Immunity to viruses

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Immunity to viruses Nov 30, 2009
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Immune effector modules T cells activate
discrete cell populations
Extracellular bacteria and fungi
Facultative and obligate intracellular organisms
Helminths and biting insects
Regulation of tissue damage in chronic infection
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Viruses
Obligate intracellular organisms Bypass barriers
- insects vectors, animal bites, trauma,
ulcerations Exploit mucosal M cells Co-evolution
with receptors drives narrow host
specificity Viremia needed to seed organs
required for transmission - kidneys (urine),
skin, salivary glands (secretions), digestive
tract (feces)
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Viral Life Cycle 1. Breach barriers 2.
Disseminate via lymph nodes 3. Viremia to seed
target organs 4. Shedding to new hosts
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Immune cells make good targets
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Viruses
Flavors ssRNA, dsRNA, DNA Encoded within
virally encoded capsid proteins Enveloped or
not Classes Lytic (cytopathic) (polio, flu)
versus nonlytic (hepatitis B, LCMV) Latency
special property of some lytic viruses
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Key players interferons Issacs and Lindemann,
Proc R Soc London B Biol Sci 147258-67, 1957
Type 1 interferons interferon-?/interferon-?
(14) Type 2 interferon interferon-? Hybrid
interferons interferon-? (3) IL-28A, IL-28B,
IL-29
Auto-enforcing loop IRF-3 gt IFNb gt
Stat1/2 IRF-9 gt IRF7 gt IFNas
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Amplification by Type 1 interferons
IFNAR
IRF-3
IFN-b
IRF-3

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Amplification by Type 1 interferons
IFN-b
IFNAR
IRF-3
Stat 1,2,4
IFN-b
Human Stat1-deficiency lethal viral infection
IFN-b
IFNa4
IRF-3
PKR
RNAseL

IFNa1
IFNa2
IFNa5
Anti-Viral State
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Auto-amplification in the Type 1 interferon
response
Tyk2
JAK1

IRF-3
Stat 2
Stat1
P
Stat1
Stat1
P
Stat1
Stat2
CBP/p300
P
GAS
IRF-9
P
NF-kB
IRF-1
IFN-b
ISRE

