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Environmental assessment for licensing infectious haematopoietic necrosis virus vaccine, DNA vaccine in Canada

For Public Release

July 5, 2005

The information in this environmental assessment was current at the time of its preparation. It is possible that the situation may have changed since that time. Please consult the VBS if you have any questions.


Table of Contents


Summary

Infectious Haematopoietic Necrosis Virus (IHNV) Vaccine, Deoxyribonucleic Acid (DNA) (Trade Name: APEX-IHN) is a plasmid DNA vaccine expressing the envelope glycoprotein (G) of IHNV. The vaccine is indicated as an aid in the prevention of Infectious Haematopoietic Necrosis Virus disease in healthy salmonids, 30 grams or larger in weight when administered intramuscularly in the area immediately anterior and lateral to the dorsal fin. The vaccine was evaluated by the Veterinary Biologics Section (VBS), Canadian Food Inspection Agency (CFIA) for licensing in Canada. As part of the requirements for licensing this product in Canada, an ‘Environmental Assessment' was conducted and a public document which contains information on the molecular and biological characteristics of the recombinant vaccine, target animal and non-target animal safety, human safety, environmental considerations and risk mitigation measures prepared

1. Introduction

1.1 Proposed Action

Veterinary Biologics Section (VBS), Animal Health and Production Division, Canadian Food Inspection Agency (CFIA) is responsible for licensing veterinary biologics for use in Canada. The legal authority for the regulation of veterinary biologics in Canada is provided under the Health of Animals Act and Regulations. Any veterinary biologic manufactured, sold or represented for use in Canada must comply with the requirements specified by the CFIA regarding safety, purity, efficacy and potency of the product. Aqua Health Ltd. has submitted the following plasmid DNA-based vaccine for licensure in Canada:

  • Infectious Haematopoietic Necrosis Virus (IHNV), DNA (Trade name: APEX-IHN), VBS File # 870VV/I4.0/A8

1.2 Background

Infectious Haematopoietic Necrosis (IHN) virus is a common viral pathogen of both wild and farmed salmonids, in particular Pacific salmonids (Oncorhynchus sp.), rainbow trout (Oncorhynchus mykiss), and Atlantic salmon (Salmo salar). IHN virus is enzootic to the Pacific Northwest, however it has varying effects on different Pacific salmonids. Coho and chinook salmon are considered resistant to IHNV while Atlantic salmon farmed on the coast of British Columbia are very susceptible. The virus first appeared in farmed Atlantic salmon in 1992 (Armstrong et al, 1993). Four waves of outbreaks (1995, 1996, 1997 and 2001) have occurred since that time, resulting in the destruction of millions of smolts as a disease management measure. Mortality rates in older fish (2-3 kg) tend to range from 10-20%; in smolts the mortality rate often exceeds 85%. The average cumulative mortality following an outbreak is estimated at 47% (St.-Hilaire et al., 2002). The outbreak in 2001 affected 36 Atlantic salmon farms from five areas on both the east and west coast of Vancouver Island; the highest mortalities (70%) were reported in salmon of less than 1 kg (Saksida, 2003). Data collected from sites that had vaccinated their salmon with an autogenous vaccine showed variable protection as cumulative mortality was equal or lower than the average mortality for fish of a similar size range (Saksida, 2003). Currently no efficacious and licensed vaccine is available to vaccinate BC farmed Atlantic salmon against IHNV disease. BC is the fourth largest producer of farmed salmon in the world. In 2003, 72,700 tonnes of farmed salmon were harvested, of which 55,600 tonnes consisted of Atlantic salmon worth approximately $213 million.

2. Purpose and need for proposed action

2.1 Significance

This vaccine is required for the prevention and management of IHNV in commercial Atlantic salmon culture facilities in British Columbia. At present there are no licensed treatments or efficacious licensed vaccines for the control or prevention, respectively, of this disease.

