Challenges and opportunities in development of efficious fish ...
Challenges and opportunities in development of efficious fish ...
Challenges and opportunities in development of efficious fish ...
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<strong>Challenges</strong> <strong>and</strong> <strong>opportunities</strong> <strong>in</strong><br />
<strong>development</strong> <strong>of</strong> <strong>efficious</strong> <strong>fish</strong> vacc<strong>in</strong>es<br />
Edel Anne Norderhus<br />
Director Product Development R&D<br />
PHARMAQ
PHARMAQ`s bus<strong>in</strong>ess idea<br />
• We provide environmentally sound,<br />
safe <strong>and</strong> efficacious health products to<br />
the global aquaculture <strong>in</strong>dustry through<br />
targeted research <strong>and</strong> the commitment<br />
<strong>of</strong> dedicated people<br />
2
PHARMAQ company<br />
PHARMAQ company<br />
• Head <strong>of</strong>fice <strong>in</strong> Norway, subsidiaries <strong>in</strong> the UK, Chile, Turkey <strong>and</strong><br />
Vietnam<br />
• Numbers <strong>of</strong> employees per September 141 persons<br />
• Global market share <strong>in</strong> salmonids more than 50 %. Ma<strong>in</strong> competitor<br />
Novartis <strong>and</strong> Intervet Sher<strong>in</strong>g Plough<br />
• Approximately NOK 80 mill allocated to R&D <strong>in</strong> 2011
Contents Agenda<br />
• General <strong>in</strong>formation<br />
about <strong>fish</strong> vacc<strong>in</strong>es<br />
• <strong>Challenges</strong> for <strong>fish</strong><br />
vacc<strong>in</strong>e <strong>development</strong><br />
- Cost-effective vacc<strong>in</strong>e<br />
- Long-last<strong>in</strong>g protection<br />
- Safe<br />
- Authorities requirement to<br />
documentation<br />
- New technology
Pathogens <strong>in</strong> licensed vacc<strong>in</strong>es<br />
Type<br />
Bacteria<br />
Intracellular<br />
bacteria<br />
Species<br />
Aeromonas salmonicida,<br />
Listonella (Vibrio) anguillarum O1 & O2<br />
Photobacterium damsela subsp. piscicida,<br />
Moritella viscosa,<br />
Vibrio salmonicida.<br />
Vibrio ordalii<br />
Yers<strong>in</strong>ia ruckeri<br />
Streptococcus <strong>in</strong>iae<br />
Streptococcus agalactiae<br />
Edwardsiella ictaluri<br />
Edwardsiella tarda<br />
Lactococcus garvieae<br />
Flavobacterium phsycrophilium<br />
Piscirickettsia salmonis<br />
Francisella sp<br />
Virus<br />
Infectious pancreatic necrosis virus (IPNV)<br />
Infectious salmon anemia virus (ISAV)<br />
Pancreas disease virus (PDV)<br />
Infectious Heamtopoietic Necrosis Virus (IHNV)
Geographical areas where vacc<strong>in</strong>ation are<br />
common practice<br />
Vacc<strong>in</strong>es are available ma<strong>in</strong>ly where aquaculture is commerzilisedareas
Different types <strong>of</strong> vacc<strong>in</strong>es <strong>in</strong> Norway 1987-2003<br />
30,000<br />
180,000<br />
Immersion vacc<strong>in</strong>e liters<br />
25,000<br />
20,000<br />
15,000<br />
10,000<br />
5,000<br />
160,000<br />
140,000<br />
120,000<br />
100,000<br />
80,000<br />
60,000<br />
40,000<br />
20,000<br />
Injection vacc<strong>in</strong>e doses<br />
immersion (liters)<br />
oil-based <strong>in</strong>jection<br />
water-based <strong>in</strong>jection<br />
