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Figure 11-1 Experimental Basis of Immunology April 1, 2009 Simon Barratt-Boyes, BVSc, PhD Infectious Diseases & Microbiology and Immunology Center for Vaccine Research [email_address] Immune Evasion
Immune evasion strategy Examples (viral or bacterial) Secreted modulators or toxins ligand mimics Modulators on the pathogen surface complement inhibitors Hide from immune surveillance latency; avoid phagolysosome fusion Antigenic hypervariability numerous Subvert or kill immune cells infect and kill immune system cells Block acquired immunity downregulate MHC; block antigen processing Inhibit complement soluble inhibitors of complement cascade Inhibit cytokines/interferons/chemokines ligand/receptor signaling inhibitors Modulate apoptosis inhibit or accelerate cell death Interfere with Toll-like receptors (TLR) block or hijack TLR signaling Block antimicrobial small molecules prevent iNOS induction Block intrinsic cellular pathways regulate RNA editing Adapted from Finlay BB and McFadden G (2006). Anti-Immunology: Evasion of the host immune system by bacterial and viral pathogens. Cell 124: 767-782
Persistent virus infection: Herpesviruses are paradigmatic Latency Viral genomes are transcriptionally silenced and not available as targets for immune effectors Active immune evasion mechanisms HCMV encodes at least 25 proteins with immune modulating functions 5 of these can impair antigen presentation via MHC class I to CD8+ T cells
Figure 11-4 Latency Herpes simplex virus (HSV) infects epithelial cells and spreads to the sensory neurons where it persists in a latent state in the trigeminal ganglion The virus expresses few proteins in this state (such as poorly immunogenic EBNA1 and/or LMP2A proteins), so infection is not recognized by antigen-specific T cells Neurons express low levels of MHC class I - so any expression of antigen is poorly presented to T cells Virus is reactivated by stress or immunosuppressive drugs and will produce infectious virions which will reinfect skin innervated by the infected neuron
Pathogen avoidance of phagolysosomal degradation. Mycobacterium tuberculosis blocks acidification and uses mannose-lipoarabinomannan (ManLAM) to prevent interactions with other endosomal compartments. Salmonella resides in an acidified compartment that resembles late endosomes but blocks the acquisition of NADPH oxidase, inducible nitric oxide synthase (iNOS) and degradative lysosomal enzymes, and uses the bacterial type III effector proteins SifA and SseJ to modify the vacuolar membrane composition. FimH+ Escherichia coli engages alternative receptors to enter macrophages using lipid rafts and avoids the oxidative burst. Legionella pneumophila secretes proteins through the Dot secretion system to establish a replicative organelle resembling rough endoplasmic reticulum. Yersinia and enteropathogenic E. coli (EPEC) express virulence proteins (such as Yops) to inhibit phagocytosis altogether. From Rosenberger CM and Finlay BB (2003) Nat Rev Mol Cell Biol (4) 385-396.
Figure 11-1 Antigenic hypervariability - 84 types of Streptococcus pneumoniae with antigenically distinct capsular polysaccharides Antibodies opsonize bacteria for phagocytosis - antibody against one type does not cross react with another type, so immunity to one type does not provide protection from other types
Figure 11-3 Antigenic hypervariability - Rearrangement of DNA of pathogen Trypanosomes are insect-borne protozoa causing sleeping sickness in humans Have surface antigens called variant-specific glycoprotein (VSG) which elicit potent protective antibody responses that rapidly clear the parasites The genome contains 1000 VSG genes, each encoding a protein of distinct antigenic properties. Only one version is expressed at a time The VSG gene can be changed by putting a new gene into the 'active expression site' The parasites that can evade the antibody response have a survival advantage and they populate the individual, inducing a new protective immune response
Figure 11-2 Antigen mutation - Antigenic drift and shift in influenza viruses Drift is caused by point mutations in hemagglutinin and neuraminidase that allow virus to evade neutralizing antibodies or T cell responses. Cause of mild epidemics as still some cross-reactive immunity to new isolates Shift is the result of reassortment of segmented RNA encoding viral genes amongst different viruses, notably between human and avian species. This causes global pandemics as the resulting virus is recognized poorly or not at all by existing immunity
Antigenic mutation - CTL escape by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) Plasma virus levels increase (a) and CD4 T cell counts drop (b) associated with a loss of Mamu-A*01/p11C tetramer binding cells (c) in an SIV infected monkey - p11C is an immunodominant CTL epitope present in SIV Gag protein. By week 20 post infection the p11C peptide sequence has mutated in all virus species isolated from the animal's plasma. Peptide binding competition assays show that mutant peptide does not compete with radioactive wild type peptide for binding to Mamu-A*01 MHC class I molecules - equates with escape from CTL recognition Barouch DH et al. (2002) Nature 415: 335-339.
