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2013 International Symposium on Ranaviruses by Jacques Robert
Citation preview
THE HOST IMMUNE SYSTEM: A DOUBLE-EDGED SWORD CONTROLLING RANAVIRUS INFECTION BUT PROMOTING
VIRAL PERSISTENCE
Jacques Robert, Francisco DeJesús Andino, Leon GrayferMicrobiology and Immunology, U. Rochester
Medical Center, Rochester NY
FV3FV3
APCAPC
CD8CD8
BB
CD4CD4
Class I
Class II
Co-stim(B7, CD40
CTLCTL
ThTh
Activation Expansion Maturation
Effectorphase
Kill class I+ targets
MemoryMemoryCD8CD8
MemoryMemoryCD4CD4
Ags + “danger”
signals
Apoptosis
Apoptosis
Plasma Plasma cellscells
Memory
Cytokinerelease
Cytokinerelease
TLRs
Anti-viral immune responses
Innate immunityPhagocytosis
Antiviral molecules (IFN, iNOS)
MøDC
Ranavirus (FV3), large double stranded DNA viruses infecting with increasing prevalence frogs, fishes and reptiles
Xenopus, instrumental laboratory model to study immunity and pathogenesis of RVs such as FV3
Adults: critical involvement of CD8 T cells and antibodies established
Immunological memory: faster recovery, viral clearance upon 2nd infection, protective anti-FV3 Ab, faster CD8 T cell response
Larvae: more immature immune system, more susceptible to FV3 infection. Not not just immature B and T cell responses, but delayed and poorer innate anti-FV3 response
Xenopus-FV3 model
Re-infection
FV3 load
1 3 6 9 1 3 6 9
Days post infection
- 10
- 2
5-
Num
ber
cells
x 1
02
Effective Ab response
PFU[x102]
First infection
Innate Immune cells1-
Infiltrating CD8 T cells
Xenopus adult immune response kinetics in infected kidneys
Chen & Robert, 2011, Viruses, 3:2065
10 m 2 2 mm
What are the specific roles played byXenopus tadpole and adult macrophages
in host immunity against FV3?
CD8T cell
• Antigen processingand presentation
• Co-stimulation• Activation of CD8
& CD4 T cellsMø
Adaptive Immune cell effector
Innate Immune cell effector
Permissive cell for viral quiescence
•Virus transcriptionally inactive
MHC class I
MHC class II
• Phagocytosis
• Pinocytosis
Release of:• Cytokines (TNF, IL-1)• Chemokines (IL-8)• Toxins (NO) Infection
Complex role of macrophages in Xenopus host defenses against RV
Chen & Robert, 2011, Viruses, 3:2065
IFN
TNF
Mx1
IL-1
Delayed and low innate immune response in larval kidney during early FV3 infection
* *
*
* *
RQ
RQ
RQ
RQ
N=3
De Jesús Andino et al. (2012). Virology 432:435
Delayed and low inflammatory response by larval PLs during early FV3 infection
Days post-infection
IL-1TNF IFN
1 3 6 1 3 6 1 3 6
Heat-killedE. coli
Fol
d in
crea
seIL-1TNF IFN-
FV3
Fol
d in
crea
se
Days post-infection
N=3 De Jesús Andino et al. (2012). Virology 432:435
Days post-infectionC 2 3C 2 3
*
Cel
l Nb/
tadp
oles
x 1
03
53R*
20 m
%
Increase of infected and total numbers of larval peritoneal phagocytes during FV3 infection
Days post-infection
15/group
The interferon response
IFNs are important antiviral agents produced and released by all cell typesThey allow for communication between cells to trigger the protective defenses of the immune system Reptiles, birds and mammals possess IFN, etc. Fish, amphibians, monotremes and marsupials do not have IFN, …Instead they possess unique type I IFNsWe have identified and cloned a X. laevis type I IFN, Produced a recombinant X. laevis type I IFN (rXlIFN)
Type I IFN gene expression during FV3 infection
Kidney Macrophages
RQ
(fo
ld c
hang
e in
exp
ress
ion
0 1 3 6 9
Days post-FV 3 infection
0 1 3 6 9
rXlIFN interferes with FV3 infection of A6 cells(3 day post infection)
10m
53R composite
10m
vector
rXlIFN 53R composite
mockA6 cells pretreated with rXlIFN for 8h; infected with 0.3 MOI FV3
FV
3 co
py
nu
mb
er
(pe
r 50
ng
DN
A)
RQ
(fo
ld c
ha
nge
in e
xpre
ssio
n)
- FV3 - FV3+ Vector + rIFN
Kidneys
PLs
Virus load Viral transcription
- FV3 - FV3+ Vector + rIFN
Pre-treatment of tadpoles with rXlIFN, 8 hrs prior FV3 infection, results in lower viral load and viral activity
