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Environmental assessment for Merck Animal Health's RNA Particle Prescription Products for Swine Influenza and other disease agents

July 24, 2018

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

Summary

Merck Animal Health has applied to the CCVB to market in Canada a series of prescription products based on their ribonucleic acid (RNA) replicon particle platform. Much like autogenous vaccines, these prescription products would be a tool veterinarians could use in situations where currently approved commercial vaccines are unavailable or seem ineffective in controlling a disease. The RNA replicon particle platform is designed to take the RNA sequence corresponding to typically one gene of the causative pathogen, and incorporate this into a piece of RNA known as a replicon. The replicon is then packaged into propagation-defective virus like particles to facilitate expression of the antigenic gene product in cells of the vaccinated animal to stimulate an immune response against the pathogen.

As part of the requirements for licensing this series of biotechnology-derived products in Canada, the CCVB conducted an environmental assessment on the vaccine organism. This public environmental assessment summary document contains information on the molecular and biological characteristics of the vaccine organism, target animal and non-target animal safety, human safety, environmental considerations, and risk mitigating measures.

1. Introduction

1.1 Proposed action

The CCVB 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 the Health of Animals Regulations. Any veterinary biologic manufactured, sold or represented for use in Canada must comply with the requirements specified by the CFIA regarding the safety, purity, potency, and efficacy of the product. In certain situations, fully validated commercial vaccines are unavailable to control an emerging disease, or do not provide adequate protection due to strain differences and antigenic drift. To provide veterinarians with options for managing disease outbreaks, the CCVB authorises licensed manufacturers to produce autogenous vaccines or prescription products for restricted use by veterinarians on specific farms. Such authorisations are granted under 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 efficacy of the vaccines authorised under this provision has not been fully established. They have also not undergone large-scale safety studies; instead the expectation of safety is based on vaccine composition, production processes and small-scale testing of each batch in mice or the target animal species.

Merck Animal Health (Ames, Iowa, USA) has applied to license the following vaccine in Canada:

Prescription Product, RNA Particle, Swine Influenza Virus Vaccine; CCVB File 920PL/S1.0/I6.2, USDA Product Code 9PP0.00

By licensing this file, the company will be allowed to produce swine influenza vaccines, using its RNA replicon particle platform, for use on specific farms under veterinary prescription and supervision. The hemagglutinin (HA) gene sequence from the swine influenza virus (SIV) isolated from the infected premise will be used to create the farm-specific vaccine.

This Environmental Assessment was prepared by the CCVB as part of the overall assessment for licensing the above vaccine file in Canada. It is based on information provided by the vaccine's manufacturer, as well as information obtained by the CCVB through searching published literature. Much of what is known about Merck Animal Health's RNA replicon particle platform has been extrapolated from studies performed to characterise the company's commercial vaccine: Swine Influenza Vaccine, RNA (USDA Product Code 19A5.D0, CCVB File 880VV/S3.0/H16). This commercial SIV vaccine contains the HA gene of a Clade IV H3N2 SIV clinical isolate from the US Midwest.

Merck Animal Health plans to eventually license additional prescription product files targeting different animal pathogens. For these, genes expressing key antigenic proteins of the pertinent disease agents will be incorporated into the replicon in place of the SIV HA gene. The present environmental assessment will also apply to these future prescription product files. Based on the properties of the technology platform, it is expected that all vaccines created with the platform will share the same fundamental safety characteristics.

1.2 Background

The RNA replicon particle platform is based on a piece of RNA known as a replicon packaged into virus-like RNA replicon particles (VRPs) with the capsid and envelope glycoproteins of the attenuated TC-83 vaccine strain of Venezuelan equine encephalomyelitis 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 an antigenic protein of the disease agent (for example, the HA protein of SIV). 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 the pathogen protein. This foreign antigen is then recognized by the animal's immune system, leading to cell-mediated and humoral immune responses against the disease agent.

2. Purpose and need for proposed action

Veterinarians have contacted the CCVB seeking access to this additional tool for controlling disease in livestock when currently approved commercial vaccines are unavailable or seem ineffective.

Similar to conventional autogenous vaccines, the following criteria must be met for licensing of the platform-based prescription products: A) the products must be produced and quality control tested in a licensed manufacturing facility; B) there must be a reasonable expectation of product efficacy; C) there must be adequate disclosure that the prescription product vaccines have not been fully validated like a commercial vaccine; and D) the CCVB must be satisfied that use of the prescription products in Canada will not endanger animal health, human health, or the environment.

Criterion A) The RNA replicon particle prescription products will be produced in the licensed manufacturing facilities of Merck Animal Health, Ames, Iowa, under US Veterinary Biologics Establishment License No. 165A.

Criterion B) In the case of the SIV prescription products, the results of a vaccination-challenge study performed using the commercial SIV RNA replicon particle vaccine provide the expectation of efficacy. Like the commercial SIV vaccine used in the trial, the SIV prescription products will be designed to express the full length HA gene of the pathogen.

Criterion C) Upon applying to import a prescription product from Merck Animal Health, the veterinarian will have to sign a declaration stating that he/she is aware, and has advised the animal owner that:

  1. the vaccine is intended for use as an interim measure in emergency situations when a suitable licensed product is not available to prevent the spread of a disease within the animals of the infected premises and associated premises where the product will be used;
  2. the efficacy of the prescription product has not been established; and
  3. the product has been prepared for use only by, or under the direction of, a licensed veterinarian

The labelling of the prescription product will additionally include the statements, "This is a prescription product for use in an emergency situation when a licensed product is not available to prevent the spread of a disease to healthy animals. Efficacy has not been determined. Recommended use shall be at the discretion of the prescribing veterinarian."

