Antibodies determine virulence of Dengue viruses

ANTIBODIES DETERMINE
VIRULENCE IN DENGUE
Scott B HALSTEAD, M.D.
DIRECTOR, Research
Pediatric Dengue Vaccine Initiative
IVI, Seoul, Korea
Global Spread of Dengue
50-100 million infections/year
Countries with active dengue + Aedes aegypti
Pathogenicity vs
Virulence
Virulence, a quantitative term:
Severe disease/total infections
Dengue hemorrhagic
fever/dengue shock
syndrome has occurred in
some (but not all) dengue
epidemics since the 1950s.
WHY?
Intuitive answer –
Differences in virus virulence
IN 1950s HAMMON DISCOVERED
NEW, “VIRULENT” DENGUE
VIRUSES THOUGHT TO BE HFASSOCIATED
PHILIPPINES DENV 3, 4:
Hammon WMcD et al Science 131:1102-3,
1960
THAILAND DENV 5, 6:
Hammon WMcD et al AJTMH 13:629-41,
1964
Virulence hypothesis
is very much alive
WHAT
FACTORS CONTROL
SEVERITY OF DENGUE?
INTRINSIC HOST FACTORS
(innate immunity)
• Humans: susceptibility
resistance.
– Race1:
– HLA2:
Caucasian/Asian
African
HLA-A*0207
HLA-A*0203
HLA-B*52
HLA-B*51
– Age3:
Children
Adults
– Nutrition4: Well nourished Malnourished
1. Guzman MG et al. AJTMH 442:179-184, 1990.
2. Stephens HA et al. Tissue Antigens 60:309-318, 2002.
3. Guzman MF et al. Int J Infect Dis 6:118-124, 2002.
4. Thisyakorn U et al. CID 16:295-297, 1993
EXTRINSIC FACTORS
VIRUS
“VIRULENT”
“NON-VIRULENT”
DEN 1, 3, 4
DEN 2 (Asian)
DEN 2 (American)1
1. Kochel T et al. The Lancet 360:1474-1479, 2002.
CRITICAL ROLE OF
ANTIBODIES
DHF
BANGKOK CHILDRENS HOSPITAL
700
600
500
1973-79
1980-89
1990-99
400
300
200
100
0
<1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
AGE (YEARS)
DSS in a 6 month-old infant with primary
dengue infection. Vietnam
Infant DHF/DSS - 5% of all cases
CHILDREN'S HOSPITAL, BANGKOK, THAILAND
40
35
1987
30
25
1990
20
15
1997
10
5
1998
0
0-2
2-4
4-6
6-8
AGE (months)
8-10
10-12
Antibody mediated
infection of macrophages (ADE)
In vitro ADE model
% Infection without serum
Ratio of % infection at PENT/ Control
Power
Control
PENT
Peak enhancement titer = titer at maximal
% infection for the serum tested
Modified from Kliks et al, Am. J.Trop. Med Hyg 1989
WHAT ABOUT VIRAL
VIRULENCE?
NON-VIRULENT DENGUE VIRUS:
IQUITOS OUTBREAK
• School children cohorts followed
from 1990 until now.
• DEN 1 transmitted in 1990 - 1994.
• DEN 2 transmitted from 1995.
• Prevalence of neutralizing antibodies
measured in 1993, 1994 and 1995
cohorts.
• In 1995, secondary DEN 2 infection
rate estimated at 60.5%
NO DHF with secondary DEN 2
(American genotype) infections
• Total population, 5 - 14 yrs-old = 81,479.
• Total 2ndary DEN 2 infections = 49,266.
• Expected hospitalized DHF = 88710247.
• Expected deaths = 18 - 204.
