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Comenius University Bratislava Aquaculture Vaccinology William (Bill) W. Kay, PhD. Prof. ( Emeritus ), Dept. of Biochemistry & Microbiology University of Victoria, Victoria, B.C. CEO & Pres. Microtek International Inc., Saanichton, B.C. A life of swimming in a changing cocktail of

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Comenius University Bratislava

Aquaculture Vaccinology

William (Bill) W. Kay, PhD.

Prof. (Emeritus), Dept. of Biochemistry & Microbiology

University of Victoria, Victoria, B.C.

CEO & Pres. Microtek International Inc., Saanichton, B.C.

recombinant subunit vaccines

A life of swimming in a changing cocktail of

microorganisms –some of which are highly virulent pathogens.

Recombinant subunit vaccines

what are the commercially important finfish species
What are the commercially important finfish species?


Atlantic salmon, Rainbow trout, Pacific salmon (Coho, Chinook).

Non salmonids:

Tilapia sp.,Channel Catfish, Striped Bass, Sea bass, Carp sp. (Koi, Grasscarp), Sablefish, Cod, Halibut, Turbot, Eel, Seabream, Seabass, Yellowtail sp., Ayu, Hamachi, Barrumundi, Milkfish, Japanese amberjack, Bluefin Tuna, Cobia etc….

It is estimated there are >300 cultivatable finfish sp.

(…and they all get sick!)

what are the commercially important microbial pathogens of fish
What are the commercially important microbial pathogens of fish?

Bacteria:Vibrio sp: (V.anguillarum Types I&II, V. salmonicida, Vibrio viscosus [Moritella viscosa]); Aeromonas sp: (A. salmonicida, A. hydrophila, A. cavia); Flavobacterium sp: (F. psychrophilum, F. columnare, F. maritimus [Tenacibacterium maritimum]); Edwardsiellasp: (E. ictaluri, E. tarda);Streptococcus sp. (S. iniae, S. agalactiae); Mycobacterium sp. (M. marinum, M. fortuitum, M. chelonae); Pseudomonas sp. (P. plecoglossida, P. anguiloseptica) Others:Yersinia ruckerii, Renibacterium salmoninarum, Piscirickettsia salmonis, Francisella sp., Photobacterium damselae, Lactococcus garvieae.………ad nauseum.

pathogens cont viruses family
pathogens (cont.)Viruses: Family:
  • IHNV* (Infectious Hematapoietic Virus) Rhabdoviridae
  • VHS (Viral Hemorrhagic Septicemia) Rhabdoviridae
  • IPNV* (Infectious Pancreatic Necrosis Virus) Birnaviridae
  • ISAV* (Infectious Salmonid Anemia Virus) Orthomyxoviridae
  • PDV (Pancreatic Disease Virus) Togaviridae
  • CCV (Channel catfish virus) Herpeviridae
  • KHV (Koi Hepes Virus) Herpeviridae
  • CPV (Carp Pox Virus) Hepesviridae
  • IRV (Iridovirus) Iridoviridae
  • NDV* (Nodavirus Disease) Nodaviridae
  • HSMI (Heart & Skeletal Muscle Inflamation Virus ) ???
pathogens cont parasites
Pathogens (cont.)Parasites:

Endoparasite: Disease Family

  • Kudoa thyrsites Soft Flesh Disease Myxosporean
  • Ceratomyxa shasta Ceratomyxosis Myxosporean
  • PKX Pancreatic Disease Myxosporean
  • Myxobolus cerebalus Whirling Disease Myxosporean


  • Gyrodactylus salaris GyrodactylosisMonogean
  • Lepeophtheirus salmonisSea lice Caligean
  • Caligus sp. (C. elongatus, C. teres, C. rogercressyi, etc.)

What is a Vaccine?

  • Anything (antigen) derived from a pathogen that when administered to a target animal (fish):
    • elicits a specific protective response against a foreign body = IR (immune response)
    • specific response must also have a memory component = DOI (duration of immunity)
how do we administer vaccines to fish
How do we administer vaccines to fish?
  • IP or IM injection (by hand or machine)
  • Immersion.
  • Oral:
    • Dried with feed
    • encapsulated
traditional vaccine testing clinical trials
Traditional vaccine testing & clinical trials:

The “White Box”

(i.e. The Wetlab)

Direct immersion


Lethal Challenge

giving rise to RPS


SL attachment

The ‘Black Box’

(i.e.The “Field” )


ld 50 or r elative p ercent s urvival rps
LD50 or Relative Percent Survival (RPS)

How do we measure efficacy?

