DNA Vaccines for Fish Diseases

Hakim Mudasir Maqsood Ph.D. (Animal Biotechnology)

Div. of Biotechnology, FVSc and AH SKUAST-K

Outline

o Aquaculture for affordable animal protein

o Hurdles in intensive farming

o Vaccinology in Aquaculture industry

o DNA vaccines (current status & future prospects)

Overview of Aquaculture production

• Global fish production = 158 million tonnes* • Global Aquaculture production (food fish) = 70.5

mt* • Per capita consumption 9.9Kg in 1960s 19.2kg in 2015* • India (total culture production is 4.2 mt) = 0.4

mt (mariculture); 3.8 mt (inland aquaculture)** • J & K = 20.03K tonnes (Trout 262 tonnes) *** Seed: carp = 440 lac; trout = 90 lac ***

*SOWFA, FAO, 2015 **FAO Year Book, 2012

***State Fisheries Department, 2016

World aquaculture production continues to grow

Global fish utilization and supply

Growth 3.2%

1.6%

Conti…

• ~ 870 million people were chronically undernourished in

2010-12 (FAO, 2014)

• In order to produce more we have to shift from Extensive to

industrial scale INTENSIVE FARMING.

• But with Intensification comes problems!!!

Fish: affordable source of animal protein

• 16.7% of global population intake of animal protein • 6.5% of all protein consumed • 150g of fish = 60% daily protein requirement FAO, 2015

Problem in Intensive Culture: Diseases

• Intensive culture = More animals per Unit area

• Increase in Horizontal Transfer of diseases

• Disease outbreaks affects:

production,

trade and

economy

• Annual losses in 16 Asian countries >USD 3 billion. (OIE, 2012)

• World’s shrimp industry suffered losses of ~US$10 billion since 1990 due to WSSV and IMV

• Vietnam alone reports loss of US$1 billion per year on average.

• Chilean salmon farming industry suffered from Infectious Salmon Anaemia in 2007 350-400 K tonnes of fish, US$2 billion of revenue and 30,000 jobs.

• Cost of IHNV disease in 2001 to 2003 ~ CAD $200 million

Addressing fish disease issues is a necessary condition for

securing new private investment in aquaculture.

OIE listed disease, 2016 (Fish) • Epizootic haematopoietic necrosis disease

• Infection with Aphanomyces invadans (EUS)

• Infection with Gyrodactylus salaris

• Infectious Salmon Anaemia Virus (ISA)

• Infection with Salmonid Alphavirus

• Infectious Haematopoietic Necrosis (IHN)

• Koi Herpesvirus Disease

• Spring Viraemia of Carp (SVC)

• Red sea bream Iridoviral Disease

• Viral Haemorrhagic Septicaemia (VHS)

Profitable Aqua Farming

Minimal use of Antibiotics

More Fish

Less Disease

Use of Antibiotics, Chemotherapy

Immunoprophylaxis

Sustainable Aquaculture

Disease prevention and control are crucial to maintain a sustainable aquaculture

Vaccinology in Aquaculture Industry

Vaccinology in Aquaculture Industry

• First report of disease prevention in carp using

vaccine is by Sniezko et al.,1938 = A. punctata

• Duff (1942) = A. salmonicida (Trout)

• First commercial vaccine against ERM, 1976

• First commercial vaccine against SVC, 1982

• First report of DNA vaccination against IHNV.*

• In 2005, Apex-IHN® (Novartis Animal Health) the

first DNA vaccine ever to be cleared for

marketing.* * Anderson et al., 1996 *Canadian Food inspection Agency

During the 1980s, salmon farming in Norway experienced huge losses due to bacterial diseases (mostly Vibrio spp.) and a total crash in the industry was only prevented by the use of vast

amounts of antibiotics

Case Study: Grave et al., 1990, 2003

The use of vaccine resulted an immediate and permanent reduction in the use of antibiotics, concurrent with a three fold increase in fish

production

Conti… Case Study: Grave et al., 1990, 2003

Types of Fish Vaccine Formulation!

• Bacterins

• Live attenuated vaccines

• Multivalent vaccines

• rDNA vaccines

Administration

Brudeseth et al., 2013

Green = vaccination is commonly used Yellow = vaccination is used but not fully implemented Red = fish vaccination is under development

Current status of fish vaccines

Brudeseth et al., 2013

Major producers of licensed fish vaccines

Major bacterial fish diseases in relation to vaccine availability

Bacterial disease/pathogen Major fish species affected Primary region(s)/country (s)

Vibriosis (Listonella anguillarum and V. spp.)

