Environmental Assessment for the Canadian licensing of Merial’s Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera
March 20, 2018
The information in this environmental assessment was current at the time of its preparation. It is possible that the situation may have changed since that time. Please consult the Canadian Centre for Veterinary Biologics (CCVB) if you have any questions.
Table of Contents
- 1 Introduction
- 2 Purpose and need for proposed action
- 3 Alternatives
- 4 Molecular and biological characteristics of parental and recombinant organisms
- 4.1 Identification, sources and strains of parental organisms
- 4.2 Source, description and function of foreign genetic material
- 4.3 Method of accomplishing genetic modification
- 4.4 Genetic and phenotypic stability of the vaccine organism
- 4.5 Horizontal gene transfer and potential for recombination
- 4.6 Host range/specificity, tissue tropism and shed/spread capabilities
- 4.7 Comparison of the modified organisms to parental properties
- 4.8 Route of administration/transmission
- 5 Human safety
- 6 Animal safety
- 6.1 Previous safe use
- 6.2 Fate of the vaccine in target and non-target species
- 6.3 Potential of shed and/or spread from vaccinate to contact target and non-target animals
- 6.4 Reversion to virulence resulting from back passage in animals
- 6.5 Effect of overdose in target and potential non-target species
- 6.6 The extent of the host range and the degree of mobility of the vector
- 7 Affected environment
- 8 Environmental consequences
- 9 Mitigative measures
- 10 Monitoring
- 11 Contacts
- 12 Conclusions and actions
Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera (Trade Name: Prevexxion Rn) consists of a Marek's disease virus, serotype 1, chimeric virus further modified to contain two long terminal repeats (LTR) of reticuloendotheliosis virus (REV). The vaccine is recommended for use in healthy, 18- to 19-day-old chicken embryos and one-day-old chicks, as an aid in the prevention of Marek's disease caused by very virulent Marek's disease virus. The CCVB of the Canadian Food Inspection Agency (CFIA) evaluated this vaccine for licensing in Canada. As part of the requirements for licensing this product in Canada, the CCVB B conducted an Environmental Assessment, and prepared a public document containing information on the molecular and biological characteristics of the live genetically modified organism, target animal and non-target animal safety, human safety, environmental considerations, and risk mitigating measures.
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 CCVB regarding the safety, purity, potency, and efficacy of the product. The CCVB received the following vaccine licensing application:
Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera (Trade Name: Prevexxion Rn), CCVB File No. 800VV/M1.4/R2.1, USDA Product Code 16L1.R0
This Environmental Assessment was prepared by the CCVB as part of the overall assessment for licensing the above vaccine in Canada.
Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera is manufactured by the company Merial, Inc. (US Veterinary Biologics Establishment License No. 298), and was licensed for sale in the U.S. on March 27, 2017. This avian vaccine consists of a live chicken herpesvirus, Marek's disease, serotype 1, RN strain, containing an insertion of the long terminal repeat (LTF) of the reticuloendotheliosis virus (REV). The vaccine is intended for use in healthy 18- or 19-day-old chicken embryos and healthy one-day-old chicks as an aid in the prevention of Marek's disease caused by very virulent Marek's disease virus.
Marek's disease is a highly contagious, ubiquitous disease of chickens caused by a Gallid herpesvirus-2 (Marek's disease virus, serotype 1). Chickens predominantly become infected with Marek's disease during the first few weeks of life and carry the infection throughout their lives. Clinical Marek's disease generally afflicts birds after four to six weeks of age, causing paralysis, carcass condemnation at slaughter, and/or mortality. Internally, Marek's disease is characterized by lymphoid cell infiltration and proliferation forming tumours (lymphomas) in various organs, including the liver, kidneys, heart, gonads, and spleen, and/or lesions or enlargement of peripheral nerves. Bursal tumours are relatively rare, helping to differentiate Marek's disease from avian lymphoid leukosis.
2 Purpose and need for proposed action
The labelling for Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera, (Prevexxion Rn) indicates that the product is recommended for the in ovo vaccination of healthy 18- to 19-day-old embryonated chicken eggs, or for subcutaneous vaccination of healthy one-day-old chickens, as an aid in the prevention of Marek's disease.
The CCVB evaluates veterinary biologic product submissions for licensure under the Health of Animals Act and the Health of Animals Regulations. The general criteria for licensing are as follows: a) the product must be pure, safe, potent and efficacious; b) vaccine components must be relevant to Canadian disease conditions; c) foreign products must be licensed in the country of origin; and d) the product must be produced and tested in accordance with generally accepted "good manufacturing practices." This U.S. origin vaccine meets these general criteria for evaluation by the CCVB.
The two options being considered are: a) to issue aPermit to Import Veterinary Biologicsto Merial Canada, Inc. (Burlington, ON) allowing the importation of Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera, if all licensing requirements are satisfactory; or b) not to issue a Permit to Import Veterinary Biologics if licensing requirements are not met.
