Environmental Assessment for the Emergency Use of Harrisvaccines' Unlicensed Porcine Epidemic Diarrhea Vaccine, iPED+

February 11, 2014

Prepared and revised by the Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency (CFIA), this environmental assessment includes information that was current at the time of its preparation. It is possible, however, that the situation may have changed since that time. Please consult CCVB, if you have any questions.

Table of contents


Harrisvaccines' iPED+ porcine epidemic diarrhea vaccine consists of a propagation-defective virus-like RNA replicon particle capable of expressing the Spike (S) protein of porcine epidemic diarrhea virus (PEDv). The vaccine is not yet licensed in any jurisdiction, and the efficacy of the vaccine has not been established. Since the first case of PED in Canada was reported on January 22, 2014, the Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency has received multiple requests from Canadian veterinarians for emergency access to this unlicensed veterinary biologic. This environmental assessment (EA) has been prepared by the CCVB as part of the assessment for whether to allow the emergency use of this biotechnology derived vaccine in Canada.

1. Introduction

1.1 Proposed Action – The Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency (CFIA) is responsible for regulating the manufacture, importation, release and use of veterinary biologics in Canada, under the authorities of the Health of Animals Act and Health of Animals Regulations. Ordinarily, veterinary biologics must be shown to be pure, potent, safe and effective before the CCVB will authorise their use in Canada. However, in emergency situations, the CCVB may authorise the use of a veterinary biologic that does not yet meet all the requirements for licensing, in accordance with section 131.1(1) of the Regulations:

131.1(1) Where an emergency exists with respect to the availability of and need for a veterinary biologic, the Minister may exempt that veterinary biologic from the application of any of the provisions of these Regulations during the period of the emergency.

The CCVB has received applications from Canadian veterinarians requesting permission to import and use an unlicensed porcine epidemic diarrhea vaccine known as iPED+, manufactured by Harrisvaccines in the USA.

This Environmental Assessment (EA) was prepared by the CCVB as part of the assessment for whether to authorise the importation and release of the abovementioned biotechnology derived vaccine in Canada for emergency use. It is based on information provided by the vaccine's manufacturer, as well as information obtained by the CCVB through searching published literature.

1.2 Background – On January 22, 2014, the first suspected Canadian case of PED was reported in south-western Ontario. Since then, at least 10 other cases have been confirmed.

PED is a viral disease of pigs caused by a member of the family Coronaviridae. Its main symptoms are diarrhea and vomiting in pigs of all ages, but the effects are most severe in baby pigs (90-100% mortality typically seen in piglets less than 21 days old). It is a highly contagious disease, spreading to healthy pigs through contact with faeces of infected pigs, or indirectly by contaminated trucks, equipment, footwear and other fomites. Since May 2013, when the United States Department of Agriculture (USDA) confirmed the first case on PED in North America, the disease has spread in that country to 23 States, resulting in significant animal and economic loss. Canadian swine producers are naturally concerned about what the disease could do to their operations.

The iPED+ vaccine manufactured by Harrisvaccines is not yet licensed in any jurisdiction. The company is in the process of completing the requirements for a conditional license in the USA, and until that is granted, is distributing the vaccine directly to veterinarians under the exemptions of 9 CFR 107.1 and the US Export Reform and Enhancement Act.

The iPED+ vaccine consists of a piece of RNA known as a replicon which is packaged into virus-like RNA replicon particles (VRPs) with the capsid and envelope glycoproteins of the attenuated TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV). The VRPs are propagation-defective, as they lack the genetic information needed to produce their structural proteins, and thus cannot form progeny VRPs within a vaccinated animal. The replicon packaged within the vaccine's VRPs contains the information necessary for expression of the antigenic spike (S) protein of PEDv. Following vaccination, the capsid and glycoproteins of the VRP help the replicon gain entry into cells of the vaccinated animal, where the replicon can then direct the expression of high levels of its PEDv S protei. This foreign antigen is then recognized by the animal's immune system, leading (presumably) to cell-mediated and humoral immune responses against the S protei component of PEDv.

