Publications by authors named "Andrew G Brooks"

142 Publications

MARCH8 Restricts Influenza A Virus Infectivity but Does Not Downregulate Viral Glycoprotein Expression at the Surface of Infected Cells.

mBio 2021 Sep 14:e0148421. Epub 2021 Sep 14.

Department of Microbiology and Immunology, The University of Melbournegrid.1008.9 at the Peter Doherty Institute for Infection and Immunity, Victoria, Australia.

Membrane-associated RING-CH8 (MARCH8) impairs the cell surface expression of envelope glycoproteins from different viruses, reducing their incorporation into virions. Using stable cell lines with inducible MARCH8 expression, we show that MARCH8 did not alter susceptibility to influenza A virus (IAV) infection, but virions released from infected cells were markedly less infectious. Knockdown of endogenous MARCH8 confirmed its effect on IAV infectivity. The expression of MARCH8 impaired the infectivity of both H3N2 and H1N1 strains and was dependent on its E3 ligase activity. Although virions released in the presence of MARCH8 expressed smaller amounts of viral hemagglutinin (HA) and neuraminidase (NA) proteins, there was no impact on levels of the viral HA, NA, or matrix 2 (M2) proteins detected on the surface of infected cells. Moreover, mutation of lysine residues in the cytoplasmic tails of HA, NA, and/or M2, or in the viral M1 protein, did not abrogate MARCH8-mediated restriction. While MARCH1 and -8 target similar immunological ligands and both restrict HIV-1, only MARCH8 inhibited IAV infectivity. Deletion of the N-terminal cytoplasmic (N-CT) domain of MARCH8 confirmed it to be a critical determinant of IAV inhibition. Of interest, deletion of the MARCH1 N-CT or its replacement with the MARCH8 N-CT resulted in acquisition of IAV restriction. Together, these data demonstrate that MARCH8 restricts a late stage in IAV replication by a mechanism distinct to its reported activity against other viruses. Moreover, we show that the N-CT of MARCH8 is essential for anti-IAV activity, whereas the MARCH1 N-CT inhibits its ability to restrict IAV. The antiviral activity of MARCH8 has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions. Here, we show that MARCH8 restricts IAV at a late stage in virus replication, but this was not associated with reduced expression of IAV envelope glycoproteins on the surface of infected cells, pointing to a distinct mechanism of antiviral activity. Our studies also demonstrate the differential ability of MARCH1 and -8 to restrict IAV infectivity, highlighting the critical role of the N-CT domain of each protein in modulating IAV restriction. Overall, these studies provide novel insights regarding the mechanisms by which MARCH proteins contribute to cell-intrinsic immunity against IAV.
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http://dx.doi.org/10.1128/mBio.01484-21DOI Listing
September 2021

IFITM proteins that restrict the early stages of respiratory virus infection do not influence late-stage replication.

J Virol 2021 Jul 28:JVI0083721. Epub 2021 Jul 28.

Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Melbourne, Victoria 3000, Australia.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2 and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus (PIV)-3 infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilise distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours post-infection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. siRNA-mediated knockdown of endogenous IFITM1, IFITM2 and IFITM3 expression, in the presence or absence of pre-treatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2 or IFITM3 immediately after infection did not impact titres of infectious virus released from IAV or PIV-3 infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways, but do not influence the late stages of replication for either virus. Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses, however their potential to modulate the later stages of virus replication has not been explored. In this study we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early or late stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2 and IFITM3 against influenza A virus (IAV) but not parainfluenza virus (PIV)-3 during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways, yet do not influence the late stages of replication for either virus.
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http://dx.doi.org/10.1128/JVI.00837-21DOI Listing
July 2021

CD8 T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph.

Nat Commun 2021 05 18;12(1):2931. Epub 2021 May 18.