RPS = {1- (%Mort. Vaccinates/%Mort. Controls)}100







Aquaculture Vaccines - History

  • Furunculosis - Proof of concept - Duff (1942)
  • Commercial bacterial vaccines (mid 1970’s)
  • Oil adjuvanted injectable vaccines (early 1990’s)
  • Commercial viral vaccines (mid 1990’s)
  • Commercial recombinant vaccines (late 1990’s)
  • Commercial DNA vaccine (2005)
  • Reverse engineered & multiepitope vaccines (2007-)

Impact of vaccines on salmon culture

From Markestad and Grave, 1997

traditional vaccine development or grow em and show em

Whole organism

Traditional vaccine development or….“Grow ‘em and Show ‘em”


Formulations with




preservatives etc.


inactivation &


Ex.Advantigen 5.1



Advantigen 5.1

EU Serial Release - 3 serials: CEFAS

* Suggested figure - no monograph standard.

complications of whole cell vaccines
Complications of whole cell vaccines
  • Selection of representative strains
  • Maintenance of immunogenicity
  • Safety and potency
  • Interference
  • Inactivations and formulations may differ
  • QC/QA issues
  • Shelf life issues
  • Regulatory Issues
  • Cost of goods
complications to multivalent vaccines
Complications to multivalent vaccines
  • Reproducible disease models must be established for each disease ($$$$).
  • Intermittent fish availability is a problem.
  • Indirect disease criteria are difficult to convince regulators.
  • COGs is high since separate fermentations are required for each component.
  • EU GMP (human vaccine) production standards.
how has biotechnology changed this targeting specific antigens for subunit vaccines


G protein

Matrix proteins

(M1 & M2)


L = polymerase

N = nucleocapsid






or S-Layer

How has biotechnology changed this?(Targeting specific antigens for “subunit” vaccines)

Ex. viruses vs bacteria or

~5 vs ~50 targets.



classical subunit vaccines

Classical subunit vaccines

Polysaccharides and proteins, purified from pathogenic organisms, and detoxified toxins, are examples of subunit immunogens.

The subunits of pathogenic origin are safe to use as vaccines, provided that extraction procedure or detoxifying methods gives a pure product.

Production of subunit vaccines generally requires large-scale, complicated down-stream processing of pathogenic organisms.

These vaccines need adjuvants or various conjugates to render them more immunogenic.

recombinant subunit vaccines demystified
Recombinant subunit vaccines – demystified.

Cut DNA


Insert gene

into a plasmid


Amplify in E. coli

Produce in E. coli


E. coli

Introduce into E. coli


Ex.Sea lice proteins

made by harmless,

boring, old E. coli


Upside of recombinant subunit vaccines

  • Pathogens can be entirely excluded from production = safety.
  • Multi-epitope vaccines engineered to a single strain = lower COGs.
  • Suppressor genes can be eliminated = detoxified.
  • Immune response can be tuned with pTCEs = more efficacious.
  • Better antigens can be further engineered for ex. through invitro evolution = more efficacious.
  • Vaccines have longer half-lives and can be stored frozen prior to formulations (clock starts at formulation) = lower COGs.
  • Genome sequencing projects provide advance information= more antigens.

Downside of recombinant subunit vaccines

  • The development is time consuming and more expensive than live attenuated or killed vaccines - ($$$$).
  • Requires highly skilled (expensive) R&D teams - ($$$).
  • Some regulatory agencies are slow to accept rDNA products - (time & $$$$).
  • Multiple antigens may be required - ($$$$).

Recent subunit vaccines

  • Via Recombinant DNA Technology:
    • Protein Engineering
    • Nucleic Acid Vaccines

Antigenic fragment from

A. Salmonicida OMP.

OMP from E. coli


A Model Case:Piscirickettsia salmonis

  • A gram negative, rickettsia bacterial pathogen
  • Aetiological agent of salmonid rickettsial septicaemia (SRS)
  • First recognized and isolated in 1989 from a coho salmon (Oncorhynchus kisutch)
  • SRS afflicts global aquaculture of salmon
  • A devastating problem in Chile, emerging in NA & EU.

