Publications by authors named "Patrick C Reading"

101 Publications

A second external quality assessment of isolation and identification of influenza viruses in cell culture in the Asia Pacific region highlights improved performance by participating laboratories.

J Clin Virol 2021 Jul 7;142:104907. Epub 2021 Jul 7.

WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. Electronic address:

Influenza viruses must be amplified in cell culture for detailed antigenic analysis and for phenotypic assays assessing susceptibility to antiviral drugs or for other assays. Following on from the first external quality assessment (EQA) for isolation and identification of influenza viruses using cell culture techniques in 2016, a follow up EQA was performed in 2019 for National Influenza Centres (NICs) in the World Health Organization (WHO) South East Asia and Western Pacific Regions. Nineteen WHO NICs performed influenza virus isolation and identification techniques on an EQA panel comprising 16 samples, containing influenza A or B viruses and negative control samples. One sample was used exclusively to assess capacity to measure a hemagglutination titer and the other 15 samples were used for virus isolation and subsequent identification. Virus isolation from EQA samples was generally detected by assessment of cytopathic effect and/or hemagglutination assay while virus identification was determined by real time RT-PCR, hemagglutination inhibition and/or immunofluorescence assays. For virus isolation from EQA samples, 6/19 participating laboratories obtained 15/15 correct results in the first EQA (2016) compared to 11/19 in the follow up (2019). For virus identification in isolates derived from EQA samples, 6/19 laboratories obtained 15/15 correct results in 2016 compared to 13/19 in 2019. Overall, NIC laboratories in the Asia Pacific Region showed a significant improvement between 2016 and 2019 in terms of the correct results reported for isolation from EQA samples and identification of virus in isolates derived from EQA samples (p=0.01 and p=0.02, respectively).
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http://dx.doi.org/10.1016/j.jcv.2021.104907DOI Listing
July 2021

Safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2: a randomised, double-blind, placebo-controlled, phase 1 trial.

Lancet Infect Dis 2021 Apr 19. Epub 2021 Apr 19.

Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.

Background: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]).

Methods: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 μg, 15 μg, or 45 μg, or one dose of sclamp vaccine at 45 μg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933.

Findings: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 μg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 μg dose (7492, 4959-11 319), and the two 45 μg dose cohorts (8770, 5526-13 920 in the two-dose 45 μg cohort; 8793, 5570-13 881 in the single-dose 45 μg cohort); 4 weeks after the second dose (day 57) with two 5 μg doses (102 400, 64 857-161 676), with two 15 μg doses (74 725, 51 300-108 847), with two 45 μg doses (79 586, 55 430-114 268), only a single 45 μg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 μg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 μg doses (GMT 228, 95% CI 146-356), two 15 μg doses (230, 170-312), and two 45 μg doses (239, 187-307).

Interpretation: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response.

Funding: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.
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http://dx.doi.org/10.1016/S1473-3099(21)00200-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055208PMC
April 2021

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

Clin Transl Immunology 2021 5;10(4):e1269. Epub 2021 Apr 5.

CSIRO Manufacturing Parkville VIC Australia.

Objectives: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia).

Methods: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat.

Results: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4 and cytotoxic CD8 T cells . In the Syrian hamster challenge model ( = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level.

Conclusion: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.
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http://dx.doi.org/10.1002/cti2.1269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021130PMC
April 2021

TLR2-mediated activation of innate responses in the upper airways confers antiviral protection of the lungs.

JCI Insight 2021 Mar 8;6(5). Epub 2021 Mar 8.