Hewitt EW (2003). The MHC class I antigen presentation pathway: strategies for viral immune evasion. Immunology 110: 163-169
TAP Herpes simplex virus ICP47 has an affinity for TAP 10-1000 times greater than most peptides, it acts as a competitive inhibitor and binds directly to peptide binding site. It is not translocated across ER and remains associated with TAP, and no ATP hydrolysis is initiated Human cytomegalovirus US6 is an ER localized type I membrane protein which interacts with TAP and prevents peptide translocation but does not interfere with peptide binding. It instead prevents conformational rearrangement of TAP that is normally associated with peptide binding MHC class I heavy chain HCMV encodes US2 and US11 promote degradation of MHC class I heavy chain in ER US2 is type I membrane protein in ER that interacts with heavy chain somehow leading to the extraction of heavy chain out of the lumen and into the cytosol where it is degraded by the proteasome
Retaining and diverting MHC class I Adenovirus gene E3/19K (E19) is a type I membrane protein that interacts with MHC in the ER and prevents transport to the plasma membrane The cytosolic tail of E19 has a dilysine motif that inhibits trafficking by acting as an ER retention motif. E19 also binds to TAP and prevents TAP/MHC class I association, presumably by acting as a tapasin mimic HCMV US3 and US10 proteins are ER resident membrane proteins that inhibit peptide loaded MHC class I export from the ER They may interact with a resident ER protein to hold MHC in ER or may interfere with recruitment of MHC to membrane of ER for export to Golgi Human herpesvirus 7 U21 prevents MHC class I from reaching the cell surface by binding to MHC and targeting it to the lysosome. Here both U21 and MHC are degraded. Mechanism unknown
MHC class I down-regulation Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3 and K5 - expression of either protein leads to a rapid down-regulation of surface MHC class I. K5 can down-regulate the costimulatory molecule CD86 K3 interacts with and promotes ubiquitination of MHC class I molecules. Ubiquitin is a small protein that attaches to proteins and generally targets them for degradation by the proteasome. In this case ubiquitination leads to internalization of MHC from the surface and subsequent sorting into the late endosomal pathway HIV-1 Nef has multiple immune escape properties including down-regulation of CD4 and MHC class I. Nef accelerates GTPase mediated internalization of MHC class I, which is involved in the normal recycling of MHC class from the surface Internalized MHC class I molecules are sequestered in the transGolgi network
EBV G-protein coupled receptor targets MHC class I for degradation PLoS Pathogens Jan 2009 5: e1000255; Zuo et al Lytic cycle gene – underscores importance of the need for EBV to be able to evade CD8+ T cell responses during the lytic cycle at a time when a large number of potential viral targets is expressed (A) BILF1 is predominantly localized at the cell surface. 293 cells stably transduced with BILF1 retrovirus were grown on glass slides, fixed and permeabilized, then stained with rat anti-HA (3F10). (B) BILF1 and MHC class I molecules co-precipitate at the cell surface. 293 cells stably transduced with control (C) or BILF1 (B) retrovirus were incubated with antibodies specific for MHC class I (W6/32), TfR (H68.4) or HA tagged BILF1 (3F10). the cells were lysed with NP40 detergent buffer, then precipitated with protein A/G beads and subjected to western-blotting as in Fig. 6B. (C) BILF1 increases the rate of internalization of MHC class I, but not class II, from the cell surface. MJS cells stably transduced with control or BILF1 retrovirus were incubated with mAb to MHC class I (W6/32; top graph) or MHC class II (L234; bottom graph), then washed and incubated at 37°C for different periods of time. The cells were subsequently stained with PE-conjugated goat anti-mouse IgG antibody, and analyzed by flow cytometry. (D) BILF1 increases the rate of internalization, but not the rate of appearance, of MHC class I at the cell surface. Top graph: 293 cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32), then treated exactly as for the internalization assay performed with MJS cells in panel C. Bottom graph: replicate aliquots of the saturated W6/32-bound cells were harvested at the indicated time points, and the appearance of new MHC class I molecules was assayed by staining with PE-conjugated W6/32 antibody.
