Fish health: Immune system and vaccine development

Vaxxinova Norway and IMR

Sonal Patel

Workshop on Atlantic halibut aquaculture, 11.-12. September

Co-funded by the Seventh

Framework Programme

of the European Union

Thanks to colleagues at IMR and Vaxxinova

Audun

Rolf HariAina

Ann Cathrine

Ina

Ingrid SisselJoachimElin

Stig

Kristian

Eivind

Colleagues at Austevoll research station

Prevention strategies

Knowledge about the immune system and disease/pathogen:

•When?•How? •For which pathogens?

Egg Adult

Diseases in halibut

•Bacterial

•Viral

•Parasties

Parasites

• Myxosporidia –High mortality in 2015, no detection since 2016

• Ichthyobodo spp. (”Costia”),Trichodina sp

• Combined with- bacterial gill health problems

Formalin treatment

Diseases

Fiskehelserapportene, VI

Disease 2012 2013 2014 2015 2016 2017

Notifiable

VER 1 1 0 0 0 0

Non-notifiable

IPNV 2

Moritella viscosa X - - - - -

ARV X? X

• Vibriosis, IPN and atypical furunculosis: three most important diseases

• Atypical furunkulosis - Can be difficult to completely get control over in landbased facilities

Furunculosis 1 - - X? 9 7

Bacterial diseases

• Atypical Aeromonas salmonicida – type II

• Vibrio logei

• Vibrio (Allivibrio) wodanis

• Vibrio splendidus

• Vibrio tapetis

Viral diseases

• Viral haemorrhagic septicaemia virus (VHSV)

• Infectious pancreatic necrosis virus (IPNV)

• Nervous necrosis virus (NNV)

• Aquareovirus (ARV)1

0 d

ph

45

dp

h

66

dp

h

94

dp

h

12

2 d

ph

15

9 d

ph

24

dp

h

Nodavirus

Aquareovirus

Aquareovirus (AHRV)

• Norway:

• First aquareovirus from a marine coldwater fish spp.

• Clinical signs: Lethargy, darkening

• Necrosis in the liver and pancreas

• 90–100 days after fertilization

• 80-90% mortality

• Canada (1998): 100 mg – 1 g, ~56% mortality, +bacterial infections

• Scotland (2003): Weaned, 1000 dd, >95% mortality

Cusack et al, 2001, Ferguson 2003, Blindheim et al, 2015

Nervous necrosis virus (NNV)

• Betanodavirus, Nodaviridae family

• Viral encephalopathy and retinopathy (VER)

• Central nervous system (CNS) and retina

• Abnormal swimming pattern and loss of appetite

• Larvae and juvenile stages affected by VER

Grotmol et al. 1999: Bath challenged halibut yolk-sac larvae

Size: 25-30 nmGenom: RNA1 3100nt

RNA2 1400nt

Pathogen transmission

• Horisontal

• Vertical

• Screening of broodstock

Immunity : Innate and Adaptive

• Innate immunity → non-specific

•Adaptive immunity→ specific→memory

The cells in the immune system

Pluripotent stem cell

Myeloid stem cell Lymphoid progenitor

Y

Red blood cells

Megakaryocyte

Dendritic cell

Mast cell

Eosinophil

Neutrophil

Basophil

Natural

killer cell

Macrophage Plasma cell

Dendritic

cell

T helper

cell

Platelets

T cytotoxic

cell

B

lymphocyte

T lymphocyte

• Developement of lymphoid organs

• Adaptive immunity:

• Detection of specific B- and T-cell markers – RNA

• Detection of IgM - protein

egg adult

Sequential sampling over 6 months

Development of immune system

Days post hatching

Halibut larvae during metamorphosis

0

10

20

30

40

50

60

70

80

90

100

59 66 73 80 87 94 102 108 115

Dis

trib

uti

on

of

ind

ivid

ua

ls(%

)

Sæle et al, 2004

A

Development of Spleen

59 dph

C

94 dph

B

80 dph

A

S

B

Development of anterior kidney

1 dph 49 dph

94 dph

D

BG

66dph

C

In situ hybridization and IHC - IgM

66 dph

ADMMC

C

A B

1

Spleen

HK

94 dph

Hematopoietic precursor

CD8+ CD4+

CD8+ TC-cell CD4+ TH-cell

RAG expression

ON

Kidney

Blood

Thymus

Blood

Peripheral tissues

33 dph

42 dph

66-73 dph

87 dph

Halibuttimeline:

T-cell development

CD3 TCRβ TCRαCD4/CD8

ETP

DN1

DN2 + +

DN3 + +

DN4 + +

DP + + + +

Immunocompetence ?