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

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8 T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8 T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2-specific CD8 T cells being cross-reactive between IAV and IBV. Memory CD8 T cells towards these specificities are present in blood (CD27CD45RA phenotype) and tissues (CD103CD69 phenotype) of healthy individuals, and effector CD27CD45RAPD-1CD38CD8 T cells in IAV/IBV patients. Our data show influenza-specific CD8 T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8 T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.
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http://dx.doi.org/10.1038/s41467-021-23212-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132304PMC
May 2021

Natural killer cell receptors regulate responses of HLA-E-restricted T cells.

Sci Immunol 2021 Apr;6(58)

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute, Parkville, Victoria 3010, Australia.

Human cytomegalovirus (CMV) infection can stimulate robust human leukocyte antigen (HLA)-E-restricted CD8 T cell responses. These T cells recognize a peptide from UL40, which differs by as little as a single methyl group from self-peptides that also bind HLA-E, challenging their capacity to avoid self-reactivity. Unexpectedly, we showed that the UL40/HLA-E T cell receptor (TCR) repertoire included TCRs that had high affinities for HLA-E/self-peptide. However, paradoxically, lower cytokine responses were observed from UL40/HLA-E T cells bearing TCRs with high affinity for HLA-E. RNA sequencing and flow cytometric analysis revealed that these T cells were marked by the expression of inhibitory natural killer cell receptors (NKRs) KIR2DL1 and KIR2DL2/L3. On the other hand, UL40/HLA-E T cells bearing lower-affinity TCRs expressed the activating receptor NKG2C. Activation of T cells bearing higher-affinity TCRs was regulated by the interaction between KIR2D receptors and HLA-C. These findings identify a role for NKR signaling in regulating self/non-self discrimination by HLA-E-restricted T cells, allowing for antiviral responses while avoiding contemporaneous self-reactivity.
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http://dx.doi.org/10.1126/sciimmunol.abe9057DOI Listing
April 2021

Structural plasticity of KIR2DL2 and KIR2DL3 enables altered docking geometries atop HLA-C.

Nat Commun 2021 04 12;12(1):2173. Epub 2021 Apr 12.

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

The closely related inhibitory killer-cell immunoglobulin-like receptors (KIR), KIR2DL2 and KIR2DL3, regulate the activation of natural killer cells (NK) by interacting with the human leukocyte antigen-C1 (HLA-C1) group of molecules. KIR2DL2, KIR2DL3 and HLA-C1 are highly polymorphic, with this variation being associated with differences in the onset and progression of some human diseases. However, the molecular bases underlying these associations remain unresolved. Here, we determined the crystal structures of KIR2DL2 and KIR2DL3 in complex with HLA-C*07:02 presenting a self-epitope. KIR2DL2 differed from KIR2DL3 in docking modality over HLA-C*07:02 that correlates with variabilty of recognition of HLA-C1 allotypes. Mutagenesis assays indicated differences in the mechanism of HLA-C1 allotype recognition by KIR2DL2 and KIR2DL3. Similarly, HLA-C1 allotypes differed markedly in their capacity to inhibit activation of primary NK cells. These functional differences derive, in part, from KIR2DS2 suggesting KIR2DL2 and KIR2DL3 binding geometries combine with other factors to distinguish HLA-C1 functional recognition.
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http://dx.doi.org/10.1038/s41467-021-22359-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041999PMC
April 2021

The Role of the HLA Class I α2 Helix in Determining Ligand Hierarchy for the Killer Cell Ig-like Receptor 3DL1.

J Immunol 2021 02 13;206(4):849-860. Epub 2021 Jan 13.

