Research Area:	Immunology
Technology Exchange:	Cellular immunology and Vaccine production and evaluation
Keywords:	virus infection, protective immunity, T cells, B cells, HIV-1 and influenza A

In collaboration with scientists from China (the epicentre for EID) and other institutions, my research interest focuses mainly on HIV and influenza A infection in humans, aiming to learn which type(s) of immune responses provide protective immunity (as opposed to immunopathology) and applying this knowledge to the development of powerful immunotherapies as well as effective vaccines. We study samples from either patients infected naturally by viruses or healthy people immunized by vaccines. Given that immune correlates of protection are multi-factorial in nature, the spectrum of our research covers both innate and adaptive immune responses at the molecular, cellular and population levels. In addition, access to clinical samples from clinical trials and cohort studies is essential to the success of our research.

Name	Department	Institution	Country
Prof Sir Andrew J McMichael	Experimental Medicine Division	Oxford University	UK
Gavin Screaton		Imperial College, London	UK
Daniel Douek		Vaccine Research Centre, NIH	USA
Mike Lenardo		Immunology, NIH	USA
Yuelong Shu		China CDC	China
Hualan Chen		Harbin Veterinary Research Institute	China
Hao Wu		Beijing You-An Hospital	China
HuiPing Yan	Capital Medical University	BeiJing You'An Hospital	China
Dr Tao Dong	Experimental Medicine Division	Oxford University	UK
Tom Wilkinson		Southampton University School of Medicine	UK
Sir John Skehel		National Insitute of Medical Research	UK
Alan Hay		National Insitute of Medical Research	UK
Anne Johnson		UCL	UK

Shu Y, Li CK, Li Z, Gao R, Liang Q, Zhang Y, Dong L, Zhou J, Dong J, Wang D, Wen L, Wang M, Bai T, Li D, Dong X, Yu H, Yang W, Wang Y, Feng Z, McMichael AJ, Xu XN et al. 2010. Avian influenza A(H5N1) viruses can directly infect and replicate in human gut tissues. J Infect Dis, 201 (8), pp. 1173-1177. Read abstract | Read more

The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues. Hide abstract

Freundt EC, Yu L, Park E, Lenardo MJ, Xu XN. 2009. Molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein. J Virol, 83 (13), pp. 6631-6640. Read abstract | Read more

Viruses such as hepatitis C and the severe acute respiratory syndrome coronavirus (SARS-CoV) encode proteins that are distributed between mitochondria and the nucleus, but little is known about the factors that control partitioning between these sites. SARS-CoV encodes a unique accessory gene called open reading frame (ORF) 3b that, like other unique accessory genes in SARS-CoV, likely contributes to viral pathogenicity. The ORF 3b protein is 154 amino acids and is predicted to express from the second ORF in subgenomic RNA3. In this report, we have characterized the molecular components that regulate intracellular localization of the ORF 3b protein. We demonstrate unique shuttling behavior of ORF 3b, whereby the protein initially accumulates in the nucleus and subsequently translocates to mitochondria. Following nuclear localization, ORF 3b traffics to the outer membrane of mitochondria via a predicted amphipathic alpha-helix. Additionally, ORF 3b contains a consensus nuclear export sequence, and we demonstrate that nuclear export and thus mitochondrial translocation are dependent on a leptomycin B-sensitive nuclear export mechanism. We further show that ORF 3b inhibits induction of type I interferon induced by retinoic acid-induced gene 1 and the mitochondrial antiviral signaling protein. Our observations provide insights into the cellular localization of ORF 3b that may enhance our understanding of the mechanisms by which ORF 3b contributes to SARS-CoV pathogenesis. The findings reported here reveal that for multilocalized proteins, consideration of the spatiotemporal distribution may be crucial for understanding viral protein behavior and function. Hide abstract

Li D, Wang L, Yu L, Freundt EC, Jin B, Screaton GR, Xu XN. 2009. Ig-like transcript 4 inhibits lipid antigen presentation through direct CD1d interaction. J Immunol, 182 (2), pp. 1033-1040. Read abstract

NKT cells recognize lipid Ags presented by CD1d molecules and play an important role in the regulation of innate and adaptive immune responses. In this study, we report the identification of a membrane-associated protein, Ig-like transcript 4 (ILT4), as a novel human CD1d receptor that inhibits CD1d-mediated immune responses. We found that native CD1d tetramer generated by mammalian cells was able to specifically bind human monocytes in the peripheral blood, and this binding was at least partly mediated by monocyte-expressed ILT4. The interaction between ILT4 and CD1d involves the two N-terminal domains of ILT4 and the Ag-binding groove of CD1d (alpha1/alpha2 domain). This interaction has been identified on the cell surface as well as in the endosomal and lysosomal compartments. Functional analysis showed that ILT4 could block the loading of lipid Ags such as alpha-GalCer, and consequently inhibited NKT recognition. The interaction between ILT4 and CD1d may provide new insights into the regulation of NKT-mediated immunity. Hide abstract

