Challenges and opportunities in development of efficious fish ...

Challenges and opportunities in
development of efficious fish vaccines
Edel Anne Norderhus
Director Product Development R&D
PHARMAQ

PHARMAQ`s business idea
• We provide environmentally sound,
safe and efficacious health products to
the global aquaculture industry through
targeted research and the commitment
of dedicated people
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PHARMAQ company
PHARMAQ company
• Head office in Norway, subsidiaries in the UK, Chile, Turkey and
Vietnam
• Numbers of employees per September 141 persons
• Global market share in salmonids more than 50 %. Main competitor
Novartis and Intervet Shering Plough
• Approximately NOK 80 mill allocated to R&D in 2011

Contents Agenda
• General information
about fish vaccines
• Challenges for fish
vaccine development
- Cost-effective vaccine
- Long-lasting protection
- Safe
- Authorities requirement to
documentation
- New technology

Pathogens in licensed vaccines
Type
Bacteria
Intracellular
bacteria
Species
Aeromonas salmonicida,
Listonella (Vibrio) anguillarum O1 & O2
Photobacterium damsela subsp. piscicida,
Moritella viscosa,
Vibrio salmonicida.
Vibrio ordalii
Yersinia ruckeri
Streptococcus iniae
Streptococcus agalactiae
Edwardsiella ictaluri
Edwardsiella tarda
Lactococcus garvieae
Flavobacterium phsycrophilium
Piscirickettsia salmonis
Francisella sp
Virus
Infectious pancreatic necrosis virus (IPNV)
Infectious salmon anemia virus (ISAV)
Pancreas disease virus (PDV)
Infectious Heamtopoietic Necrosis Virus (IHNV)

Geographical areas where vaccination are
common practice
Vaccines are available mainly where aquaculture is commerzilisedareas

Different types of vaccines in Norway 1987-2003
30,000
180,000
Immersion vaccine liters
25,000
20,000
15,000
10,000
5,000
160,000
140,000
120,000
100,000
80,000
60,000
40,000
20,000
Injection vaccine doses
immersion (liters)
oil-based injection
water-based injection
0,000
0,000
1985
1987
1989
1991
1993
1995
1997
1999
2001
2003
Sommerset et al. Expert Rev Vaccines 2005;4:89-101

Challenges in fish vaccine development
• Low cost
• Efficacious
– Long-lasting protection
– Multivalent: Efficacious towards several
diseases
• Safe
– Low side effect levels (inactivated)
– Not revert to virulence (live)
– GMO (live and DNA vaccines)

Fish health represents a small portion of total farming cost
- but willingness to spend more when needed (e.g. lice, PD)
Cost structure in the Norwegian salmon farming industry
NOK/kg 2006 2007 2008 2009 2010e 2011e
Smolt 1,76 2,36 2,33 2,16 2,16 2,16
Feed 9,29 10,08 10,85 10,94 11,24 11,34
Insurance 0,18 0,17 0,17 0,15 0,15 0,15
Salaries 1,59 1,54 1,59 1,43 1,43 1,43
Depreciation 0,82 0,93 1,18 1,10 1,10 1,10
Other operating cost * 2,48 2,12 3,20 3,22 3,52 3,62
Net finance 0,26 0,48 1,04 0,42 0,42 0,42
Production cost 16,37 17,68 20,34 19,42 20,02 20,22
Slaughter 2,32 2,50 2,59 2,61 2,61 2,61
Total cost 18,69 20,18 22,93 22,03 22,63 22,83
* Whereof
Vaccines ** 0,20 0,17 0,19 0,29 0,33 0,32
% of total cost 1,1 % 0,8 % 0,8 % 1,3 % 1,5 % 1,4 %
Sea lice treatment ** 0,76 0,79 0,81
% of total cost 3,4 % 3,5 % 3,5 %
** Rough estimates based on statements from industry players and must not be considered accurate
Source: Norwegian Directorate of Fisheries, FishPool, Kverva Research
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From idea to licensed product
Identification - process development – production –
validation – documentation - License

Development of a process for a new virus
• How to achieve high yield ... within a limit time
and have a reproducible process
Cost effective product
CHALLENGES :
• Select of cell line
• Achieve a high yield
• Choose the right technology
to culture the virus
• Possible to up-scale
Cell
Replication

Water in oil emulsion
• Water in oil is a on the most efficient vaccine formulations
for ensuring long term protection by a single injection.
• Factor contributing to efficacy
• .
– 1) Depot: Retention and slow release of antigen
– 2) Presentation: Uptake of vaccine droplets by APCs
– 3) Immunostimulation: Bacteria contain immunostimulatory
compounds such as lipopolysaccharides, peptidoglycan and
procaryotic DNA motifs.

Vaccine administration
Labour
Efficacy
Oral Bath Immersion Injection
Slide 13
Figures cortesy of Ida Skaar

Oral vaccines
• Ideal delivery method (no labour)
• Poor and short lasting protection
– Exceptions: Yersinia ruckeri and Vibrio
anguillarum.
• Gastric degradation can affect
protective antigen
Day 3
Day 1

Adverse reactions
Reactions after oil
based injection in
salmon and sea bass
Photo: Trygve T Poppe.
Adverse reactions are species
dependent
Photo: Thanos Prapas

Extended safety
Fish in holdingtank
Individual
vaccination in
abdominal cavity
Immunization
sampling
sampling
6 weeks 12 weeks
Randomly selected
fish transfered to
experimental units
Acclimation for min
one week:
Temperature 17 o C
water quality,
light..
Adhesions and melanin
evaluation
Weight