Department of Microbiology and Immunology, the University of Melbourne, the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

The impact of respiratory virus infections on global health is felt not just during a pandemic, but endemic seasonal infections pose an equal and ongoing risk of severe disease. Moreover, vaccines and antiviral drugs are not always effective or available for many respiratory viruses. We investigated how induction of effective and appropriate antigen-independent innate immunity in the upper airways can prevent the spread of respiratory virus infection to the vulnerable lower airways. Activation of TLR2, when restricted to the nasal turbinates, resulted in prompt induction of innate immune-driven antiviral responses through action of cytokines, chemokines, and cellular activity in the upper but not the lower airways. We have defined how nasal epithelial cells and recruitment of macrophages work in concert and play pivotal roles to limit progression of influenza virus to the lungs and sustain protection for up to 7 days. These results reveal underlying mechanisms of how control of viral infection in the upper airways can occur and support the implementation of strategies that can activate TLR2 in nasal passages to provide rapid protection, especially for at-risk populations, against severe respiratory infection when vaccines and antiviral drugs are not always effective or available.
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http://dx.doi.org/10.1172/jci.insight.140267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021123PMC
March 2021

Influenza, but not SARS-CoV-2, infection induces a rapid interferon response that wanes with age and diminished tissue-resident memory CD8 T cells.

Clin Transl Immunology 2021 26;10(1):e1242. Epub 2021 Jan 26.

Department of Microbiology and Immunology Peter Doherty Institute for Infection and Immunity The University of Melbourne Melbourne VIC Australia.

Older individuals exhibit a diminished ability to respond to and clear respiratory pathogens and, as such, experience a higher rate of lung infections with a higher mortality rate. It is unclear why respiratory pathogens impact older people disproportionately. Using human lung tissue from donors aged 22-68 years, we assessed how the immune cell landscape in lungs changes throughout life and investigated how these immune cells respond following exposure to influenza virus and SARS-CoV-2, two clinically relevant respiratory viruses. While the frequency of most immune cell subsets profiled in the human lung remained stable with age, memory CD8 T cells declined, with the tissue-resident memory (Trm) CD8 T-cell subset being most susceptible to age-associated attrition. Infection of lung tissue with influenza virus resulted in an age-associated attenuation in the antiviral immune response, with aged donors producing less type I interferon (IFN), GM-CSF and IFNγ, the latter correlated with a reduction of IFNγ-producing memory CD8 T cells. In contrast, irrespective of donor age, exposure of human lung cells to SARS-CoV-2, a pathogen for which all donors were immunologically naïve, did not trigger activation of local immune cells and did not result in the induction of an early IFN response. Our findings show that the attrition of tissue-bound pathogen-specific Trm in the lung that occurs with advanced age, or their absence in immunologically naïve individuals, results in a diminished early antiviral immune response which creates a window of opportunity for respiratory pathogens to gain a greater foothold.
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http://dx.doi.org/10.1002/cti2.1242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837404PMC
January 2021

Host Cell Restriction Factors of Paramyxoviruses and Pneumoviruses.

Viruses 2020 12 2;12(12). Epub 2020 Dec 2.

Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne VIC 3000, Australia.

The paramyxo- and pneumovirus family includes a wide range of viruses that can cause respiratory and/or systemic infections in humans and animals. The significant disease burden of these viruses is further exacerbated by the limited therapeutics that are currently available. Host cellular proteins that can antagonize or limit virus replication are therefore a promising area of research to identify candidate molecules with the potential for host-targeted therapies. Host proteins known as host cell restriction factors are constitutively expressed and/or induced in response to virus infection and include proteins from interferon-stimulated genes (ISGs). Many ISG proteins have been identified but relatively few have been characterized in detail and most studies have focused on studying their antiviral activities against particular viruses, such as influenza A viruses and human immunodeficiency virus (HIV)-1. This review summarizes current literature regarding host cell restriction factors against paramyxo- and pneumoviruses, on which there is more limited data. Alongside discussion of known restriction factors, this review also considers viral countermeasures in overcoming host restriction, the strengths and limitations in different experimental approaches in studies reported to date, and the challenges in reconciling differences between in vitro and in vivo data. Furthermore, this review provides an outlook regarding the landscape of emerging technologies and tools available to study host cell restriction factors, as well as the suitability of these proteins as targets for broad-spectrum antiviral therapeutics.
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http://dx.doi.org/10.3390/v12121381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761617PMC
December 2020

Airway Exosomes Released During Influenza Virus Infection Serve as a Key Component of the Antiviral Innate Immune Response.