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

HLA class I molecules that represent ligands for the inhibitory killer cell Ig-like receptor (KIR) 3DL1 found on NK cells are categorically defined as those HLA-A and HLA-B allotypes containing the Bw4 motif, yet KIR3DL1 demonstrates hierarchical recognition of these HLA-Bw4 ligands. To better understand the molecular basis underpinning differential KIR3DL1 recognition, the HLA-A family of allotypes were investigated. Transfected human 721.221 cells expressing HLA-A*32:01 strongly inhibited primary human KIR3DL1 NK cells, whereas HLA-A*24:02 and HLA-A*23:01 displayed intermediate potency and HLA-A*25:01 failed to inhibit activation of KIR3DL1 NK cells. Structural studies demonstrated that recognition of HLA-A*24:02 by KIR3DL1 used identical contacts as the potent HLA-B*57:01 ligand. Namely, the D1-D2 domains of KIR3DL1 were placed over the α1 helix and α2 helix of the HLA-A*24:02 binding cleft, respectively, whereas the D0 domain contacted the side of the HLA-A*24:02 molecule. Nevertheless, functional analyses showed KIR3DL1 recognition of HLA-A*24:02 was more sensitive to substitutions within the α2 helix of HLA-A*24:02, including residues Ile and Lys Furthermore, the presence of Thr in the α2 helix of HLA-A*25:01 abrogated KIR3DL1 NK inhibition. Together, these data demonstrate a role for the HLA class I α2 helix in determining the hierarchy of KIR3DL1 ligands. Thus, recognition of HLA class I is dependent on a complex interplay between the peptide repertoire, polymorphisms within and proximal to the Bw4 motif, and the α2 helix. Collectively, the data furthers our understanding of KIR3DL1 ligands and will inform genetic association and immunogenetics studies examining the role of KIR3DL1 in disease settings.
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http://dx.doi.org/10.4049/jimmunol.2001109DOI Listing
February 2021

Estradiol Enhances Antiviral CD4 Tissue-Resident Memory T Cell Responses following Mucosal Herpes Simplex Virus 2 Vaccination through an IL-17-Mediated Pathway.

J Virol 2020 12 9;95(1). Epub 2020 Dec 9.

McMaster Immunology Research Centre, McMaster University, Hamilton, Ontario, Canada

Estradiol (E2) is a sex hormone which has been shown to be protective against sexually transmitted infections such as herpes simplex virus 2 (HSV-2). However, few studies have examined the underlying mechanisms by which this occurs. Here, we investigated the effect of E2 on the establishment of memory T cells post-intranasal immunization with HSV-2. CD4 T cell responses first appeared in the upper respiratory tract (URT) within 3 days postimmunization before being detected in the female reproductive tract (FRT) at 7 days. E2 treatment resulted in greater and earlier T17 responses, which preceded augmented T1 responses at these sites. The CD4 T cells persisted in the URT for up to 28 days, and E2 treatment resulted in higher frequencies of memory T cells. Intranasal immunization also led to the establishment of CD4 tissue-resident memory T cells (T cells) in the FRT, and E2 treatment resulted in increased T1 and T17 T cells. When the migration of circulating T cells into the FRT was blocked by FTY720, immunized E2-treated mice remained completely protected against subsequent genital HSV-2 challenge compared to non-E2 controls, confirming that T cells alone are adequate for protection in these mice. Finally, the enhanced vaginal T1 T cells present in E2-treated mice were found to be modulated through an interleukin 17 (IL-17)-mediated pathway, as E2-treated IL-17A-deficient mice had impaired establishment of T1 T cells. This study describes a novel role for E2 in enhancing CD4 memory T cells and provides insight on potential strategies for generating optimal immunity during vaccination. Herpes simplex virus 2 (HSV-2) is a highly prevalent sexually transmitted infection for which there is currently no vaccine available. Interestingly, the female sex hormone estradiol has been shown to be protective against HSV-2. However, the underlying mechanisms by which this occurs remains relatively unknown. Our study demonstrates that under the influence of estradiol treatment, intranasal immunization with an attenuated strain of HSV-2 leads to enhanced establishment of antiviral memory T cell responses in the upper respiratory tract and female reproductive tract. In these sites, estradiol treatment leads to greater T17 memory cells, which precede enhanced T1 memory responses. Consequently, the T cell responses mounted by tissue-resident memory cells in the female reproductive tract of estradiol-treated mice are sufficient to protect mice against vaginal HSV-2 challenge. This study offers important insights regarding the regulation of mucosal immunity by hormones and on potential strategies for generating optimal immunity during vaccination.
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http://dx.doi.org/10.1128/JVI.01206-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737739PMC
December 2020

Cytomegalovirus replication is associated with enrichment of distinct γδ T cell subsets following lung transplantation: A novel therapeutic approach?