2.2 Rationale

The VBS evaluates veterinary biologics submissions for licensure under the Health of Animals Act and Regulations. General criteria for licensure are (a) the product must be pure, safe, potent and efficacious, (b) the product must be licensed in the country of origin, (c) vaccine components must be relevant to Canadian disease conditions and (d) the product must be produced and tested in accordance with generally accepted “good manufacturing practices”. This Canadian origin vaccine meets the general criteria and therefore was evaluated for licensure by VBS.

3. Alternatives

The alternatives would be (a) to issue a Canadian Veterinary Biologics Product License to Aqua Health Ltd. for the production and distribution of APEX-IHN, if all licensing requirements are satisfactory, (b) not to issue a Canadian Veterinary Biologics Product License for the above product, if licensing requirements are not met.

4. Molecular and biological characteristics of parental and recombinant organisms

4.1 Parental Organisms

Plasmid Vector: The original parental plasmid is a non-commercial, synthetic plasmid modified to become a eukaryotic expression vector by insertion of an intermediate-early promoter and a polyadenylation signal. The parental plasmid also carries a specific origin of replication (ori) allowing it to replicate to high copy numbers in a commercially available E. coli expression system.

Infectious Haematopoietic Necrosis Virus (IHNV): IHNV is a single stranded negative ribonucleic acid (RNA) virus in the family Rhabdoviridae identified as the most important rhabdoviral disease of salmon and trout in North America. The viral surface glycoprotein (G) is the only protein capable of eliciting neutralizing antibody and the production of a protective immune response.

4.2 Recombinant Organism

The virus strain of IHNV used for cloning the G gene was isolated from sockeye salmon caught in the Fulton River, BC, Canada. The complementary deoxyribonucleic acid (cDNA) sequence containing the open reading frame for the G protein of IHNV was generated by the reverse transcription of the gene from single stranded viral RNA, and subsequent amplification by polymerase chain reaction (PCR) using specific primers designed according to a published sequence. The recombinant pUK-ihnG plasmid (corresponding to the nucleic acid vaccine) was then constructed by cloning the isolated cDNA sequence into the parental plasmid at specific restriction sites. Details of the methods used in the construction of pUK-ihnG are on file with VBS. Aqua Health Ltd. has established a Master Seed Stock of the recombinant plasmid; this stock has been tested for extraneous agents, purity and safety according to recognized tests. This data has been reviewed and is on file at VBS.

5. Human Safety

Human safety risk factors associated with the use of this vaccine include consumption of food products derived from vaccinated fish and, in rare instances, self-injection of the vaccine by vaccinators.

5.1 Assessment by Health Canada

As part of the safety assessment, the Bureau of Microbial Hazards, Microbiology Evaluation Division, Health Canada, reviewed the human safety data including the Human Health Risk Assessment (Dec. 1, 2004) supplied by Aqua Health Ltd. Following the assessment, the Bureau of Microbial Hazards indicated that there is no risk to the safety of Canadians in regard to exposure to food products for human consumption derived from fish immunized with this vaccine.

5.2 Previous Safe Use

Plasmid based vaccines have been used in human phase I clinical trials to vaccinate against a range of diseases with no adverse reactions reported attributable to the vaccine, for example MacGregor et al., 2002, McConkey et al., 2003, Wang et al., 2001. The amount of plasmid DNA administered in a single human dose is at least 100-300 µg, which is 10X-30X the dose given to the fish.

5.3 Probability of Human Exposure

(a) Consumption of Food Products: Studies generated by the manufacturer, Aqua Health Ltd., have shown that the vaccine APEX-IHN is rapidly cleared from primary organs when Atlantic salmon were injected with 2X the recommended dose. The vaccine persists at the injection site; under laboratory conditions less than 0.01% of the injected vaccine remains after one year. In field conditions the number of plasmids is reduced from 1.8 X 10E12 plasmid copies per dose to 32 plasmid copies per mg injection site muscle tissue per fish at 252 days post-exposure. This amount of remaining plasmid DNA is negligible when compared to the amount of genomic DNA that is present. Further it has been established that plasmid DNA is rapidly degraded in the stomach and duodenum due to gastric acid, and pancreatic and bile secretions respectively (Maturin and Curtiss, 1977). Thus any plasmid DNA that remains at the site of injection of harvested fish, if any at all, would be expected to be rapidly degraded in the human gastrointestinal tract upon ingestion.

