vaccination in inducing cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.
Full-text · Article · Oct 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Background. During the 2009 H1N1 influenza pandemic older individuals were partially protected from severe disease. It is not known whether pre-existing antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed.Methods. We tested sera from 182 individuals aged 1-72 years collected either immediately prior to, or following, the 2009-H1N1 pandemic for ADCC antibodies to the A(H1N1)pdm09 haemagglutinin(HA) protein.Results. A(H1N1)pdm09 HA-specific ADCC antibodies were detected in almost all individuals in the >45 age group (28/31 subjects) prior to the 2009-H1N1 pandemic. Conversely, only approximately half of the individuals aged 1-14 (11/31) and 15-45 (17/31) had cross-reactive ADCC antibodies prior to the 2009-H1N1 pandemic. The A(H1N1)pdm09-specific ADCC antibodies were able to efficiently mediate the killing of influenza-infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC to a range of seasonal H1N1 HA proteins, including from the 1918 virus, compared to younger individuals.Conclusions. ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009-H1N1 pandemic. This work has significant implications for improved vaccination strategies for future influenza pandemics.
Full-text · Article · Jun 2013 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.
[Show abstract][Hide abstract] ABSTRACT: There is an urgent need for a human immunodeficiency virus (HIV) vaccine that induces robust mucosal immunity. CD8+ cytotoxic T lymphocytes (CTLs) apply substantial antiviral pressure, but CTLs to individual epitopes select for immune escape
variants in both HIV in humans and SIV in macaques. Inducing multiple simian immunodeficiency virus (SIV)-specific CTLs may
assist in controlling viremia. We vaccinated 10 Mane-A1*08401+ female pigtail macaques with recombinant influenza viruses expressing three Mane-A1*08401-restricted SIV-specific CTL epitopes and subsequently challenged the animals, along with five controls, intravaginally with
SIVmac251. Seroconversion to the influenza virus vector resulted and small, but detectable, SIV-specific CTL responses were induced.
There was a boost in CTL responses after challenge but no protection from high-level viremia or CD4 depletion was observed.
All three CTL epitopes underwent a coordinated pattern of immune escape during early SIV infection. CTL escape was more rapid
in the vaccinees than in the controls at the more dominant CTL epitopes. Although CTL escape can incur a “fitness” cost to
the virus, a putative compensatory mutation 20 amino acids upstream from an immunodominant Gag CTL epitope also evolved soon
after the primary CTL escape mutation. We conclude that vaccines based only on CTL epitopes will likely be undermined by rapid
evolution of both CTL escape and compensatory mutations. More potent and possibly broader immune responses may be required
to protect pigtail macaques from SIV.
Full-text · Article · Jan 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.
No preview · Article · Jan 2013 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: To estimate population attack rates of influenza A(H1N1)pdm2009 in the Southern Hemisphere during June-August 2009, we conducted several serologic studies. We pooled individual-level data from studies using hemagglutination inhibition assays performed in Australia, New Zealand, and Singapore. We determined seropositive proportions (titer >40) for each study region by age-group and sex in pre- and postpandemic phases, as defined by jurisdictional notification data. After exclusions, the pooled database consisted of, 4,414 prepandemic assays and 7,715 postpandemic assays. In the prepandemic phase, older age groups showed greater seropositive proportions, with age-standardized, community-based proportions ranging from 3.5% in Singapore to 11.9% in New Zealand. In the postpandemic phase, seropositive proportions ranged from 17.5% in Singapore to 30.8% in New Zealand, with highest proportions seen in school-aged children. Pregnancy and residential care were associated with lower postpandemic seropositivity, whereas Aboriginal and Torres Strait Islander Australians and Pacific Peoples of New Zealand had greater postpandemic seropositivity.
No preview · Article · Jan 2013 · Emerging Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Background Serological studies can detect infection with a novel influenza virus in the absence of symptoms or positive virology, providing useful information on infection that goes beyond the estimates from epidemiological, clinical and virological data. During the 2009 A(H1N1) pandemic, an impressive number of detailed serological studies were performed, yet the majority of serological data were available only after the first wave of infection. This limited the ability to estimate the transmissibility and severity of this novel infection, and the variability in methodology and reporting limited the ability to compare and combine the serological data. Objectives To identify best practices for conduct and standardisation of serological studies on outbreak and pandemic influenza to inform public policy. Methods/Setting An international meeting was held in February 2011 in Ottawa, Canada, to foster the consensus for greater standardisation of influenza serological studies. Results Best practices for serological investigations of influenza epidemiology include the following: classification of studies as pre-pandemic, outbreak, pandemic or inter-pandemic with a clearly identified objective; use of international serum standards for laboratory assays; cohort and cross-sectional study designs with common standards for data collection; use of serum banks to improve sampling capacity; and potential for linkage of serological, clinical and epidemiological data. Advance planning for outbreak studies would enable a rapid and coordinated response; inclusion of serological studies in pandemic plans should be considered. Conclusions Optimising the quality, comparability and combinability of influenza serological studies will provide important data upon emergence of a novel or variant influenza virus to inform public health action.
Full-text · Article · Apr 2012 · Influenza and Other Respiratory Viruses