Vaccine responses
• Killed vaccines
– Humoral (antibody)
responses
• Live, attenuated and
DNA vaccines
– Cytotoxic T-cell
response
• New adjuvant systems
aid in activating both
arms of the adaptive
immune system
Fusogenic
liposomes
Virosomes
ISCOMs
Mineral salts ,
Emulsions
Polymer particles

Fish Vaccines
Past Current Future
Waterbased
Aluminium
Emulsions
DNA
Live Bacterial
What is the
opportunities and
challenges for
future vaccine
technology in
aquaculture
Live viral
Reverse genetics
PLGA particles, VLPs,
ISCOM, Virosomes,
Liposomes
1970 1980 1990 2000 2010

DNA vaccines
• Advantages
– presentation by MHC-I
elicits cytotoxic response
as well
– low cost, stable
– multivalent or multigenic
– can be modified to include
co-stimulatory molecules
• Disadvantages
– delivery by i.m. Injection
– Persistence of plasmid DNA in
muscle
– GMO issues
• Viral epitope
inserted in
plasmid
• Taken up by
host cell –
viral protein
antigen
produced in
host cells
• Mimics
natural
infection
Poland et al. 2002, BMJ 324;1315-1319

Experimental DNA vaccines in fish
Pathogen Viral fam Gene Protection
Infectious Hematopoietic Necrosis Virus (IHNV)
Viral Hemorrhagic Septicemia Virus (VHSV),
Hirame rhabdovirus (HIRRV)
Spring viremia of carp virus (SVCV)
Rhabdoviridae G-gene High
Infectious pancreatic necrosis virus (IPNV)
Birnaviridae
Seg. A large ORF
& VP2
Infectious salmon anemia virus (ISAV)
Orthomyxoviridae
Hemaglutinin
esterase HE
Moderate
Red seabream iridovirus (RSIV)
Iridoviridae
Major capsid
protein, ORF569
Channel catfish virus (CCV) Herpesviridae ORF 6&59 No
Atlantic halibut nodavirus (AHNV) Nodaviridae Capsid protein No
Kurath 2008

Live vaccines
• Advantages
– High efficacy (elicits
cytotoxic response)
– Low production cost,
compared to inactivated
– Can be administered by
dip/bath or oral delivery
(natural pathway of
infection)
• Challenges
– Safety: Must not revert to
virulence
– Document that vaccine is safe
– The safest vaccines are
produced by use genetic
modification (GMO issues)
Methods of attenuation
• Attenuation
by passage
in cells.
• Virus lose
virulent
properties by
adapting to
growth in cell
lines (virulent
properties not
selected)
• What is risk
of reversion
• Attenuation
by use of
genetic tools
(reverse
genetics)
• Removal
genes vital
for growth or
virulence is a
safe method
of attenuation
• GMO issues

Live attenuated bacterial vaccine
• The first licensed modified live bacterial vaccines
for fish. Catfish vaccine for US marked.
– Edw. Ict. AQUAVAC-ESC
– Fla. Col. AQUAVAC-COL
(Intervet Schering-Plough)
• Live Arthrobacter sp. nov., cross protective
property against Renibacterium salmoninarum.
For use in salmonids by injection
– Renogen®
(NOVARTIS ANIMAL HEALTH CANADA Inc)
Slide 22

Basic requirements for live vaccines
• Contain the necessary antigenic
determinants to induce protective
immunity
• Possess a high immunogenic potential
• Safe for administration without risk of
clinical infection in the vaccinee
• Safe for humans
• Induce a serological response that
allows differentiation of vaccinated
from infected animals
• Stable for long-term storage
• Not to be contraindicated for release in
the environment
• Traceable in the animal population and
in the environment

Effect of a live vaccine
120
Cumulative mortality
100
80
60
40
20
Vaccine
shedders
Cumulative mortality
100
80
60
40
20
Test vaccine 1
Test vaccine 2
PBS
Test vaccine 3
AJ 3000
Shedders
Test vaccine 4
0
0 4 8 12 16 20 24 28 32 36 40 44
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
Experiment A. The Vaccine is
based upon new technology
Experiment B. Test vaccine 3 and 4
are based upon new technology
Cohabitant challenge model: Natural way of infection. 20 %
shedders used. Rather low reproducibility in the experiments. The
new vaccine technology always show good protection even the
other test vaccines are variable.

Virus-like particles
(VLPs)
• Subunit vaccines
• Viral capsid proteins selfassemble
into virion-like
structures
• Expression systems:
Baculovirus,yeast,plants
Poland et al. 2002,
BMJ 324;1315-1319
Iscoms
• ISCOM (Immune
stimulating complex)
• Spherical open cage like
structures (40 nm)
• Spontaneously formed
when mixing together antigen,
cholesterol, phospholipids and
Quillaia saponins
• Immunopotentiating
properties
• Facilitates interaction with
membranes
Liposomes and
virosomes
• Liposomes: spherical,
structures of concentric
bilayers enclosing an aqueous
phase.
– Antigen associated .
– Small (~100 nm), unilamellar or
large (~1 µm), multilamellar
• Virosomes: empty virus
envelope
– No genetic material
– Fusion active proteins in
membrane (HA )

Summary: Future challenges in vaccine
development
– Identify protective antigen
– Develop cost effective production
– Obtain challenge model for
vaccine efficacy
– Reduce vaccine side effects
– Develop more multivalent
vaccines without affecting efficacy
of each component
– Willingness of customers to invest
in high quality and higher priced
vaccines. Understanding that it
will repay at end of fish production
– New diseases that will need new
vaccine technologies for vaccine
efficacy

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