J Heart Lung Transplant 2020 Aug 26. Epub 2020 Aug 26.

Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia; Lung Transplant Service, The Alfred Hospital, Melbourne, Victoria, Australia. Electronic address:

Background: Anti-viral treatments to control cytomegalovirus (CMV) after lung transplantation (LTx) are associated with toxicity and anti-viral resistance. Cellular immunotherapy with virus-specific cytotoxic T cells has yielded promising results but requires donor/recipient matching. γδ T cells are involved in anti-viral immunity and can recognize antigens independently of major histocompatibility complex molecules and may not require the same level of matching. We assessed the phenotype of circulating γδ T cells after LTx to identify the candidate populations for CMV immunotherapy.

Methods: Peripheral blood mononuclear cells were isolated from lung transplant recipients before transplantation and at routine bronchoscopies after LTx. Patients were stratified by risk of CMV disease into moderate risk (recipient CMV seropositive, n = 15) or high risk (HR) (recipient CMV seronegative/donor CMV seropositive, n = 10). CMV replication was classified as polymerase chain reaction positive (>150 copies/ml) in blood and/or bronchoalveolar lavage within the first 18 months. The phenotype of γδ T cells was assessed by multicolor flow cytometry, and T-cell receptor (TCR) sequences were determined by deep sequencing.

Results: In HR lung transplant recipients with CMV replication, we observed striking phenotypic changes in γδ T cells, marked by an increase in the proportion of effector Vδ1+ γδ T cells expressing the activating natural killer cell receptor NKG2C. Moreover, we observed a remarkable increase in TCR diversity.

Conclusions: NKG2C+ Vδ1+ γδ T cells were associated with CMV replication and may indicate their potential to control infection. As such, we propose that they could be a potential target for cellular therapy against CMV.
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http://dx.doi.org/10.1016/j.healun.2020.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448790PMC
August 2020

Harnessing HLA-E-restricted CD8 T lymphocytes for adoptive cell therapy of patients with severe COVID-19.

Br J Haematol 2020 08 19;190(4):e185-e187. Epub 2020 Jun 19.

Central Laboratory for Advanced Diagnosis and Biomedical Research, University of Palermo, Palermo, Italy.

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http://dx.doi.org/10.1111/bjh.16895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301003PMC
August 2020

The molecular basis of how buried human leukocyte antigen polymorphism modulates natural killer cell function.

Proc Natl Acad Sci U S A 2020 05 13;117(21):11636-11647. Epub 2020 May 13.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIC 3010, Australia;

Micropolymorphisms within human leukocyte antigen (HLA) class I molecules can change the architecture of the peptide-binding cleft, leading to differences in peptide presentation and T cell recognition. The impact of such HLA variation on natural killer (NK) cell recognition remains unclear. Given the differential association of HLA-B*57:01 and HLA-B*57:03 with the control of HIV, recognition of these HLA-B57 allomorphs by the killer cell immunoglobulin-like receptor (KIR) 3DL1 was compared. Despite differing by only two polymorphic residues, both buried within the peptide-binding cleft, HLA-B*57:01 more potently inhibited NK cell activation. Direct-binding studies showed KIR3DL1 to preferentially recognize HLA-B*57:01, particularly when presenting peptides with positively charged position (P)Ω-2 residues. In HLA-B*57:01, charged PΩ-2 residues were oriented toward the peptide-binding cleft and away from KIR3DL1. In HLA-B*57:03, the charged PΩ-2 residues protruded out from the cleft and directly impacted KIR3DL1 engagement. Accordingly, KIR3DL1 recognition of HLA class I ligands is modulated by both the peptide sequence and conformation, as determined by the HLA polymorphic framework, providing a rationale for understanding differences in clinical associations.
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http://dx.doi.org/10.1073/pnas.1920570117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261055PMC
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

HLA-E-restricted CD8 T Lymphocytes Efficiently Control and HIV-1 Coinfection.

