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