0,000<br />
0,000<br />
1985<br />
1987<br />
1989<br />
1991<br />
1993<br />
1995<br />
1997<br />
1999<br />
2001<br />
2003<br />
Sommerset et al. Expert Rev Vacc<strong>in</strong>es 2005;4:89-101
<strong>Challenges</strong> <strong>in</strong> <strong>fish</strong> vacc<strong>in</strong>e <strong>development</strong><br />
• Low cost<br />
• Efficacious<br />
– Long-last<strong>in</strong>g protection<br />
– Multivalent: Efficacious towards several<br />
diseases<br />
• Safe<br />
– Low side effect levels (<strong>in</strong>activated)<br />
– Not revert to virulence (live)<br />
– GMO (live <strong>and</strong> DNA vacc<strong>in</strong>es)
Fish health represents a small portion <strong>of</strong> total farm<strong>in</strong>g cost<br />
- but will<strong>in</strong>gness to spend more when needed (e.g. lice, PD)<br />
Cost structure <strong>in</strong> the Norwegian salmon farm<strong>in</strong>g <strong>in</strong>dustry<br />
NOK/kg 2006 2007 2008 2009 2010e 2011e<br />
Smolt 1,76 2,36 2,33 2,16 2,16 2,16<br />
Feed 9,29 10,08 10,85 10,94 11,24 11,34<br />
Insurance 0,18 0,17 0,17 0,15 0,15 0,15<br />
Salaries 1,59 1,54 1,59 1,43 1,43 1,43<br />
Depreciation 0,82 0,93 1,18 1,10 1,10 1,10<br />
Other operat<strong>in</strong>g cost * 2,48 2,12 3,20 3,22 3,52 3,62<br />
Net f<strong>in</strong>ance 0,26 0,48 1,04 0,42 0,42 0,42<br />
Production cost 16,37 17,68 20,34 19,42 20,02 20,22<br />
Slaughter 2,32 2,50 2,59 2,61 2,61 2,61<br />
Total cost 18,69 20,18 22,93 22,03 22,63 22,83<br />
* Where<strong>of</strong><br />
Vacc<strong>in</strong>es ** 0,20 0,17 0,19 0,29 0,33 0,32<br />
% <strong>of</strong> total cost 1,1 % 0,8 % 0,8 % 1,3 % 1,5 % 1,4 %<br />
Sea lice treatment ** 0,76 0,79 0,81<br />
% <strong>of</strong> total cost 3,4 % 3,5 % 3,5 %<br />
** Rough estimates based on statements from <strong>in</strong>dustry players <strong>and</strong> must not be considered accurate<br />
Source: Norwegian Directorate <strong>of</strong> Fisheries, FishPool, Kverva Research<br />
9
From idea to licensed product<br />
Identification - process <strong>development</strong> – production –<br />
validation – documentation - License
Development <strong>of</strong> a process for a new virus<br />
• How to achieve high yield ... with<strong>in</strong> a limit time<br />
<strong>and</strong> have a reproducible process<br />
Cost effective product<br />
CHALLENGES :<br />
• Select <strong>of</strong> cell l<strong>in</strong>e<br />
• Achieve a high yield<br />
• Choose the right technology<br />
to culture the virus<br />
• Possible to up-scale<br />
Cell<br />
Replication
Water <strong>in</strong> oil emulsion<br />
• Water <strong>in</strong> oil is a on the most efficient vacc<strong>in</strong>e formulations<br />
for ensur<strong>in</strong>g long term protection by a s<strong>in</strong>gle <strong>in</strong>jection.<br />
• Factor contribut<strong>in</strong>g to efficacy<br />
• .<br />
– 1) Depot: Retention <strong>and</strong> slow release <strong>of</strong> antigen<br />
– 2) Presentation: Uptake <strong>of</strong> vacc<strong>in</strong>e droplets by APCs<br />
– 3) Immunostimulation: Bacteria conta<strong>in</strong> immunostimulatory<br />
compounds such as lipopolysaccharides, peptidoglycan <strong>and</strong><br />
procaryotic DNA motifs.