Front Immunol 2020 12;11:887. Epub 2020 May 12.

Department of Microbiology and Immunology, The University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.

Exosomes are extracellular vesicles secreted by cells that have an important biological function in intercellular communication by transferring biologically active proteins, lipids, and RNAs to neighboring or distant cells. While a role for exosomes in antimicrobial defense has recently emerged, currently very little is known regarding the nature and functional relevance of exosomes generated , particularly during an active viral infection. Here, we characterized exosomes released into the airways during influenza virus infection. We show that these vesicles dynamically change in protein composition over the course of infection, increasing expression of host proteins with known anti-influenza activity, and viral proteins with the potential to trigger host immune responses. We show that exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigen that can be utilized by antigen presenting cells to drive the induction of a cellular immune response. Moreover, we show that attachment factors for influenza virus, namely α2,3 and α2,6-linked sialic acids, are present on the surface of airway exosomes and these vesicles have the ability to neutralize influenza virus, thereby preventing the virus from binding and entering target cells. These data reveal a novel role for airway exosomes in the antiviral innate immune defense against influenza virus infection.
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http://dx.doi.org/10.3389/fimmu.2020.00887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236881PMC
April 2021

Influenza A virus interactions with macrophages: Lessons from epithelial cells.

Cell Microbiol 2020 05 18;22(5):e13170. Epub 2020 Feb 18.

Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

Influenza viruses are an important cause of respiratory infection worldwide. In humans, infection with seasonal influenza A virus (IAV) is generally restricted to the respiratory tract where productive infection of airway epithelial cells promotes viral amplification, dissemination, and disease. Alveolar macrophages (MΦ) are also among the first cells to detect and respond to IAV, where they play a pivotal role in mounting effective innate immune responses. In contrast to epithelial cells, IAV infection of MΦ is a "dead end" for most seasonal strains, where replication is abortive and newly synthesised virions are not released. Although the key replicative stages leading to productive IAV infection in epithelial cells are defined, there is limited knowledge about the abortive IAV life cycle in MΦ. In this review, we will explore host factors and viral elements that support the early stages (entry) through to the late stages (viral egress) of IAV replication in epithelial cells. Similarities, differences, and unknowns for each key stage of the IAV replicative cycle in MΦ will then be highlighted. Herein, we provide mechanistic insights into MΦ-specific control of seasonal IAV replication through abortive infection, which may in turn, contribute to effective host defence.
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http://dx.doi.org/10.1111/cmi.13170DOI Listing
May 2020

IFITM3 and type I interferons are important for the control of influenza A virus replication in murine macrophages.

Virology 2020 01 5;540:17-22. Epub 2019 Nov 5.

Department of Microbiology and Immunology, The University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia; WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia.

Abortive infection of macrophages serves as a "dead end" for most seasonal influenza A virus (IAV) strains, and it is likely to contribute to effective host defence. Interferon (IFN)-induced transmembrane protein 3 (IFITM3) restricts the early stages of IAV replication in epithelial cells, but IFITM3 restriction of IAV replication in macrophages has not been previously investigated. Herein, macrophages isolated from IFITM3-deficient mice were more susceptible to initial IAV infection, but late-stage viral replication was still controlled through abortive infection. Strikingly, IFNα/β receptor (IFNAR)-deficient macrophages infected with IAV were not only more susceptible to initial infection, but these cells also supported productive viral replication. Significantly, we have established that abortive IAV infection in macrophages is controlled through a type I IFN-dependent mechanism, where late-stage IAV replication can proceed in the absence of type I IFN responses. These findings provide novel mechanistic insight into macrophage-specific processes that potently shut down IAV replication.
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http://dx.doi.org/10.1016/j.virol.2019.11.003DOI Listing
January 2020

Downregulation of MHC Class I Expression by Influenza A and B Viruses.

