2/28/11 VIRAL IMMUNOLOGY. Overview of immune system Innate immune response Adaprive immunity

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1 2/28/11 VIRAL IMMUNOLOGY Sergei Nekhai, Ph.D. Objectives: Overview of immune system Innate immune response Adaprive immunity

2 Immune Response to Viruses Continuous struggle with the invading microbes Vaccination eradicated smallpox, polio, measles, mumps, rubella etc. New viruses appear: HIV, Hendra, Dengue, Sars, Avian Flu, etc. Reappearance of old viruses with higher h mortality (measles, flu) Association of viral infection with cancer (HPV service carcinoma, HHV 8 - Kaposi s sarcoma, HBV- hepatocellular acarcinoma, etc.)

3 Immune Response cont. Effector function Carried by cells (natural killer (NK), T cells) cellular immunity Fluid-born humoral immunity (antibodies, chemokines, cytokines, complement, etc) Antigen Specificity Antigen-specific (adaptive) has memory Non-antigen specific (innate) no memory

4 The Immune System Bacteria Parasite in red blood cell SARS virus Fungus

5 Markers of Self Epithelial cell Muscle cell Leukocyte Nerve cell Class I MHC self-marker protein

6 Markers of Non-Self Bacteria SARS virus Antigen Epitope Antibody Non-self nerve cell Non-self leukocyte Antigen Epitope Class I MHC protein Antibody

7 Markers of Self: Major Histocompatibility Complex Viral infection Antigenic peptide MHC Class I Antigenic peptide MHC Class I Antigenic peptide MHC Class II Infected cell Antigen-presenting cell uses MHC Class I or II Cell membrane

8 Endogenous antigen processing: MHC class I peptide presentation Intracellular proteins of host and virus are marked for degradation by ubiquitination and are degraded by the Proteasome. The resulting viral peptides are transported into the ER lumen by the Tap1-Tap2 Tap2 heterodimeric transporter. In the ER lumen, viral peptides associate with newly synthesized MHC class I molecules. MHC class I-peptide complex is transported to the cell surface via the golgi compartments. On the cell surface the MHC class I peptide complex On the cell surface, the MHC class I-peptide complex interacts with the T- cell receptor of a Tc cell carrying the CD8 coreceptor.

9 Endogenous antigen processing: MHC class I peptide presentation

10 Exogenous antigen processing: MHC class II peptide presentation MHC class II complex is prevented from binding to viral peptides in the ER by association with the invariant chain. The complex is transported t through h golgi where the invariant chain is removed, activating the MHC class II complex. The peptides are derived from extracellular proteins that enter the cell by endocytosis. Viral proteins are degraded in the lysosomes by proteases that are activated by low ph. Endosomes fuse with vesicles containing MHC class II. On the surface of the cell the MHC class II complex p interacts with the T cell receptor of a Th cell carrying the CD4 coreceptor.

11 Exogenous antigen processing: MHC class II peptide presentation

12 Organs of the Immune System Tonsils and adenoids Lymph nodes Lymphatic vessels Thymus Lymph nodes Spleen Appendix Bone marrow Peyer s patches Lymph nodes Lymphatic vessels

13 Sergei Nekhai: ANATOMY OF THE IMMUNE SYSTEM Thymus glandular organ near the heart where T cells learn their jobs Bone marrow blood-producing tissue located inside certain bones blood stem cells give rise to all of the different types of blood cells Spleen serves as a filter for the blood removes old and damaged red blood cells removes infectious agents and uses them to activate cells called lymphocytes Lymph nodes small organs that filter out dead cells, antigens, and other stuff to present to lymphocytes Lymphatic vessels collect fluid (lymph) that has leaked out from the blood into the tissues and returns it to circulation

14 Lymph Node Incoming lymphatic vessel Germinal center Follicle Paracortex Medulla Cortex Vein Artery Outgoing lymphatic vessel

15 Cells of the Immune System Bone graft Macrophage Mast cell Marrow Eosinophil Erythrocytes Basophil Bone Hematopoietic stem cell Monocyte Megakaryocyte Multipotential stem cell Myeloid progenitor cell Neutrophil Platelets Lymphoid progenitor cell T lymphocyte Dendritic cell Natural killer cell B lymphocyte

16 B Cells Antigen-specific B cell receptor Class II MHC and processed antigen are displayed Antigen B cell Antibodies Lymphokines Plasma cell Antigen-presenting bacteria Activated helper T cell