It is important to note that much of what is written in this EA about iPED+ is being extrapolated from information gathered for the first fully characterised vaccine produced using Harrisvaccine's RNA replicon particle platform, Swine Influenza Vaccine, RNA (USDA Product Code 19A5.D0, CCVB File 880VV/S3.0/H16). Based on the properties of the technology platform, we have no reason to believe that the iPED+ replicon vaccine will not share the same safety characteristics as Swine Influenza Vaccine, RNA. Due to the urgent nature of the requests for iPED+ vaccine, and the fact that this vaccine is only in the early stages of development, comprehensive information about the efficacy of the iPED+ vaccine is not available at this time.

2. Purpose and Need for Proposed Action

2.1 Significance – Biosecurity measures have proven insufficient to stop the spread of PEDv in North America. Vaccination with a PED vaccine may help prevent, or reduce the severity of, PED cases, should exposure to the virus occur subsequent to vaccination.

2.2 Rationale – The CCVB evaluates applications for emergency access to veterinary biologics requested under the provisions of Section 131.1(1) of the Health of Animals Regulations on a case-by-case basis. The main criteria for granting emergency use access are as follows: a) veterinarians must acknowledge that they will use the unlicensed product at their own risk; b) there must be a reasonable expectation that the vaccine is efficacious; and c) the CCVB must be satisfied that use of the vaccine in Canada will not endanger animal health, human health, or the environment.

Criterion A) The CCVB has received responses from veterinarians in Canada indicating that they are prepared to use the unlicensed vaccine, at their own risk.

Criterion B) In emergency use situations, the reasonable expectation of efficacy criterion is generally satisfied by demonstration of at least one of the following:

  1. vaccine components are consistent with what the vaccine is recommended to protect against;
  2. product licensing by a foreign regulatory authority; and/or
  3. preliminary experimental data.

Information provided by Harrisvaccines about the design and production methods of iPED+ satisfied item i above, and the results of a small scale study performed by the manufacturer showing that the vaccine induces PED virus neutralizing antibodies in pigs (n=4) following vaccination was sufficient to satisfy item iii. Taken together, this information was acceptable to satisfy criterion B.

Criterion C) The purpose of this EA is to determine whether criterion C can also be satisfied.

3. Alternatives

The two alternative options being considered are: a) to issue a Permit to Import Veterinary Biologics to applying veterinarians to allow them to import and use iPED+ at their own risk if the CCVB is satisfied that use of the unlicensed vaccine in Canada is unlikely to be a risk to animal health, human health or the environment; or b) not to issue a Permit to Import Veterinary Biologics if significant risks are identified that cannot be mitigated.

4. Molecular and Biological Characteristics of Parental and Recombinant Organisms

4.1 Source, Description and Function of Parental Organisms and Foreign Genetic Material – The vaccine "organism" consists of a piece of RNA known as a replicon which is packaged into a VRP with the capsid and envelope glycoproteins of an attenuated strain of VEEv. The replicon contains the genetic information necessary for the expression of the PEDv S protei in vaccinated animals. The VEEv capsid and glycoproteins encapsulating the replicon serve to help the replicon gain entry into cells of the vaccinated animal.

The genetic material for the S protei component of the replicon originates from a Chinese isolate of PEDv. However, sequence analyses have determined that the Chinese isolate shares more than 99% nucleotide sequence identity with PEDv isolates circulating in the US Midwest.

The remaining sequences of the replicon include: the 5'-UTR and genes for non-structural proteins 1-4 from the TC-83 attenuated vaccine strain of VEEV, a VEEv derived 26S subgenomic promoter and 3'-UTR, and a multiple cloning site.

The capsid and envelope glycoproteins comprising the VRPs within which the replicon RNA is packaged are produced from sequences also originating from the TC-83 vaccine strain of VEEV.

The TC-83 attenuated VEE virus was created in 1961 by serial passage of the Trinidad donkey strain of VEEv in guinea pig heart cells (OIE Terrestrial Manual; Paessler et al., 2006). It was originally developed for the protection of humans at high risk of occupational exposure; however, the TC-83 strain has also been used in countries outside of Canada for the vaccination of horses against VEEV. In Canada, vaccination of horses against VEEv is not permitted, as Canada is considered free of VEE disease.

The lipid envelope portion of the VRPs originates from the VERO African green monkey kidney cell line used to manufacture the vaccine.