Vacc<strong>in</strong>e adm<strong>in</strong>istration<br />
Labour<br />
Efficacy<br />
Oral Bath Immersion Injection<br />
Slide 13<br />
Figures cortesy <strong>of</strong> Ida Skaar
Oral vacc<strong>in</strong>es<br />
• Ideal delivery method (no labour)<br />
• Poor <strong>and</strong> short last<strong>in</strong>g protection<br />
– Exceptions: Yers<strong>in</strong>ia ruckeri <strong>and</strong> Vibrio<br />
anguillarum.<br />
• Gastric degradation can affect<br />
protective antigen<br />
Day 3<br />
Day 1
Adverse reactions<br />
Reactions after oil<br />
based <strong>in</strong>jection <strong>in</strong><br />
salmon <strong>and</strong> sea bass<br />
Photo: Trygve T Poppe.<br />
Adverse reactions are species<br />
dependent<br />
Photo: Thanos Prapas
Extended safety<br />
Fish <strong>in</strong> hold<strong>in</strong>gtank<br />
Individual<br />
vacc<strong>in</strong>ation <strong>in</strong><br />
abdom<strong>in</strong>al cavity<br />
Immunization<br />
sampl<strong>in</strong>g<br />
sampl<strong>in</strong>g<br />
6 weeks 12 weeks<br />
R<strong>and</strong>omly selected<br />
<strong>fish</strong> transfered to<br />
experimental units<br />
Acclimation for m<strong>in</strong><br />
one week:<br />
Temperature 17 o C<br />
water quality,<br />
light..<br />
Adhesions <strong>and</strong> melan<strong>in</strong><br />
evaluation<br />
Weight
Vacc<strong>in</strong>e responses<br />
• Killed vacc<strong>in</strong>es<br />
– Humoral (antibody)<br />
responses<br />
• Live, attenuated <strong>and</strong><br />
DNA vacc<strong>in</strong>es<br />
– Cytotoxic T-cell<br />
response<br />
• New adjuvant systems<br />
aid <strong>in</strong> activat<strong>in</strong>g both<br />
arms <strong>of</strong> the adaptive<br />
immune system<br />
Fusogenic<br />
liposomes<br />
Virosomes<br />
ISCOMs<br />
M<strong>in</strong>eral salts ,<br />
Emulsions<br />
Polymer particles
Fish Vacc<strong>in</strong>es<br />
Past Current Future<br />
Waterbased<br />
Alum<strong>in</strong>ium<br />
Emulsions<br />
DNA<br />
Live Bacterial<br />
What is the<br />
<strong>opportunities</strong> <strong>and</strong><br />
challenges for<br />
future vacc<strong>in</strong>e<br />
technology <strong>in</strong><br />
aquaculture <br />
Live viral<br />
Reverse genetics<br />
PLGA particles, VLPs,<br />
ISCOM, Virosomes,<br />
Liposomes<br />
<br />
1970 1980 1990 2000 2010
DNA vacc<strong>in</strong>es<br />
• Advantages<br />
– presentation by MHC-I<br />
elicits cytotoxic response<br />
as well<br />
– low cost, stable<br />
– multivalent or multigenic<br />
– can be modified to <strong>in</strong>clude<br />
co-stimulatory molecules<br />
• Disadvantages<br />
– delivery by i.m. Injection<br />
– Persistence <strong>of</strong> plasmid DNA <strong>in</strong><br />
muscle<br />
– GMO issues<br />
• Viral epitope<br />
<strong>in</strong>serted <strong>in</strong><br />
plasmid<br />
• Taken up by<br />
host cell –<br />
viral prote<strong>in</strong><br />
antigen<br />
produced <strong>in</strong><br />
host cells<br />
• Mimics<br />
natural<br />
<strong>in</strong>fection<br />
Pol<strong>and</strong> et al. 2002, BMJ 324;1315-1319
Experimental DNA vacc<strong>in</strong>es <strong>in</strong> <strong>fish</strong><br />
Pathogen Viral fam Gene Protection<br />
Infectious Hematopoietic Necrosis Virus (IHNV)<br />
Viral Hemorrhagic Septicemia Virus (VHSV),<br />