17 Antibody Heavy chain Light chain Antigen-binding region Constant region Assembled antibody molecule

18 Immunoglobulins IgG, IgD, IgE, and IgA IgA IgM

19 Type Number of ag binding sites Site of action Functions IgG 2 Blood Tissue fluid CAN CROSS PLACENTA Increase macrophage activity Antitoxins Agglutination IgM 10 Blood Agglutination Tissue fluid IgA 2or4 Secretions (saliva, tears, Stop bacteria small intestine, vaginal, adhering to host cells prostate, nasal, breast Prevents bacteria milk) forming colonies on mucous membranes IgE 2 Tissues Activate mast cells HISTAMINE Worm response

20 T Cells Resting helper T cell Resting cytotoxic T cell Activated helper T cell Activated killer cell

21 Cytokines Lymphokines Monokines Mature helper T cell Macrophage

22 Killer Cells: Cytotoxic Ts and NKs Killer cell Target cell Surface contact Target-oriented t granules

23 Phagocytes and Their Relatives Monocyte Eosinophil Mast cell Macrophage Dendritic cell Neutrophil Basophil

24 Phagocytes in the Body Brain: microglial cells Lung: alveolar macrophages Liver: Kupffer cells Kidney: mesangial phagocytes Lymph node: resident and recirculating macrophages Spleen: macrophages Blood: monocytes Precursors in bone marrow Joint: synovial A cells

25 Complement C2 C3 C3a C5a C7 C1 IgG C6 C5b C8 Antigen C4 Enzyme C3b C5 C5b C9

26 Mounting an Immune Response Lymphokines T cell Antibodies B cell Killer cell Complement Macrophage Virus

27 YOUR ACTIVE IMMUNE DEFENSES Innate Immunity -invariant a (generalized) e ed) - early, limited specificity - the first line of defense Adaptive Immunity - variable (custom) - later, highly specific - remembers infection

28 The innate immune response: Can be activated t rapidly and functions within hours of a viral infection. Continued activity is damaging to the host. Considerable interplay occurs between the adaptive and innate immune defenses. Important components are: -cytokines -complement -collectins -natural killer (NK) cells

29 Initiation of Immune Responses Field s Virology, Fifth Edition

30 Activation of INFs and Cytokines by Viral Infection Field s Virology, Fifth Edition

31 Recognition of Foreign Nucleic Acids

32 Assembly of MyD88/IRAK complex Lin and Wu, Nature 2010

33 Interferons IFn-γ is induced only when certain lymphocytes are stimulated to replicate and divide after binding a foreign antigen IFn-α and IFn-β are induced by viral infection of any cell type

34 Interferons IFN is induced by accumulation of double stranded RNA (dsrna). IFN induces gene expression at the transcriptional level after binding to specific cell surface receptors. A cell that is bound to interferon and responds to it is in an antiviral i state. t IFN induces expression of more that 100 genes, products of many of these genes possess broad spectrum antiviral activity. They lead to cell death by apoptosis or programmed cells death, limiting cell to cell spread of virus. Production of large amounts if IFN causes common g symptoms such as fever, chills, nausea, etc.

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36 Interferon induced antiviral responses: Both viral and cellular protein synthesis stops in IFN treated cells. This is dues to two cellular proteins, ds-rna activated protein kinase (PKR) and ribonuclease L (RNase L). PKR is a serine/threonine kinase that has antiviral properties, as well as antiproliferative and antitumor functions. Activated PKR phosphorylates the alpha subunit of the translation initiation factor eif2, inhibiting translation. RNase L is a nuclease that can degrade cellular and viral RNA; its concentration increases after Ifn treatment.

37 Interferon induced antiviral responses: RNase L concentration ti increases 10-1, fold after Ifn treatment, but is inactive unless 2-5 -oligo(a) synthetase is produced oligo(a) synthetase produces 2, 5 oligomers of adenylic acid, only when activated by dsrna. These poly(a) oligomers then activate RNase L, which degrades all host and viral mrna in the cell. RNase L participates not only in Ifn-mediated antiviral defense, but also in apoptosis. Ifn is a broad spectrum, highly effective antiviral agent. However, viruses have developed numerous mechanisms for inhibiting interferon action.