4.2 Method of Accomplishing Genetic Modification – Using standard molecular laboratory techniques, three DNA plasmids were created, which serve as the master sequences for vaccine production. One plasmid contains the replicon sequence, a second plasmid contains the sequences necessary for expression of the VEEv capsid protein, and the third plasmid contains the sequences necessary for the production of VEEv glycoproteins E1, E2, E3 and 6KD. The plasmids each contain a T7 promoter upstream of the replicon/VEEv sequences, to allow for in vitro transcription of the desired regions. The plasmids also contain a kanamycin resistance gene; however, this gene is not present in the vaccine due to restriction enzyme digestion prior to in vitro transcription.

During vaccine manufacturing, VERO cells are co-transfected with the purified RNA transcripts produced by in vitro transcription from the T7 promoters on the three plasmids. Within the co-transfected cells, the replicon RNA directs the production of multiple copies of itself, while the two helper RNA sequences are translated and processed into capsid and envelope glycoproteins. The newly synthesised capsid and glycoproteins assemble themselves into a VRP, incorporating the replicon RNA at its core. The pieces of helper RNAs present within the cell (i.e., those encoding the capsid and glycoproteins) are not packaged within the VRPs due to their lack of the necessary virus packaging signals. The VRPs are then purified from the cell culture fluids for quality control testing and formulation into the final vaccine.

Details of the methods used to create the master sequence plasmids and produce the vaccine are on file at CCVB.

4.3 Genetic and Phenotypic Stability of the Vaccine Organism – The manufacturer performed studies using their SIV replicon particle vaccine to confirm the stability of the propagation defective attribute of the VRPs. In one study, pigs were concurrently administered two doses of vaccine at 50 times the normal swine dose: one dose by the intramuscular route and one dose intravenously. Blood, nasal swabs and rectal swabs were collected at 3, 7, 10 and 14 days post injection and were analysed for the presence of replication competent particles by applying them to a monolayer of VERO cells (1hr adsorption, cultures washed before new media added), then transferring media from these cultures (24hr post-inoculation) to fresh VERO cell monolayers, and finally examining these second passage VERO cultures for cytopathic effect (CPE). Any replication competent particles present in the samples should have infected the first passage VERO cells and resulted in the release of progeny into the culture media to infect and cause CPE in the second passage VERO cells. No CPE was detected in the second passage cultures indicating that the defects that render the VRPs non-propagative are stable, even following injection into animals.

Each batch of vaccine is also tested for the presence of propagation competent virus. This is similarly achieved by inoculating a culture of VERO cells with a sample of the vaccine, washing, then after 24hr transferring the culture media to a new uninfected culture of VERO cells. The test is satisfactory if no CPE is detected in the second VERO cell culture after three days. Again, should any replication competent viruses be present in the vaccine, their progeny would be released into the culture media from the first culture, and would infect cells in the second culture, causing detectable CPE.

4.4 Horizontal Gene Transfer and Potential for Recombination – The vaccine production system has been designed to minimize the potential for recombination events leading to replication competent virus. A split helper system is employed to help ensure that genetic material encoding the structural proteins of VEEv is not present within the vaccine particles. That is, the helper RNAs encoding the VEEv structural proteins are supplied as two separate pieces of RNA, meaning a minimum of two independent recombination events with the replicon RNA must take place to generate a piece of RNA with all the necessary information for propagation competent virus production. Further hindering the chances of successful recombination is the fact that the helper RNAs are promoterless, and thus cannot act as independent transcriptional units. This means that not only must at least two recombination events occur, but they must occur such that the helper RNAs are integrated in a certain order. The capsid coding sequence must integrate downstream of the 26S subgenomic promoter on the replicon but 5' to the glycoproteins, so that the capsid protein's autocatalytic activity can cleave the capsid from the rest of the polyprotein. The glycoprotein RNA must integrate not only downstream of the capsid protein but also in the same codon reading frame as the capsid protein to avoid a frame-shift mutation. Finally the recombination events must be able to abrogate the stop codon added to the end of the capsid helper RNA and restore the capsid's autocatalytic cleavage site, which was purposely destroyed when the researchers created the capsid helper RNA (Vander Veen et al., 2012; Kamrud et al, 2010).