Criterion D) The purpose of this EA is to determine whether criterion D 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 the prescription products on specific farms if the CCVB is satisfied that their use 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 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 desired antigen (for example, the SIV HA protein) 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 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 starting material for vaccine production. One plasmid contains the replicon sequence. This plasmid is modified each time by inserting the farm-specific gene sequence for the desired antigen (for example, SIV HA). The other two plasmids do not change. They contain the sequences necessary for expression of the VEEV capsid protein, and the sequences necessary for the production of VEEV glycoproteins E1, E2, E3 and 6KD, respectively. The three 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 (for example, 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 commercial 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 (for example, 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 106 to 108 particles. According to manufacturer data generated for the commercial 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 the commercial SIV replicon particle 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 commercial 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 arthropods 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 106 – 108 VRPs injected per animal 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 the parents

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 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 the commercial 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 livestock 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).

While swine influenza viruses can cause disease in humans, the vaccine contains only one piece of the virus, namely the HA (hemagglutinin) gene and protein. Humans are commonly exposed to HA genes and proteins through exposure to human influenzas and influenza vaccines. The HA gene and protein alone, in isolation from the rest of the influenza virus, are not pathogenic to 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 prescription products will not undergo large scale field safety testing. However, each batch will be tested in either mice or the target animal.

Merck Animal Health's commercial SIV vaccine based on the RNA replicon particle platform was field safety tested in approximately 900 commercial pigs at three geographically separate locations in the USA. Pigs were approximately 3 weeks of age at the time of first vaccination, and each received two doses of the vaccine, three weeks apart. The vaccine was well-tolerated. Systemic adverse reactions were not observed, and localized injection site reactions were reported for only four pigs.

A few thousand doses of Merck Animal Health's unlicensed RNA replicon particle vaccine against porcine epidemic diarrhea have been used in Canada since 2014. No safety issues have been reported to the CCVB.

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 pathogen protein (e.g., the SIV HA protein). This foreign antigen is then responsible for invoking an immune response in the vaccinee against the disease agent. 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 commercial 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 commercial 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 commercial 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 commercial 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 Merck Animal Health's platform 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 commercial SIV replicon particle vaccine which indicate that the VRPs are completely inactivated by 7 days at 37°C and largely inactivated after about 21 days at 27°C, 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 livestock 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 these prescription products is that they might help protect livestock in Canada against disease when commercially available products are unavailable or seemingly ineffective.

The primary risk of the vaccine is the risk of the propagation defective VRPs somehow gaining the ability to spread from animal to animal or recombine with another virus to create a VEE chimeric virus capable of spreading from animal to animal. While it is believed that the chances of such an event are exceedingly low, the potential implications are perhaps higher in Canada than some other countries, as Canada is recognized as being free of VEE. Should a recombinant virus be created that can infect equines, it could lead to misdiagnosis of VEE and a costly foreign animal disease investigation by animal health authorities. It should be noted, however, that the vaccine contains genetic material only for the non-structural proteins of VEEV. Typically, serological diagnosis of VEE is based on neutralizing antibodies against VEEV structural proteins. However, RT-PCR diagnostic assays may target VEEV sequences present in the replicon RNA. After consultation with the CFIA Foreign Animal Disease section, it was decided that the potential benefits of the vaccines outweigh this theoretical risk.

It should also be noted that administration of Merck Animal Health's RNA replicon particle vaccines to animals will quite likely result in the animals producing antibodies specific for the VEE capsid and envelope glycoproteins present in the vaccine. Consequently, administration to horses in Canada should be avoided to ensure these horses do not react on VEE serological diagnostic assays. A positive reactor could initiate a costly foreign animal disease investigation, and perhaps even temporary trade restrictions and economic repercussions. The Canadian labelling for these prescription products will warn users against administering the product to horses or other equine species.

8.2 Relative safety compared to other vaccines

Current swine influenza vaccines are killed virus products. The present vaccine platform does not require chemical inactivation during vaccine manufacturing like a killed vaccine, so there is no risk of vaccine-induced disease due to incomplete chemical inactivation. The safety of the different variations of the platform vaccines will not be established in the types of large-scale safety studies normally conducted for commercial vaccines. Thus less may be known about their risks of less common adverse events.

9. Mitigative measures

9.1 Worker safety

Individuals responsible for administering the vaccine to animals are at 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 livestock 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 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

Individual veterinarians will have to apply to the CCVB to import a prescription product from Merck Animal Health. One of the permit conditions will be the requirement for the veterinarian to report to the CFIA any information concerning, or evidence of, a significant deficiency in the safety of the product within 15 days of becoming aware of an adverse event. The CCVB will then typically ask the manufacturer to investigate and prepare a report on the suspected adverse event for review by the CCVB.

10.2 Human

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

10.3 Animal

Veterinarians must report any suspected adverse reactions to the CCVB as indicated above. Form CFIA/ACIA 2205 – Notification of Suspected Adverse Events to Veterinary Biologicscan be used for this purpose. Vaccinators and producers may also report suspected adverse reactions using the same form.

11. Conclusions and actions

Based on our assessment of the available information, the CCVB has concluded that the importation and use of Merck Animal Health's prescription products based on the RNA replicon particle platform would not be expected to have a significant adverse effect on the environment, when manufactured and tested as described in the approved Outline of Production, and used according to label directions.

Following this assessment and the completion of the Canadian veterinary biologics licensing process, individual veterinarians may apply for a Permit to Import Veterinary Biologics to import and use the following product in Canada:

Prescription Product, RNA Particle, Swine Influenza Virus Vaccine; CCVB File No. 920PL/S1.0/I6.2, USDA Product Code 9PP0.00

All serials of this product must be released by the USDA prior to importation into Canada. All conditions described on the Permits to Import Veterinary Biologics issued to individual veterinarians must be followed with respect to the importation and use of this product.

12. References

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