UNITED STATES DEPARTMENT OF AGRICULTURE

ANIMAL AND PLANT HEALTH INSPECTION SERVICE

THE CENTER FOR VETERINARY BIOLOGICS

Environmental Assessment

For Field Testing of a

Swine Influenza Vaccine, RNA

August 2011

Prepared by:

United States Department of Agriculture

Animal and Plant Health Inspection Service

The Center for Veterinary Biologics

1920 Dayton Avenue

Ames, IA 50010

___________________________________________________________________________________________________________________

Environmental Assessment - Field Trial of an Experimental Swine Influenza Vaccine, RNA
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USDA-APHIS-Center for Veterinary Biologics

Environmental Assessment

For Field Testing 

Swine Influenza Vaccine, RNA

I. Proposed Action

The United States Department of Agriculture (USDA)-Animal and Plant Health 

Inspection Service (APHIS)-Center for Veterinary Biologics (CVB) is considering 

granting authorization to ship an unlicensed Swine Influenza Vaccine, RNA, for field

safety testing in pigs. Harrisvaccines, Inc., Ames, Iowa, has requested authorization to 

conduct field tests under conditions of husbandry that are typical of the commercial 

swine industry in the United States (US).

Under the provisions of the Virus-Serum-Toxin Act of 1913, as amended in 1985, the 

USDA-APHIS-VS-CVB must ensure that veterinary biologics are pure, safe, potent, and 

efficacious and not worthless, contaminated, dangerous, or harmful. Accordingly, the 

CVB has conducted a risk analysis and has concluded that, regarding use of this swine 

influenza vaccine, the safety risks to animals, public health, and the environment are low. 

A copy of the firm’s risk analysis with confidential business information removed is 

available upon request. The CVB risk analysis includes that document, but also 

incorporates review of all the firm’s studies and tests related to the vaccine, the 

confirmatory testing completed by CVB personnel on three recombinant master plasmids

to confirm purity, identity, and expression of immunogenic protein, consultation with 

subject matter experts as needed, results of literary and regulatory review, and evaluation 

in light of other previously tested or licensed products.

Based on the CVB risk analysis and other relevant data, we have reached a preliminary

determination that field testing this veterinary vaccine will not have a significant impact 

on the quality of the human environment, and that an environmental impact statement 

need not be prepared. We intend to authorize shipment of this vaccine for field testing 

following the close of the comment period of the Federal Register Notice announcing the 

pending trial unless new substantial issues bearing on the effects of this action are 

brought to our attention. 

II. Background

Swine influenza virus (SIV) infects domestic swine and may also infect humans, causing 

respiratory disease of varying severity. In pigs, SIV is very contagious, with rapid onset 

and spread throughout a herd. Field isolates display variable virulence which may be due 

to co-infection with secondary organisms; mortality typically ranges from 1 to 4%.1 

Reassortant and mutated viruses can arise through antigenic ‘shift’ and ‘drift’ changes in 

viruses, with potential to quickly cause significant and widespread disease. Treatment 
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options are limited. The best strategy is prevention using vaccination, biosecurity, and 

good management.

The experimental vaccine to be used in the proposed field trial is generated from three 

recombinant master plasmids that produce an encapsidated RNA vaccine against SIV, 

subtype H3. One recombinant plasmid contains the hemagglutinin (HA) gene from a 

Clade IV H3N2 SIV strain. This plasmid also encodes the nonstructural proteins 1-4

(nsp1-4) from the live attenuated TC-83 vaccine strain of Venezuelan equine encephalitis 

virus (VEEV). The nsp1-4 genes are responsible for transcription and replication of the 

viral RNA.

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 There are also two recombinant helper plasmids each containing a gene 

derived from the TC-83 vaccine strain of VEEV. One helper plasmid contains a 

modified version of the glycoprotein (GP) gene; the other helper plasmid contains a 

highly modified version of the capsid gene. The GP and capsid structural proteins are 

produced in trans and are able to encapsidate the viral RNA. 