IRF-E
ISRE
PRD
NF-kB
ISG15ISG54IP-10
iNOS
IFN-a
IRS-7 PKR OAS

PRD-LE
P
IRS-7
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IFN-a/b
IFNAR
Induced Synthesis
2-5(A) Synthetase
dsRNA-dependent protein kinase, PKR
(inactive)
dsRNA
ATP
ATP
2-5(A)
dsRNA
PKR, phosphorylated (active)
RNaseL RnaseL (inactive)
(active)
eIF2 a, eIF2 a Phosphorylated
ATP
AUG AAAAA
GEF
Degraded mRNA
eIF2-GTP, Phosphorylated
mRNA
eIF2-GDP, phosphorylated
KO with increased viral susceptibility
Inhibition of Protein Synthesis
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TRIF/TRAM Anti-viral TLR Adapters
TLR 3, 7, 9 TLR 4
TLR 1, 2, 4, 6
TLR 5, 7, 9
NF-kB JNK
NF-kB JNK AP-1 IRF-3
Interferons, RANTES
IL-1, TNF, IL-6, IL-8, antimicrobial peptides
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Cytosolic dsRNA detectors - RNA helicases
Cell 122645-7, 2005
dsRNA (5-triP-ssRNA for flu) binds cytosolic
RIG-I and/or Mda5, exposes CARD domain, binds
MAVS/Cardif/IPS-1, activates kinase complexes
leading to phosphorylation of IRF-3/IRF-7 and
I?B?. MAVS targeted by HCV.
Seth RB et al., Identification and
characterization of MAVS, a mitochondrial
antiviral signaling protein that activates NF-?B
and IRF3. Cell 122669-82, 2005.Kawai T et al.,
IPS-1, an adaptor triggering RIG-1 and
Mda5-mediated type 1 interferon induction.
Nature Immunol 6981-8, 2005.Meylan E et al.,
Cardif is an adaptor protein in the RIG-1
antiviral pathway and is targeted by hepatitis C
virus. Nature 4371167-72.
NOTE Dispensable in plasmacytoid DCs
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Cytosolic RNA/DNA recognition pathways
ASCInflammasome
AIM2
NF?B
DAI (DNA-dependent activator of IFN-regulatory
factors)
DNA
STING
TBK1IRF3
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Innate response to viruses - nucleic acid
recognition
Pichlmair and Reis e Sousa, Immunity
27370, 2007
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Plasmacytoid DCs
Produce early IFN?/? after incubation of PBMC
with virus (independent of RNA helicase
pathway) Prevalence 0.1-0.3 PBMC Phenotype
CD4, CD11c-, IL3R, CD62L Biology Migrate to
HEV and protect transiting naïve
lymphocytes
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TLRs activate the virus recognition response
early in pDCs
Plasmacytoid DC
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pDCs use components of autophagy pathway for TLR7
ssRNA detection
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IFN? activates anti-viral cellular miRNAs
Activation by IFN? is time- and dose-dependent
Expression of IFN?-inducible miRNAs inhibits
viral replication
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Defects in UNC-93B abolish TLR3, 7, -8, -9
signaling against viruses. Abolishes
cross-priming. Human mutations in UNC-93B and
dominant-negative TLR3 associated with HSV
encephalitis.
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Innate response to viruses - protein and nucleic
acid recognition
Pichlmair and Reis e Sousa, Immunity
27370, 2007
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Anti-viral Cytokines
Type 1 interferons Anti-viral NK
cytotoxicity T cell survival, DC
maturation IL-6 Systemic response Cell
recruitment IL-12, IL-18 Type 1
immunity NK cell activation IL-15 CTL
and NK growth, survival
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NK Cells and Anti-Viral Host Immunity
Mechanism IFN-a/b Cytotoxicity,
Anti-viral IL-12, IL-18 Cytokines
(IFN-g, TNF, LT, TRAIL, TWEAK) Antibody
ADCC
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NK cells target herpesviruses
Genetic Evidence 'NK-deficient' girl with
severe primary herpesvirus infections Murine
klra8 (Ly-49H) deficiency Unable to clear
murine CMV Human SAP (SLAM-assoc. prot.)
deficiency Loss of functional 2B4 (CD244) NK
activating receptor with fatal EBV infections
(also no NKT cells)
Caution only 'pure' NK cell deficiency in
humans is bone marrow reconstituted ?c- and
JAK3-deficient patients frequently reconstitute T
and B cells but not NK cells over long-term
follow-up and have no overt immunodeficiency.
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Direct recognition of murine CMV-encoded proteins
by NK receptors
Suggests NK receptor diversity may be driven by
herpesviruses
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Direct recognition of murine CMV-encoded proteins
by NK receptors
NK cell
Ly49P
H-2Dk/MCMV
MCMV-infected cells
Desrosiers M-P et al. Nature Genetics 37593-99.
Are NK cells simply antiviral T cells?
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Duncans Syndrome
Early death due to primary progressive EBV
infection. Mutation in SAP (SLAM-associated
protein), an X-linked adapter protein, or rarely
in XIAP (inhibitor of apoptosis).
2B4 ?
EBV-infected cell
SAP
NK Cell
2B4 ?
EBV-infected cell
SAP
Mutant SAP transduces negative instead of
positive signals from engaged 2B4 receptors.
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NK Cells Localize Anti-Viral Immunity
MCMV
NK
NK
Liver
IFN-g
MIP-1a
Mig
IFN-g
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Major Viral Effectors CD8 CTL
cytolysis
IFN ( )
cytolysis
0 5
10
Time (Hrs)
Virus peptide Remove peptide
Replace peptide
Dogma Non-cytopath. virus CTL Cytopath. Virus
Ab
Infectious Virions 3-10 Hrs.
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CD8 Response to LCMV
Clonal Apoptosis Memory a Burst Size


Burst (IFNg)
(perforin, IL-15, antigen)
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Peptide-specific Activation
All Tetramer-Positive CTL Have Effector
Function No Bystander
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So much time, so little space
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Antibodies Prevent Re-infection
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LCMV
Cant maintain CD8s without CD4 Help
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Viruses attack common cellular defense pathways
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Viruses block activation of cellular apoptosis
pathways
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CMV attacks MHC class I pathways at multiple
levels
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Large DNA viruses (herpesviruses, poxviruses)
encode additional proteins to mitigate host
defense and sustain infectivity
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Induction of MIC-A at foci of CMV in infected
lungs
MIC-A CMV
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NKG2D enhances cytolytic activity after TCR
engagement
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IFN-g
TNF
IL-2
IL-4
Decreasing Peptide
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Joe Bob Briggs three dead bodies, two dead
birds, multiple seagull divebomb attacks,
playground crow attack, bird migration, bird
flocking, exploding gas station, two car crashes,
crow kung-fu, kamikaze seagull 4 Stars. A
classic. Check it out!
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Influenza - Obligate Virology
Orthomyxovirus Negative-sense ssRNA,
eight-segmented genome Types A (avian, humans,
responsible for pandemics), B (avian, humans,
seals), C (avian, pigs, humans rare) 10
proteins PB1(-F2), PB2, PA Heterotrimeric
polymerase (?mitochondrial apoptosis) HA Ho
motrimeric binding and fusion element NA Homot
etrameric enzymatic release factor NP Nucleopro
tein (nucleocapsid packaging) M1 Transport of
viral RNPs M2 Homotetrameric cation channel
pore NS1 Binds RNA interdicts host
translational machinery and defense (PKR,
cytokines) NS2 Nuclear export of viral RNPs
Neuraminidase inhibitor target Amantadine
target
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Relevant Life Cycle Issues
1. An intestinal infection of wild waterfowl. 2.
Crosses to mammals through close contact. 3.
Multiple crosses enhance capacity to establish
mutants and reassortment variants
adapted to mammalian hosts. 4. HA species
specificity sialic acid a-2,3 galactose
linkage (avian intestine) sialic acid a-2,6
galactose linkage (human trachea) both (pig
trachea) 5. NA compatibility human viruses gain
a-2,6 activity stalk length (longer NA
enhances activity in humans) 6. HA, NA
Adaptations HA glycosylation HA1/HA2 fusion
domain (expanded basic amino acid repeat in
highly pathogenic chicken H5/H7/H9 flu -HPAI-
enhances spectrum of proteases that can
activate HA fusion event may explain
pathogenicity of co-infection with bacteria)

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Mutation and reassortment drive influenza A
epidemics and pandemics
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Asian Live-Animal Markets - The Great Zoonotic
Mixer
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Influenza Pandemics
Year Common Name Subtype
Origin Deaths
1889 - H2N2 ?Europe 6
million 1898 - H3N2 ?Europe 0.5
million 1918 Spanish Flu H1N1 ?Eurasia
40 million 1957 Asian Flu H2N2 China
4 million 1968 Hong Kong Flu
H3N2 China 2 million 1977
Russian Flu H1N1 China/Russia
? 2009 Swine Flu H1N1 N.
America ?
Contained elements from avian viruses
Laboratory-derived from frozen stock (persons
pre-50s immune) Antigenic variants continue to
co-circulate
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Relevant Immunology
Innate immunity type 1 IFNs, TNF-a, Mx
proteins HA antibodies Neutralize infectivity,
protective NA antibodies Restrict viral
spread Cytotoxic CD8 T cells M2, PB2, HA, NP
specificity common M2 specificity almost
universal
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The Most Common Human TCR in the World
CD8 TCR a/b chains Vb17/Va10.2
Influenza A Matrix Protein amino acids
58-66
HLA-A2 (A0201)
Stewart-Jones et al. Nature Immunol 7657, 2003
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Antigenic Drift - 2003/04
HA1 A/Panama/2007/99 HA1
A/Fujian/411/2002
Treanor, NEJM 350218, 2004
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Influenza NS1 protein sequesters viral ss RNA to
block cellular anti-viral defense
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Why do they die?
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Verified H5N1 influenza through October 2006

Human deaths/cases 152/256 (59)
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Verified H5N1 influenza in birds since 2003

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Verified human H5N1 influenza in 2007

Mortality 69
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A new pandemic influenza virus, H1N1/09
USA estimates 22 million infected, 3900 deaths

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Human Immunodeficiency Virus
Worldwide 35 million infected 29
million dead 14,000 new infections/day
2/3 infected persons in Africa U.S.
1 million infected including 400,000 dead
(appeared 1983)
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Worldwide Estimates of Numbers of HIV-Infected
Persons
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HIV Origins - Primate Lentiviruses
SIVcpz - West equatorial Africa M
group Cameroon N group Gabon O
group HIV-2 SIVsm (sooty mangabey)
Infection/Disease in areas of active bushmeat
trade.
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HIV Origins
SIVcpz - Asymptomatic infection of chimpanzees
(up to 1 in areas of west Central Africa) HIV-1
M group consists of 11 clades Last
common ancestor entered human population around
1890 ( 30 yrs)
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Prevalent HIV clades
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HIV is a primate lentivirus
Lentiviruses can infect nondividing
cells Replication driven from long terminal
repeats Structural genes - gag, pol,
env Regulatory genes - tat, rev Accessory genes -
vif, vpr, vpu, nef
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HIV life-cycle
APOBEC
TRIM5a
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HIV vif sequesters APOBEC enzymes from the
budding virions
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HIV Pathogenesis
1. Entry at sites of M cells or trauma (STDs)
M
2. Transit to LN via C-type lectins on
dendritic cells
DC-SIGN, MR, Langerin
DC-SIGN
3. Peak CD4 T cell infection days 4-7
4. Viremia peaks day 14
5. All lymphoid tissues infected by day 23
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HIV infection occurs predominantly at
mucosa Dendritic cells mediate transit of virus
to regional lymph nodes via CLRs Massive loss of
mucosa-associated lymphocytes of the small
intestine precedes systemic CD4 T cell loss
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Proposed epithelial damage mediates sustained
activation of mucosal T cells after HIV infection
Brenchley, Price Douek, Nat Immunol 7235, 2006
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HIV Receptors
CD4
1o Infection M-tropic, CCR5
R5
Progressive CD4 T cell destruction CXCR4
T-tropic Syncytium-forming
X4
Turnover 1010 virions/day
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Natural History of Untreated HIV Infection
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Natural HIV Resistance

• 1. CCR5D32 - slow progression if infected
• 20 W. European Caucasians heterozygous
• 1 homozygous
• HLA class I homozygosity - rapid progression
• Rare HLA class I alleles - slow progression
  (suggests virus near mutational threshold)

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Why no HIV vaccine?

• Escape variants/altered peptide ligands - virus
  operates near mutational threshold
• Neutralizing antibodies low-affinity, arise late
  (conformationally hidden, glycan shielding,
  mutational escape, evolutionary escape from
  natural antibodies, polyclonal B cell
  activation may impede)
• Loss of CD4 help required for CD8, antibody
  responses
• Immune exhaustion with PD-1 expression on CD4 and
  CD8 anti-HIV T cells

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The End