Immunology 334Y Host Response to Viral Infections

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Immunology 334YHost Response to Viral Infections

• G A Levy
• glfgl2_at_attglobal.net
• April 2 2009

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• Viral Infections
• Viruses
• - intracellular pathogens
• - route of infection
• blood
• secretions
• - eliminated largely by cell mediated immunity
• - CD4 (TH1)- INFa and IL-2
• - CD8 (CTL)-primary elimination
• - B cell (IgG) TH1 secondary (memory)

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Viral Pathogenesis

• Viral Factors
• Virus hijacks immune system

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Figure 11-5
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Figure 11-4
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Figure 11-2
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Influenza
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Influenza

• Is a global contagion
• 20 of children and 5 of adults develop
  symptomatic influenza A or B each year
• Illness ranges from asymptomatic infection to
  various respiratory syndromes, to multiorgan
  involvement and death
• Spread by
• large droplets and contact
• airborne

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Influenza viruses

• Subtyping of influenza A
• based on the antigenicity of two surface
  glycoproteins, hemagglutinin (HA) and
  neuraminidase (NA).
• 16 HA and 9 NA subtypes have been identified
  among influenza A viruses
• The amino acid sequences of the HA1 region, which
  is responsible for HA antigenicity, differ from
  subtype to subtype by 30 or more (i.e., H3 from
  H5)

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Influenza viruses

• Belong to the Orthomyxoviridae family
• Classified as A, B, and C
• based on antigenic differences in their
  nucleoprotein (NP) and matrix (M1) protein.

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Viral Architecture

• Hemagglutinin binds the virus to the target
  epithelial cell receptor
• Neuraminidase degrades the receptor, permitting
  the virus to enter the cell
• The RNA core genome of the virus is composed of
  multiple, segmented single strands of nucleic
  acids.

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H 16
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• human viruses bind to receptors containing
  2-6-linked sialic acid receptors
• avian influenza viruses bind to cell-surface
  2-3-linked sialic acid receptors

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a, Nasal mucosa b, paranasal sinuses c,
bronchus d, bronchiole e, alveolus. Res,
respiratory bronchiole (adjacent to alveoli)
Ter, terminal bronchiole (distal to alveoli)
Alv, alveolus. Green, reaction with Sambucus
nigra lectin, indicating the presence of sialic
acid linked to galactose by an 2,6-linkage (SA
2,6Gal). Red, reaction with Maackia amurensis
lectin, indicating the presence of SA 2,3Gal.
Cells were counterstained with DAPI
(4,6-diamidino-2-phenylindole).
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a, Extensive infection of bronchial epithelial
cells by human A/Kawasaki/173/01 (H1N1) virus. b,
Infection of alveolar cells by the human virus.
c, Bronchial epithelial cells are not infected by
A/duck/Mongolia/301/01 (H3N2) virus. d, Infection
of alveolar cells by the avian virus. The results
obtained with other human viruses,
A/Yokohama/2057/03 (H3N2) and A/Kawasaki/176/02
(H1N1), were similar to those obtained with
A/Kawasaki/173/01, and those obtained with the
avian viruses A/duck/Czechoslovakia/56 (H4N6) and
A/duck/Vietnam/5001/05 (H5N1) were similar to
those obtained with A/duck/Mongolia/301/01.
Infected cells are stained brown.
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HA Cleavage as a Major Determinant of
Pathogenicity
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HA0
HA1
HA2

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HA Cleavage as a Major Determinant of
Pathogenicity

• nonpathogenic viruses the HA generally contains a
  single arginine residue at the site of cleavage,
  which is recognized by extracellular trypsin-like
  proteases
• highly pathogenic viruses have additional basic
  residues at the site of cleavage (R-X-R/K-R)

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HA Cleavage as a Major Determinant of
Pathogenicity

• Of the viruses that have circulated in humans
  over the past century, none contain polybasic
  sequences at the HA cleavage site that provide
  furin recognition motifs
• H5N1 strains of 1997
• All contained polybasic sequences at the cleavage
  site
• Pathogenic strains had five mutations 2 in the
  PB1 and one each in the PB2, NA, and M1 proteins

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Adaptation of Infuenza

• Antigenic drift
• A/California/04
• Antigenic shift
• H5N1 and pandemic influenza

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Antigenic Drift

• The multigenic influenza A virus undergoes
  antigenic changes through an accumulation of
  point mutations during propagation in the human
  host species
• These changes provide a means for immunologic
  escape and for propagative success in partially
  and variably immune populations in which there
  are continuous selection pressures resulting from
  constraints imposed by developing herd immunity

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Pandemic Influenza

• originate from birds
• both initial human infections by avian viruses
  and the emergence of pandemic viruses are
  restricted by a limited fitness of avian viruses
  in humans
• In the case of virus genes (other than the HA
  gene) this restriction can be overcome by gene
  reassortment between avian and human viruses

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Pandemic Influenza
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Recent avian influenza activity