• DHF cases observed = 0
Watts DM et al Lancet 354:1431-4, 1999
BUT, DENV 2 INFECTIONS ARE
MODULATED BY DENV 1ANTIBODIES
34 DEN 1- IMMUNE HUMAN SERA NEUTRALIZE
AMERICAN GENOTYPE DEN 2 VIRUSES
DEN-2 genotypes
Virus DEN-1 AMER SE
SE
ASIA ASIA
Strain Venez IQT Venez Thai
GMT
9842
2124
8041
16681
875
262
32
29
Kochel T, et al Lancet 360:310, 2002
ONE-WAY CROSS:
17 DENGUE 2-IMMUNE SERA
DO NOT NEUTRALIZE DENGUE-1 VIRUSES
DEN 1 viruses
DEN 2 viruses
Genotype
Strain
SE Asia Venez
SE Asia Peru
16007
16681
GMT
20
OBS
9842
27
635
IQT
2913
417
PLAUSIBLE HYPOTHESIS:
HETEROTYPIC DENGUE ANTIBODIES EITHER
PREVENT OR
ENHANCE SECOND DENGUE INFECTIONS
“NON-VIRULENT”
Neutralizing
“VIRULENT”
Non-Neutralizing
ACTIVELY ACQUIRED (TWO INFECTIONS)
or PASSIVELY TRANSFERRED IN UTERO1
1. Kliks S et al. AJTMH 40:444-451, 1989.
Another example of viral
“virulence”
CUBAN DHF, 1997
accelerated “virulence”
DHF/DSS
DEATHS
CONF DF
DHF/DF
DEAD/T
CFR
MAY
37
1
705
5.2
0.14
2.7
JUNE
132
6
1785
7.4
0.33
4.5
JULY
29
5
244
11.9 P<0.01
2.04 P<0.01
17.2 P<0.05
Guzman MG et al, Lancet 355: 1902-3, 2000
SHIFT FROM THREONINE TO
SERINE AT NS1 aa 186
• All patients had secondary dengue
infections.
• Based on 5 complete and 19 partial
genome sequences.
• Reference strain: DENV 2 Jam 1407/83
Rodriguez-Roche R et al J Gen Virol (in
press)
- Increased severity only
occurs during secondary
dengue infections.
- That is, severity is antibodydependent
- Perhaps, due to increased
“fitness” for viral
replication?
INTRINSIC ADE
• Infectious immune complexes
(IgG + virus) directly regulate
viral infections in
macrophages.
Th1/Th2 responses in
macrophages
• Ag presentation to M Φ ► IL-12 causes
T cells to produce IFN γ (Th1 response)
• Ag + Ab presentation to M Φ ► IL-10
causes T cells to produce IL- 4 (Th2
response).
Anderson et al J Immunol 168:3957, 2002
Suppression of antiviral transcription factors
(STAT-1 and NF-KB complexes) by ADE
infection of macrophages by Ross River virus.
Mahalingham S & Lidbury BA. PNAS 2002 99: 1392
• Polyclonal mouse anti-RRV used at 10-3
• ADE infectivity of mouse macrophage cell line
increased 25-fold by 12 hours.
• Nearly 50% of cells infected by viral RNA.
• RRV plus simultaneous addition of irrelevant immune
complex did not increase infectivity.
• Ablated or suppressed: NO production, IFN-1, IP-10 +
three IFN factors. IL-10 production increased.
• Transcription factors shut down in bystander cells.
• IL-10 thought to mediate this latter effect.
Dengue virus ADE upregulates the production
of anti-inflammatory cytokines but suppresses
anti-dengue free radical and pro-inflammatory
cytokine production in THP-1 cells.
Charoensrisuthigul S et al J Gen Virol 2007 88:265
• Human polyclonal antibodies from 2ndary DENV 3
DHF case incubated at 1:100,000 with 16681.
• DENV 2 infection of individual THP-1 cells increased
by 10-fold at 24 hours.
• Suppression of STAT-1 phosphorylation and IRF-1
gene expression may be mechanism by which ADE
infection down-regulates innate immunity.
• ADE facilitated production of IL-10 and decreased NO
production.
• Transient suppression of TNF α.
