Vaccination against VHS in rainbow trout:

Experiments and perspectives related to practical fish farming

Niels LorenzenDanish Institute for Food and Veterinary Research

Nv ~ non-structural protein

Topics

• Traditional VHS vaccines

• DNA vaccines –Principle

–Safety

• Results from the lab

• Testing under fish farm conditions

• What is the origin of VHS?

Bacterial vaccines are produced by fermentation in simplegrowth substrate.

Bacteria in substrate

Fermentation

Vaccines against viruses?

Vaccines against viruses

• Traditional vaccines:Inactivated virus or attenuated virus

- usually too expensive or not safe enoughfor aquaculture

DNA vaccines: the principle

Virus particle Surface protein (G)

Gene cloning

Vaccine plasmid

De bedøvede fisk vaccineres med plasmid i rygmuskularturen

Vaccination

DNA vaccines: the principle

Virus particle Surface protein (G)

Gene cloning

Vaccine plasmid

De bedøvede fisk vaccineres med plasmid i rygmuskularturen

Vaccination

Plasmid DNA is easy to produce in bacteria

Fermentation

Purification

E.coli

DNA vaccines: the principle

Virus particle Surface protein (G)

Gene cloning

Vaccine plasmid

De bedøvede fisk vaccineres med plasmid i rygmuskularturen

Vaccination

GMO

Fermentation

Purification of plasmid

Not GMO

E.coli

DNA vaccination: Princip

Virus particle Surface protein (G)

Gene cloning

Vaccine plasmid

Vaccination byIntramuscularinjection

Fermentation +Purification

DNA vaccination of fish: intramuscular injection

Production of VHSV G-protein in fish cells

Fishcell

Transport to cell surface

Muscle of fish vaccinated with overdose of DNA vaccine -3 weeks after vaccination -

E. Lorenzen et al. Fish Shellfish Immunol.( 2005) 19:27-41

-5 weeks after vaccination -

E. Lorenzen et al. Fish Shellfish Immunol.( 2005) 19:27-41

Advantages of DNA vaccines

• High safety – no risk of disease, no GMO • Rapid and long-lasting protection • High stability• Simple to procedure

Disadvantages of DNA vaccines

• Injection vaccination not practical for small fish.

• Licensing/commercialisation complicated/expensive.

• Consumers acceptance important (in some countries).

Challenge experiments with DNA-vaccinated fish

in the laboratory

Challenge of vaccinated fish

VHS virus

Efficacy of the VHS DNA vaccine when tested in rainbow trout fingerlings at 12°C:

•Time-course•Specificity

0

20

40

60

80

100

cum

ulat

ive

mor

talit

y (%

)

0 5 10 15 20 days post challenge

8d,vhsG 19d,vhsG 28d,vhsG 61d,vhsG

112d,vhsG 168d,vhsG 168d,pcDNA3

Time-course studyBased on E. Lorenzen et al J. Aquat. Anim. Health. 12, 167-180, 2000

Time-course of protection following DNA vaccination against VHSV and IHNV

0

20

40

60

80

100

0 4 8 12 16 20 24Weeks post vaccination

RP

S

Non-specific mechanismsSpecific mechanismsTotal protection

Weeks postVaccination

IFN↑ Antibody ↑

106

N Lorenzen et al. Fish & Shellfish Immunology (2002) 12: 439-453.

The VHS DNA vaccine protects turbot against nodavirus challenge 8 days post vaccination

0

10

20

30

40

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60

0 5 10 15 20 25

Time post challenge (days)

Cum

ulat

ive

mor

talit

y (%

) pVHSV-GpcDNA3.1(Contr.)pAHNV-C

Sommerset et al, Vaccine, 2003

Conclusions :

• The VHS DNA vaccine induces early non-specific as well as long-lasting specific protection.

• The VHS DNA vaccine seems to have an adjuvant- like effect

Field trialExperimental setup

• Vaccination at two different VHS-freefarms

Field trialExperimental setup

• Vaccination at two different VHS-freefarms

• Transfer of vaccinated fish to net-cages on farms with clinical VHS.

Collaborative project

• DFVF• Danish Aquaculture Association• GeneCare• The Danish Veterinary and Food Administration

Supported by Danish Ministry of Food, Agriculture and Fisheries

The Henriksen Foundation

Results from field-challenge with VHS:

High variability among cages: - high survival of vaccinated fish in

some cages- high mortality of all fish in other cages (other diseases than VHS)

Conclusions

• The DNA vaccine works under field conditions – but does not protect against other diseases.

• Experimental setup with net cages is not optimal.

• Next experiment should include vaccination of all fish in several ponds or all fish on selected farms in endemic zones.

Dansk AkvakulturVHS

First observation in Denmark in 1950

1965:520 Farms infected

2004:41 Farms infected

In Denmark, the strategy is still VHS-eradication byStamping-out

Very important to avoid re-introduction of virus

What is the origin of VHS virus?

VHS virus has been found more than 70 marine fish species

Based on Skall et al J Fish Dis. 2005 Sep;28(9):509-29

VHS in Europe might originate from the Baltic Sea

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Who was the ancestor of VHSV in Europe?

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EU

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50 years

500 years

50 years 50 years

K. Einer-Jensen et al. J Gen Virol. (2004) 85:1167-79.

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In order to avoid VHS:Always be careful when fish are

moved from one place to another!

Acknowledgements

• European Commission• The Danish Ministry for Food,

Agriculture and Fisheries• The Henriksen Foundation