**UNITED STATES DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE**  **THE CEN**  **TER FOR VETERINARY B IOLOGICS** 



# Environmental Assessment **For Field Testing of a**  **Sw ine Influenza Vaccine, RNA** 

August 2011 Prepared by: United States Department of Agriculture Animal and Plant Health Inspect ion Service The Center for Veterinary Biologics 1920 Dayton Avenue Am es, IA 50010

_____________________________________________ ______________________________________________________________________ Environmental Assessment
- Field Trial of an Experimental Swine Influenza Vaccine, RNA1 **USDA -**  **APHIS -**  **Center for Veterinary Biologics**

**Environmental Assessment**  **For Fi**  **eld Testing Swine Influenza V**  **accine, 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 safet y testing in pigs
. Harrisvaccines, Inc., Ames , Iowa , has requested authorization to conduct field tests under conditions of husbandry that are t ypical 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
- A PHIS
- VS
- CVB must ensure that veterinary biologics are pure, safe, potent, and efficacious and not worthless, contaminated, dangerous, or h armful. Accordingl y, the CVB has conducted a risk anal ys is and has concluded that , regarding use of this s wine influe nza vaccine, the safety risks to animals, public health, and the environment are low. A cop y of the firm’s risk anal ys is with confidential business information removed is available upon request. The CVB risk anal ys is includes th at document, but also inco rporates review of all the firm’s studies and tests related to the vaccine , the confirmat ory

testing completed by

CVB personnel on thre e recombinant m aster plasmids to confirm purity , identity , and expression of immunogenic protein , consultation with subje ct matter e xperts as needed , results of literary and regulatory review, and evaluation in light of other previously tested or licensed products. Based on the CVB risk analy sis 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 c lose of the comment period of the Federal Register N otice 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 ( S IV) infects domestic swine and may also infect humans, causing respiratory

disease of varyingseverity
. In pigs, SIV is very contagious , with rapid onset and spread through out a herd
. F ield isolates display variable virulence which may be due to co - infection with secondary organism s; mortality

t ypicall y 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 s ignificant and widespread disease. T reatment2

options are limited. The best strategy is prevention using v accination, bio securit y, and good management
. The experimental vaccine to be used in the proposed field trial is generated from three rec ombinant master plasmids that produce an encapsi dated RNA vaccine against SIV, subty pe H3. One recombinant plasmid contains the hemagglutinin (HA) gene from a Clade IV H3N2 SIV strain . This plasmid also encodes the nonstructural protein s 1
- 4 (ns p 1
- 4) from the live attenuated TC
- 83 vaccine strain of Venezuelan equine encephalitis virus (VEE V) . The nsp 1
- 4 genes are responsible for transcription and replication of the viral RNA
. 2 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 highl y 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 pla smids described above are amplified in a commerciall y - 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 R NA. Th e transcribed RNA is purified and used to transfect b y ele ctroporation 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 propag ation
- def

ective “RNA partic les” (RP) , also known in the literature as “replicon particle s” . Onl y 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 single
cycle vectors for high levels of heterologous protein expression that target dendritic cell s. 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. 5 The potential for escape and dispersal of the experimental vaccine from the proposed release sites is low. The personnel to con duct the study are experienced in swine health management. **III. Need for the Proposed Action**  Currentl y, the onl y 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
.3

**IV. Areas of Concerns**  The three areas of concern to the CVB regarding use of this recombinant vac cine are: 1) animal safet y, 2) public health, and 3) environmental safet y. The CVB has conducted its own risk anal ys is to assess whether risks are associated with the proposal to field test this experimental vaccine. T he safet y characteristics of the va ccine have been thoroughl y 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. 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 pol ym erase chain reaction (RT
- PCR) assay s were used to test for the presence of vaccine RP in serum, injection site, to nsils, lung, spleen, liver, kidney , closest ly mph 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 stud ies did not seroconvert at an y time, indicating that the SIV RP vaccine is not shed nor spread to co mmingled 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. She d/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 b y a cy topathic effect (CPE) assay . These back passage and shed
- spread studies demonstr ated that the vaccine virus would be very

unlikely to replicate, revert to virulence, or be shed after injection in pigs. 4. Regarding SIV , o nl y 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 conve ntionally 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 cell4

m onolayers . In addition, restriction endonuclease anal ys is 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
. Onl y 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 safet y of this specific RP vaccine has not been evaluated in humans and is therefore unknown, a related RP vacc ine for human cy tomegalovirus 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 th e 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 producti on. TC
- 83 has been tested in human volunteers, producing seroconversion in about 80% and causing mild to moderate flu
- like s ymptoms in some vaccinates. However, use of its proteins in replicons (or RP) is not considered harmful
. 7 **C. Environmental Safety**  The risk to the environment is lo w. 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 s ys tem has several safet y 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 recombi nant. 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 must5

occur in specific places in the RP RNA and in a specific order. This would be extremely unlikel y to occur. 4. As an additional pre caution, 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 b y a lipid membrane wi th projecting glycoproteins (or RNA within a lipid membrane particle). These are readil y disrupted in the environment. 6. The firm determined that SIV RP are not stable at room temperature, with 88% reduction in potency

after 14 day s. No functional SIV RP were detected after seven day s at 37 o C. There are no apparent substantial issues with adverse environmental impacts concerning this vaccine. **V. Alternatives**  Two alternatives were considered. T he onl y alternative considered other than the preferred acti on 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 an y potential environmental impact. Based upon the results of our risk analy sis and the potential applications for this vaccine in disease control, APHI S adopts the alternative that the proposed field tests be approved. **VI. Conclusion**  Based upon the ri sk analy sis documented in this risk assessment , APHI S has det ermined 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. R espiratory Diseases of Pigs: Swine Influenza . pp. 1 228
- 1 229 . 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. Ray ner, 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 r eplicon vaccine for influenza. Vaccine 25(48):8180
- 8189.6

4. Bernstein, D. I., Reap, E. A., Katen, K., et al . 2010. Randomized, double
- blind, Phase 11 trial of an alphavirus replicon vaccine for cy tomegalovirus 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 o f 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 S uppl 4:D80
- D85.