Host immune system gene targeting by a viral miRNA Science 20 July 2007 Vol 317 p 376, Stern-Ginossar et al Virally encoded microRNA encoded by HCMV – developed algorithm for prediction of miRNA targets and identified MHC class I related chain B (MICB) as top candidate of hcmv-miR-UL112. MICB is stress-induced ligand of NK cell activating receptor NKG2D and is critical for NK killing of virus-infected cells. Hcmv-miR-UL112 down-regulates MICB expression during viral infection leading to decreased binding of NKG2D ad reduced killing by NK cells . Fig. 1. hcmv-miR-UL112 specifically down-regulates MICB expression and reduces NK cytotoxicity. ( A ) The predicted duplex of hcmv-miR-UL112 (red) and its target site (blue) in the 3'UTR of MICB (top) and MICA (bottom). ( B ) Ectopic expression of hcmv-miR-UL112 down-regulates MICB expression. Various human cell lines were transduced with lentiviruses expressing GFP either with hcmv-miR-UL112 (black histogram) or miR-control (open gray histogram). Expression levels of MHC class I, MICA, and MICB were assessed by FACS. ( C ) Ectopic expression of hcmv-miR-US5-1 does not affect MICB expression. The histogram plots of (B) and (C) were gated only on the GFP-positive cells. Background levels for (B) and (C) were measured by using only the secondary Cy5-conjugated Ab (gray solid histogram).
( D ) Reduced binding of NKG2D to cells expressing hcmv-miR-UL112. Binding of NKG2D-Ig to the RKO cells expressing miR-control, hcmv-miR-US5-1, or hcmv-miR-UL112 was assessed by FACS using NKG2D-Ig and the control CD99-Ig (Control-Ig) in various concentrations. ( E ) The reduced NKG2D-Ig binding is due to reduced MICB expression. The expression level of the various NKG2D ligands was assessed by FACS in RKO cells expressing hcmv-miR-UL112 (open red histogram), miR-control (open gray histogram), or hcmv-miR-US5-1 (open black histogram). The histogram plots are gated only on the GFP-positive cells. The background was measured as in (B) and(C) (solid gray histogram). ( F ) Reduced killing of RKO cells expressing hcmv-miR-UL112. Bulk NK cells were preincubated either with anti-NKG2D mAb (white) or with isotype-match control mAb (gray). Labeled RKO cells expressing miR-control or hcmv-miR-UL112 were then added and incubated for 5 hours at the indicated effector:target (E:T) ratios. The differences between the killing of the RKO cells expressing miR-control and those expressing hcmv-miR-UL112 in the presence of the isotypematched control mAb were significant ( P < 0.01, t test). Error bars represent standard deviation of replicates.
Fernandez-Sesma et al J Viology 2006, 80: 6295-6304 Role of influenza virus protein NS1 in suppression of dendritic cell responses Influenza virus and NDV induce different degrees of maturation in human DCs. Human DCs were infected on day 5 or 6 of culture with influenza virus (PR8, Texas, or Moscow) or NDV (NDVB1) at an MOI of 0.5. Supernatants from infected cells (at 18 h postinfection) were tested by ELISA for the release of (B) IFN-alpha and IFN-ß. (C) After infection, cells were incubated at 37°C for 18 h and stained for flow cytometry analysis of the expression of HLA-DR (left panels) or CD86 (right panels). Filled histograms represent uninfected cells, and open histograms represent infected cells (with NDVB1 [top panels] and influenza virus PR8 [bottom panels]).
The NS1 protein of influenza virus abolishes the release of proinflammatory cytokines and IFN-alpha/ß by human DCs after infection with influenza virus. Human DCs were infected with influenza virus PR8 or DeltaNS1 or mock infected. Supernatants from infected DCs were tested by ELISA for the release of (A) IFN-alpha (white bars) and IFN-ß (black bars) or (B) TNF-alpha (black bars) and IL-6 (white bars) at different times after infection. Error bars are for triplicate samples. Data are representative of at least three independent experiments.
Pichlmair et al Science 2006, 314: 997 The NS1 protein of influenza A virus interacts with RIG-I but not MDA5. (A and B) 293T cells were transfected with pGFP–RIG-I (A) or with pHA–RIG-I or pHA-MDA5 (B), and 12 hours later they were infected or not infected with influenza virus, as indicated. At 24 hours, cells were lysed and analyzed by Western blot (WB) for the presence of NS1 and GFP (A) or NS1 and HA (B) in total cell lysates (lower panels) or after immunoprecipitation (IP) with an antibody to NS1 (upper panels). (C) 293T cells were transfected with GFP–RIG-I, infected with influenza virus at 16 hours, and stained for NS1 at 24 hours. Shown are GFP–RIG-I, NS1, and the merged image.