**UNITED STATES DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE** 

**VETERINARY SERVICES THE**  **CEN TER FOR VETERINARY B**  **IOLOGICS**



# Environmental Assessment **For Issuance of a Permit for**  **Distribution and Sale of**  **a n**  **Imported Infectious Hematopoietic Necrosis** 

**Virus Vaccine**  **, DNA** 

August 201 3 Prepared by: United States Department of Agriculture Animal and Plant Health Inspection Service

Veterinary Services The Center for Veterinary Biologics

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__________________ Environmental Assessment Permit for Distribution and Sale of an Imported Infectious Hematopoietic Necrosis Virus Vaccine , DNA**Environmental Assessment For**  **Issuance of a Permit for Distribution and Sale of an Imported Infectious Hematopoietic Necrosis Virus Vaccine, DNA** 
**I**
. **Proposed Action** APHI S is con sidering granting a permit for distribution and sale in the United States of an Infectious Hematopoietic Necrosis Virus Vaccine , DNA , for use in healthy salmonids weighing at least 30 grams (1.058 ounce)
. Novartis Animal Health US , Inc., Larchwood , Io w a, has requested authorization to provide the vaccine as an aid in the prevention of d isease caused b y infectious hematopoietic necrosis virus (IHNV) in salmon and trout
. Under the provisions of the Virus
- Serum
- Toxin Act of 1913, as amended in 1985, the USDA must ensure that veterinary biologics are pure, safe, potent, and efficacious and not worthless, contaminated, dangerous, or h armful. Biological products produced in other countries may

be imported into the United States for research and evaluation, transit shipment, or general sale and distribution, in accordance with Title 9, Code of Federal Regul ations, Part 104. Products imported under a Permit for Distribution and Sale must meet the same requirements as those required for licensed (domestically produced) products
. Accordingly , APHI S has conducted a risk analy sis of this product and has conclud ed that the safety risks to animals, public health, and the environment are low. A copy of the firm’s risk analy sis with confidential business information re dact ed is available online and b y request, as indicated in the Federal Register Notice announcing the proposed issuance of a permit
. The APHIS risk anal ys is cover s th e firm’s document , but also in clude s review of other studies and tests conducted b y the firm , the confirmat ory

testing completed by

APHI S personnel on the Master Seed to confirm the vacci ne’s purity , identity, and expression of immunogenic protein , and results of literary and r egulatory review regard ing this and similar p reviously tested or licensed products. **II. Background**  IHNV causes an acute rhabdovirus inf ection of salmonids resulting in high mortality among fry

and fingerlings (young or small fish) at water temperatures less than 12 o C (53.6 o F). IHNV is not considered to be pathogenic for people. In this vaccine, only the gl ycoprotein (G) gene of I HNV is present, expressing the surface protein of the virus envelope. It is not associated with virulence and does not encode a toxin , enzy me, or allergen. The confirmatory

testing at the Center for Veterinary Biologics (CVB) laboratory for the Master Seed (MS) bacterial host cont aining the recombinant plasmid was satisfactory
. CVB lab oratory characterization of the MS included identity testing, sequence analy sis, gene expression, and purity testing. The vaccine made from the plasmid DNA is licensed in Canada
. Aqua Health Ltd. , a division of Novartis Animal Health Canada, Inc., developed the vaccine (trade nameAPEX
- IHNTM). It has been commercially available in Canada since 2005 and has been

used in field trials there since 2003. The imported IHNV vaccine to be used under permit for distribution and sale is a r eplication
- incompetent plasmid which has been geneticall y modified to contain an immunogen ic gene. The route of administration for this vaccine is intramuscular , given by trained personnel in the area immediatel y ant erior and lateral to the dorsal fin. Plasmid DNA ha s been used previousl y to express foreign genes for vaccine development. Examples of USDA
- licensed plasmid DNA vaccine s include West Nile Virus Vaccine licensed in 2005, and Canine Melanoma Vaccine licen sed in 2006
. No safet y issues have arisen from the use of these plasmid DNA vaccines. **III. Need for the Proposed Action**  There are no infectious hematopoietic necrosis virus vaccines approved for use in fish
. A replication
- incompetent DNA vaccine can potentially be both safe and efficacious, inducing a protective immune response (Kurath, et al . 2006; Garver, et al. 2005)
. **IV. Areas of Concerns**  The three areas of concern to APHI S are: 1) animal safet y, 2) public health, and 3) environmental safety . APHI S has conducted its own risk anal ys is to assess whether risks are associated with the proposal to issue a permit for this imported vaccine. Th e safet y characteristics of the vaccine have been thoroughly evaluated. The conclusions derived from the risk anal ys is for each of the areas of concern are summarized below. **A. Animal Safety**  The risk to animals is low.  In a contained safety study conducted in Maine, nine hundred salmon weighing between 10 and 20 grams were vaccinated. Six buckets of 25 fish each were netted and placed into each of 6 holding tanks. After a 31
- day acclimation period, the 150 fish from each tank were anaesthetized and randomly assigned to be injected intramuscularl y with either saline control or one of two serials. The salmon were observed for 21 day s after vaccination. There were no adverse events attributable to vaccination.  There is a long history of safe use in salmonids in Canada. The firm reports that from 2003 to 2010, nearly 53 million fish in Canada were vaccinated without significant adverse effects
.  The vaccine is not a risk for target or non
- target species because it cannot replicate in eukaryotic cells , is not infectious, and is highly purified. Reversion to virulence and shedding are not safety concerns applicable to this DNA vaccine. Should inadvertent ingestion occur, the DNA is readily degraded without negative effect to the host or non - target spe cies
. Regarding an y potential for integration, it has been estimated that the probability of a DNA molecule to integrate into the chromosome of muscle cells after intramuscular injection is at least 3000x less than the natural mutation rate (Martin , et al
. 1999 ; Ledwith, et al . 2000).  In persistence studies conducted b y the firm, within fourteen days following a 2x dose vaccination, no plasmid DNA was detected in major tissues. The tissues tested included the liver, spleen, head, kidney , gonad, and gut lumen. In muscle tissue at the site of injection, the cop y number of plasmid (1.8 x 1012) decreased greatl y so that b y day 251, only 0.01% of the plasmid remained. A 1x dose resulted in a 2.5
- fold less copy number at day