Am J Respir Cell Mol Biol 2020 04;62(4):430-439

Central Laboratory for Advanced Diagnosis and Biomedical Research.

We investigated the contribution of human leukocyte antigen A2 (HLA-A2) and HLA-E-restricted CD8 T cells in patients with and human immunodeficiency virus 1 (HIV-1) coinfection. HIV-1 downregulates HLA-A, -B, and -C molecules in infected cells, thus influencing recognition by HLA class I-restricted CD8 T cells but not by HLA-E-restricted CD8 T cells, owing to the inability of the virus to downmodulate their expression. Therefore, antigen-specific HLA-E-restricted CD8 T cells could play a protective role in and HIV-1 coinfection. HLA-E- and HLA-A2-restricted -specific CD8 T cells were tested for cytotoxic and microbicidal activities, and their frequencies and phenotypes were evaluated in patients with active tuberculosis and concomitant HIV-1 infection. HIV-1 and coinfection caused downmodulation of HLA-A2 expression in human monocyte-derived macrophages associated with resistance to lysis by HLA-A2-restricted CD8 T cells and failure to restrict the growth of intracellular . Conversely, HLA-E surface expression and HLA-E-restricted cytolytic and microbicidal CD8 responses were not affected. HLA-E-restricted and -specific CD8 T cells were expanded in the circulation of patients with /HIV-1 coinfection, as measured by tetramer staining, but displayed a terminally differentiated and exhausted phenotype that was rescued by anti-PD-1 (programmed cell death protein 1) monoclonal antibody. Together, these results indicate that HLA-E-restricted and -specific CD8 T cells in patients with /HIV-1 coinfection have an exhausted phenotype and fail to expand in response to antigen stimulation, which can be restored by blocking the PD-1 pathway using the specific monoclonal antibody nivolumab.
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http://dx.doi.org/10.1165/rcmb.2019-0261OCDOI Listing
April 2020

The complex existence of γδ T cells following transplantation: the good, the bad and the simply confusing.

Clin Transl Immunology 2019 17;8(9):e1078. Epub 2019 Sep 17.

Lung Transplant Service The Alfred Hospital Melbourne VIC Australia.

Gamma delta (γδ) T cells are a highly heterogeneous population of lymphocytes that exhibit innate and adaptive immune properties. Despite comprising the majority of residing lymphocytes in many organs, the role of γδ T cells in transplantation outcomes is under-researched. γδ T cells can recognise a diverse array of ligands and exert disparate effector functions. As such, they may potentially contribute to both allograft acceptance and rejection, as well as impacting on infection and post-transplant malignancy. Here, we review the current literature on the role and function of γδ T cells following solid organ and hematopoietic stem cell transplantation.
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http://dx.doi.org/10.1002/cti2.1078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748302PMC
September 2019

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

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

Classical Type 1 Dendritic Cells Dominate Priming of Th1 Responses to Herpes Simplex Virus Type 1 Skin Infection.

J Immunol 2019 02 31;202(3):653-663. Epub 2018 Dec 31.

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

CD4 T cell responses are crucial for the control of many intracellular pathogens, yet the requirements for their induction are not fully understood. To better understand the role that various dendritic cell (DC) subtypes play in CD4 T cell priming, we compared in vivo T cell responses to skin inoculation of mice with infectious or UV-inactivated HSV type 1. Localized infection elicited a Th1 response that was primed in skin-draining lymph nodes involving Ag presentation by migratory dermal and lymph node-resident DC. However, expansion and Th1 differentiation was impaired in response to UV-inactivated virus (UV-HSV), and this defect correlated with a restriction of Ag presentation to migratory CD103 dermal DC. A similar differentiation defect was seen in infected mice lacking CD8α and CD103 classical type 1 DC (cDC1). Finally, Th1 differentiation after UV-HSV inoculation was rescued by targeted Ag delivery to CD8α and CD103 cDC1 using an anti-Clec9A Ab construct. This suggests that Ag presentation by cDC1 is crucial for optimal Th1 immunity to HSV type 1 infection and potentially other pathogens of the skin.
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http://dx.doi.org/10.4049/jimmunol.1800218DOI Listing
February 2019

Enrichment of Cytomegalovirus-induced NKG2C+ Natural Killer Cells in the Lung Allograft.