• Pennsylvania 1983-1985
• avirulent H5N2
• 17 M birds culled
• Mexico 1993
• avirulent to virulent H5N2
• Italy 1999-2000
• H7N1

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Antigenic Drift in H5N1 Viruses from Asia
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H5N1-the next pandemic?
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Human Hepatitis Viruses

• Virus Family Genome Disease Spectrum
• HAV picornavirus RNA acute/fulminant
• HBV hepadnavirus DNA acute/fulminant/
• chronic/ cirrhosis/ cancer
• HCV flaviviridae RNA acute/chronic/ cirrhosis/
• cancer
• HDV deltaviridae RNA acute/chronic/ cirrhosis
• HEV calciviridae RNA acute/fulminant

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Hepatitis B
Torresi, Gastroenterology 2000
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HBV is a Global Healthcare Concern
Percentage long-term HBV carriers (350 million
long-term carriers)
lt 2 - Low
2-7 - Intermediate
gt 8 - High
Margolis et al 1991
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Natural History
Gow, BMJ 2001
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HBV Genotypes Geographical Distribution
F
D
A, B, C, D
C
A
D
C
D
Bj
D
Ba
D
D
E
F
A, B, C, D
A
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HBV Genotypes Clinical Course

• HBV Virus Load C gt B Lindh et al., J Viral Hep
  2000 D gt A,B,C Yuen et al., Hepatology 2003
• Spontaneous HBeSeroconversion B gt C Lindh et
  al., J Infect Dis 1999 A gt D Kao et al.,
  Gastroenterology 2000
• Progression to liver cirrhosis C gt B Sumi,
  Hepatology 2003 D gt A Sanchez-Tapias.,
  Gastroenterology 2002
• Development of HCC C gt B Orito, Intervirology
  2003

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Hepatitis B serology

• anti-HBc? exposure (IgM acute)
• HBsAg ? infection (carrier)
• anti-HBs ? immunity
• HBeAg ? viral replication
• anti-HBe ? seroconversion
• HBV-DNA ? viral replication

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HCV Infection Worldwide Prevalence
lt112.42.54.9510gt10No data
available
WHO. Wkly Epidemiol Rec. 20007518-19.
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HCV Infection Worldwide Genotype Distribution
1a, 1b 2a, 2b, 2c, 3a
1a, 1b 2a, 2b, 3a
1b
2a
4
1b, 6
1b, 3a
4
3b
1a, 1b, 2b, 3a
1b, 3a
5a
Fang JWS et al. Clin Liver Dis. 19971493-514.
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Progression to Cirrhosis
Effect of age
Age at infection
lt 40 YRS gt 40 YRS
Cirrhosis 7.7 23.5 Time to
cirrhosis 13.5 6.5 8.4 4.7
Pol S, Fontaine H, Carnot F, Nalpas B, et al.
Predictive Factors for Development of Cirrhosis
in Parenterally Acquired Chronic Hepatitis C A
Comparison Between Immunocompetent and
Immunocompromised Patients. J. Hepatology.
199812-19.
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Progression to Cirrhosis
Alcohol use
100 80 60 40 20 0
HCV HCVALC
85
64
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Cirrhosis
40
31
18
12
6
10
20
30
40
Years after exposure
Wiley et al. Hepatology, 1998
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Progression to Cirrhosis
Effect of HIV coinfection
Ten-year incidence of cirrhosis ()
Mean time to cirrhosis(years)
23.2
25
14.9
16
20
14
P lt 0.001
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P lt 0.01
15
10
Time (years)
Frequency ()
8
10
6.9
6
2.6
4
5
2
0
0
HIV -
HIV
HIV -
HIV
n 431 116
n 431 116
Soto et al. J Hepatol 1997261-5
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Hepatocellular Carcinoma in HCV
0.6
0.5
Cumulativeincidence
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
8
9
10
0
Years
Yoshida H et al. Ann Intern Med. 1999131178.
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Pathogenesis
Th2
Boosting Th1 or inhibiting Th2 responses may
potentially be associated viral clearance.
IL-4, IL-5 IL-6, IL-10
IL-2, TNF INF-gamma
Th1