During vaccine production, the DNA plasmids described above are amplified in a 

commercially-available Escherichia coli K-12 strain, the Escherichia coli are harvested, 

and the plasmid DNAs are purified. The purified plasmid DNAs are linearized and then 

transcribed, producing positive strand RNA. The transcribed RNA is purified and used to 

transfect by electroporation certain Vero cells. Once in the Vero cells, the GP and capsid 

proteins are produced to form a unique coat around the RNA which is packaged into 

propagation-defective “RNA particles” (RP), also known in the literature as “replicon

particles”. Only the RNA from the plasmid containing the SIV HA and the nsp1-4 is 

packaged into RP, because the helper RNAs lack the required packaging sequence. The 

RP are harvested and, when used to vaccinate pigs by intramuscular route, are singlecycle vectors for high levels of heterologous protein expression that target dendritic 

cells.3,4 The RP allow for HA protein expression in the cells into which they have been 

introduced, but do not spread to other cells. 

The proposed field safety test will be conducted in three different geographical locations 

and the product will be used according to instructions on the product circular, and in 

accordance with standard requirements published in Title 9, Code of Federal Regulations 

(CFR), Part 103.3, concerning shipment of experimental biological products for use in 

animals.5The potential for escape and dispersal of the experimental vaccine from the 

proposed release sites is low. The personnel to conduct the study are experienced in 

swine health management.

III. Need for the Proposed Action

Currently, the only vaccines available for SIV are inactivated vaccines. This 

experimental SIV vaccine represents an attempt by Harrisvaccines, Inc. to produce an 

efficacious and safe vaccine that stimulates strong cellular and humoral immunity, to aid 

in the prevention of clinical disease in pigs due to SIV. 
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IV. Areas of Concerns

The three areas of concern to the CVB regarding use of this recombinant vaccine are: 1) 

animal safety, 2) public health, and 3) environmental safety. The CVB has conducted its 

own risk analysis to assess whether risks are associated with the proposal to field test this 

experimental vaccine. The safety characteristics of the vaccine have been thoroughly 

evaluated. The conclusions derived from the risk analysis for each of the areas of 

concern are summarized below.

A. Animal Safety

The risk to animals is low. 

1. The firm has conducted studies wherein the SIV RP has been given to pigs at 200 

times the normal dose by intramuscular and intravenous route. No untoward effects 

were observed. Reverse transcriptase polymerase chain reaction (RT-PCR) assays 

were used to test for the presence of vaccine RP in serum, injection site, tonsils, lung, 

spleen, liver, kidney, closest lymph node to injection site, heart, brain, intestine, and 

nasal and rectal swabs. All results were negative for SIV RP-specific RNA.

2. Placebo and strict (no treatment) control pigs in several of the firm’s studies did not 

seroconvert at any time, indicating that the SIV RP vaccine is not shed nor spread to 

commingled cohorts.

3. No reversion to virulence or shed/spread was detected in studies by the firm of 

vaccinated porcine host and murine non-host animals. Shed/spread was evaluated by

RT-PCR testing of serum, tissues, nasal and rectal swabs. Serum, tissues, nasal and 

rectal swabs were also tested for reversion to virulence by a cytopathic effect (CPE) 

assay. These back passage and shed-spread studies demonstrated that the vaccine 

virus would be very unlikely to replicate, revert to virulence, or be shed after injection 

in pigs.

4. Regarding SIV, only the HA gene is incorporated into the RP. The RP has been 

shown to cause no adverse clinical signs or lesions and can elicit an immune response 

that protects swine from virulent SIV challenge. Antibody to the remaining influenza

virus proteins is not generated and this can be used to differentiate RP vaccinates 

from SIV-exposed animals or those vaccinated with conventionally prepared 

vaccines.

5. Other RP vaccines, both prophylactic and therapeutic, have been evaluated in 

numerous rodents (including immunocompromised mice), guinea pigs, nonhuman 

primates, sheep, chickens, and humans without significant adverse effects.2,3,4

6. The three purified plasmids as well as purified RP supplied by the firm were tested at 

the CVB. Expression of the SIV H3 from RP after infection of swine tissue culture 

was confirmed by Western blot and fluorescent antibody immunostaining of cell 
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monolayers. In addition, restriction endonuclease analysis and genetic sequencing 

confirmed the cloning sites and recombinant sequences of SIV H3 and VEE strain 

TC-83 genes in the three plasmids. 

B. Public Health

The risk to public health is low. 

1. There are no indications that special safety measures should be taken to conduct this 

study. Only qualified personnel will be administering the recombinant vaccine and 

will follow appropriate vaccine handling precautions. They will be at low risk since 

the SIV RP vaccine is propagation-defective and is not shed.

