crayfish survivors that are exposed to the same

(1)

A “Correct cloud” of Dscam is induced in long-term crayfish survivors that are exposed to the same

pathogen a second time

1 Speaker: Irene Tze Hann Ng

Advisor: Dr. KC Han-Ching Wang

(2)

Vertebrate

 Adaptive immunity

 Only vertebrates can be vaccinated

2

(3)

 No adaptive immunity

Invertebrate

 Shrimp can be vaccinated against viral infection

 No consistent efficiency

 Immune response with specificity and memory?

 Parameter of the protective immune response?

To figure out~

3

(4)

Pattern recognition proteins (PRP)--

Down syndrome cell adhesion molecule (Dscam)

 A member of immunoglobulin (Ig) superfamily receptors

 The typical domain architecture

 10 Ig domains, 6 fibronectin (FN) domains and 1 TM domain

 Multiple forms of Dscam are generated by alternative splicing

 In Drosophila -- 38,016 isoforms

1 3 4 5 6 7 8 9 10

1 1 2

Ig-like domain Fibronectin domain Transmembrane domain

N C

The extracellular region The cytoplasmic tail

4

(5)

Dscam --- Antibody-like molecule?

5

(6)

6 Shrimp Down Syndrome Cell Adhesion Molecule

 The first Dscam from decapod (shrimp, Litopenaeus vannamei )

LvDscam PmDscam

Litopenaeus vannamei Marsupenaeus japonicus Penaeus monodon

MjDscam

1 3 4 5 6 7 8 9 10

1 1 2

N C

The extracellular region The cytoplasmic tail

Variable regions Possible combinations

Ig2 Ig3 Ig7

LvDscam 32 79 23 > 58,144

PmDscam 28 43 19 > 22,879

MjDscam 30 46 14 > 19,320

Subsequent signal transduction

Variable pathogen recognition

(7)

 Membrane-bound Dscam

 Tail-less Dscam

2 Dscam forms

Litopenaeus vannamei

Secreted directly

7 7

(8)

Tail-less Dscam is increased after pathogen challenge

8

hemocytes Shrimp Shrimp

Seawater V. harveyi

Specific V. harveyi- induced Dscam

PBS Pathogen

10 tissues

Membrane Dscam Tail-less Dscam

Hcy

Gill PL

Hemocyte

Tail-less Dscam

Dscam-mediated immune response

(9)

Tail-less/secreted Dscam in crayfish hemolymph

9 Hemocyte

Hemolymph Hemolymph

Secreted Dscam is actually in the

hemolymph of crayfish who have

survived an infection

(10)

10 Pathogen specificity ?

 After pathogen invasion, tail-less Dscam starts to be produced in large quantity.

 Does these Dscam isoforms show specificity against pathogen?

(11)

Dscam – immune specificity?

11 Dscam – immune memory?

Dscam – “correct cloud” of isoform?

(12)

Recombinant protein + bacteria

Pull-down

Binding assay

Mixed overnight Coating

Inactivated Vibrio harveyi (2.29 x 10⁷CFU / well)

Blocking & rLvDscam Binding rLvDscam: N1, N2, V2, V5, V9

(serial dilution: 0.2578, 0.515, 1.03, 2.06, 4.125, 8.25, 16.5, 33 μg / ml )

Labeling 1° Antibody & 2 ° Antibody

1° Ab: Rabbit-Anti-His 2° Ab: Anti-Rabbit-IgG conjugated with AP 1° Ab

2° Ab

Adding pNPP &

Reading O.D. 405 nmabsorbance at 450 nm TMB

HRP

ELISA

Vibrio harveyi

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

b

c

d cd

a

Escherichia coli

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

ab

c c

b a

Staphylococcus aureus

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

b

c

b

b a

WSSV

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

b

b b

a

Gram-negative bacteria binding

Gram-positive bacteria binding Virus binding

• V5 may be specific to V. harveyi.