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39 The eif2α-mediated Translational Control Heme Controlled Repressor (HCR) is an eif2 kinase Reticulocytes (red blood cell precursors) translate hemoglobin messages very efficiently i Globin is of no use without heme Heme prevents HCR from phosphorylating/inactivating eif2 Interferon induced by viral (ds)rna Interferon induces eif-2 kinase eif-2 kinase activated by dsrna 2,5 A synthetase 2,5 A activates RNAse L;RNAse L degrades RNA

40 Heme Regulates the Activity of Heme-regulated Kinase

41 Interferon-induced Repression of Translation

42 Heme Regulates the Activity of Heme-regulated Kinase J.J. Chen

43 Iron Transport in Humans Drakesmith and Prentice, 2008

44 Iron Homeostasis in Humans Drakesmith and Prentice, 200

45 Multiple Protein Families Possessing dsrbms Motifs Representative PKR Protein Function Interferon-induced kinase Antiviral responses ADAR2, ADAR1 pre-mrna editing deaminase Potential viral defense Dicer, RNase III Staufen RNA helicase A TRBP RNA interference, RNA-nuclease activity mrna trafficking RNA and DNA helicase activity PKR inhibitor, TAR RNA-binding; a DICER co-factor E3L NF90 PKR inhibitor RNA metabolism

46 Other Intrinic Antivirail Responces Autophagy A t h Formation of specialized membrane compartmetnts related to lysosomes Epigenetic silencing Defence against DNA containing viruses, formation hromatin structure RNA silencing Ssequence-specific RNA degradation Cytosine Deamination (APOBEC) C s to U s convertion TRIM Proteins Targeting capsid protein by TRM5a protein Tetherin Inability of the virions to bud

47 Micro RNA Founding members of mirnas, 22 nt and 61 nt RNAs coded by C.elegans Lin-1 gene complementary to 3 UTR of Lin-14 gene that blocked translation of Lin-14 Control of cell proliferaton, cell death and fat metabolism in flies Modulation of fhematopoietic ti lineage differentiation in mammals Leaf and flower development in plants Majority of mirnas are transcribed independently Some e(quarter) e) mirna are aederived from intrones mirna are conserved

48 Formation of RISCs and Other Silencing Complexes Pratt and McRae, JBC, 2009

49 Examples of Metazoan mirnas

50 Maturation of mirna Cleavege with Drosha Plants Metazoa Animals Additional Cleavage with Dicer Complex with RISC (RNA-induced Silencing complex)

51 Actions of Small Silencing RNAs mrna celavage Translational repression Transcriptional silencing

52 Regulation of Natural Killer (NK) Cells Field s Virology, Fifth Edition

53 Activation of Adaptive by Innate Immune Responces Field s Virology, Fifth Edition

54 Cytokine-mediated Immune Responces Field s Virology, Fifth Edition

55 The adaptive immune response: Humoral response Consists of lymphocytes of the B-cell lineage Interaction of a specific receptor on precursor B lymphocytes with antigens promotes differentiation into antibody secreting cells (plasma cells). Cell-mediated response Consists of lymphocytes of the T-cell lineage Cytotoxic T cells (Tc cells) and T-helper cells (Th cells) are the key effectors of this response.

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57 The antigen receptors on the surface of B and T cells B cells have about 100,000 molecules of a single antibody receptor per cell, which has specificity for one antigen epitope. T cells bearing the surface membrane protein CD4 always recognize peptides bound to MHC class II proteins and function as Th cells. T cells bearing the surface membrane protein CD8 always recognize peptide antigens bound to MHC class I proteins and function as cytotoxic T cells.

58 Antibody Activities in Viral Infection Field s Virology, Fifth Edition

59 Maturation of CD4+ and CD8+ T cells Field s Virology, Fifth Edition

60 TCR α:β TCR γ:δ Lymphocyte Subsets Dominant T-cells Responding to Viral Infection CD8+ recognize MHC I viral pepide complex CD4+ recognize MHC I viral peptide complex, regulate B- cell differentiation and inflammation T H 1 produce antiviral cytokines (IFNγ) T H 2 produce cytokines for allergic response (IL-4, IL-5) Innate-like effectors cells express CD3 but not CD4 or CD8 recognize products of stressed cells NKT Treg innate-like immune effectrs early in the immune response express CD4, but not CD8 Control T- and B-responses Express CD4 and CD25 Usually suppress T-and B-cell responces

61 Cell-mediated response cont. T lymphocytes recognize antigens on the surface of self cells. The antigens on self cells can be recognized only by a receptor on the surface of T cells when they are bound to the MHC family of membrane proteins. The Th cells recognize antigens bound to MHC class II molecules and produce powerful cytokines that affect other lymphocytes (B and T cells) by promoting or inhibiting cell division and gene expression. Once activated by Th cells, Tc cells differentiate into CTLs that can kill virus infected cells.

62 Regulatory T Cells T cells compete for same antigen T cells compete for cytokine signals Cytotoxic T cell Mature dendritic cell Regulatory T cell Regulatory T cells Proliferation

63 Questions: