Transcript Kurt-Jones, Evelyn A. et al. (2004) Proc. Natl. Acad. Sci. USA 101

Introduction to Viral Immunology
Part I
Dr. David J. Topham
Portions adapted from Dr. Colin R.A. Hewitt
References/Texts
•
Principles of Virology: Molecular Biology, Pathogenesis, and Control
Author(s)/Editor(s): S.J. Flint, L.W. Enquist, R.M. Krug, V.R. Racaniello, and
A.M. Skalka ASM Press, December 2003
– Chapter 14
•
Immunobiology:The Immune System in Health and Disease (6th Edition)
Authors/Editors: C.A. Janeway, Jr., P. Travers, M. Walport, M.J. Schlomchik
Garland Science Publishing, 2005.
– Chapters 2, 5, 10
•
Viruses and Human Diseases
Authors: James H. Strauss and Ellen G. Strauss. Elsevier.
Outline
• Innate mechanisms of antiviral immunity
– Toll like receptors
– Type I interferons
– Natural Killer cells
• Antigen-specific recognition of virus and virus
infected cells
– Antigen processing and presentation
– Dendritic cells
– Cytotoxic CD8 T cells and Helper CD4 T cells
Preventing infection
• Physical barriers
– Skin, mucosa
• Neutralization
– Antibodies
• requires immunization
• Limit replication
– Interferons (Type I and Type 2)
– Defensins
•
MULTIPLE ROLES OF ANTIMICROBIAL DEFENSINS, CATHELICIDINS, AND EOSINOPHIL-DERIVED NEUROTOXIN IN HOST
DEFENSE De Yang, Arya Biragyn,DavidM. Hoover,Jacek Lubkowski, and Joost J. Oppenheim Annu. Rev. Immunol. 2004. 22:181–215
• Mostly restricted to bacterial and fungal pathogens
Recognition of a virus infection
• Toll like receptors (TLR)
– TLR 2, 3, 7, 9
– MyD88, MAL/TIRAP,TRIF, TRAM
• NK Receptors
– Activation and Inhibitory receptors
• ITAM and ITIM intracellular signaling motifs
• Dendritic cells
– Linking innate and adaptive immune responses
• Antigen processing and presentation of viral
peptides
– Class I and class II MHC
Toll Like Receptors
Innate immunity to viruses
• Pattern recognition
– Recognize conserved molecular structures
unique to pathogens
• Germline encoded
• Expressed on a variety of immune and
non-immune cells
• Instruct Dendritic Cells (DC)
Phylogenetic tree of murine TLRs and
their representative ligands.
RSV?
Herpesvirus
glycoproteins
Viral DNA
Murine TLR family members are aligned according to their amino acid
structures. Because mice do not have TLR10, human TLR10 is displayed in the
figure. Their representative ligands are also shown with dotted arrows.
Signaling through Toll like receptors
Signaling through Toll like receptors
• TIR-Toll/Interleukin-1 Receptor
• *MyD88-myeloid differentiation factor 88
• MAL/TIRAP-MyD88 Adaptor-like/TIR-associated protein
• *TRIF-Toll Receptor Interferon Factor
• TRAM-Toll Receptor Associate Molecule
Specificity in the response is mediated through adaptor proteins
The pattern of TLR expression
by DC’s depends on the type of
DC and its development
ssRNA viruses are recognized by TLR7
pDC response to VSV
BM DC response to influenza
Lund, J. M., Alexopoulou, L., Sato, A., Karow, M.,
Adams, N. C., Gale, N. W., Iwasaki, A., and Flavell, R.
A. (2004). Recognition of single-stranded RNA viruses
by Toll-like receptor 7. Proc Natl Acad Sci U S A
101(15), 5598-603.
Recognition of RNA viruses by TLR7 requires endosomal
acidification
WT bone marrow cells were pretreated for 2 h with media alone or chloroquine at the indicated
concentrations. (A) After the addition of 5 x 106 pfu/ml VSV, cells were cultured for an additional 18 h,
and supernatants were collected and IFN levels were measured by ELISA
TLR3,7,8, & 9 ligand recognition occurs in endosomes
Lund, Jennifer M. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 5598-5603
Copyright ©2004 by the National Academy of Sciences
Tlr9 deficient, MyD88-/-, and Tlr3-/- mice are
hypersusceptible to viral infections
TLR3–/–
TLR9–/–
Tabeta, Koichi et al. (2004) Proc. Natl. Acad. Sci. USA 101, 3516-3521
Copyright ©2004 by the National Academy of Sciences
TLR3 mediates response to dsRNA
TLR3 transfected cells
RAW264 M
Figure 1 TLR3 specifically signals for NF-B activation in response to poly(I:C). a, 293T cells
were transiently transfected with 50 ng of human TLR3, TLR2 or empty pcDNA3 vector
together with an NF-B luciferase reporter. Luciferase activity in cells treated with 25 µg ml-1
poly(I:C) or 10 µg ml-1 PGN or untreated (media) cells was measured. b, Luciferase activity in
CaCo-2 cells transiently transfected with 500 ng of empty vector or TLR3 DNA, together with
200 ng NF-B luciferase reporter and stimulated with 25 µg ml-1 poly(I:C). c, 293T cells
transiently transfected with expression vector for TLR3 or empty vector, together with NF-B
luciferase reporter and, where indicated, 1 µg of dominant negative (DN) TLR3 or DN TLR2
DNAs. NF-B-induced luciferase activity in cells treated with 25 µg ml-1 poly(I:C) or untreated
cells was measured. d, Transfection of RAW 264.7 macrophages with a NF-B luciferase
reporter. Luciferase activity in cells treated with 20 µg ml-1 poly(I:C), poly(A:U), poly(C) or
poly(dI:dC), or untreated cells.
Nature 413, 732 - 738 (18 October 2001) Recognition of
double-stranded RNA and activation of NF-B by Toll-like
receptor 3 LENA ALEXOPOULOU, AGNIESZKA
CZOPIK HOLT, RUSLAN MEDZHITOV & RICHARD A.
FLAVELL
Cells from TLR3 deficient mice have impaired responses
to synthetic and viral dsRNA
Figure 3 Impaired responses to poly(I:C) from TLR3-/cells. a, Bone-marrow-derived macrophages (M) or total
splenocytes (SP) from wild-type or TLR3-/- mice were
stimulated with poly(I:C) or LPS for 24 h, and
concentrations of IL-6, IL-2 p40/p70 and TNF- in the
culture supernatants were measured by ELISA. Data are
representative of three independent experiments. b, Bonemarrow-derived macrophages from wild-type or TLR3-/mice were stimulated with 100 µg ml-1 poly(I:C), 10 µg ml1 LPS, 10 µg ml-1 PGN, 100 µg ml-1 zymosan or
10 µg ml-1 LTA for 24 h, and concentrations of IL-6 in the
culture supernatants were measured by ELISA. Data are
representative of two independent experiments. UD,
undetected. c, Splenocytes were cultured for 24 h with
100 µg ml-1 poly(I:C), 5 ng ml-1 LPS or 30 µg ml-1 viral
dsRNA, or left untreated. Cells were collected, stained and
analysed by flow cytometry. Histograms show expression
levels of CD69, CD86 and CD80 in B220+ (B cells) gated
lymphocyte populations. d, Bone-marrow-derived
macrophages from wild-type or TLR3-/- mice were
stimulated with 20 µg ml-1 poly(I:C) or 5 ng ml-1 LPS. At
the indicated points, cells were lysed; nuclear translocation
of NF-B was visualized by EMSA, and degradation of I-B
was analysed by western blot. e, Bone-marrow-derived
macrophages from wild-type or TLR3-/- mice were
stimulated with 100 µg ml-1 poly(I:C) or 1 µg ml-1 LPS, or
left untreated. After 4 h, total RNA was isolated and
expression of IFN-, IFN- and HPRT was determined by RTPCR
Nature 413, 732 - 738 (18 October 2001) Recognition of
double-stranded RNA and activation of NF-B by Toll-like
receptor 3 LENA ALEXOPOULOU, AGNIESZKA
CZOPIK HOLT, RUSLAN MEDZHITOV & RICHARD A.
FLAVELL
dsRNA/TLR3 signals via the Toll like receptor pathway
Requires:
MyD88
TRAF6
Tollip
Nature 413, 732 - 738 (18 October 2001) Recognition of
double-stranded RNA and activation of NF-B by Toll-like
receptor 3 LENA ALEXOPOULOU, AGNIESZKA
CZOPIK HOLT, RUSLAN MEDZHITOV & RICHARD A.
FLAVELL
HSV activates cells through TLR2
Transfected HEK293 cells
PEC
(A) HEK293 cells expressing human TLR2,
TLR3, or TLR4 MD2 were transfected with an
NF- B-driven firefly luciferase reporter plasmid
and were stimulated for 6 h with HSV-1 (KOS
strain) at a moi of 100 or with IL-1 (100 ng ml)
as a positive control. Luciferase activity was
calculated in relative light units as a ratio of NFB-dependent firefly luciferase activity to NFB-independent Renilla luciferase activity. The
results are shown as the mean SD of triplicate
wells. Each cell line was tested in 3–10
independent experiments. (B) HEK293 cells
expressing human TLR2 or TLR9 were
challenged with HSV-1 KOS (a moi of 3–100),
CpG DNA (0.1–3 M), GpC control DNA (0.1–3
M), or medium alone. NF- B luciferase
activity was measured as above. (C) Peritoneal
exudates cells from wild-type, TLR2 / , or
TLR4 / mice were stimulated with medium
alone or with HSV-1 KOS at mois of 1, 10, and
100. IL-6 levels were measured in 16-h
supernatants. The results are shown as the
mean
SD of duplicate wells. Each mouse
strain was tested in at least three independent
experiments. (D) Wild-type, TLR6 / , or
TLR2 /
peritoneal exudates cells were
challenged with HSV-1 KOS (a moi of 100),
Pam2CSK4 (100 ng ml, a TLR2 TLR6 ligand),
or LPS (10 ng ml, a TLR4 ligand). IL-6 levels
were measured as above.
Kurt-Jones, Evelyn A. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 1315-1320
Copyright ©2004 by the National Academy of Sciences
Adult TLR2 knockout mice are resistant to lethal HSV-1
intracerebral challenge
Kurt-Jones, Evelyn A. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 1315-1320
Copyright ©2004 by the National Academy of Sciences
Neonatal TLR2 knockout mice are resistant to lethal
HSV-1 challenge
Kurt-Jones, Evelyn A. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 1315-1320
Copyright ©2004 by the National Academy of Sciences
Toll like receptors
• Innate recognition of pathogen-specific
molecular motifs
• Initiation of inflammatory response
– IL6, IFN-, IL8, IL12, chemokines, IL1, etc.
• Maturation of dendritic cells
– Promote activation of naïve T cells
– Promote Th1 like responses
• Initiate antiviral IFN- response
Limiting viral replication
• Type I interferons: IFN-, IFN-
– Direct inhibition of viral gene expression
– Activate NK cells
– Many viral evasion strategies target interferon
• Ex. Influenza NS1 protein (Influenza A Virus Lacking the NS1 Gene
Replicates in Interferon-Deficient Systems Adolfo Garcı´a-Sastre,*,1 Andrej Egorov,†,2
Demetrius Matassov,* Sabine Brandt,†,‡ David E. Levy,§ Joan E. Durbin,¶ Peter Palese,*,1
and Thomas Muster†,‡ VIROLOGY 252, 324–330 (1998)
– Promotes Th1 response
IFN-
– Released in response to TLR ligation by viral
products (ssRNS, dsRNA, glycoproteins)
– Acts in paracrine and autocrine fashion
– Inhibit viral gene expression/replication
• Jak1/Stat-1 upregulation of 2’5’oligoadenylate synthetase
which polymerizes ATP and activates an
endoribonuclease
• P1 Kinase activation causes phosphorylation of eIF2
• Induces MxA required for influenza resistance
INHIBITION OF VIRAL GENE EXPRESSION
INTERFERONS
2’,5’-oligo A synthetase
p68 Kinase
(inactive)
(n+1)ATP
vRNA
mRNA
rRNA
RNAse L
active
p68 Kinase
(active)
(2’,5’)pppA(pA)
+ nPPi
RNAse L
inactive
eIF2
eIF2-PO4
active
inactive
INHIBITION OF
TRANSLATION
INITIATION
Type I IFN Modulates Innate and Specific Antiviral Immunity
FIGURE 1. Stat1-/- mice are competent to produce influenza
virus-specific CTL. Wild-type (WT) () and Stat1-/- C57BL6/J
mice () were immunized with a sublethal dose of influenza
virus A/PR/8, and splenocytes were assayed for cytotoxic
activity against EL4 target cells either without (,  ) or pulsed
with NP366-374 peptide (,  ). Specific target cell lysis was
measured at various E:T ratios, as indicated, and results from
one representative mouse of a group of four of each genotype
are shown
Joan E. Durbin, Ana Fernandez-Sesma, Chien-Kuo Lee, T. Dharma
Rao, Alan B. Frey,Thomas M. Moran, Stanislav Vukmanovic, Adolfo
Garcia-Sastre, and David E. Levy. The Journal of Immunology,
2000, 164: 4220–4228.