Salmonid Rickettsial Septicaemia (SRS)

  • P. salmonis causes a systemic infection
  • obligate intracellular pathogen
  • up to 40% of net pen stock can be lost annually ($150M-200M in Chile)
  • SRS is poorly managed by antibiotic treatment
  • natural reservoir or vector is unknown
growth of p salmonis
Growth of P. salmonis
  • grown in salmonid cell culture @15ºC
  • 14-18 days for full growth
  • exceptionally low yields (~1/10 other Rickettsias)
  • grows within cytoplasmic vacuoles of host cells
large scale growth of p salmonis
Large scale growth ofP. salmonis
  • A very large monolayer surface area is required
  • 3 months minimum from freezer to harvest.
  • More complex than growing viruses.
genomic dna library construction
Genomic DNA library construction
  • Isolated P. salmonis DNA free of host cell chromosomal DNA contamination.
  • Obtained ~250 µg DNA from 12,000 cm2 purification
  • Constructed expression library in λZAP II
library screening
Library screening:
  • Goal: to identify immunoreactive clones
  • Only available tool for screening:
    • rabbit Pabs
    • Salmon Pabs
  • Obtained several strongly immunoreactive clones

Trialing Putative SRS Vaccines

Weak Challenge

Poor RPS and DOI

protein engineering introducing immunostimulatory peptide sequences
Protein Engineering: Introducing Immunostimulatory Peptide Sequences

Two different immunostimulatory sequences were added to the original OspA fusion protein resulting to three constructs:




immune response to promiscuous t cell epitopes in ospa
Immune response to promiscuous T-cell epitopes in OspA.

Stimulation Index (SI) = ratio of specific stimulation (Pvacc/Pcon)


1 - preinduced sample

2 - 1 hour of induction

3 - 2 hours of induction

4 - 3 hours of induction

The Bratislava connection -Large scale production of a recombinant SRS vaccine



Jan Burian, Ph.D. VP R&D Microtek.

other applications of this technology
Other applications of this technology
  • Viruses: IHNV, IPNV, ISAV
  • Flavobacterial diseases
  • Kudoa thyrsites
  • Sea lice (Lepeotherius salmonis & Caligus rogercresseyi)
subunit vaccines for parasitic diseases of salmon


Subunit vaccines for parasitic diseases of salmon



B Ectoparasite


Parasiticide control is costly and environmentally questionable.

Concern over developing resistance.

Concern over transmission to wild species.

Vaccine control was previously considered to be impossible.

Sea lice


Discovering sea lice antigens

  • Proteomics and genomics approaches to reveal target antigens.
  • Target genes subcloned and DNA sequenced.
  • Cloned target antigens expressed and formulated for trials

SDS PAGE of SL Clones

Immunoblot of SL Clones


Testing of prospective Sea Lice antigens

  • 3 Targets/19:
  • Attachment
  • Fertility
  • Feeding


These 3 targets have been engineered into a trivalent single vaccine and is in trials.



  • Aquaculture vaccines are available for a variety of
  • microbial diseases.
  • Vaccines can be comprised of monvalent-multivalent
  • components which engage the immune system of fishes
  • Genetic engineering has enhanced our capacity to
  • design effective vaccines.
  • New-age vaccines for a variety of previously intractible
  • diseases are now becoming available.
  • Vaccines for sea lice and ISAV are now within reach
  • and close to commercial development.

The Current Microtek Team

Research and Development

William Kay, PhD Jan Burian, PhD

Sharon Clouthier, PhD Eric Anderson, PhD Elizabeth Crump, PhD

Joe Barlow, BSc; Kyle Clarke, BSc; Chuong Hyunh, BSc, Jan Burian Jr. BSc.

Development , Production & Regulatory

Steve Carlos, BSc Emily Coble, BSc ADA

Sales and Marketing Patty Byrne, BSc, ADA

Brandi Tudor, BSc.

Fish Health Services

Tim Hewison, BSc Hernan Pizarro, BSc

Jesse Kelter, BSc. MSc.


Norm Johnson, BSc., Stephanie Scheraga, BSc.

the historical u victoria team
The Historical U. Victoria Team
  • Trevor Trust, Ph.D.
  • Bill Kay, Ph.D.
  • Ed Ishiguro, Ph.D.
  • Terry Pearson, Ph.D
  • Bob Olafson, Ph.D.
  • Fran Nano, Ph.D.
  • Innovation and Development Centre (Technology Transfer)

Happiness is a healthy fish!


The Microtek Team

Research and Development

William Kay, PhD Julian Thornton, PhD

Jan Burian, PhD Sharon Clouthier, PhD

Eric Anderson, PhD Michael Kuzyk, PhD

Elizabeth Crump, PhD Shannon Balfrey, PhD

Dave Machander, MSc. John Drennan, MSc

Daphne Dolhane, BSc. Kathy Gurgul, BSc.

Iqbal Kathrada, BSc.

Development and Production

Steve Carlos, BSc Patty Byrne, BSc, ADA

Tim Hewison, BSc Steven Gale

Fish Health Services

Joe Barlow, BSc Emily Coble, BSc

Hernan Pizarro, BSc Steve Cameron, MSc.


Melanie Sheppard, BSc, ADA Mike Norris, BSc, ADA

Joe Klimik, BSc. MSc.