Increased number of cells and intrinsic viral
production with ADE in Mature DC
% DV infected cells
>2-log
2.5x
Serum dilution factor
Marovich, M. personal communication
IL-10 suppresses secondary cytokine
responses via SOCS-3 activity
Ubol S et al. JID 2008
Suppression of IFN synthesis in DENV-ADE
infected THP-1
Ubol S et al. JID 2008
Global PBMC gene expression
secondary DF vs secondary DHF
1.DHF (enhanced infection) has stronger influence on
the gene expression profile than DF (partially
protected secondary infection).
2. 17 genes in immune response category are
more strongly upregulated in DF PBMCs
than in DHF PBMCs. 40% of them are genes
of the interferon system.
Ubol S et al. JID 2008
Conclusion
1. Dengue enhancing antibodies not only
facilitate entry into target cells but also modify
post-entry events.
2. An outcome is the suppression of innate and
adaptive intracellular anti-viral mechanisms.
3. These are mediated through IL-10 activity and
via the mechanism upstream of MDA-5 and
RIG-I activation.
4. Immune complexes directly regulate dengue
virus infection productivity.
DEN 3
VIREMIA
●--● DSS
▲- ▲DHF
□--□ DF
LIBRATY DH et al JID 185:1213, 2002
Sequential and cumulative viral distribution in tissues
during dengue infection in 31 rhesus monkeys.
PEAK CELLULAR
INFECTION
OCCURS AT OR
AFTER
DEFERVESCENCE
Marchette et al
JID 1973 128:23
Halstead SB
In Schlesinger W
Togaviruses
Academic Press
1980: 107-173
DV
lysis
Ab
T8
MHC I
+
Mo
MHC II
T cell receptor
TNFα
activ
ation
DV peptides
lys
is
+
DV receptor
T4
FcγR
IFNγ
IL-2
TNFα
TNFβ
T4
lysis
Mo
T8
Mo
EC
endothelial cell
EC
C3a
C5a
plasma
leakage
complement
TNFα
EC
After Rothman, 2001
monocyte
TAKE HOME MESSAGE
• Intrinsic factors place many humans at
reduced risk to severe dengue disease.
• In at risk humans antibodies (passive or
actively acquired) regulate the severity
(virulence) of dengue infections:
– Homologous antibodies provide complete
protection.
– Heterotypic neutralizing antibodies down-regulate
severe disease, usually with inapparent outcome.
– Enhancing antibodies suppress innate antiviral
defenses leasing to increased infected cell mass
and increased disease severity.
VIRULENCE = ANTIBODIES
THE GREATER THE NUMBER
OF DENGUE-INFECTED CELLS,
THE GREATER THE
T- CELL RESPONSE, THE
GREATER THE CYTOKINE
STORM, THE MORE SEVERE
THE DISEASE
FOR THE LEISHMANIACS
Virulence = visceral
leishmaniasis
Leishmania-macrophage interactions….
L. amazonensis amastigotes have host IgG on their surface
Lesion-derived amastigotes
are coated with host Ig
Axenic
Lesion-derived
Control
o
2 Ab
o
Count
1 Ab
o
+2 Ab
LOG FLOURESCENCE
Mosser, D personal communication
IgG Reconstitution of JH Mice and the Effect of α-IL10R
5
JH + IgG
1010
109
108
107
JH + IgG
G
Ig
JH
3
G
Ig
JH
+
IL
α0R
-1
Lesion size (mm)
4
JH + IgG + α-IL-10R
Parasite Burdens
JH
2
1
JH + IgG + α-IL10R
0
0
5
10
15
20
Days post-infection
25
30
Mosser , D. pers comm., Miles SA et al JEM 201:747-54, 2005
footpad swelling (mm)
IL-10-/- (BALB/c) mice control
lesion development during L. major infection
BALB/c
IL-10 -/(BALB/c)
*
6
4
C57BL/6
2
0
* euthanized
0
10
20
30
40
50
Days post-infection
60
IL-10 induction following FcγR Ligation
3000
LPS
LPS + E-IgG
IL-10 (pg/ml)
2500
2000
1500
1000
500
0
0
0.25
0.5
1
2
5
10
100
1000
0
0.25
LPS (ng/ml)
0.5
1
2
5
10
100
1000