Transplantation 2019 08;103(8):1689-1699

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

Background: In lung transplant recipients, immunosuppressive medications result in impaired antiviral immunity and a propensity for cytomegalovirus (CMV) reactivation within the lung allograft. Natural killer (NK) cells play a key role in immunity to CMV, with an increase in the proportion of NK cells expressing activating CD94-NKG2C receptors in the blood being a strong correlate of CMV infection. Whether a similar increase in NKG2C NK cells occurs in lung transplant recipients following CMV reactivation in the allograft and if such cells contribute to viral control remains unclear.

Methods: In this pilot study, we longitudinally assessed the frequency and phenotype of NKG2C NK cells in the blood and bronchoalveolar lavage (BAL) of lung transplant recipients and stratified recipients based on their risk of developing CMV disease.

Results: We observed an increase in the proportion of NKG2C NK cells in the blood and BAL of CMV high-risk patients, coincident with both the cessation of antiviral prophylaxis and subsequent detection of actively replicating CMV in the blood and lung allograft. Additionally, these NKG2C NK cells expressed killer-cell immunoglobulin-like receptors distinct from those of other NK subsets and BAL NKG2C NK cells possessed an activated phenotype. Finally, the frequency of NKG2C NK cells in the BAL may be inversely correlated with CMV blood titers.

Conclusions: Monitoring the phenotype of NK cells postlung transplant may be a useful biomarker for monitoring patient levels of CMV immunity.
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http://dx.doi.org/10.1097/TP.0000000000002545DOI Listing
August 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

Killer cell immunoglobulin-like receptor 3DL1 variation modifies HLA-B*57 protection against HIV-1.

J Clin Invest 2018 05 3;128(5):1903-1912. Epub 2018 Apr 3.

Cardiff University School of Medicine, Heath Park, University Hospital of Wales, Cardiff, United Kingdom.

HLA-B*57 control of HIV involves enhanced CD8+ T cell responses against infected cells, but extensive heterogeneity exists in the level of HIV control among B*57+ individuals. Using whole-genome sequencing of untreated B*57+ HIV-1-infected controllers and noncontrollers, we identified a single variant (rs643347A/G) encoding an isoleucine-to-valine substitution at position 47 (I47V) of the inhibitory killer cell immunoglobulin-like receptor KIR3DL1 as the only significant modifier of B*57 protection. The association was replicated in an independent cohort and across multiple outcomes. The modifying effect of I47V was confined to B*57:01 and was not observed for the closely related B*57:03. Positions 2, 47, and 54 tracked one another nearly perfectly, and 2 KIR3DL1 allotypes differing only at these 3 positions showed significant differences in binding B*57:01 tetramers, whereas the protective allotype showed lower binding. Thus, variation in an immune NK cell receptor that binds B*57:01 modifies its protection. These data highlight the exquisite specificity of KIR-HLA interactions in human health and disease.
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http://dx.doi.org/10.1172/JCI98463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919796PMC
May 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

A conserved energetic footprint underpins recognition of human leukocyte antigen-E by two distinct αβ T cell receptors.

J Biol Chem 2017 12 25;292(51):21149-21158. Epub 2017 Sep 25.

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

αβ T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8 T cell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8 T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of a TRBV9 TCR in complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9 TCR adopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14 TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells.
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http://dx.doi.org/10.1074/jbc.M117.807719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743087PMC
December 2017

The molecular basis for peptide repertoire selection in the human leucocyte antigen (HLA) C*06:02 molecule.