In situ hybridisation, thymus:

Figure modified from Kindt, 2007

RAG1 42dph

CD4 66dph

CD8α 87dph

Immunity development

LIVE FEED LARVAL STAGE DRY FEED JUVENILEYOLK-SAC LARVAL STAGE

RAG1

CD3ε

CD3ζ

TCRβ

Lck

CD4-2

CD3γδ

TCRα

CD8α

ZAP-70

CD4

CD8β

mR

NA

leve

l rel

ated

to

0 d

ph

(2

-

Ct ) m

RN

A level related

to 0 d

ph

(2-

Ct)

0 3 7 10 14 17 21 24 28 31 35 38 42 45 49 52 59 66 73 80 87 94 102 108 115 122 129 144 1590

100

200

300

400

500

0

1000

2000

3000

4000

5000RAG1

TCRa

TCRß

CD3d

CD3e

CD3

CD4

CD4-2

CD8a

CD8ß

Lck

ZAP-70

dph

RAG1 42dph

CD4 66dph

CD8α 87dph

IgM

66dphIgM

94 dph

IHC

1

Patel et al, 2009;

Øvergård et al 2011

Vaccine trials - Laboratory

Trials:

1. Larvae ~70 dph – tolerance trial

2. Juveniles (25 g) – recombinant recCP by injection

3. Larvae/juveniles (100 dph) – several different formulations – oral

and injection

Vaccine and tolerance trial -larvae

Larvae ~70 dph, recCP, no adjuvant

Unfortunately....

• 99% larvae died – handling stress!!

• Other methods!?

• Trials with bath treatment

• Trials with oral uptake

• ……and analyse uptake of vaccine

Assess protection against nodavirusJuveniles (25 g)

PBS PBS Adjuvant

10 μg recCP

Adjuvant

50 μg recCPAdjuvant

Sampling: 12 weeks post vaccination

Challenge: 15 weeks post vaccination

Sampling: 2, 4, 6, and 8 wpc

Ab production and protection

N = 6

*

*

Time post vaccination

EL

ISA

Ab

so

rban

ce (

OD

49

0)

2 w 5 w 8 w 10 w 12 w0.0

0.4

0.8

1.2

PBS

Adjuvant

recCP(10)

recCP(50)

PBS

Time post challenge

Ct

valu

es (

RN

A2)

2 w 4 w 6 w 8 w

20

25

30

35

40

Adjuvant

Time post challenge

Ct

valu

es (

RN

A2)

2 w 4 w 6 w 8 w

20

25

30

35

40

recCP(10)

Time post challengeC

t valu

es (

RN

A2)

2 w 4 w 6 w 8 w

20

25

30

35

40

recCP(50)

Time post challenge

Ct

valu

es (

RN

A2)

2 w 4 w 6 w 8 w

20

25

30

35

40

Nodavirus RNA2

WP 26 Atlantic halibut

Objective: Determine the effect of delivering recombinant capsid protein during late larval stages on protection to nodavirus (VNN)

Task 26.1 Production of VNN capsid protein

Task 26.2 Monitor and assess immune response and protection

Assessment of vaccine

Protein expression in different systems

E.coli Leishmania tarentolae Tobacco leavesPichia pastoris

Production of antigen

Delivery and challenge

Le

ish

ma

nia

tare

nto

lea

E.c

oli

Fluorescence OverlayLight

Test with GFP expressing organisms

1. purified recCP expressed in E. coli

2. E. coli expressing capsid protein

3. L. tarentolae expressing capsid

protein

4. Pischia expressing capsid protein.

Uptake of protein by Artemia

Day 1 Day 2Pos

ctrl

Lab trial – oral and injection delivery

Ct

valu

e

Protection?

Notes from lab trials

• Vaccination trials should not be initiated before 100 dph

• A balanced diet during development could be important

• Best practice – hygiene during early phases

• Both E.coli and L.tarantolae are filtered from seawater and taken up by Artemia

Vaccines in practice –Administration and strategy at site

• Which pathogen is causing disease? Choosing the

right isolate

• Farm/farm history….

• When is protection needed (onset and duration of

immunity)?

• Handling of fish – stress

• Effect vs. stress and side effects

• Choose strategy (Bath, immersion/dip, injection)

Depending on the situation at the specific farm

02.10.2018

• Atypical Aeromonas salmonicida

• Vibrio logei

• Vibrio splendidus

• Vibrio tapetis

Relevant bacterial diseases

Vaccine as a solution

02.10.2018

Example from use at a commercial site

Bath, immersion/dip, injection

02.10.2018

Smitte/sykdom

Size/time

Challenge/disease

Imm

ersi

on

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--

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--

--

--

-

--

--

--

--

--

--

--

--

--

--

--

--

--

-

IP-injection? Harvest

Imm

ersi

on

--

--

--

--

--

--

--

--

--

--

--

--

--

-

First feeding

~1 gram Approx. 25 gram

Vibrio logeiVibrio splendidus

Atypical Aeromonas salmonicidaVibrio logei

Imm

ersi

on

3-5 gram

02.10.2018

Intraperitoneal injection (i.p)

Site of injection

• Site of injection is close to the

basis of the ventral fins posterior

to the urogenital papilla

• The dose is deposited in the buccal

cavity.

02.10.2018

Intramuscular injection (i.m)

Site of injection

• Half way between the basis of the

dorsal fin and the lateral line.

• Only done in trials!

• Experienced up to 20% with vaccine in the intestine

• Loss of herd immunity

• Disease, and lack of vaccine effect

• Need a solution

• Ongoing trial to check for safety – Trial ends on 20.Sep

Vaccine injected into the intestine

Challenges

02.10.2018

Change: administration, adjuvant, regime

In need of a solution!

02.10.2018

• Injection site (i.p, i.m)? Is it safe?

• Adjuvant (water, oil)

• A.salm needs oil adjuvant for duration of

immunity

• Two injections, different adjuvant?

• Water based i.m injection, small fish

• Oil based i.p injection, large fish

• Theory in lab (ILAB) vs reality at site!

Thank you!