Taken together, there are many barriers in place to ensure that replication competent recombinants are not created during vaccine production. Consistent with this is the vaccine manufacturer's declaration that they have never detected a replication competent recombinant. VRPs devoid of the helper RNA sequences cannot produce additional capsid and envelope glycoproteins in the cells of the vaccinated animal, and these structural proteins are needed for assembly of new VRPs and dissemination of the infection from one cell to other cells within the animal.

Within the vaccinated animal, the genetic material that is present in the VRP (i.e., the replicon RNA) is restricted in its ability to participate in horizontal gene transfer events or recombine with other viruses primarily due to the fact that the VRPs cause only a single-cycle infection and their numbers do not amplify within the host. The only source of VRPs is that injected in the volume of vaccine, approximately 3x108 particles. According to manufacturer data generated for the SIV replicon vaccine, the level of the VRPs in the blood, nasal secretions and feces of vaccinated animals is below assay detection limits by 3 days post-vaccination (earliest time point examined). Consistent with this finding, a published paper reported that RNA from a different VEEV-based VRP was undetectable in the blood, liver, brain and spinal cord 24hr post intramuscular injection of mice (Kowalski et al., 2007). In the Kowalski study, replicon RNA was detected in muscle/skin samples taken from the injection sites; however, other studies suggest that VRPs may be rapidly cleared from the injection site and transferred to draining lymph nodes via the migration of infected dendritic cells (Laust et al., 2007; MacDonald and Johnston, 2000). Regardless, the low presence of the vaccine soon after vaccination, aside from some residual RNA in the draining lymph nodes and perhaps injection site, should hamper opportunities to recombine with other pathogens. For RNA recombination to occur there would have to be coinfection of the same cell with both the VRP and another RNA virus. In addition, RNA recombination between non-segmented RNA viruses tends to occur more frequently when there is homology between the two viruses (Simon-Loriere and Holmes, 2011); there are no alphaviruses that routinely infect swine in Canada.

The replicon RNA replicates in the cytoplasm, and without a DNA intermediate. This alleviates concerns about nuclear recombination with the vaccinated animal's DNA, and insertional mutations or aberrant gene expression resulting from integration within the chromosomal DNA.

4.5 Host Range/Specificity, Tissue Tropism and Shed/Spread Capabilities – Like wild type VEEV, VEEV-based VRPs have been shown to have tissue tropism for dendritic cells, such as the Langerhans cells residing in the skin and monocyte-derived inflammatory dendritic cells, which upon infection migrate to the lymph node draining the injection site (Tonkin et al., 2012; Gardner et al., 2008; Nishimoto et al 2007; MacDonald and Johnston, 2000). In studies conducted with Harrisvaccines' SIV replicon vaccine, there was no evidence of VRP shedding in nasal secretions or faeces from pigs at 3, 7, 10 or 14 days post vaccination, or in the feces of mice at 1, 3, 7, 14-19, 21, 28 or 42 days post vaccination. Should small amounts of the VRPs be eliminated intact from a vaccinated animal that escaped detection in the manufacturer's studies, the VRPs are known to be unstable at room temperature or above outside of a host, and VEE viruses are generally sensitive to desiccation, exposure to sunlight, acidic pH and various common disinfectants (OIE Technical Disease Card).

Studies performed by the manufacturer using its SIV replicon particle vaccine demonstrated that the VRPs cannot directly spread from a vaccinated pig to in-contact pigs, or from vaccinated mice to in-contact mice.

Wild type VEE viruses are typically spread by mosquito vectors that become infected following oral ingestion of the blood of a viremic animal (Weaver et al., 2004). After infecting cells of the insect's midgut, the infection must spread to the salivary glands for the mosquito to transmit the virus to another vertebrate host through salivary secretions. Mosquitos or other hematophagous arthopods known to facilitate indirect animal-to-animal transmission of wild type VEEv were not known to be present during the manufacturer's studies designed to detect spread of the SIV vaccine 'organism' between animals. However, it is not anticipated that mosquito vectors can spread the VRPs from a vaccinated pig to other animals. Should a mosquito happen to ingest a few VRPs, the VRPs are still only capable of producing a single cycle infection in the insects. Following oral ingestion of blood containing VEEV-based VRPs, VRP infected cells appear to be localized in the mosquito's midgut and not the salivary glands (Smith et al., 2007). Moreover, it is possible that a bloodmeal may need to contain at least 105 VRPs per mL to reliably find VRP infected midgut cells in mosquitoes of at least one species, Aedes taeniorhynchus (Smith et al., 2007). With only approximately 3x108 VRPs injected per pig during vaccination, it is doubtful that this titre could be attained in the blood of a vaccinated animal, aside from possibly near the injection site immediately after vaccination.

