Sergei Nekhai, Ph.D. Objectives - Home | Howard University Immunology 201… · Immune Response to...

02/25/13 VIRAL IMMUNOLOGY

Sergei Nekhai, Ph.D.

Objectives:

• Overview of immune system

•Intrinsic antiviral response

• Innate immune response

•Adaptive immunity

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 mortality

(measles, flu)

•Association of viral infection with cancer

(HPV – service carcinoma, HHV 8 - Kaposi’s

sarcoma, HBV- hepatocellular carcinoma, etc.)

Host Defense Systems

• First line of defense (physical and chemical):

skin, mucosa, tears, acidic and basic pH, surface cleansing mechanisms (cells dying or shedding)

• Second line of defense (immediate)

cell-autonomous, intrinsic defense; detection of unusual macromolecules, changes in metabolism, parasites; production of interferon's, cytokines, block of early steps of infection.

• Third line: Innate and adaptive response

Immediate response, amplified by cytokines and interferons;

Activation of cytotoxic T cells and helper T cells

Production and maintenance of memory B-cells and T-cells

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

The Immune System

SARS virus

Parasite in red blood cell

Fungus

Bacteria

Markers of Self

Muscle cell

Nerve cell

Epithelial cell

Leukocyte

Class I MHC self-marker protein

Markers of Non-Self

Non-self leukocyte

Antibody

Epitope Class I MHC protein

Epitope

Antibody

Antigen

Antigen

Bacteria

Non-self nerve cell

SARS virus

Markers of Self: Major Histocompatibility Complex

Antigenic peptide

Antigen-presenting cell uses MHC Class I or II

Cell membrane

MHC Class II

Antigenic peptide

Viral infection

Infected cell

MHC Class I

Antigenic peptide

MHC Class I

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 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

Endogenous antigen processing: MHC class I peptide presentation

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 through 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 interacts with the T cell receptor of a Th cell

Exogenous antigen processing: MHC class II peptide

presentation

Organs of the Immune System

Tonsils and adenoids

Lymph nodes

Bone marrow

Appendix

Lymphatic vessels

Lymph nodes

Thymus

Peyer’s patches

Spleen

Lymphatic vessels

Lymph nodes

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

Lymph Node

Germinal center

Vein

Cortex

Paracortex

Incoming lymphatic vessel

Outgoing lymphatic vessel

Artery

Medulla

Follicle

Cells of the Immune System Bone graft

Multipotential stem cell

Hematopoietic stem cell

Platelets

Macrophage

Erythrocytes

Eosinophil

Neutrophil

Megakaryocyte

Mast cell

Basophil

T lymphocyte

Natural killer cell

Dendritic cell

B lymphocyte

Lymphoid progenitor cell

Myeloid progenitor

cell

Monocyte

Marrow

Bone

B Cells

Plasma cell

Class II MHC and processed antigen are displayed

Antigen-presenting bacteria

Antigen

Antigen-specific B cell receptor

Antibodies B cell

Activated helper T cell

Lymphokines

Antibody

Assembled antibody molecule

Heavy chain

Antigen-binding region

Constant region

Light chain

Immunoglobulins

IgA

IgM

IgG, IgD, IgE, and IgA

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

•Tissue fluid

Agglutination

IgA 2 or 4 •Secretions (saliva, tears,

small intestine, vaginal,

prostate, nasal, breast

milk)

•Stop bacteria

adhering to host cells

•Prevents bacteria

forming colonies on

mucous membranes

IgE 2 Tissues •Activate mast cells

HISTAMINE

•Worm response

T Cells

Activated killer cell Activated helper T cell

Resting cytotoxic T cell Resting helper T cell

Activation of T Cells: Helper


Monokines

MHC Class II

T cell receptor

Antigen-presenting cell

CD4 protein

Antigenic peptide

Antigen is processed

Resting helper T cell

Class II MHC

Lymphokines

Helper T cell receptor recognizes processed antigen plus Class II MHC

Macrophage

Processed antigen and Class II MHC are displayed Antigen

Helper T cell

Activation of T Cells: Cytotoxic

Processed antigen and Class I MHC

Lymphokines

Class I MHC

Class II MHC

Processed antigen and Class II MHC are displayed Antigen

Resting helper T cell receptor recognizes processed antigen plus Class II MHC

Macrophage

Monokines

Cyto

tox

ic T

ce

ll

Infe

cte

d c

ell

MHC Class I

Antigenic peptide T cell receptor

CD8 protein

Resting helper T cell

Cytotoxic T cell

Cytotoxic T cell becomes activated

Antigen (virus)

Processed antigen (viral protein)