Front Immunol 2019 29;10:1158. Epub 2019 May 29.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, VIC, Australia.

Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8 T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8 T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8 T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection . This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses.
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http://dx.doi.org/10.3389/fimmu.2019.01158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548845PMC
July 2020

Unraveling the role of the MOV10 RNA helicase during influenza A virus infection.

Biochem J 2019 03 27;476(6):1005-1008. Epub 2019 Mar 27.

Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia

Moloney leukemia virus 10 (MOV10) is an interferon-inducible RNA helicase that has been implicated in a broad range of cellular functions, including modulating the replication of a diverse range of viruses. However, the mechanisms by which MOV10 promotes or inhibits the replication of particular viruses have not been well defined. A recent paper published in the by Li et al. [Biochem. J. (2019) , 467-481] provides insight regarding the mechanisms by which MOV10 restricts influenza A virus (IAV) infection in host cells. First, the authors confirm that MOV10 binds to the viral nucleoprotein (NP) and sequesters the viral ribonucleoprotein complex in cytoplasmic granules called processing (P)-bodies, thus inhibiting IAV replication. Second, they demonstrate that the non-structural (NS)1 protein of IAV can act as an antagonist of MOV10, inhibiting the association of MOV10 with NP and promoting MOV10 degradation through the lysosomal pathway. Further research will determine if cellular RNA helicases such as MOV10 represent suitable targets for the development of novel anti-IAV therapies.
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http://dx.doi.org/10.1042/BCJ20190018DOI Listing
March 2019

Rapid interferon independent expression of IFITM3 following T cell activation protects cells from influenza virus infection.

PLoS One 2019 16;14(1):e0210132. Epub 2019 Jan 16.

Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

Interferon-induced transmembrane protein 3 (IFITM3) is a potent antiviral protein that enhances cellular resistance to a variety of pathogens, including influenza virus. Classically defined as an interferon-stimulated gene, expression of IFITM3 on cells is rapidly up-regulated in response to type I and II interferon. Here we found that IFITM3 is rapidly up-regulated by T cells following their activation and this occurred independently of type I and II interferon and the interferon regulatory factors 3 and 7. Up-regulation of IFITM3 on effector T cells protected these cells from virus infection and imparted a survival advantage at sites of virus infection. Our results show that IFITM3 expression on effector T cells is crucial for these cells to mediate their effector function and highlights an interferon independent pathway for the induction of IFITM3 which, if targeted, could be an effective approach to harness the activity of IFITM3 for infection prevention.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210132PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334895PMC
September 2019

Unique Transcriptional Architecture in Airway Epithelial Cells and Macrophages Shapes Distinct Responses following Influenza Virus Infection .

J Virol 2019 03 5;93(6). Epub 2019 Mar 5.

Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia

Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 h postinfection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with the virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity, whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon (IFN) regulatory factor 3/7 (IRF3/7) and/or type I IFN signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent of and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection. Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activities, consistent with distinct physiological roles in the airways. Not surprisingly, these cells also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architectures of airway cells from two different lineages drive transcriptional responses to IAV infection.
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http://dx.doi.org/10.1128/JVI.01986-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401432PMC
March 2019

MAIT cells contribute to protection against lethal influenza infection in vivo.

Nat Commun 2018 11 9;9(1):4706. Epub 2018 Nov 9.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, VIC, 3000, Australia.

Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulate and are activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post-infection. Activation is modulated via cytokines independently of MR1. MAIT cell-deficient MR1 mice show enhanced weight loss and mortality to severe (H1N1) influenza. This is ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2γC mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.
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http://dx.doi.org/10.1038/s41467-018-07207-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226485PMC
November 2018

CSF3R/CD114 mediates infection-dependent transition to severe asthma.

J Allergy Clin Immunol 2019 02 9;143(2):785-788.e6. Epub 2018 Oct 9.