• V2 specific for Gram negative bacteria in general

• N2 might therefore be a “super-isoform” involved in general immune surveillance

• N1 appeared to has no specific binding affinity

V. harveyi-induced LvDscam isoform V5 appears to be specific for V. harveyi

12 Hung et al., 2013

(13)

Recombinant protein + bacteria

Pull-down

Binding assay

Mixed overnight Coating

Inactivated Vibrio harveyi (2.29 x 10⁷CFU / well)

Blocking & rLvDscam Binding rLvDscam: N1, N2, V2, V5, V9

(serial dilution: 0.2578, 0.515, 1.03, 2.06, 4.125, 8.25, 16.5, 33 μg / ml )

Labeling 1° Antibody & 2 ° Antibody

1° Ab: Rabbit-Anti-His 2° Ab: Anti-Rabbit-IgG conjugated with AP 1° Ab

2° Ab

Adding pNPP &

Reading O.D. 405 nmabsorbance at 450 nm TMB

HRP

ELISA

Vibrio harveyi

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

b

c

d cd

a

Escherichia coli

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

ab

c c

b a

Staphylococcus aureus

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

b

c

b

b a

WSSV

A450

N1 N2 V2 V5

2 1.6 1.2 0.8 0.4

0 EGFP

b

b b

a

Gram-negative bacteria binding

Gram-positive bacteria binding Virus binding

• V5 may be specific to V. harveyi.

• V2 specific for Gram negative bacteria in general

• N2 might therefore be a “super-isoform” involved in general immune surveillance

• N1 appeared to has no specific binding affinity

V. harveyi-induced LvDscam isoform V5 appears to be specific for V. harveyi

13 --- Immune Specificity---

Pathogen-specific recognizing Dscam can be generated after corresponding pathogen challenge.

Hung et al., 2013

(14)

Dscam – immune specificity?

14 Dscam – immune memory?

Dscam – “correct cloud” of isoform?

(15)

15  Super-survivor Dscam –

 a significantly stronger binding ability to WSSV

 WSSV neutralization ability

ELISA assay

At 2 months post-challenge --

In vitro neutralization test

Ng et al., 2014

---Prolonged immune protective effect---

WSSV-induced crayfish Dscam shows durable

immune behavior

(16)

16 What we still do not know….

How Dscam response when

re-encountered a previous pathogen?

WSSV

(17)

 Dscam response in crayfish survivors do not reach significant change

Dscam mRNA

17 Lower dose of 1 ^st immune stimulation

Will these crayfish “remember” something from first stimulation?

0d 14d

1^ststimulation

14d 5d

2^ndstimulation

(18)

 When survivors were challenged again 14 days later with a second dose of WSSV

 CqDscam involved in WSSV tolerance

18 WSSV Dscam

Hemocyte mRNA Hemocyte mRNA Hemolymph protein

0d 14d

1^ststimulation

14d 5d

2^ndstimulation

2 ^nd immune stimulation after 14 days

14d 5d

2^ndstimulation

(19)

 When survivors were challenged again 1 month later with a second dose of WSSV

 CqDscam can be induced

19 2 ^nd immune stimulation after 1 month

0d 14d

1^ststimulation

14d 5d

2^ndstimulation

(20)

20  CqDscam response in crayfish survivors –

 Dscam expression reached a peak 2 days after the 2nd challenge

2 ^nd immune stimulation after 1 month

0d 14d

1^ststimulation

14d 5d

2^ndstimulation

(21)

21  CqDscam response in crayfish survivors –

 No consistent Dscam response pattern

Lower dose of 1 ^st immune stimulation

0d 14d

1^ststimulation

14d 5d

2^ndstimulation

(22)

22  When survivors were challenged again 2 months later with a second dose of WSSV

 There was no consistent Dscam response in the survivors

Rel ati v e ex press ion of Ds c am (Ds c am /EF1 α )

Day post WSSV 2 ^nd challenge

2 ^nd immune stimulation after 2 months

0d 14d

1^ststimulation

14d 5d

2^ndstimulation

(23)

Dscam – immune specificity?

23 Dscam – immune memory?

“Correct cloud” of Dscam was induced

in response to WSSV re-exposure?

(24)

PBS control groups and WSSV survivors

0 14 5

Day after 1

^st

injection Day after 2

^nd

injection

Population of Dscam isoforms after WSSV re-exposure

 Diversity of the Ig2 and Ig3 variable exons

24 1 3 4 5 6 7 8 9 10

1 1 2

Ig-like domain Fibronectin domain Transmembrane domain

N C

The extracellular region The cytoplasmic tail

(25)

Crayfish survived after WSSV 1 ^st & 2 ^nd challenges Crayfish died after WSSV 1st challenge

DEAD 2

0

1

^st

challenge

WSSV

1 0 14 2 DEAD

WSSV

1

^st

challenge

WSSV

2

^nd

challenge

2 0 14 5

Survivor WSSV

1

^st

challenge

WSSV

2

^nd

challenge

Samples were collected according to crayfish

survival status

(26)