Cell dies

Infected cell

Activated cytotoxic T cell


Antigen is processed

Cytokines

Mature helper T cell

Monokines Lymphokines

Macrophage

Killer Cells: Cytotoxic Ts and NKs

Killer cell

Target-oriented granules

Surface contact

Target cell

Phagocytes and Their Relatives

Monocyte

Dendritic cell

Eosinophil

Neutrophil

Basophil

Mast cell

Macrophage

Phagocytes in the Body

Brain: microglial cells

Joint: synovial A cells

Precursors in bone marrow

Lymph node: resident and recirculating

macrophages

Blood: monocytes

Kidney: mesangial

phagocytes

Spleen: macrophages

Liver: Kupffer cells

Lung: alveolar

macrophages

Complement

C9

Enzyme

C2

C5 C3b

C3a C3

C4 Antigen

IgG

C1 C8

C7

C6

C5b

C5b

C5a

Mounting an Immune Response

Complement

Virus

Killer cell

B cell

Antibodies

T cell

Lymphokines

Macrophage

YOUR ACTIVE IMMUNE DEFENSES

INDUCTION OF AN IMMUNE RESPONSE

Foreign invaders - viruses, bacteria, allergens, toxins and parasites- constantly bombard our body.

Innate Immunity - invariant (generalized)

- early, limited specificity

- the first line of defense

Adaptive Immunity - variable (custom)

- later, highly specific

- ‘‘remembers’’ infection

The innate immune response:

• Can be activated 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

Field’s Virology, Fifth Edition

Initiation of Immune Responses


Activation of INFs and Cytokines by Viral Infection

Recognition of Foreign Nucleic Acids

Toll-like Receptor Signaling

Assembly of MyD88/IRAK complex

Lin and Wu, Nature 2010

Toll-like Receptors Recognition Patterns

Toll-like Receptor Pattern Recognized

TLR1 Triacyl lipoproteins

TLR2 Lipoproteins, viral glycoproteins, gram-positive

peptidoglycans

TRL3 double-stranded RNA

TLR4 Lipopolysaccharids, viral glycoproteins

TLR5 Flagellin

TLR6 Diacyl lypoproteins

TLR7 Single-stranded RNA

TLR8 Single-stranded RNA (siRNA)

TLR9 CpG DNA, unmethylated CpG oligonucleotides

TLR10 Unknown

TLR11 profilin

Recognition of RNA by RigI and Mda5

Toll-like Receptors Recognition Patterns

Toll-like Receptor Pattern Recognized

TLR1 Triacyl lipoproteins

TLR2 Lipoproteins, viral glycoproteins, gram-positive

peptidoglycans

TRL3 double-stranded RNA

TLR4 Lipopolysaccharids, viral glycoproteins

TLR5 Flagellin

TLR6 Diacyl lypoproteins

TLR7 Single-stranded RNA

TLR8 Single-stranded RNA (siRNA)

TLR9 CpG DNA, unmethylated CpG oligonucleotides

TLR10 Unknown

TLR11 profilin

Three Primary Classes of Cytokines

Function Members Activity

Proinflammatory IL-1, TNF-α, IL-6, IL-12 Activation of leukocytes

Anti-inflammatory IL-10, IL-4, TGF-β Supress activity of pro-

inflammatory cytokines

Chemokines IL-8 Early stages rectrutiment

of immune cells

IFn-g is induced only when certain lymphocytes are

stimulated to replicate and divide after binding a foreign

antigen

IFn-a and IFn-b are induced by viral infection of any

cell type

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 state.

• 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

symptoms such as fever, chills, nausea, etc.

Interferons

Interferon induced antiviral responses:

• Both viral and cellular protein synthesis stops in IFN

treated cells.

• This is due 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.

• RNase L concentration increases 10-1,000 fold after

Ifn treatment, but is inactive unless 2’-5’-oligo(A)

synthetase is produced.

• 2’-5’-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.

Interferon induced antiviral responses:

Interferon-induced Repression of Translation

Viral dsRNAs Regulate PKR Activity

Virus

RNA

Effect on PKR

Adenovirus

VA RNA I

Inhibition of PKR

Epstein-Barr

EBER-1

Inhibition of PKR

HIV-1

TAR RNA

Activation and

inhibition of PKR

Human hepatitis

virus

Human hepatitis agent

Inhibition of PKR

Reovirus

Reovirus S1 gene, 1463

nucleotide mRNA

Activation of PKR

T-cell leukemia

virus type 1

(HTLV-I)

Rex-RE RNA

Activation and

inhibition of PKR

Viral Proteins Inhibit PKR

Virus

Protein

Effect on PKR

Hepatitis C

virus

E2 protein

Pseudosubstrate inhibition of PKR

Hepatitis C

virus

Viral nonstructural 5A

protein (NS5A).