4.6 Comparison of the Modified Organisms to Parental Properties – A few nucleotide substitutions were introduced into the nsp1 gene sequence in the replicon RNA, allowing replicon derived sequences to be distinguishable from the parental TC-83 vaccine sequence.

4.7 Route of Administration/Transmission – The vaccine is to be administered by the intramuscular route. No transmission is expected based on the design of the VRPs and manufacturer data.

5. Human Safety

5.1 Previous Safe Use – The TC-83 VEE vaccine strain from which much of the vaccine is derived was originally developed for use in personnel involved in high-risk VEE research (OIE Terrestrial Manual). The live TC-83 virus has been administered to thousands of humans, primarily laboratory workers and military personnel (Pittman et al., 1996).

VEEV-based replicon particle vaccines similar to the present PEDv vaccine have been administered to at least 140 healthy human volunteers (typically receiving 3 doses each) as part of various Phase I clinical trials (Wecker et al., 2012; Bernstein et al., 2010). A VEEV-based replicon vaccine has also been administered to 12 subjects with prostate cancer, with each patient receiving up to 5 doses of the experimental cancer vaccine (Slovin et al., 2013). No safety concerns were identified in any of these studies.

5.2 Probability of Human Exposure – The probability of human exposure to the vaccine's VRP is expected to be low due to the fact that the VRPs do not propagate in a vaccinated animal, and do not appear to be shed from vaccinated pigs to a significant degree, if at all. This is based on attempts to detect the VRPs after 3 days post-vaccination, and the lack of spreading to susceptible pigs comingled with vaccinates beginning 24hr post-vaccination with Harrisvaccines' SIV replicon vaccine. In addition, the VRPs, due to their known sensitivity to heat, and presumed sensitivity (based on characteristics of VEEV) to desiccation, sunlight and common cleaning agents, would not be expected to persist in the environment should a limited amount of shedding, or an undetected spill of the vaccine, occur.

Biosecurity protocols in place at nearly all modern commercial swine production operations normally restrict access of the general public to the animals and facilities.

5.3 Possible Outcomes of Human Exposure – Human exposure to the vaccine or vaccinated animals is not expected to be a safety concern. The VRPs do not have the capacity to propagate themselves, due to the absence of genetic material for the production of additional structural proteins, and this deficiency is not species dependent.

5.4 Pathogenicity of Parent Microorganisms in Humans – The parental TC-83 strain is attenuated and can be administered to humans as a live vaccine. When TC-83 vaccine is administered to humans, it is reported that approximately 25% of people experience adverse reactions following vaccination; however, these tend to be mostly flu-like and short-lived (Pittman et al., 1996).

PED viruses are not known to cause disease in humans.

5.5 Effect of Gene Manipulation on Pathogenicity in Humans – The genetic manipulations to the VEEv TC-83 parental virus serve to attenuate pathogenicity. By removing the genetic material for the virus' structural proteins, the manufacturer has created a virus-like RNA replicon particle, which has lost its ability to propagate like the parental virus.

5.6 Risk Associated with Widespread Use of the Vaccine – Risks associated with the widespread use of the vaccines have not been identified.

6. Animal Safety

6.1 Previous Safe Use – The iPED+ vaccine has not yet undergone large scale field safety testing. The manufacturer has administered the vaccine to a small number pigs, including naïve pigs, as part of R&D studies, and has not reported any safety concerns. Harrisvaccines has also distributed over 20,000 doses of the unlicensed vaccine to veterinarians in the USA for use on farms under their care.

Harrisvaccines' distributed more than 700,000 doses of its first generation PED replicon particle vaccine, iPED, which expressed only the S1 region of the spike protein, for use in pigs in the USA.