Salmonids/Cod/Halibut/Sea bass/bream Amberjack/yellowtail

Globally

Coldwater vibriosis (V. salmonicida) Salmonids Northern Europe,

Furunculosis (A. salmonicida) Salmonids Northern Europe, Canada/USA

ERM/Yersiniosis (Yersinia rukeri) Salmonids (FW) Europe, Chile, Canada/USA

BGD (Flavobacterium branchiophilum) Salmonids and Carp Europe, Chile, Canada/USA

Rainbow trout fry syndrome (F. psychrophilum)

Salmoinds Europe, Chile, Canada/USA

BKD (R. salmoninarum) Salmoinds Europe, Chile, Canada/USA, Japan

Streptococciosis (S. iniae) Tilapia Asia

Major viral fish diseases in relation to vaccine availability

Viral disease/pathogen Major fish species affected Primary region(s)/country(s)

IPN/IPNV Salmonids Globally

Pancreases disease /PDV Salmonids UK, Ireland, Norway

Infectious salmon anemia/ISAV

Salmonids Canada/USA (East), Norway, UK

IHN/IHNV Salmonids Canada/USA (West)

VHS/VHSV Rainbow trout, brown trout, Japanese flounder

Europe and USA

Channel cat fish virus CCV Channel cat fish USA

SVC Carp species Europe

Grass carp hemorrhagic disease / GCHDV

Grass carp China

DNA Vaccination

• DNA vaccination is defined as the intentional transfer of genetic material (DNA/RNA) to somatic cells for the purposes of influencing the immune system.*

• For DNA vaccination, a short-term expression is sufficient for evoking an immune response.

• It is different from Gene therapy!

*The Norwegian Biotechnology Advisory Board

DNA vaccine in Fish!

Promoter = pCMV or pIRF1A in case of IHNV

Distribution of pDNA in Fish

Jensen et al., 2008

Tonheim and Dalmo, 2008

Immune response against DNA vaccine

Exogenous Pathway Endogenous Pathway

Endogenous Exogenous

Antigen (Viral for example) entry in host cells

DNA Vaccine against IHNV

Plasmid DNA CMV promoter-enhancer, bovine growth hormone polyA signal, Kanamycin viral G protein gene

Factors influencing transfection and gene expression

• pDNA vector design (super coiled DNA, choice of promoter, polyA signals)

• pDNA concentration

• Dosage (ng)

• Age and size of fish

• Water temperature

• Route of administration

Structure (A)Mechanism of action (B)of suicidal DNA vaccine for IHNV

Alanso et al., 2014

Suicidal DNA vaccines: Improved safety

Gene construct Action

Overview of studies performed with DNA vaccines encoding viral or bacterial antigens in fish

Tonheim and Dalmo, 2014

Conti…

Advantages of DNA vaccines demonstrated in Fish

• Activation of both humoral and cellular immunity • Intrinsic immunostimulatory property due to CpG

motifs • Multivalent vaccination possible by simply mixing

of DNA vaccines • More effective when given at the early life stage • Effective cross-protection • Temperature-independent protection (Poikilothermic)

• Inexpensive and easy to produce • Stable as dry powder or in solution (no storage

problems)

Lorenzen and LaPatra, 2010

Disadvantages/current concerns

• Difficulty/cost of delivery is high!

• Development of myosists (myocytes becoming targets of CTLs)

• A relatively high antigenic mass needed in most fish vaccines compared with similar vaccines used in higher vertebrates.

• Environmental release of pDNA!!! • Regulatory Issues*

Antibiotic resistance genes may then spread to various bacterial populations in intestine of fish or

soil and water. *Norwegian Gene Technology Act

1. Leakage of DNA from administration site 2. By consumption of pDNA residues in the meat of

vaccinated animals 3. By spills or waste of DNA vaccine from production

process 4. pDNA may find its way to intestine where bacteria

may be taken up and released with faeces

Future prospects of DNA vaccinology in Aquaculture Industry

• Out of 35000 fish species known, 600 species are cultured as food fishes and this number will increase

• More number of Species specific DNA vaccines need to be developed

• Better understanding of fish immune system will help to design more efficient DNA vaccines

• Use of multiple epitopes in a single vaccine

Future prospects of DNA vaccinology in Aquaculture Industry

Conti…

• Immunoprophylaxis against fish parasites at industrial scale needs attention.

• Need for new strategies for mass vaccination of small fish

• Recombinant live feed!!!

Environmental safety concerns currently hinder the development and use of DNA vaccines in fish.

@ Mudasir