(b) Self-injection by the Operator: The probability of human exposure is limited to the administration of the vaccine by experienced operators (vaccinators). Past studies have documented that self-injection can occur once for every 480,000 fish injected (Dyrkorn et al., 1993, cited in Leira and Baalsrud, 1997). There has been a trend towards fewer accidental self-injections due to increased experience and training of the vaccinators and the use of finger guards attached to the repeating syringe. The label on the vaccine advises the operators to seek immediate medical attention in case of self injection. Safety studies carried out in mice using the recommended dose or a 10X dose by the company demonstrated that the vaccine was safe without any adverse effects. Following the application of 6.1 million doses of this vaccine during monitored field safety studies as required for the licensure of this product, no incidents of vaccinator self-injection were reported by Aqua Health Ltd.

5.4 Possible Outcome of Human Exposure

  • The plasmid vaccine APEX-IHN is not pathogenic to humans according to current knowledge and no adverse outcomes are anticipated.
  • The plasmid vaccine APEX-IHN is a non-infectious, non-replicating agent. There is no possibility of reversion to virulence or inadequate inactivation.
  • The plasmid vaccine APEX-IHN does not contain adjuvants such as aluminium phosphate or mineral oil which are known to cause post-vaccination peritonitis and other inflammatory complications.
  • The dose per fish (10 µg) of the plasmid vaccine APEX-IHN is considerably lower than the dose of similarly produced plasmid vaccines normally used in human clinical trials (100-300+ µg) with no adverse events.
  • Nucleic acid vaccines have been studied in a wide range of animal species for safety and efficacy and numerous DNA vaccines have entered Phase I clinical trials in humans.
  • The injection needles used for the vaccination of the fish would not be sterile; therefore there is a risk of injecting naturally occurring surface bacteria from the fish into the hand of an operator. This could result in infection and, in rare instances, cellulitis (Weinstein et al., 1997). However, this risk is not different and would be lower than the risk posed with the use of any of the currently licensed injectable oil-adjuvanted bacterin vaccines used in aquaculture.

5.5 Risk Associated with Widespread Use of the Vaccine

The widespread use of the vaccine is not expected to have any public health significance.

6. Animal Safety

6.1 Target Animal

  • The manufacturer has submitted results of safety studies carried out in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).
  • The manufacturer has submitted study results demonstrating that the vaccine is not shed from vaccinated fish.
  • The nucleic acid vaccine pUK-ihnG is a non-virulent, non-replicating agent, therefore reversion to virulence is not applicable.
  • In the safety trial, the manufacturer has evaluated this vaccine in a 2X overdose vaccination scenario with no adverse effects.
  • The manufacturer has submitted study results demonstrating that the plasmid vaccine is not integrated in the fish genomic DNA.
  • In monitored field safety studies for investigation and emergency use, a total of 6,601,567 doses were used to vaccinate farmed Atlantic salmon at 7 sites/hatcheries on the BC coast. No adverse events in the farmed salmon related to the use of APEX-IHN were reported at any of the sites.
  • Relative safety - Nucleic acid vaccines are considered safer than conventional vaccines (inactivated whole virus, with or without oil adjuvant, or attenuated live virus). The vaccine is not formulated with oil adjuvant which is known to cause post-vaccination peritonitis (Lillehaug et al., 1992; Midtlyng et al., 1996). Inactivated whole virus vaccines may contain unknown impurities and trace amounts of inactivating agents. Live attenuated vaccines may cause a risk of infection by mutants or may revert to virulence.
  • Extensive literature review (on file at VBS) has supported the efficacy and safety of DNA vaccines for numerous viral diseases of various fish species including salmonids, rainbow trout, goldfish, Japanes flounder, glass catfish, and spotted sand bass.