Inhibition by direct binding to PKR

HIV-1

Tat

Inhibition by dsRNA-dependent and independent mechanism; pseudosubstrate inhibition of PKR

Parapox virus

Parapox virus orf OV20.0L

gene product

Inhibition by binding to PKR

Porcine group

C rotavirus

NSP3 protein

Binding to dsRNA and inhibition of

PKR

Reovirus

Sigma3 virion outer shell

protein

Binding to dsRNA and inhibition of

PKR

Swinepox

virus

C8L gene product


Vaccinia virus

E3L gene product, pE3

Inhibition by binding to dsRNA and to

PKR

Vaccinia virus

K3L gene product, pK3


Multiple Protein Families Possessing dsRBMs Motifs

Representative Protein Function

PKR Interferon-induced kinase

Antiviral responses

ADAR2, ADAR1 pre-mRNA editing deaminase

Potential viral defense

Dicer, RNase III RNA interference, RNA-nuclease activity

Staufen mRNA trafficking

RNA helicase A RNA and DNA helicase activity

TRBP PKR inhibitor, TAR RNA-binding;

a DICER co-factor

E3L PKR inhibitor

NF90 RNA metabolism

Drakesmith and Prentice, 2008

Iron Transport in Humans

Drakesmith and Prentice, 2008

Iron Homeostasis in Humans

Activation of Heme Oxygenase (HO)-1 and Ferroportin (FPN) in

Marophages

Beaumont,. Haematologica 2010

HO-1 Effects on Microbial and

Viral Infections

Chung, Hall and Perrella, Cell Mirobiol. 2009

Apoptosis

• Controlled cell death

• Uses regulators to ensure cells die off – Regulators include various

proteins that either inhibit or promote certain parts of the caspase cascade.

– Caspases (which originally exist as procaspases) which act as proteases and initiate the caspase cascade which causes cell death.

Blebbing

Mechanisms of Apoptosis Activation

Viral inhibition of apoptosis

Other Intrinsic Antivirail Responces

•Autophagy Formation of specialized membrane

compartments related to lysosomes

•Epigenetic silencing Defense against DNA

containing viruses, formation of chromatin structure

•RNA silencing Sequence-specific RNA

degradation

•Cytosine Deamination (APOBEC) C’s to U’s

conversion

•TRIM Proteins Targeting capsid protein by

TRM5a protein

•Tetherin Inability of the virions to bud

Nitric oxide synthase

Directs synthesis of NO in NK cells

Cytotoxic

Inhibits poxvirus and herpesvirus replication

Ubiquitin-proteosome pathway components

Proteins tagged with ubiquitin are targeted to the proteosome for

degradation

Other Intrinsic Antivirail Responces-cont.

Retrovirus Capsid Sensing by TRIM5

Pertel et al., Nature 2011

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 hematopoietic lineage differentiation in mammals

•Leaf and flower development in plants

•Majority of miRNAs are transcribed independently

•Some (quarter) miRNA are derived from intrones

•miRNA are conserved

Formation of RISCs and Other Silencing Complexes

Pratt and McRae, JBC, 2009

Examples of Metazoan miRNAs

Maturation of miRNA

Plants Metazoa Animals

Cleavege

with Drosha

Additional

Cleavage

with Dicer

Complex with

RISC (RNA-induced

Silencing complex)

Actions of Small Silencing RNAs

mRNA celavage Translational repression Transcriptional silencing


Regulation of Natural Killer (NK) Cells


Activation of Adaptive by Innate Immune Responces


Cytokine-mediated Immune Responces

• 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.

The adaptive immune response:

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.


Antibody Activities in Viral Infection


Maturation of CD4+ and CD8+ T cells

Lymphocyte Subsets

TCR

a:b

Dominant T-cells Responding to Viral Infection

CD8+ recognize MHC I viral pepide complex

CD4+ recognize MHC II viral peptide complex,

regulate B- cell differentiation and inflammation

TH1 produce antiviral cytokines (IFNg)

TH2 produce cytokines for allergic response (IL-4, IL-5)

TCR g:

“Innate-like” effectors cells

express CD3 but not CD4 or CD8

recognize products of stressed cells

NKT

“innate-like” immune effects early in the immune response

express CD4, but not CD8

Treg

Control T- and B-responses

Express CD4 and CD25

Usually suppress T-and B-cell responces

• 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.

Cell-mediated Response

Antigen Receptors

Killer cell

Infected cell Antigen-presenting cell Antigen-presenting cell

CD8 protein

Cell membrane MHC

Class I MHC Class I

Antigenic peptide

T cell receptor

CD4 protein

Cell membrane MHC

Class II

Antigenic peptide

T cell receptor

Cell membrane

Antigen

Antigen-specific receptor

Helper T cell B cell

Regulatory T Cells

Regulatory T cells

Mature dendritic

cell

Regulatory T cell

Proliferation

T cells compete for cytokine signals

T cells compete for same antigen

Cytotoxic T cell

Questions:

[email protected]

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