Defective early control of LCMV in IFN Receptor
deficient mice
SPLEEN
LIVER
Fig. 5. LCMV viral titers in IFN-/R KO as compared with WT mice. WT and IFN-/R KO mice were infected on day 0 with
2×104 PFU LCMV Armstrong strain. Spleens and livers were harvested on 1.5,3,4.5,7, 8,9.5,11,14,21,28,or 35d after
infection, for quantitation of LCMV titers in plaque assays. Data shown are means for three mice per group +/- SEM, and the
solid lines across the graphs represent the lower limits of detection.
Two Roads Diverged: Interferon  and Interleukin 12–mediated Pathways in Promoting T Cell Interferon-g Responses during Viral
Infection Leslie P. Cousens,Ron Peterson,Sang Hsu,Andrew Dorner, John D. Altman,Rafi Ahmed,and Christine A. Biron
J. Exp. Med. Volume 189, Number 8, April 19, 1999 1315–1327
Alpha/Beta Interferon Protects Adult Mice from Fatal Sindbis Virus
Infection and Is an Important Determinant of Cell and Tissue Tropism KATE D.
RYMAN,1* WILLIAM B. KLIMSTRA,1 KHUONG B. NGUYEN,2 CHRISTINE A. BIRON,2 AND ROBERT E. JOHNSTON1 JOURNAL OF VIROLOGY, Apr.
2000, p. 3366–3378 Vol. 74, No. 7

IFNR +/+
H IFNR +/–
 IFNR –/–
Effects of IFN- on the
replication and dissemination of
Sindbis virus TR339 in vivo.
129 Sv/Ev IFN-/R+/+ mice, IFN/R+/ heterozygous mice, and IFN/R/ mice were inoculated s.c. with
100 PFU of TR339 and sacrificed at
various times p.i., and virus titers
from serum (A), brain (B), spleen
(C), and liver (D) were determined.
Values represent the geometric mean
virus titer (log10 PFU/ml or g) for
two (IFN-/R+/) or three (IFN-/R/
and 129 Sv/Ev) mice as determined
on BHK cells. Datum points are
shown ± the standard deviation
(SD), where n = 3. The lower limit
of detection is indicated (broken
line).
Impaired Antiviral Response and Alpha/Beta Interferon Induction in Mice
Lacking Beta Interferon RAJ DEONARAIN, ANTONIO ALCAMI´, MARIA ALEXIOU, MARGARET J. DALLMAN, DIRK R. GEWERT, *
1
AND ANDREW
2
1
3
4
C. G. PORTER1* JOURNAL OF VIROLOGY,Apr. 2000, p. 3404–3409 Vol. 74, No. 7
Vaccinia Virus
 IFN- +/+
 IFN- –/–
Vaccinia virus infection in IFN-+/+ and IFN-/ mice. Groups of 7- to 9-week-old IFN-+/+ (open circles) or IFN-/ mice (closed circles) were intranasally infected with 103, 104,
or 105 PFU of vaccinia virus strain Western Reserve. Every day, mice were individually weighed and monitored for signs of illness, scored from zero to four (ruffled fur,
arched backs, and reduced mobility), or death. The mean percentage weight loss of each group ± the standard error of the mean, relative to the weight immediately preceding
the infection, and the mean value of signs of illness ± the standard error of the mean in groups of mice infected with the indicated doses of virus, are shown. The horizontal
bars indicate those days in which differences were statistically significant when analyzed by Student's t test, and the P values are shown. The number of mice per group that
either died or were sacrificed due to severe infection is shown in the insets.
Cell, Vol. 93, 373–383, May 1, 1998
Disruption of the Jak1 Gene Demonstrates Obligatory and Nonredundant
Roles of the Jaks in Cytokine-Induced Biologic Responses
Scott J. Rodig,* Marco A. Meraz,*J. Michael White,* Pat A. Lampe,†Joan K. Riley,* Cora D. Arthur,*Kathleen L.
King,‡ Kathleen C. F. Sheehan,*Li Yin,* Diane Pennica,‡ Eugene M. Johnson, Jr.,†and Robert D. Schreiber*§
Figure 2. Jak1-/- Cells Are Unresponsive to Ligands that Utilize Class II Cytokine Receptors (B)
Immortalized EFs were stimulated with IFN or IFNg and cultured with vesicular stomatitis virus. After 48 hr,
surviving cells were quantitated by vital dye staining.