6.2 Fate of the Vaccine in Target and Non-Target Species – Following injection into the musculature of a pig, the VRPs are believed to preferentially infect dendritic cells near the site of inoculation, such as the Langerhans residing in the skin and monocyte-derived inflammatory dendritic cells (Tonkin et al., 2012; Nishmoto et al., 2007; MacDonald and Johnston, 2000). Infected/activated dendritic cells rapidly migrate to the lymph node draining the injection site. The results of one study suggest that a low dose (103) of a VEEV-based VRP, when injected subcutaneously into mice, can be cleared from the injection site within 1hr, with VRPs being detectable in the draining lymph node within 30min of injection (MacDonald and Johnston, 2000).

Within an infected cell, the positive-sense, single-stranded RNA replicon is translated to produce non-structural proteins 1-4. These proteins then direct the replication of the replicon, the production of the 26S subgenomic transcript, and the translation of the subgenomic transcript to yield high levels of the PEDv S protei. This foreign antigen is then responsible for invoking an immune response in the vaccinee against the S protei component of PEDv. As mentioned previously, although the replicon RNA is able to replicate within the infected cells, new (progeny) VRPs cannot be formed and released from the infected cells to infect other cells, due to the absence of genetic material encoding the necessary structural proteins.

Activated dendritic cells are typically eliminated from the body via apoptotic cell death (Granucci and Zanoni, 2009). In the study mentioned above, VRPs remained detectable in the draining lymph node for up to 5 days (MacDonald and Johnston, 2000).

Consistent with the notion that VRPs may be rapidly cleared from an animal, results provided by the manufacturer generated using its SIV replicon particle vaccine indicate that at 14 days post-vaccination, no VRPs can be detected by their RT PCR assay in tissue samples from the injection site musculature, tonsils, lung, spleen, liver, kidney, heart, brain, intestine, and lymph node closest to the injection site.

6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non Target Animals – The manufacturer examined the ability of the VRPs to shed and spread from vaccinated pigs and mice using their SIV replicon particle vaccine.

In the pig study, pigs were concurrently administered two doses of the SIV vaccine at 50 times the normal swine dose: one dose by the intramuscular route and one dose intravenously. Nasal and rectal swabs were collected at 3, 7, 10 and 14 days post injection, and the extracted RNA from each of these samples analysed for the presence of replicon RNA by RT-PCR. No replicon RNA could be detected, indicating that by 3 days post-injection, the level of the VRPs is below the detection limit of the assay. This suggests that VRPs are not shed in nasal secretions or feces from 3 days post-vaccination. The manufacturer also failed to detect VRPs in blood, nasal swabs and rectal swabs taken at days 3, 7, 10, and 14 from pigs comingled with vaccinated pigs beginning 24hr after vaccination. Whereas vaccinates all seroconverted for SIV HA, none of the in-contact control pigs showed anti-HA antibodies, further supporting the notion the vaccine cannot spread from vaccinated pigs to in-contact pigs (Vander Veen et al., 2012).

A similar study was performed with mice, which showed that the VRPs are not shed in the feces of mice and cannot spread from vaccinated mice to in-contact mice.

As mentioned in section 4.5, it is not known whether hematophagous arthropods known to be capable of transmitting wild type VEEv from animal to animal were present in the manufacturer's studies. Nonetheless, it is not anticipated that the VRPs can spread to any animal to cause productive infection in the indirectly exposed animal, in light of the genetic makeup of the VRPs.

6.4 Reversion to Virulence Resulting from Back Passage in Animals – The manufacturer performed studies using their SIV replicon particle vaccine to confirm that the propagation defective property of the VRP is maintained, even when the VRPs are injected into animals. In one study, pigs were concurrently administered two doses of vaccine at 50 times the normal swine dose: one dose by the intramuscular route and one dose intravenously. Blood, nasal swabs and rectal swabs were collected at 3, 7, 10 and 14 days post injection and were analysed for the presence of replication competent particles by applying them to a monolayer of VERO cells, washing, then transferring media from these cultures (24hr post-inoculation) to fresh VERO cell monolayers, and finally examining these second passage VERO cultures for CPE. Any replication competent particles present in the samples should have infected the first passage VERO cells and resulted in the release of progeny into the culture media to infect and cause CPE in the second passage VERO cells. No CPE was detected in the second passage cultures indicating that the defects that render the VRPs non-propagative are stable, even following injection into animals.