• Interferons
• Interleukin-2
• DNA-based vaccines

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An immunomodulatory role for CD4CD25
regulatory T lymphocytes in hepatitis C virus
infectionRoniel Cabrera , Zhengkun Tu , Yiling
Xu, Roberto J. Firpi, Hugo R. Rosen, Chen Liu,
David R. NelsonHepatology Vol 40(5)November
20041062
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Figure 1. An ex vivo comparison of peripheral
blood CD4CD25 T cell frequency in spontaneous
resolution (n 15), chronic infection (n 30),
and normal control (n 15) subjects. Data are
expressed as box plots, in which the horizontal
lines illustrate the 25th, 50th, and 75th
percentiles of the frequencies of the CD4CD25 T
lymphocytes as assessed by flow cytometry. The
vertical lines represent the 10th and 90th
percentiles, and circles denote values outside
these percentiles. The P values were calculated
by using the nonparametric Mann-Whitney U test.
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Figure 6. CD4CD25 T cells in chronic infection
(n 30) produce TGF- 1 and IL-10. Enzyme-linked
immunosorbent assay cytokine analysis for TGF- 1
and IL-10 was performed on culture supernatants
from the 3 PBMC cultures representing whole PBMC,
PBMC depleted of CD4CD25 cells (PBMC -
CD4CD25), and PBMC supplemented with CD4CD25
cells (PBMC CD4CD25). Bars represent the
difference in cytokine production after HCV
antigen and negative control antigen stimulation.
(A) Lower levels of TGF- 1 are found in CD4CD25
depleted PBMC cultures and higher levels noted in
PBMC cultures supplemented with CD4CD25 T cells
in comparison with whole PBMC cultures in
subjects with chronic infection. (B) IL-10 levels
are also lower in PBMC - CD4CD25 T cell
cultures and higher in PBMC CD4CD25 T cell
cultures when compared with whole PBMC. P lt .05
and P lt .01 calculated using the Mann-Whitney U
test. TGF- , transforming growth factor beta
PBMC, peripheral blood mononuclear cells IL,
interleukin.
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Figure 3. CD4CD25 T cells suppress HCV-specific
IFN- secretion. HCV-specific IFN- responses
were assessed with the IFN- ELISpot using 3 PBMC
cultures (PBMC, PBM - CD4CD25 PBMC
CD4CD25) that were mixed with HCV antigens,
positive controls (PHA, TT), or negative controls
(medium, HIV) in subjects with (A) chronic
infection (n 30) and (B) spontaneous recovery
(n 15). The magnitude of the bars represent the
mean number of IFN- spots per 2 105
mononuclear cells from the 3 PBMC cultures after
subtracting the number of IFN- spots from their
negative control wells. Although depletion of
CD4CD25 T cells (PBMC - CD4CD25 T cells)
enhances HCV-specific IFN- responses,
supplementation of whole PBMC with CD4CD25 T
cells (PBMC CD4CD25 T cells) suppresses
HCV-specific IFN- responses when compared with
whole PBMC. P lt .05 and P lt .001 calculated
with the Mann Whitney U test. PBMC, peripheral
blood mononuclear cells PHA, phytohemagglutinin
antigen HIV, human immunodeficiency virus TT,
tetanus toxoid.
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Figure 4. CD4CD25 T cell immunosuppression of
HCV-specific CD4 T cell proliferation is dose
dependent. CD4CD25- T cells from subjects with
chronic infection (n 30) were used as effector
cells to examine HCV-specific CD4 T cell
proliferation as measured by 3H-thymidine
incorporation in response to pooled HCV antigens
in coculture with CD4CD25 T cells at various
ratios (100, 101, and 21). Positive (PHA) and
negative controls (HIV antigens) are included.
Results are expressed as mean counts per minute
(cpm 103) after subtracting the cpm in the
absence of antigen. CD4CD25 T cells suppress
HCV-specific CD4CD25- proliferation in a
dose-dependent manner. To determine whether the
suppression was cytokine mediated and/or
cell-contact requiring, transwell studies were
performed. Suppression of HCV-specific CD4CD25-
proliferation was abolished after separating the
2 cell fractions (CD4CD25- and CD4CD25) with a
transwell insert, suggesting that suppression is
cell-cell contact dependent. P lt .05, P lt .01
by the Mann-Whitney U test. PHA,
phytohemagglutinin antigen HIV, human
immunodeficiency virus HCV, hepatitis C virus.
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Figure 5. CD4CD25 T cells suppress HCV-specific
CD8 T cell proliferation. CFSE assay for a
representative patient with chronic infection.
CD4CD25 T cells suppress proliferation of
HCV-specific tetramer CTLs. Two
HLA-A2-restricted peptides (NS3 and NS5B) were
used to generate CTLs from pure CD8 cells of
HLA-A2 subjects. HCV-specific CD8 T cells
proliferation were evaluated with CFSE, anti-CD8,
and PE-HLA-A2 tetramers. (A) CFSE-labeled
tetramer CTLs as detected after a 7-day culture
with unlabeled CD4 T cells, with unlabeled
CD4CD25- T cells and with unlabeled CD4CD25 T
cells. CD8 T cells were cocultured with the
various CD4 fractions at 101 ratio (1 106
CD80.1 106 CD4 fraction).The CFSE signal in
the left upper quadrant represents the percentage
of HCV-specific CTLs that have proliferated
during the 7-day culture. Dot plots were
generated by gating for lymphocytes. (B)
Histogram plot showing the CFSE signal intensity
of the CD8 T cells in the presence of CD4,
CD4CD25-, and CD4CD25 T cells when using the
NS3 and NS4 tetramers.
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Viruses

• Intracellular pathogens
• Cause broad spectrum of diease in humans
• Clearance TH1-CTL (CD8)