Explanation to interpret Ig2 & Ig3 isoforms diversity

Survival status

Survivor

Day 0 Day 1 Day 5 Day 14 Day 1

1

^o

2

^o

1

^o

2

^o

Day 0 Day 1 Day 5 Day 14 Day 1

1

^o

2

^o

Hemocyte samples Ig2+3 PCR

amplification Sequencing

(27)

Ig2 Ig3

Explanation to interpret Ig2 & Ig3 isoforms diversity

Survival status

Survivor

Day 0 Day 1 Day 5 Day 14 Day 1 Day 0 Day 1 Day 5 Day 14 Day 1

1

^o

2

^o

1

^o

2

^o

1

^o

2

^o

Vi: variable isoform number Each row = one type of isoform

No.: Number of colony

(28)

Ig2 Ig3

Explanation to interpret Ig2 & Ig3 isoforms diversity

Survival status

Survivor

Day 0 Day 1 Day 5 Day 14 Day 1 Day 0 Day 1 Day 5 Day 14 Day 1

1

^o

2

^o

1

^o

2

^o

1

^o

2

^o

Dscam isoform diversity after 1 ^st WSSV challenge

Dscam isoform diversity after 2 ^nd WSSV challenge

The highest the bar, the highest the

isoform diversity

(29)

Ig2 Ig3

Explanation to interpret Ig2 & Ig3 isoforms diversity

Survival status

Survivor

Day 0 Day 1 Day 5 Day 14 Day 1 Day 0 Day 1 Day 5 Day 14 Day 1

1

^o

2

^o

1

^o

2

^o

1

^o

2

^o

Same Dscam isoforms are highlighted

with same color

(30)

Crayfish died after WSSV 1st challenge

1  Crayfish died after 2 days of 1 ^st infection

 failed to undergoes isoforms selection process

39

Ig2 Ig3

Survival status

Day 0 Day 1 Day 5 Day 14 Day 1 Day 0 Day 1 Day 5 Day 14 Day 1

#9

#8

#12

(31)

Crayfish survived after WSSV 1 ^st & 2 ^nd challenges

2  Dscam isoforms that appeared after 1 ^st challenge were specifically

expressed in the crayfish after 2 ^nd challenge

(32)

Crayfish survived after WSSV 1 ^st & 2 ^nd challenges

2  Dscam isoforms that appeared after 1 ^st challenge were specifically

expressed in the crayfish after 2 ^nd challenge

(33)

Crayfish survived after WSSV 1 ^st & 2 ^nd challenges

2 An unknown selection process was generating a

“correct cloud” of Dscams against a previously encountered pathogen?

Hypothesis

0 14 2 DEAD

WSSV

1

^st

challenge

WSSV

2

^nd

challenge

0 14 5

Survivor WSSV

1

^st

challenge

WSSV

2

^nd

challenge

(34)

Dscam isoforms that appeared after the 1 ^st challenge were specifically expressed in the crayfish after 2 ^nd challenge.

Will these isoforms have higher WSSV binding ability?

WSSV-selected isoforms W13 & W14

(Ig2-11+Ig3-35) (Ig2-11+Ig3-16)

(35)

W13 and W14

• the selected isoforms have high WSSV binding ability

“Correct cloud” of Dscams show high WSSV binding ability

35 Negative control

(36)

 Identify WSSV antigen that binds with these

selected Dscam isoforms for vaccine development

(37)

Identify WSSV antigen that binds with selected Dscam isoforms

 Establish platform for WSSV-specific vaccine

 Antigen search

 Far-Western blotting

VP28&VP24

Envelope protein

VP26

Tegument

protein

(38)

Identify WSSV antigen that binds with selected Dscam isoforms

 Establish platform for WSSV-specific vaccine

 Antigen search

 Far-Western blotting  LC-MS/MS

Sample Protein Coverage(%) Location

VP41 VP41B 27% ENV

VP41A 18% ENV

VP39

VP39B 16% ENV

VP36A 13% TEQ

VP38A 13% ENV

VP36

VP36B 19% NC

VP36A 6% TEQ

VP11 4% ENV

VP28 ENV

VP24 VP24 24% ENV

(39)

SOP for shrimp vaccine development

 Establish platform for pathogen-specific shrimp vaccine development

(40)

• Dscam-mediated immunity

– Diversity

– Immune specificity

 Pathogen-specific recognizing Dscam can be generated after corresponding pathogen challenge

(Hung et al., 2013)