2. Accidental human injection is not expected to cause adverse effects. Although the 

safety of this specific RP vaccine has not been evaluated in humans and is therefore 

unknown, a related RP vaccine for human cytomegalovirus has been tested in adult 

humans without causing illness.4

3. The HA sequence in the SIV RP vaccine is from a swine-origin Clade 4 H3N2 strain. 

Although certain SIV H3N2 strains have been associated with human disease, only

the HA gene is present in the vaccine and the encoded protein is not pathogenic. 

4. The attenuated TC-83 vaccine strain of VEE is the source of the helper proteins used 

to package the SIV HA RNA into RP during vaccine production. TC-83 has been 

tested in human volunteers, producing seroconversion in about 80% and causing mild 

to moderate flu-like symptoms in some vaccinates. However, use of its proteins in 

replicons (or RP) is not considered harmful.

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C. Environmental Safety

The risk to the environment is low. 

1. Shed-spread and backpassage studies conducted by the firm in pigs and mice indicate 

that the SIV RP are not shed, thus not spread into the environment.

2. The vaccine is for intramuscular (IM) use in pigs. Given the lack of RP replication

and limited spread of the vaccine, IM administration in pigs will not result in 

environmental release.

3. The RP vaccine system has several safety features to prevent the generation of a 

replication-competent recombinant. One is the “split helper” design, requiring RNA 

transcribed from two separate plasmids to be transfected. Another is the lack of a 26S 

promoter on RNAs transcribed from the helper plasmids, which has been shown to 

decrease the probability of double recombination needed to produce a functional 

recombinant.6 Also, a stop codon at the 3’ end of the capsid gene has been 

introduced. The full result of this is that two independent recombination events must 
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occur in specific places in the RP RNA and in a specific order. This would be 

extremely unlikely to occur.

4. As an additional precaution, each bulk lot produced is tested using the CPE assay, 

which can detect one virus in more than a billion RP.

5. SIV RP consist of RNA encapsidated to form an icosehedral nucleocapsid core 

surrounded by a lipid membrane with projecting glycoproteins (or RNA within a lipid 

membrane particle). These are readily disrupted in the environment.

6. The firm determined that SIV RP are not stable at room temperature, with 88% 

reduction in potency after 14 days. No functional SIV RP were detected after seven 

days at 37oC.

There are no apparent substantial issues with adverse environmental impacts concerning 

this vaccine.

V. Alternatives

Two alternatives were considered. The only alternative considered other than the 

preferred action alternative is not to approve the proposed field tests, the “no action” 

alternative. 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 analysis and the potential applications for this vaccine in disease 

control, APHIS adopts the alternative that the proposed field tests be approved.

VI. Conclusion

Based upon the risk analysis documented in this risk assessment, APHIS 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. Dee, S.A. 2005. Respiratory Diseases of Pigs: Swine Influenza. pp. 1228-1229. In: 

The Merck Veterinary Manual, Ninth Edition. C.M. Kahn, (Ed), Merck & Co., Inc., 

Whitehouse Station, N.J. Available online: 

http://www.merckvetmanual.com/mvm/index.jsp

2. Rayner, J. O., Dryga, S. A., Kamrud, K. I. 2002. Alphavirus vectors and vaccination. 

Rev Med Virol 12:279-296.

3. Hubby, B., Talarico, T., Maughan, M., et al. 2007. Development and preclinical 

evaluation of an alphavirus replicon vaccine for influenza. Vaccine 25(48):8180-

8189.
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4. Bernstein, D. I., Reap, E. A., Katen, K., et al. 2010. Randomized, double-blind, Phase 

11 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative 

adult volunteers. Vaccine 28:484-493.

5. Code of Federal Regulations, Washington, DC. Available online: 

http://www.gpoaccess.gov/cfr/index.html

6. Kamrud, K. I., Alterson, K., Custer, M., et al. 2010. Development and 

characterization of promoterless helper RNAs for the production of alphavirus 

replicon particle. J Gen Vir 91:1723-1727.

7. Paessler, S., Weaver, S. C. 2009. Vaccines for Venezuelan equine encephalitis. 

Vaccine 27 Suppl 4:D80-D85.