J Biol Chem 2017 10 30;292(42):17203-17215. Epub 2017 Aug 30.

From the Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton 3800, Victoria, Australia,

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.
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http://dx.doi.org/10.1074/jbc.M117.806976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655500PMC
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

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

MHC-I peptides get out of the groove and enable a novel mechanism of HIV-1 escape.

Nat Struct Mol Biol 2017 04 20;24(4):387-394. Epub 2017 Feb 20.

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

Major histocompatibility complex class I (MHC-I) molecules play a crucial role in immunity by capturing peptides for presentation to T cells and natural killer (NK) cells. The peptide termini are tethered within the MHC-I antigen-binding groove, but it is unknown whether other presentation modes occur. Here we show that 20% of the HLA-B*57:01 peptide repertoire comprises N-terminally extended sets characterized by a common motif at position 1 (P1) to P2. Structures of HLA-B*57:01 presenting N-terminally extended peptides, including the immunodominant HIV-1 Gag epitope TW10 (TSTLQEQIGW), showed that the N terminus protrudes from the peptide-binding groove. The common escape mutant TSNLQEQIGW bound HLA-B*57:01 canonically, adopting a dramatically different conformation than the TW10 peptide. This affected recognition by killer cell immunoglobulin-like receptor (KIR) 3DL1 expressed on NK cells. We thus define a previously uncharacterized feature of the human leukocyte antigen class I (HLA-I) immunopeptidome that has implications for viral immune escape. We further suggest that recognition of the HLA-B*57:01-TW10 epitope is governed by a 'molecular tension' between the adaptive and innate immune systems.
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http://dx.doi.org/10.1038/nsmb.3381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900914PMC
April 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

Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C.

J Biol Chem 2016 09 6;291(36):18740-52. Epub 2016 Jul 6.

the Department of Immunology and Pathology, Central Clinical School, Monash University, Melbourne, Victoria 3004, Australia

Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 μm) than that observed between Ly49C and MHC-Ia (H-2K(b)/H-2D(d), both ∼1 μm), and this recognition could be prevented by cis interactions with H-2K in situ To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated β2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2K(b) Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2K(b) possess similar energetic footprints focused around residues located within the Ly49C β4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules.
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http://dx.doi.org/10.1074/jbc.M116.737130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009249PMC
September 2016

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

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

Virology 2016 07 26;494:143-57. Epub 2016 Apr 26.

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

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.
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http://dx.doi.org/10.1016/j.virol.2016.01.024DOI Listing
July 2016

Killer cell immunoglobulin-like receptor 3DL1 polymorphism defines distinct hierarchies of HLA class I recognition.

J Exp Med 2016 05 4;213(5):791-807. Epub 2016 Apr 4.

Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia

Natural killer (NK) cells play a key role in immunity, but how HLA class I (HLA-I) and killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1) polymorphism impacts disease outcome remains unclear. KIR3DL1 (*001/*005/*015) tetramers were screened for reactivity against a panel of HLA-I molecules. This revealed different and distinct hierarchies of specificity for each KIR3DL1 allotype, with KIR3DL1*005 recognizing the widest array of HLA-I ligands. These differences were further reflected in functional studies using NK clones expressing these specific KIR3DL1 allotypes. Unexpectedly, the Ile/Thr80 dimorphism in the Bw4-motif did not categorically define strong/weak KIR3DL1 recognition. Although the KIR3DL1*001, *005, and *015 polymorphisms are remote from the KIR3DL1-HLA-I interface, the structures of these three KIR3DL1-HLA-I complexes showed that the broader HLA-I specificity of KIR3DL1*005 correlated with an altered KIR3DL1*005 interdomain positioning and increased mobility within its ligand-binding site. Collectively, we provide a generic framework for understanding the impact of KIR3DL1 polymorphism on the recognition of HLA-I allomorphs.
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http://dx.doi.org/10.1084/jem.20152023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854737PMC
May 2016
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