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
Bacteria Parasite in red blood cell

SARS virus Fungus

 Markers of Self

Epithelial Muscle cell

cell

Leukocyte

Nerve
cell

Class I MHC self-marker protein

 Markers of Non-Self
Bacteria SARS virus

Antigen Epitope

Antibody

Non-self nerve cell Non-self leukocyte

Antigen Epitope
Class I MHC protein

Antibody
 Markers of Self:
Major Histocompatibility Complex
Antigenic Antigenic Antigenic
peptide peptide peptide
Viral
infection
MHC MHC MHC
Class I Class I Class II

Antigen-presenting cell
Infected cell uses MHC Class I or II

Cell
membrane
 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.
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
Exogenous antigen processing: MHC class II peptide
presentation
 Organs of the Immune System

Tonsils and adenoids

Lymph nodes
Lymphatic vessels

Thymus
Lymph nodes

Spleen

Peyer’s patches
Appendix
Lymph nodes
Bone marrow Lymphatic vessels
 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

Incoming lymphatic vessel

Germinal
center

Follicle
Paracortex

Cortex
Medulla

Outgoing lymphatic vessel

Vein
Artery
Cells of the Immune System
Bone graft
Macrophage
Mast cell

Eosinophil
Marrow Erythrocytes
Basophil

Monocyte Megakaryocyte
Bone
Hematopoietic
stem cell

Multipotential
stem cell Myeloid
progenitor Neutrophil
cell
Platelets

Lymphoid progenitor cell

T lymphocyte Dendritic cell

B lymphocyte
Natural killer cell
 B Cells

Antigen-specific Class II MHC and

B cell receptor processed antigen
are displayed

Antigen

B cell Antibodies

Lymphokines Plasma cell

Antigen-presenting Activated
bacteria helper T cell
 Antibody
Heavy chain

Light chain

Antigen-binding
region

Constant region

Assembled antibody molecule

Immunoglobulins

IgG, IgD, IgE, and IgA

IgA

IgM
Type Number of Site of action Functions
ag binding
sites
IgG 2 •Blood •Increase macrophage
•Tissue fluid activity
•CAN CROSS •Antitoxins
PLACENTA •Agglutination

IgM 10 •Blood Agglutination

•Tissue fluid

IgA 2 or 4 •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
 T Cells
Resting helper T cell Resting cytotoxic T cell

Activated helper T cell Activated killer cell

 Activation of T Cells: Helper
Antigen is Processed antigen
processed and Class II MHC
Antigen are displayed

Macrophage

Class II MHC Helper T cell receptor

recognizes processed
Monokines antigen plus Class II
MHC
Antigen-presenting cell
Resting helper T cell

Lymphokines

MHC Class II
Activated helper T cell
Antigenic peptide
T cell receptor

CD4 protein

Helper T cell
 Activation of T Cells: Cytotoxic
Antigen is Processed antigen
processed and Class II MHC
Antigen are displayed

Macrophage

Class II MHC Resting helper T cell

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

Lymphokines
Activated helper T cell

Class I MHC
Processed antigen and Class I MHC
Infected cell Cytotoxic T cell
becomes activated
Antigen (virus)
MHC Class I CD8 protein
Cytotoxic T cell

Activated
Infected cell

cytotoxic T cell

Processed antigen
(viral protein)

Cell Cytotoxic T cell

dies
Antigenic peptide T cell receptor
 Cytokines

Lymphokines Monokines

Mature helper T cell Macrophage

Killer Cells: Cytotoxic Ts and NKs

Killer cell Target cell

Target-oriented
granules
Surface contact
 Phagocytes and Their Relatives

Monocyte

Eosinophil

Mast cell

Macrophage Dendritic cell

Neutrophil

Basophil
 Phagocytes in the Body
Brain:
microglial cells

Lung:
alveolar
macrophages
Liver:
Kupffer cells Spleen:
macrophages
Kidney:
mesangial Blood:
phagocytes monocytes
Lymph node:
resident and
Precursors in bone
recirculating
marrow
macrophages

Joint:
synovial A cells
 Complement

C2 C3 C3a C5a C7

C1
C6 C8
C5b
IgG

C5b
C4 Enzyme C3b C5
Antigen
C9
Mounting an Immune Response

Lymphokines Complement
T cell
Antibodies Macrophage
B cell

Killer cell
Virus
reign invaders - viruses, bacteria, allergens, toxins and
rasites- constantly bombard our body.
YOUR ACTIVE IMMUNE DEFENSES

Innate Immunity Adaptive Immunity

- invariant (generalized) - variable (custom)
- early, limited specificity - later, highly specific
- the first line of defense - ‘‘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
Initiation of Immune Responses

Field’s Virology, Fifth Edition

Activation of INFs and Cytokines by Viral Infection

Field’s Virology, Fifth Edition

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
 Interferons

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.
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.
 Interferon induced antiviral responses:
• 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 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 Human  hepatitis agent Inhibition of PKR

virus
Reovirus Reovirus S1 gene, 1463 Activation of PKR
nucleotide mRNA

T-cell leukemia Rex-RE RNA Activation and

virus type 1 inhibition of PKR
(HTLV-I)
 Viral Proteins Inhibit PKR
Virus Protein Effect on PKR

Hepatitis C E2 protein Pseudosubstrate inhibition of PKR

virus
Hepatitis C Viral nonstructural 5A Inhibition by direct binding to PKR
virus protein (NS5A).
HIV-1 Tat Inhibition by dsRNA-dependent and
independent mechanism;
pseudosubstrate inhibition of PKR
Parapox virus Parapox virus orf OV20.0L Inhibition by binding to PKR
gene product
Porcine group NSP3 protein Binding to dsRNA and inhibition of
C rotavirus PKR
Reovirus Sigma3 virion outer shell Binding to dsRNA and inhibition of
protein PKR
Swinepox C8L gene product Pseudosubstrate inhibition of PKR
virus
Vaccinia virus E3L gene product, pE3 Inhibition by binding to dsRNA and to
PKR
Vaccinia virus K3L gene product, pK3 Pseudosubstrate inhibition of PKR
 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

Iron Transport in Humans

Drakesmith and Prentice, 20

Iron Homeostasis in Humans

Drakesmith and Prentice, 2008

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
Other Intrinsic Antivirail Responces-cont.
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
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

Field’s Virology, Fifth Edition

Activation of Adaptive by Innate Immune Responces

Field’s Virology, Fifth Edition

Cytokine-mediated Immune Responces

Field’s Virology, Fifth Edition

 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.
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
Antibody Activities in Viral Infection

Field’s Virology, Fifth Edition

Maturation of CD4+ and CD8+ T cells

Field’s Virology, Fifth Edition

 Lymphocyte Subsets
TCR Dominant T-cells Responding to Viral Infection
a:b 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
 Cell-mediated Response

• 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.
 Antigen Receptors

B cell Killer cell Helper T cell

Antigen-
specific T cell T cell
receptor receptor receptor
CD8 CD4
Antigen protein Antigenic protein Antigenic
peptide peptide
Cell Cell Cell
membrane MHC membrane MHC membrane MHC
Class I Class I Class II

Antigen-presenting cell Infected cell Antigen-presenting cell

 Regulatory T Cells
T cells compete T cells compete for
for same antigen cytokine signals

Cytotoxic
T cell

Mature
dendritic
cell

Regulatory
T cells
Regulatory
T cell

Proliferation
 Questions:
snekhai@howard.edu