RNA isolated from these same blood, nasal swab, and rectal swab samples were also analysed for the presence replicon RNA by RT-PCR. No replicon RNA could be detected, indicating that by 3 days post-injection, the level of the VRPs is below the detection limit of the assay. Tissue samples from the injection site musculature, tonsils, lung, spleen, liver, kidney, heart, brain, intestine, and lymph node closest to the injection site taken at 14 days post-vaccination were similarly tested for the presence of VRPs by RT-PCR and found to be negative. Should the VRPs of the vaccine have gained the ability to propagate and amplify to high titres within the host, one would expect to detect their presence at least at one of the time points tested.

Taken together, these data confirm that the VRPs do not gain the ability to propagate in pigs following vaccination. Restoration of propagative capacity would be a prerequisite for the vaccine particles to revert to a virulent virus; otherwise infection cannot spread beyond the first infected cell.

The manufacturer was unable to perform a standard back-passage study as VRPs could not be recovered from the first group of vaccinated animals.

6.5 Effect of Overdose in Target and Potential Non-Target Species – The manufacturer tested the safety of a vaccine overdose by administering ten pigs at approximately six weeks of age two doses of the SIV vaccine, each at 50-times the normal pig dose. One dose was given by the recommended intramuscular route, while the other dose was concurrently given intravenously. Adverse events related to the administration of the vaccine were not reported.

The manufacturer also administered the SIV vaccine intraperitoneally to mice at approximately 1/10 the swine dose with no signs of toxicity.

VEEV-based VRPs similar to the Harrisvaccines' SIV and PEDv vaccines have been tested in guinea pigs, rats, rabbits, cattle, rhesus macaques, cynomolgus macaques, and humans. No safety concerns have been identified (Herbert et al., 2013; Loy et al., 2013; Wecker et al., 2012; Bernstein et al., 2010; Hubby et al., 2007; Laust et al., 2007).

6.6 The Extent of the Host Range and the Degree of Mobility of the Vector – The degree of mobility of the vector is restricted. The VRPs are capable of only a single round of infection; following replicon RNA delivery to the first cell, they cannot propagate to produce additional VRPs to infect other cells.

7. Affected Environment

7.1 Extent of Release into the Environment – The extent of release of VRPs from the vaccine will be quite limited. The VRPs lack the genetic information necessary to produce progeny VRPs. Therefore, the maximum amount of VRPs that can potentially be released into the environment is that contained within the vaccine. Following vaccination of an animal, it is presumed that the vast majority of the injected VRPs are taken up by cells and degraded by normal protein and nucleic acid metabolism pathways before elimination of the constituents into the environment.

7.2 Persistence of the Vector in the Environment and Cumulative Impacts – The VRPs are enveloped structures like parental VEEV. This makes them relatively sensitive to heat, desiccation, and detergents, and thus less able to survive in the environment outside of a host cell (OIE Technical Disease Card; Sagripanti et al, 2010; Harper, 1961). The manufacturer provided data generated using its SIV replicon vaccine which indicate that the VRPs are completely inactivated by 7 days at 37ºC and largely inactivated after about 21 days at 27ºC, even when kept in the protected environment of its sterile vaccine vial. VEE viruses are also susceptible to inactivation by solar UV radiation (Lytle and Sagripanti, 2005).

7.3 Extent of Exposure to Non-Target Species – Most swine in Canada is reared within biosecure production facilities, so relatively few non-target species can potentially be exposed. As mentioned previously, the non-propagative nature of the VRPs will largely restrict exposure.

8. Environmental Consequences

8.1 Risks and Benefits – The potential benefit of this vaccine is that it might help protect swine in Canada against disease caused by PEDv, by either reducing the number or severity of cases of PED. Many believe that the risk of exposure to PEDv is currently very high in Canada based upon the virus' spread in North America. Since the virus' introduction less than one year ago to the USA, PEDv has spread across 23 US states and now into one Canadian province, despite heightened awareness in the industry to adhere to biosecurity protocols. Vaccination may be a tool to help reduce the impacts of PED in Canada.