School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria, Australia; Department of Pharmacology & Therapeutics, Lung Health Research Centre, The University of Melbourne, Parkville, Victoria, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2018.10.001DOI Listing
February 2019

Changes in Gut Microbiota Prior to Influenza A Virus Infection Do Not Affect Immune Responses in Pups or Juvenile Mice.

Front Cell Infect Microbiol 2018 12;8:319. Epub 2018 Sep 12.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIC, Australia.

Previous studies demonstrated that oral antibiotic (ABX) treatment prior to and during influenza A virus (IAV) infection of adult mice profoundly altered gut microbiota (GM) and was associated with increased susceptibility and impaired immunity to IAV. We examined the impact of ABX during critical times relevant to the establishment of GM in early life (using perinatal treatment of neonates and direct treatment of juvenile mice) and asked whether cessation of ABX treatment in early life had lasting effects on GM composition and anti-IAV immunity. ABX treatment significantly changed GM composition in juvenile mice and in ABX-treated dams. However, if ABX treatment ceased at the time of infection, neither neonates nor juvenile mice showed enhanced susceptibility to IAV, nor were major differences detected in cellular and humoral adaptive antiviral immunity. Thus, while ABX treatment alters GM diversity in early life, cessation and subsequent re-colonization correlates with effective immunity against IAV.
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http://dx.doi.org/10.3389/fcimb.2018.00319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145060PMC
August 2019

Influenza virus detection: driving change in public health laboratories in the Western Pacific Region.

Western Pac Surveill Response J Winter 2018;9(5 Suppl 1):68-70. Epub 2018 Sep 5.

WHO Regional Office for the Eastern Mediterranean, Cairo, Egypt.

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http://dx.doi.org/10.5365/wpsar.2018.9.5.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902649PMC
March 2020

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

Infect Genet Evol 2018 10 19;64:95-104. Epub 2018 Jun 19.

Department of Virology, Institute of Experimental Medicine, 12, acad. Pavlova street, Saint Petersburg, Russia.

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.
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http://dx.doi.org/10.1016/j.meegid.2018.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330673PMC
October 2018

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

J Infect Dis 2018 07;218(3):406-417

WHO Collaborating Centre for Reference and Research on Influenza, The University of Melbourne, Melbourne.

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.
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http://dx.doi.org/10.1093/infdis/jiy184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7107400PMC
July 2018

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

J Infect Dis 2018 01;217(4):548-559

WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, Melbourne, Australia.

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.
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http://dx.doi.org/10.1093/infdis/jix509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853430PMC
January 2018

Host Cell Restriction Factors that Limit Influenza A Infection.

Viruses 2017 12 7;9(12). Epub 2017 Dec 7.

Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia.

Viral infection of different cell types induces a unique spectrum of host defence genes, including interferon-stimulated genes (ISGs) and genes encoding other proteins with antiviral potential. Although hundreds of ISGs have been described, the vast majority have not been functionally characterised. Cellular proteins with putative antiviral activity (hereafter referred to as "restriction factors") can target various steps in the virus life-cycle. In the context of influenza virus infection, restriction factors have been described that target virus entry, genomic replication, translation and virus release. Genome wide analyses, in combination with ectopic overexpression and/or gene silencing studies, have accelerated the identification of restriction factors that are active against influenza and other viruses, as well as providing important insights regarding mechanisms of antiviral activity. Herein, we review current knowledge regarding restriction factors that mediate anti-influenza virus activity and consider the viral countermeasures that are known to limit their impact. Moreover, we consider the strengths and limitations of experimental approaches to study restriction factors, discrepancies between in vitro and in vivo studies, and the potential to exploit restriction factors to limit disease caused by influenza and other respiratory viruses.
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http://dx.doi.org/10.3390/v9120376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744151PMC
December 2017

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

J Virol 2018 02 30;92(4). Epub 2018 Jan 30.

WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

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. 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.
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http://dx.doi.org/10.1128/JVI.01322-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790937PMC
February 2018

The first external quality assessment of isolation and identification of influenza viruses in cell culture in the Asia Pacific region, 2016.

J Clin Virol 2017 12 2;97:54-58. Epub 2017 Nov 2.

WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, 3000, Australia.

Background: The isolation and propagation of influenza viruses from clinical specimens are essential tools for comprehensive virologic surveillance. Influenza viruses must be amplified in cell culture for detailed antigenic analysis and for phenotypic assays assessing susceptibility to antiviral drugs or for other assays.

Objectives: To conduct an external quality assessment (EQA) of proficiency for isolation and identification of influenza viruses using cell culture techniques among National Influenza Centres (NICs) in the World Health Organisation (WHO) South East Asia and Western Pacific Regions.

Study Design: Twenty-one NICs performed routine influenza virus isolation and identification techniques on a proficiency testing panel comprising 16 samples, containing influenza A or B viruses and negative control samples. One sample was used exclusively to determine their capacity to measure hemagglutination titer and the other 15 samples were used for virus isolation and identification.

Results: All NICs performed influenza virus isolation using Madin Darby canine kidney (MDCK) or MDCK-SIAT-1 cells. If virus growth was detected, the type, subtype and/or lineage of virus present in isolates was determined using immunofluorescence, RT-PCR and/or hemagglutination inhibition (HI) assays. Most participating laboratories could detect influenza virus growth and could identify virus amplified from EQA samples. However, some laboratories failed to isolate and identify viruses from EQA samples that contained lower titres of virus, highlighting issues regarding the sensitivity of influenza virus isolation methods between laboratories.

Conclusion: This first round of EQA was successfully conducted by NICs in the Asia Pacific Region, revealing good proficiency in influenza virus isolation and identification.
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http://dx.doi.org/10.1016/j.jcv.2017.10.018DOI Listing
December 2017

Infectious Laryngotracheitis Virus Viral Chemokine-Binding Protein Glycoprotein G Alters Transcription of Key Inflammatory Mediators and .

J Virol 2018 01 14;92(1). Epub 2017 Dec 14.

Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad-range viral chemokine-binding protein conserved among most alphaherpesviruses, including ILTV. A number of studies comparing the immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and the composition of the inflammatory response, and modulation of the immune responses toward antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. infection studies using tracheal organ tissue specimen cultures and blood-derived monocytes and infection studies in specific-pathogen-free chickens showed that leukocyte recruitment to the site of infection is an important component of the induced pathology and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXC chemokine ligand 8 (CXCL8), chicken CXCLi1 and chicken CXCLi2, among other cytokines and chemokines. The results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence, and the balance of the immune response to infection. Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad-range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signaling cascade associated with infection. Here, data from and infection studies are presented. These data show that gG has a direct impact on the transcription of cytokines and chemokine ligands (such as chicken CXCL8 orthologues, among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with the dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.
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http://dx.doi.org/10.1128/JVI.01534-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730763PMC
January 2018

Extrinsically derived TNF is primarily responsible for limiting antiviral CD8+ T cell response magnitude.

PLoS One 2017 8;12(9):e0184732. Epub 2017 Sep 8.

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

TNF is a pro-inflammatory cytokine produced by both lymphoid and non-lymphoid cells. As a consequence of the widespread expression of its receptors (TNFR1 and 2), TNF plays a role in many important biological processes. In the context of influenza A virus (IAV) infection, TNF has variably been implicated in mediating immunopathology as well as suppression of the immune response. Although a number of cell types are able to produce TNF, the ability of CD8+ T cells to produce TNF following viral infection is a hallmark of their effector function. As such, the regulation and role of CD8+ T cell-derived TNF following viral infection is of great interest. Here, we show that the biphasic production of TNF by CD8+ T cells following in vitro stimulation corresponds to distinct patterns of epigenetic modifications. Further, we show that a global loss of TNF during IAV infection results in an augmentation of the peripheral virus-specific CD8+ T cell response. Subsequent adoptive transfer experiments demonstrated that this attenuation of the CD8+ T cell response was largely, but not exclusively, conferred by extrinsic TNF, with intrinsically-derived TNF making only modest contributions. In conclusion, TNF exerts an immunoregulatory role on CD8+ T cell responses following IAV infection, an effect that is largely mediated by extrinsically-derived TNF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184732PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590991PMC
October 2017

Resident memory CD8 T cells in the upper respiratory tract prevent pulmonary influenza virus infection.