6.2 Non-target Animal

  • According to the approved label, the vaccine is administered to Atlantic salmon at least 400 degree days prior to sea water transfer, at the hatchery in an artificial tank based rearing systems. All hatchery effluent water in BC is treated, by law, such that it cannot be released directly to fish-bearing waters. Consequently, the risk to freshwater and marine invertebrates and other non-target aquatic species is negligible.
  • The manufacturer has submitted study results demonstrating that the vaccine is not shed from vaccinated fish.
  • Extensive literature review (on file at VBS) has supported the effectiveness and safety of DNA vaccines for numerous viral diseases of various fish species including salmonids, rainbow trout, goldfish, Japanes flounder, glass catfish, spotted sand bass.

6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non-Target Animals

The manufacturer has submitted study results demonstrating that the vaccine is not shed from vaccinated fish.

6.4 Extent of Host Range and Degree of Motility of the Vector

The plasmid vaccine APEX-IHN is a non-infectious, non-replicating agent. Vaccine that may be released to the environment is inert as the plasmid DNA cannot replicate on its own.

7. Affected Environment

  • The vaccine is administered to Atlantic salmon at the hatchery, in artificial tank based rearing systems. By law, all hatchery effluent water in BC has to be treated; it cannot be released directly to fish-bearing waters. Consequently, the risk to the environment is considered negligible.
  • Should vaccine be shed or spread within the tank based rearing system, this is expected to occur in the first 7 days post-vaccination and well within the 400 degree days prior to sea water transfer (Garver et al., 2002).
  • The manufacturer has submitted study results demonstrating that the vaccine is not shed from vaccinated fish.
  • The disease agent IHNV is endemic to the British Columbia coast and expression of the IHN G protein already occurs in the environment naturally.
  • E. coli, the most likely non-target organism that could be implicated in transmitting the plasmid outside the target species, is not considered a natural component of the gut flora of salmonids under cultured conditions (Del Rio-Rodriguez et al., 1997) and is absent from the intestinal content of cultured fishes (Gonzalez et al., 1999).
  • The plasmid vaccine APEX-IHN is a non-infectious, non-replicating agent. Vaccine that may be released to the environment is inert; the plasmid DNA cannot replicate on its own.

8. Environmental Consequences

  • The risk to the environment is considered negligible.
  • The manufacturer has submitted study results demonstrating that the vaccine is not shed from vaccinated fish.
  • The disease agent IHNV is endemic to the British Columbia coast and expression of the IHNV glycoprotein already occurs in the environment naturally.
  • Relative Safety Compared to Other Vaccines: The vaccine contains no additives, inactivating agents or adjuvants, and therefore presents no contamination risk from these ingredients commonly found in conventional vaccines (inactivated whole virus, with or without oil adjuvant, or attenuated live virus). The DNA vaccine is a non-virulent, non-replicating agent, therefore reversion to virulence is not a risk factor as compared to inactivated vaccines.

9. Mitigative Measures

  • Only fully trained vaccinators (operators) will be handling the vaccine and vaccinating Atlantic salmon smolts.
  • The manufacturer has demonstrated that the vaccine is not shed from vaccinated fish.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all suspected adverse reactions to CFIA within 15 days of receiving notice from an owner /hatchery operator/ or a veterinarian. Veterinarians may also report suspected adverse reactions directly to the CFIA. On VBS receipt of an adverse reaction complaint, the manufacturer is asked to investigate and prepare a report for the owner's veterinarian and CFIA. If the problem is resolved to the satisfaction of the veterinarian / client, no further action is usually requested by VBS. However, if the investigation is not satisfactory, VBS may initiate regulatory action depending on the case, which may include further safety testing, temporary stop sale or product withdrawal from the market.

10.2 Animal

Veterinarians and/or hatchery operators should report any suspected adverse reactions to VBS-CFIA as indicated above. Suspected adverse reactions should be reported using the form Notification of Adverse Reactions to Veterinary Biologics (CFIA/ACIA 2205).

10.3 Human

No special monitoring of the human safety of the product will be carried out.

11. Consultation and Contacts

  • National Aquatic Animal Health, Oceans and Aquaculture Science, Department of Fisheries and Oceans (DFO), was consulted on the initial investigational use of this vaccine in farmed Atlantic salmon on the BC coast and was also consulted in June, 2005, prior to VBS licensure. DFO had no objections or concerns regarding the use of this vaccine in accordance with submitted protocols and approved Production Outlines on file at VBS.