rXlIFN pre-treatment results in slight delayed tadpoles’ death following FV3 infection
zfMpeg1:GFP and Cherry transgenic X. laevis larvae
zebrafish macrophage specific promoter mpeg
GFP
Cherry
Heterogeneity of X. laevis larval myeloid cell populations
Double transgenic X. laevis xLurp:GFP/xfMpeg1:Cherry
Green: Granulocytes (lurp+/Mpeg-)Orange/yellow: macrophage (lurp+/Mpeg+)Red: Macrophages? (lurp-/Mpeg+)
Increased numbers of total and GFP+ PLs
6 days post-FV3 infection
Total PLs GFP+ PLs
C 6dpi C 6dpi
50 m
FV3 53R Xlurp:GFP Overlay
xLurp:GFP transgenic Xenopus tadpoles
Increased susceptibility to FV3 infection of F0 Tg tadpoles with b2m KD
% s
urvi
val
Days post-infection
5 x 106 PFU FV3 in 5ml water for 1 hr
Median survival time
C = 38.5 daysTg = 19.5 days
p> 8x10-5
Median survival time
Tg-Scr = 28.0 days
Tg-B2M = 13.5 days
p> 0.002
Tg-ScTg-B2M
N=20/group
Nedelkovska et al., Biol. Open, 2013
Pre-metamorphic (st 57) OB tadpoles
Effective and specific KD of the class Ib gene XNC10 by transgenesis
V6 J39 C domain
Edholm et al., 2013, Proc Natl Acad Sci USA (In press)
% s
urv
ival
Days post-FV3 infectionFV3 i.p 10,000 pfu (Trial 1)
Increased susceptibility to FV3 infection of LG-15 F0 Tg tadpoles with XNC10 KD
Edholm et al., 2013, Proc Natl Acad Sci USA (In press)
6 dpi6 dpiCC15 dpi15 dpi 21 dpi21 dpi
MCPMCP
2-M2-M
LMPXLMPX
MCPMCP
-RT-RT
-actin-actin
IEIE
2-M2-M
MCPMCP
MCPMCP
PCRPCR
RT-PCRRT-PCR
RT-PCRRT-PCR
PCRPCR
6 dpi6 dpiCC
15 dpi15 dpi 21 dpi21 dpi
In vivo FV3 infection and transcription in adult PLs
Viral DNA
Viral transcription
6 dpi 15-21 dpi
Morales et al., 2010, J. Virol. 84:4912
Mø infected in vitro for 2 days with FV3
1 m 1 m
100 m
100 m
Morales et al., 2010, J. Virol. 84:4912
Experimental Method
FV3 infection (5 x106 PFU)
0 30
PL cells collectionRNA and DNA isolation
Virus load, viral transcription
Bacterial stimulation
32 35 days
vDNA-Pol
FV3 reactivation
+ C (3dpi)
- C (u
ninfected)
DW
30 dpi
35 dpi FV3 +
HK E. coli
DNA
cDNA
EF-1
1 2 3 4 5 6 7 8 9 10
35 cycles
1 2 3 4 5 6 7 8 9 1035 dpi FV3
+ HK E.coli
vDNA-Pol
vDNA-Pol
EF-1
Survival curve in Adult frogs after FV3 infection, followed by Bacterial Stimulation
C n=22FV3 n=23
C Exp
DNA 0/23 5/23
cDNA 0/23 8/23At 35 dpi
SummarySummary Although FV3 triggers poor type I IFN response in tadpoles, rXlIFN
pre-treatment is protective, suggesting that larval antiviral effector system is functional and capable of controlling FV3 infection.
Thus tadpole susceptibility likely stems from poor recognition system and/or highly efficient FV3 immune evasion
Although the adult frog immune system is efficient in controlling FV3 infection, FV3 persists in a fraction of otherwise healthy asymptomatic frogs
This persistence involves macrophages harboring quiescent, transcriptionally inactive virus
Preliminary evidence suggests that bacterial stimulation-mediated inflammatory signals can reactivate quiescent FV3 infections
Loss-of-function approach by Tg suggests that besides class Ia-restricted conventional CD8 T cells, class Ib-restricted invariant or innate T cells are critical for Xenopus anti-FV3 immunity, especially at larval stages
ModelFV3 targets adult macrophages to escape host immunity and persist in asymptomatic host, but targets larval macrophages to overcome immune defenses and rapidly disseminate
Jumping Frog LabLeon Grayfer, PhDEva-Stina Edholm, PhDNikesha HaynesMaureen BanachFransisco De Jesús AndinoJason SifkarovskiTina Martin
CollaboratorsBrian Ward (U. Rochester)Paige Lawrence V.G. Chinchar (U. Mississippi)
NIH R24-AI-0598R24-AI-059830
MetamorphosisMetamorphosis
Accessible to experimental manipulation
(e.g., thymus)
Absence of maternal influences on the
embryo
Immune system develop early (10 days post-
fertilization)
Larvae are immunocompetent but immature
B and T cell receptor repertoires distinct
from adult
Presence of CD8 T cells but not NK cells,
weak T cell responses