Sci Immunol 2017 Jun;2(12)

Department of Microbiology and Immunology, University of Melbourne, at Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.

Nasal epithelial tissue of the upper respiratory tract is the first site of contact by inhaled pathogens such as influenza virus. We show that this region is key to limiting viral spread to the lower respiratory tract and associated disease pathology. Immunization of the upper respiratory tract leads to the formation of local tissue-resident memory CD8 T cells (Trm cells). Unlike Trm cells in the lung, these cells develop independently of local cognate antigen recognition and transforming growth factor-β signaling and persist with minimal decay, representing a long-term protective population. Repertoire characterization revealed unexpected differences between lung and nasal tissue Trm cells, the composition of which was shaped by the developmental need for lung, but not nasal, Trm cells to recognize antigen within their local tissue. We show that influenza-specific Trm cells in the nasal epithelia can block the transmission of influenza virus from the upper respiratory tract to the lung and, in doing so, prevent the development of severe pulmonary disease. Our findings reveal the protective capacity and longevity of upper respiratory tract Trm cells and highlight the potential of targeting these cells to augment protective responses induced to respiratory viral vaccines.
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http://dx.doi.org/10.1126/sciimmunol.aam6970DOI Listing
June 2017

Endosomal NOX2 oxidase exacerbates virus pathogenicity and is a target for antiviral therapy.

Nat Commun 2017 07 12;8(1):69. Epub 2017 Jul 12.

Program in Chronic Infectious and Inflammatory Diseases, School of Health and Biomedical Sciences, College of Science, Engineering & Health, RMIT University, Bundoora, Victoria, 3083, Australia.

The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.Production of reactive oxygen species is an ancient antimicrobial mechanism, but its role in antiviral defense in mammals is unclear. Here, To et al. show that virus infection activates endosomal NOX2 oxidase and restricts TLR7 signaling, and that an endosomal NOX2 inhibitor decreases viral pathogenicity.
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http://dx.doi.org/10.1038/s41467-017-00057-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507984PMC
July 2017

Nasal-associated lymphoid tissues (NALTs) support the recall but not priming of influenza virus-specific cytotoxic T cells.

Proc Natl Acad Sci U S A 2017 05 1;114(20):5225-5230. Epub 2017 May 1.

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia;

The lymphoid tissue that drains the upper respiratory tract represents an important induction site for cytotoxic T lymphocyte (CTL) immunity to airborne pathogens and intranasal vaccines. Here, we investigated the role of the nasal-associated lymphoid tissues (NALTs), which are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage, in the initial priming and recall expansion of CD8 T cells following an upper respiratory tract infection with a pathogenic influenza virus and immunization with a live attenuated influenza virus vaccine. Whereas NALTs served as the induction site for the recall expansion of memory CD8 T cells following influenza virus infection or vaccination, they failed to support activation of naïve CD8 T cells. Strikingly, NALTs, unlike other lymphoid tissues, were not routinely surveyed during the steady state by circulating T cells. The selective recruitment of memory T cells into these lymphoid structures occurred in response to infection-induced elevation of the chemokine CXCL10, which attracted CXCR3 memory CD8 T cells. These results have significant implications for intranasal vaccines, which deliver antigen to mucosal-associated lymphoid tissue and aim to elicit protective CTL-mediated immunity.
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http://dx.doi.org/10.1073/pnas.1620194114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441821PMC
May 2017

Pattern recognition receptor immunomodulation of innate immunity as a strategy to limit the impact of influenza virus.