Hirame rhabdovirus (HIRRV)<br />
Spr<strong>in</strong>g viremia <strong>of</strong> carp virus (SVCV)<br />
Rhabdoviridae G-gene High<br />
Infectious pancreatic necrosis virus (IPNV)<br />
Birnaviridae<br />
Seg. A large ORF<br />
& VP2<br />
Infectious salmon anemia virus (ISAV)<br />
Orthomyxoviridae<br />
Hemaglut<strong>in</strong><strong>in</strong><br />
esterase HE<br />
Moderate<br />
Red seabream iridovirus (RSIV)<br />
Iridoviridae<br />
Major capsid<br />
prote<strong>in</strong>, ORF569<br />
Channel cat<strong>fish</strong> virus (CCV) Herpesviridae ORF 6&59 No<br />
Atlantic halibut nodavirus (AHNV) Nodaviridae Capsid prote<strong>in</strong> No<br />
Kurath 2008
Live vacc<strong>in</strong>es<br />
• Advantages<br />
– High efficacy (elicits<br />
cytotoxic response)<br />
– Low production cost,<br />
compared to <strong>in</strong>activated<br />
– Can be adm<strong>in</strong>istered by<br />
dip/bath or oral delivery<br />
(natural pathway <strong>of</strong><br />
<strong>in</strong>fection)<br />
• <strong>Challenges</strong><br />
– Safety: Must not revert to<br />
virulence<br />
– Document that vacc<strong>in</strong>e is safe<br />
– The safest vacc<strong>in</strong>es are<br />
produced by use genetic<br />
modification (GMO issues)<br />
Methods <strong>of</strong> attenuation<br />
• Attenuation<br />
by passage<br />
<strong>in</strong> cells.<br />
• Virus lose<br />
virulent<br />
properties by<br />
adapt<strong>in</strong>g to<br />
growth <strong>in</strong> cell<br />
l<strong>in</strong>es (virulent<br />
properties not<br />
selected)<br />
• What is risk<br />
<strong>of</strong> reversion<br />
• Attenuation<br />
by use <strong>of</strong><br />
genetic tools<br />
(reverse<br />
genetics)<br />
• Removal<br />
genes vital<br />
for growth or<br />
virulence is a<br />
safe method<br />
<strong>of</strong> attenuation<br />
• GMO issues
Live attenuated bacterial vacc<strong>in</strong>e<br />
• The first licensed modified live bacterial vacc<strong>in</strong>es<br />
for <strong>fish</strong>. Cat<strong>fish</strong> vacc<strong>in</strong>e for US marked.<br />
– Edw. Ict. AQUAVAC-ESC<br />
– Fla. Col. AQUAVAC-COL<br />
(Intervet Scher<strong>in</strong>g-Plough)<br />
• Live Arthrobacter sp. nov., cross protective<br />
property aga<strong>in</strong>st Renibacterium salmon<strong>in</strong>arum.<br />
For use <strong>in</strong> salmonids by <strong>in</strong>jection<br />
– Renogen®<br />
(NOVARTIS ANIMAL HEALTH CANADA Inc)<br />
Slide 22
Basic requirements for live vacc<strong>in</strong>es<br />
• Conta<strong>in</strong> the necessary antigenic<br />
determ<strong>in</strong>ants to <strong>in</strong>duce protective<br />
immunity<br />
• Possess a high immunogenic potential<br />
• Safe for adm<strong>in</strong>istration without risk <strong>of</strong><br />
cl<strong>in</strong>ical <strong>in</strong>fection <strong>in</strong> the vacc<strong>in</strong>ee<br />
• Safe for humans<br />
• Induce a serological response that<br />
allows differentiation <strong>of</strong> vacc<strong>in</strong>ated<br />
from <strong>in</strong>fected animals<br />
• Stable for long-term storage<br />
• Not to be contra<strong>in</strong>dicated for release <strong>in</strong><br />
the environment<br />
• Traceable <strong>in</strong> the animal population <strong>and</strong><br />
<strong>in</strong> the environment