It must be emphasized that Harrisvaccines' iPED+ vaccine has not yet been licensed in any jurisdiction because the efficacy of the vaccine has not been established. While there are anecdotal accounts of beneficial effects of the vaccine in the USA, the results of controlled vaccination challenge studies are not yet available, and the immunogenicity data supplied to date are based on serological responses in only a small number of pigs. Therefore, although the CCVB is willing to allow veterinarians access to this potential tool for use as a control against disease caused by PEDv, the CCVB cannot predict at this time how well the vaccine will perform in field applications. This is why veterinarians will have to acknowledge that they are aware that the vaccine is unlicensed, that they will use the vaccine at their own risk, and that they will inform their clients that the efficacy of the vaccine has not been established. Furthermore, veterinarians and producers must be aware that use of the vaccine should not be a substitute for continued adherence to heightened biosecurity protocols.

8.2 Relative Safety Compared to Other Vaccines – There are currently no other PED vaccines available for use in Canada. Compared to the possibility of using live attenuated PEDv strains as vaccines, which might have only a few point mutations responsible for their attenuation, the present vaccine has a much lower risk of reversion to virulence. In fact, there is virtually no risk of the iPED+ vaccine 'reverting' to virulence to cause PED disease, as the vaccine contains only a fragment of the PED virus. Compared to the possibility of using inactivated PED virus vaccines, there are no concerns about the iPED+ vaccine causing disease due to incomplete chemical inactivation, as iPED+ does not require chemical inactivation during vaccine manufacturing like a killed virus vaccine.

The VRPs contained in the iPED+ vaccine are not believed to be shed or spread between animals, so there is also less concern about dissemination of the recombinant vaccine 'organism' in the environment compared to some other potential live genetically modified vaccine designs.

9. Mitigative Measures

9.1 Worker Safety – Individuals responsible for administering the vaccine to pigs are at the greatest risk of exposure due to accidental self-injection. Should this occur, exposure to the VRPs is not expected to be a human health concern based on the fact that related VRPs have been administered to human volunteers, and the fact that the genetic mutations that render the VRPs propagation defective function irrespective of species. Moreover, since the vaccine does not contain any adjuvant, the risk of clinical problems due to accidental self-injection of oil adjuvant is removed. Should the vaccine be spilled, it can easily be inactivated by commonly available cleaning and disinfection agents.

9.2 Handling Vaccinated or Exposed Animals – In Canada, most swine are reared in biosecure facilities with restricted access to the general public. Workers handling vaccinated animals will have limited exposure to the VRPs, as manufacturer data generated using the related SIV replicon vaccine suggest that the VRPs are not shed in the feces or nasal secretions from 3 days post-vaccination (earliest timepoint examined).

10. Monitoring

10.1 General – Emergency use import permits issued for iPED+ will carry a condition indicating that the permit holder must report any information concerning or any evidence of a significant deficiency in product safety, potency or efficacy to the CFIA within 15 days from the date on which such information or evidence is known to him or her. In addition, veterinarians will be asked to provide feedback to the CCVB on their experiences with the vaccine, including its presumed performance, anecdotal or otherwise.

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

10.3 Animal – Veterinarians, vaccinators, and producers should report any suspected adverse reactions to the CCVB as indicated above. Suspected adverse reactions should be reported using Form CFIA/ACIA 2205: Notification of Suspected Adverse Events to Veterinary Biologics. The conditions on the import permit will also require veterinarians to maintain a record of on which herds/premises the vaccine was used.

11. Conclusions and Actions

Based on our assessment of the available information, the CCVB has concluded that the importation and use of Harrisvaccines' unlicensed iPED+ porcine epidemic diarrhea vaccine in Canada would not be expected to have any significant adverse effect on the environment, when manufactured and tested according to generally accepted good manufacturing practices and used according to label directions.

Following this assessment, the CCVB has decided to accept applications from veterinarians in Canada to import iPED+ for emergency use.

Each serial (batch) of the iPED+ vaccine destined for Canada must be quality control tested by the manufacturer, and the results of these tests submitted to the CCVB for review. Emergency use import permits for iPED+ issued by the CCVB to veterinarians will be restricted to only specific approved serials. The vaccine imported by a veterinarian may only be used in herds under their supervision, and may not be distributed further.

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