J Leukoc Biol 2017 04 3;101(4):851-861. Epub 2016 Nov 3.

Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Victoria, Australia; and

Influenza remains a major global health issue and the effectiveness of current vaccines and antiviral drugs is limited by the continual evolution of influenza viruses. Therefore, identifying novel prophylactic or therapeutic treatments that induce appropriate innate immune responses to protect against influenza infection would represent an important advance in efforts to limit the impact of influenza. Cellular pattern recognition receptors (PRRs) recognize conserved structures expressed by pathogens to trigger intracellular signaling cascades, promoting expression of proinflammatory molecules and innate immunity. Therefore, a number of approaches have been developed to target specific PRRs in an effort to stimulate innate immunity and reduce disease in a variety of settings, including during influenza infections. Herein, we discuss progress in immunomodulation strategies designed to target cell-associated PRRs of the innate immune system, thereby, modifying innate responses to IAV infection and/or augmenting immune responses to influenza vaccines.
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http://dx.doi.org/10.1189/jlb.4MR0716-290RDOI Listing
April 2017

DC-SIGN and L-SIGN Are Attachment Factors That Promote Infection of Target Cells by Human Metapneumovirus in the Presence or Absence of Cellular Glycosaminoglycans.

J Virol 2016 09 12;90(17):7848-63. Epub 2016 Aug 12.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

Unlabelled: It is well established that glycosaminoglycans (GAGs) function as attachment factors for human metapneumovirus (HMPV), concentrating virions at the cell surface to promote interaction with other receptors for virus entry and infection. There is increasing evidence to suggest that multiple receptors may exhibit the capacity to promote infectious entry of HMPV into host cells; however, definitive identification of specific transmembrane receptors for HMPV attachment and entry is complicated by the widespread expression of cell surface GAGs. pgsA745 Chinese hamster ovary (CHO) cells are deficient in the expression of cell surface GAGs and resistant to HMPV infection. Here, we demonstrate that the expression of the Ca(2+)-dependent C-type lectin receptor (CLR) DC-SIGN (CD209L) or L-SIGN (CD209L) rendered pgsA745 cells permissive to HMPV infection. Unlike infection of parental CHO cells, HMPV infection of pgsA745 cells expressing DC-SIGN or L-SIGN was dynamin dependent and inhibited by mannan but not by pretreatment with bacterial heparinase. Parental CHO cells expressing DC-SIGN/L-SIGN also showed enhanced susceptibility to dynamin-dependent HMPV infection, confirming that CLRs can promote HMPV infection in the presence or absence of GAGs. Comparison of pgsA745 cells expressing wild-type and endocytosis-defective mutants of DC-SIGN/L-SIGN indicated that the endocytic function of CLRs was not essential but could contribute to HMPV infection of GAG-deficient cells. Together, these studies confirm a role for CLRs as attachment factors and entry receptors for HMPV infection. Moreover, they define an experimental system that can be exploited to identify transmembrane receptors and entry pathways where permissivity to HMPV infection can be rescued following the expression of a single cell surface receptor.

Importance: On the surface of CHO cells, glycosaminoglycans (GAGs) function as the major attachment factor for human metapneumoviruses (HMPV), promoting dynamin-independent infection. Consistent with this, GAG-deficient pgaA745 CHO cells are resistant to HMPV. However, expression of DC-SIGN or L-SIGN rendered pgsA745 cells permissive to dynamin-dependent infection by HMPV, although the endocytic function of DC-SIGN/L-SIGN was not essential for, but could contribute to, enhanced infection. These studies provide direct evidence implicating DC-SIGN/L-SIGN as an alternate attachment factor for HMPV attachment, promoting dynamin-dependent infection via other unknown receptors in the absence of GAGs. Moreover, we describe a unique experimental system for the assessment of putative attachment and entry receptors for HMPV.
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http://dx.doi.org/10.1128/JVI.00537-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988148PMC
September 2016
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