  • The Bureau of Microbial Hazards, Microbiology Evaluation Division, Health Canada, was consulted on the human health perspective, via exposure through food derived from vaccinated fish. The Bureau of Microbial Hazards had no objections or concerns regarding the sale of food products derived from vaccinated fish for human consumption.

  • Aqua Health Ltd., 37 McCarville Street, Charlottetown, PEI, Canada, C1E 2A7, is the company responsible for this product.

12. Conclusions and Actions

Following this assessment and the review of documentation submitted by the manufacturer, VBS-CFIA will amend the Canadian Veterinary Biologics Product Licence to allow the manufacture and distribution by Aqua Health Ltd. of the following product:

  • Infectious Haematopoietic Necrosis Virus (IHNV), DNA (Trade name: APEX-IHN), VBS File # 870VV/I4.0/A8

13. References

Armstrong, R. et al., 1983. Infectious hematopoietic necrosis in Atlantic salmon in British Columbia. Canadian Veterinary Journal 34:312-313.

Corbeil, S. et al., 2000. Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunization. Fish & Shellfish Immunology 10:711-723.

Del-Rio Rodriguez, R.E. et al., 1997. Survival of Escherichia coli in the intestine of fish. Aquaculture Research, 28: 257-264.

Garver, K. et al., 2002. Distribution, persistence, and pathological analysis of a DNA vaccine against Infectious hematopoietic necrosis virus. Poster Presentation: Fourth International Symposium on Aquatic Animal Health, September 2-6, 2002. New Orleans, USA.

Garver, K. et al. 2005. Analysis of DNA vaccinated fish reveals viral antigen in muscle, kidney and thymus, and transient histopathological changes. Marine Biotechnology 7:540-553.

Gonzalez, C.J. et al., 1999. Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquaculture rainbow trout (Oncorhynchus mykiss). Journal of Food Protection 62:1270-1277.

Government of British Columbia - Ministry of Fisheries and Aquaculture web site including links: www.agf.gov.bc.ca/fisheries/health/IHN.htm.

Leira, H.L. and Baalsrud, K.J. 1997. Operator safety during injection vaccination of fish. In Gudding R., Lillehaug, A., Midtlyng, P.J. [eds], Fish Vaccinology. Developments in Biological Standardization 90:383-387.

Lillehaug, A. et al., 1992. Field testing of adjuvanted furunculosis vaccines in Atlantic salmon, Salmo salar L. Journal of Fish Diseases 15:485-496.

MacGregor, R.R. et al., 2002. T-cell responses induced in normal volunteers immunized with a DNA-based vaccine containing HIV-1 env and rec. AIDS 16(16):2137-2143.

Maturin, L. and Curtiss, R. 1977. Degradation of DNA by nucleases in intestinal tract of rats. Science 196:216-218.

McConkey, S.J., et al., 2003. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nature Medicine 9(6):729-735.

Midtlyng, P.J. et al., 1996. Experimental studies on the efficacy and side-effects of intraperitoneal vaccination of Atlantic salmon (Salmo salar L.) against furunculosis. Fish and Shellfish Immunology 6:335-350.

Saksida, Sonja. 2003. Investigation of the 2001-2003 IHN epizootic in farmed Atlantic Salmon in British Columbia.

St.-Hilaire, S. et al., 2002. Epidemiological investigation of infectious hematopoietic necrosis virus in salt water net-pen reared Atlantic salmon in British Columbia, Canada, 2002. Aquaculture 212:49-67.

Wang, R. et al., 2001. Induction of CD4(+) T cell-dependent CD8(+) type 1 responses in humans by a malaria DNA vaccine. Proc. Natl. Acad. Sci. U.S.A. 98(19):10817-10822.

Weinstein, M.R. et al., 1997. Invasive infections due to a fish pathogen, Streptococcus iniae. The New England Journal of Medicine 337:589-594.


Prepared and revised by:

Veterinary Biologics Section
Animal Health and Production Division
Canadian Food Inspection Agency