252, or a 2.0x1 0
- 9 % reduction in c opy number, a negligible amount
. **B. Public Health**  The risk to public health is likewise low.  The plasmid has no virulence
- associated genes, and no known mobility or integration motifs which would allow integration into the human genome.  Only qualified personnel, knowledgeable in the art of inoculation and prevention of accidental exposure, will be administering the DNA vaccine. Training, experience, and the use of finger guards attached to the repeating s yringe greatly decrease the probability

of human exposure.  Although the safet y of this specific vaccine has not been evaluated in humans and is therefore unknown, accidental human injection is not expected to cause adverse effects. Man y doses of various DNA vaccines using a plasmid vector have been administered to animals, including dogs, mice, guinea pigs, and horses (Grosenbaugh, et al. 2011; Martin, et al. 1999 ; Ledwith, et al . 2000; Manam, et al
. 2000; Davis, et al . 2001). No significant adverse events have been reported for animals receiving those vaccines or the humans administering them . Regarding this IHNV vaccine, there was one case of human self
- injection in 2009 which resulted in no negative impact to the patient.  Numerous DNA vaccines have entered human clinical trials (L iu & Ulmer, 2 005). There has been no evidence of autoimmunity , immunological tolerance, or integration in these various trials. The dose for fish (10 μg) of this vaccine is much lower than the dose of similar plasmid DNA vaccines used in human clinical trials (10x
- 30 x dose) with no adverse events.  Minute quantities of plasmid DNA in muscle tissue at the injection site were detected in the firm’s persistence studies, out to day 728 post
- vaccination, past the time of marketing. However, it is known that plasmid DNA is rapidly degraded in the stomach by

gastric acid, and in the duodenum by pancreatic and bile secretions (Maturin & Curtiss, 1977) and represents an infinitesimal amount relative to salmon genomic DNA that is routinel y consumed. **C. Environmental Safety**  The risks to the environment are low. The risks to environmental safety are low, given that the vaccine is based on highl y purified and well
- characterized plasmid DNA that does not replicate in vaccinated animals.  The vaccine, which has no adjuvant, is admi nistered to salmon ids at the hatchery in artifici al tank
- based rearing s ys tems.  In the unlikel y event the vaccine is released in the environment, the plasmid will not be maintained without kanamycin selection pressure, an antibiotic not used in agricultural or human health applications. The plasmid construct cannot readily infect bac teria without prior lab
- based chemical or phy sical treatment of the bacteria to generate cells competent to take up plasmids.  The plasmid is unstable in the environment and exposure to nucleases, pH variations, oxidation, and/or other denaturing conditio ns will result in degradation of the DNA. The information above provides support to the claims made regarding the safety of the IHNV vaccine to animals, public health, and the environment. There are no apparent substantial issues with adverse environment al impacts concerning use of this vaccine
. **V. Alternatives**  Two alternatives were considered. T he onl y alternative considered other than the preferred action alternative is not to approve the proposed permit for distribution and sale , the “no action” alt ernative. We have considered the applicants’ goals in light of the agency ’s public interest and responsibilities and any potential environmental impact. Based upon the results of our risk anal ys is and the potential applications for this vaccine in diseas e control, APHIS adopts the alternative that the proposed permit be approved. **VI. Conclusion**  Based upon the risk analysis documented in this EA, APHI S has determined that implementation of the proposal would not significantly affect the quality

of the human environment and that the preparation of an Environmental Impact Statement is not required (Finding of No Significant Impact).

**References** 1. Kurath, G., Garver, K.A., Corbeil, S., et al. 2006. Protective immunity and lack of histopathological damage two years after DNA vaccination against infectious hematopoietic necrosis virus in trout. Vaccine 24:345
- 354. 2. Garver, K.A., LaPatra, S.E., Kurath, G. 2005. Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhyn chus tshawytscha and sockeye O. nerka salmon . Dis Aquat Org 64:13
- 22.3. Martin, T., Parker, S.E., Hedstrom, R., et al . 1999. Plasmid DNA malaria vaccine: the

potential for genomic integration after intramuscular injection. Hum Gene Therap y 10:759
- 768. 4. Ledwith, B.J., Manam, S., Troilo, P.J., et al . 2000. Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirol 43:258
- 272. 5. Grosenbaugh, D.A., Leard, A.T., Bergman, P.J., et al. 2011. Safety and efficacy

of a xenogeneic DNA vaccine encoding for human tyr osinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor. Am J Vet Res 72:1631
- 8. 6. Manam, S., Ledwith, B.J., Barnum, A.B ., et al . 2000. Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirol 43:273
- 81. 7. Davis, B.S., Chang, G.J., Cropp, B., et al . 2001. West Nile virus recombinant DNA vacci ne protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzy me - linked immunosorbent assay s. J Vir 75:4040
- 4047. 8. Liu, M.A. & Ulmer, J.B. 2005. Human clinical trials of plasmid DNA vacc ines. Adv Genetics 55:25
- 40. 9. Maturin, L . & Curtiss, R. 1977. Degradation of DNA by nucleases in intestinal tract of rats. Science 196(4286):216
- 8.