Li CK, Wu H, Yan H, Ma S, Wang L, Zhang M, Tang X, Temperton NJ, Weiss RA, Brenchley JM, Douek DC, Mongkolsapaya J, Tran BH, Lin CL, Screaton GR, Hou JL, McMichael AJ, Xu XN et al. 2008. T cell responses to whole SARS coronavirus in humans. J Immunol, 181 (8), pp. 5490-5500. Read abstract

Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-gamma ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8(+) T cell responses were more frequent and of a greater magnitude than CD4(+) T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27(+)/CD45RO(+)) with a significantly higher frequency of polyfunctional CD4(+) T cells producing IFN-gamma, TNF-alpha, and IL-2, and CD8(+) T cells producing IFN-gamma, TNF-alpha, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation. Hide abstract

Chen N, McCarthy C, Drakesmith H, Li D, Cerundolo V, McMichael AJ, Screaton GR, Xu XN. 2006. HIV-1 down-regulates the expression of CD1d via Nef. Eur J Immunol, 36 (2), pp. 278-286. Read abstract | Read more

HIV-1 has evolved several strategies to subvert host immune responses to the infected cells. One is to inhibit CTL recognition by HIV-1 Nef-mediated down-regulation of MHC-I expression on the surface of infected cells. Here we report that Nef also reduces the expression of the non-classical MHC-I like CD1d molecule, a third lineage of antigen-presenting molecule, which presents lipid antigens. Nef achieves this by increasing internalization of CD1d molecules from the cell surface and retaining CD1d in the trans-Golgi-network (TGN). This effect depends on a tyrosine-based motif present in CD1 cytoplasmic tail as well as the actions of four Nef motifs, which are known to be involved in the down-regulation of MHC-I or CD4. These results suggest that Nef regulates intracellular trafficking of CD1d via a distinct but shared pathway with MHC-I and CD4. Thus, HIV-1 reduces the visibility of its infected cells not only to MHC-I-restricted T cells but also to CD1d-restricted NKT cells. Given that CD1d-restricted T cells have unique effector and regulatory functions in innate and adapted immune responses as compared with their counterpart MHC-restricted T cells, our data provide additional new insights into molecular basis of HIV-1-mediated damage to the immune system. Hide abstract

Drakesmith H, Chen N, Ledermann H, Screaton G, Townsend A, Xu XN. 2005. HIV-1 Nef down-regulates the hemochromatosis protein HFE, manipulating cellular iron homeostasis. Proc Natl Acad Sci U S A, 102 (31), pp. 11017-11022. Read abstract | Read more

The multifunctional Nef protein of HIV-1 is important for the progression to AIDS. One action of Nef is to down-regulate surface MHC I molecules, helping infected cells to evade immunity. We found that Nef also down-regulates the macrophage-expressed MHC 1b protein HFE, which regulates iron homeostasis and is mutated in the iron-overloading disorder hemochromatosis. In model cell lines, Nef reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE. This activity requires a Src-kinase-binding proline-rich domain of Nef and a conserved tyrosine-based motif in the cytoplasmic tail of HFE. HIV-1 infection of ex vivo macrophages similarly down-regulates naturally expressed surface HFE in a Nef-dependent manner. The effect of Nef expression on cellular iron was explored; iron and ferritin accumulation were increased in HIV-1-infected ex vivo macrophages expressing wild-type HFE, but this effect was lost with Nef-deleted HIV-1 or when infecting macrophages from hemochromatosis patients expressing mutated HFE. The iron accumulation in HIV-1-infected HFE-expressing macrophages was paralleled by an increase in cellular HIV-1-gag expression. We conclude that, through Nef and HFE, HIV-1 directly regulates cellular iron metabolism, possibly benefiting viral growth. Hide abstract

Xu XN, Purbhoo MA, Chen N, Mongkolsapaya J, Cox JH, Meier UC, Tafuro S, Dunbar PR, Sewell AK, Hourigan CS, Appay V, Cerundolo V, Burrows SR, McMichael AJ, Screaton GR et al. 2001. A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex. Immunity, 14 (5), pp. 591-602. Read abstract | Read more

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation. Hide abstract

Xu XN, Laffert B, Screaton GR, Kraft M, Wolf D, Kolanus W, Mongkolsapay J, McMichael AJ, Baur AS. 1999. Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain. J Exp Med, 189 (9), pp. 1489-1496. Read abstract | Read more

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV. Hide abstract