Effect <strong>of</strong> a live vacc<strong>in</strong>e<br />
120<br />
Cumulative mortality<br />
100<br />
80<br />
60<br />
40<br />
20<br />
Vacc<strong>in</strong>e<br />
shedders<br />
Cumulative mortality<br />
100<br />
80<br />
60<br />
40<br />
20<br />
Test vacc<strong>in</strong>e 1<br />
Test vacc<strong>in</strong>e 2<br />
PBS<br />
Test vacc<strong>in</strong>e 3<br />
AJ 3000<br />
Shedders<br />
Test vacc<strong>in</strong>e 4<br />
0<br />
0 4 8 12 16 20 24 28 32 36 40 44<br />
0<br />
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41<br />
Experiment A. The Vacc<strong>in</strong>e is<br />
based upon new technology<br />
Experiment B. Test vacc<strong>in</strong>e 3 <strong>and</strong> 4<br />
are based upon new technology<br />
Cohabitant challenge model: Natural way <strong>of</strong> <strong>in</strong>fection. 20 %<br />
shedders used. Rather low reproducibility <strong>in</strong> the experiments. The<br />
new vacc<strong>in</strong>e technology always show good protection even the<br />
other test vacc<strong>in</strong>es are variable.
Virus-like particles<br />
(VLPs)<br />
• Subunit vacc<strong>in</strong>es<br />
• Viral capsid prote<strong>in</strong>s selfassemble<br />
<strong>in</strong>to virion-like<br />
structures<br />
• Expression systems:<br />
Baculovirus,yeast,plants<br />
Pol<strong>and</strong> et al. 2002,<br />
BMJ 324;1315-1319<br />
Iscoms<br />
• ISCOM (Immune<br />
stimulat<strong>in</strong>g complex)<br />
• Spherical open cage like<br />
structures (40 nm)<br />
• Spontaneously formed<br />
when mix<strong>in</strong>g together antigen,<br />
cholesterol, phospholipids <strong>and</strong><br />
Quillaia sapon<strong>in</strong>s<br />
• Immunopotentiat<strong>in</strong>g<br />
properties<br />
• Facilitates <strong>in</strong>teraction with<br />
membranes<br />
Liposomes <strong>and</strong><br />
virosomes<br />
• Liposomes: spherical,<br />
structures <strong>of</strong> concentric<br />
bilayers enclos<strong>in</strong>g an aqueous<br />
phase.<br />
– Antigen associated .<br />
– Small (~100 nm), unilamellar or<br />
large (~1 µm), multilamellar<br />
• Virosomes: empty virus<br />
envelope<br />
– No genetic material<br />
– Fusion active prote<strong>in</strong>s <strong>in</strong><br />
membrane (HA )
Summary: Future challenges <strong>in</strong> vacc<strong>in</strong>e<br />
<strong>development</strong><br />
– Identify protective antigen<br />
– Develop cost effective production<br />
– Obta<strong>in</strong> challenge model for<br />
vacc<strong>in</strong>e efficacy<br />
– Reduce vacc<strong>in</strong>e side effects<br />
– Develop more multivalent<br />
vacc<strong>in</strong>es without affect<strong>in</strong>g efficacy<br />
<strong>of</strong> each component<br />
– Will<strong>in</strong>gness <strong>of</strong> customers to <strong>in</strong>vest<br />
<strong>in</strong> high quality <strong>and</strong> higher priced<br />
vacc<strong>in</strong>es. Underst<strong>and</strong><strong>in</strong>g that it<br />
will repay at end <strong>of</strong> <strong>fish</strong> production<br />
– New diseases that will need new<br />
vacc<strong>in</strong>e technologies for vacc<strong>in</strong>e<br />
efficacy
!<br />
We make aquaculture progress<br />
<strong>in</strong>novation<br />
quality<br />
team spirit<br />
dynamism<br />
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