Type I IFN
IFN-
• Limits viral gene expression
• Makes uninfected cells resistant to
infection
• Promotes Th1/IFN-g response
• Causes attrition of memory T cells and
then promotes memory T cell expansion
Figure 2-48
Innate recognition of infected
cells by Natural Killer (NK)
cells
NK and NKT cells
• NK cells: (Biron et al. Annual Review of Immunology Apr 1999, Vol. 17: 189-220)
– CD56+ (human) NK1.1+ (mouse)
– DX5+ (CD49b/VLA-2) mouse (human?)
• NKT cells: (Bendelac et al. Annual Review of Immunology Apr 1997, Vol. 15: 535-562)
–
–
–
–
Restricted  TCR repertoire (V14, V2,7,8)
CD4+ (60%) or double negative (40%)
NK1.1+
Recognize CD1 non-classical MHC class-I-like
• Presents lipids (Porcelli et al Annu. Rev. Immunol. 1999. 17:297–329)
•
Annu. Rev. Immunol. 2004. 22:405–29 THE DYNAMIC LIFE OF NATURAL KILLER CELLS Wayne M.
Yokoyama, Sungjin Kim, and Anthony R. French
NK receptors
• Killer Ig-like receptors (KIR)
• Lectin-like receptors (CD94/NKG2, NKG2D, Ly49)
• ITIMs: immunoreceptor tyrosine-based inhibitory motifs
• ITAMs: immunoreceptor tyrosine-based activation motifs
– Also present on adaptor proteins that bind to ITIM-less receptors,
ex. DAP12
Inhibitory
NK cell
receptors
Human NK Receptors
Non-inhibitory
Recognition of normal cells by NK cells
Figure 2-50 part 1 of 2
Recognition of abnormal cells by NK’s
Figure 2-50 part 2 of 2
Missing-self hypothesis
• Downregulation of MHC molecules on
infected cells triggers NK cell lysis
• Dependent on absence of MHC
signaling through NK inhibitory
receptors
NK cell recognition
of targets for killing
reflects a balance of
signals
1.
Inhibitory
2.
Stimulatory
3.
Cytokines
Recognition of infected cells
by natural killer cells
Leonidas N Carayannopoulos and
Wayne M Yokoyama Current
Opinion in Immunology 2004, 16:26–
33
Ly49H deficiency
Ly49H NK1.1
Ly49D
Science. 2001 May 4;292(5518):934-7.
MCMV
Anti-Ly49c
Anti-Ly49h
Anti-NK1.1
(B) MCMV replication in F1 hybrid offspring from DBA/2 and BXD-8. Three days
after infection [with MCMV Smith strain,2-3x104 plaque-forming units (PFUs)], organ
viral titers were assessed in tissue homogenates collected from C57BL/6, DBA/2,
BXD-8, and (DBA/2 3 BXD-8)F1 hybrid mice (five mice per group), as indicated.
Spleen titers are shown here; liver titers are available online (8). Each point
represents the average titer determined for an individual mouse. Mean viral titers
for each group are depicted as horizontal bars. For mice with titers below the level
of detection of the assay, the minimum number of detectable PFUs (102) was
assumed to determine the mean.
NK cell recognition of CMV
infected targets
• Ly49H activation receptor on NK cells
• M157 gene product from CMV
– Class I MHC like molecule expressed on infected
cells
– May have evolved to bind Ly49I, an inhibitory
receptor on NK cells
• Innate immune response encoded within the
genome
Mechanisms to detect and
eliminate virus infected cells
• Antibody dependent cellular cytotoxicity
(ADCC)
• Activated macrophage, neutrophils,
granulocytes
• NK/NKT cell mediated lysis
• Cytotoxic T cells (CTL)
ADCC
• Antibodies to pathogen or cell surface
antigen bind to NK cells via FcgRIII
receptor
• IgG1, IgG3
• Antibodies bind FcR on macrophage to
elicit TNF- secretion
Alternative modes of antibody
mediated pathogen toxicity
• IgE/FceRI
– Mast cells
• Release histamine, prostaglandins, leukotrienes, TNF-
– Basophils
• Histamine and IL4
– Eosinophils
• Toxic granule proteins and free radicals
• IgG1,IgG3/FcgRIII
• Fc
– Macrophage, neutrophils, mast cells, basophils, and eosinophils
bind pathogen surface via Fc/Ig and deliver contents of
lysosomes into membrane or on surface of pathogen
Antigen-specific recognition of
viruses
Antigen processing and
presentation through class I and
class II MHC molecules