4 Molecular and biological characteristics of parental and recombinant organisms
4.1 Identification, sources and strains of parental organisms
The CVI988 Gallid herpesvirus 2 (Marek's disease virus serotype 1) Rispens vaccine strain is the parent organism. It has been used in licensed vaccines since the 1970's.
4.2 Source, description and function of foreign genetic material
The foreign or donor genetic material is a cosmid designated B40-RM1Pac containing Marek's disease virus sequences flanking a DNA fragment of the reticuloendotheliosis virus (REV) with 2 copies of the long terminal repeat (LTR), one in each of the 2 inverted repeats flanking the US region. Details of the source, description and function of the foreign genetic material are on file at the CCVB.
4.3 Method of accomplishing genetic modification
Details of the methods used to create the chimeric virus are on file with the CCVB. In brief, the expression cassette was assembled in a cosmid containing sequences homologous to those flanking the desired site of integration in the Marek's disease virus genome. The cosmid and CVI988 DNA were co-transfected in primary chicken embryo fibroblasts (CEF) to generate a targeted insertion of the REV LTR into the exact same locus of the CVI988 genome. The insertion was confirmed by PCR. PCR assays confirmed the identity of the final viral construct. Additional characterization of the construct was done by a Southern blot.
4.4 Genetic and phenotypic stability of the vaccine organism
The manufacturer confirmed genomic stability by PCR analysis, Southern blotting and by sequencing the LTR inserts and their flanking sequences of the RN1250 master seed virus, the fifth in vitro passage from the master seed virus, and the fifth in vivo back passage in chickens. These reports are on file with the CCVB. Results were as expected confirming the genetic stability of the vaccine candidate.
4.5 Horizontal gene transfer and potential for recombination
The potential for infection and host chromosomal integration by Marek's disease viruses was previously assessed in risk assessments prepared for other CCVB licensed Marek's disease virus, live chimera vaccines, based on Marek's Serotype 3. These risk assessments may be consulted on the CCVB public web site. Although this is the first licensed use of Marek's Serotype 1, the results of those assessments would generally apply to this chimeric vaccine.
Based on a literature review of factors contributing to retrovirus integration, the presence of only REV LTR sequences in the RN1250 genome will not favor retrovirus provirus integration or genetic exchange between RN1250 and retroviruses; additionally the REV LTR cannot jump and integrate into another DNA sequence without the presence of retroviral enzymes.
Recombination between the RN1250 vaccine virus and related viruses within chicken cells to create a new virus that is pathogenic to chickens is another theoretical possibility. Such a genetic transfer requires both viruses to be infecting the same cell. As the vaccine strain is administered in ovo or by subcutaneous injection of day old chicks, while the naturally occurring Marek's disease virus isolates infect via the respiratory tract, the potential of both viruses co-infecting a cell and transferring genetic material are negligible.
The manufacturer evaluated the potential recombination between the vaccine strain and other serotype 1 or serotype 3 Marek's vaccines in day old SPF chicks. No recombination events or gross Marek's lesions (demonstrating increased virulence) were detected. (Report No. 11-069)
4.6 Host range/specificity, tissue tropism and shed/spread capabilities
Marek's disease viruses replicate exclusively in cells of avian origin (particularly chicken, turkey, duck, and quail) and attempts to infect mammalian cells have repeatedly failed (Sharma, 1998). In exposed birds, Marek's disease viruses initially infect and replicate within lymphocytes, where the virus remains cell associated. After about seven days, although lymphocytes continue to harbour the viral genome, the infection becomes latent and viral antigen expression wanes in lymphocytes. Productive infection, producing mature cell-free infectious virus, begins after about a week or more in feather follicle epithelium cells.
The manufacturer provided studies confirming that the tissue tropism of the vaccine construct and the parental CVI988 virus were similar (very low rate of isolation from spleen) but less severe and in fewer tissues than the observed tissue tropism with a currently licensed Rispens vaccine. Tissues from the spleen, liver, lung, kidney, and gonads were examined on days 7 and 28. Data was also filed to show that the Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera did not spread horizontally from vaccinates to in-contact chickens.
Marek's disease virus can be shed in feather dander, but it is not thought to readily spread horizontally between chickens (Cho, 1975). Data submitted by the manufacturer indicate that all feather follicle samples tested negative for virus recovery.
4.7 Comparison of the modified organisms to parental properties
The recombinant Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera differs genetically from the parental CVI988 strain by the integration of the two copies of the REV LTR sequences described in section 4.2. The foreign DNA is inserted into a non-coding region of the HVT genome, and does not disrupt any endogenous genes. The genetically modified organism does not contain any selectable markers such as antibiotic resistance genes. The presence of the REV LTR was shown to further reduce the virulence of the Marek's Disease vaccine strain.
4.8 Route of administration/transmission
The Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera is to be administered either in ovo to 18- or 19-days-of-embryonation eggs, or subcutaneously in the neck to one-day-old chicks.
5 Human safety
5.1 Previous safe use
The parental serotype 1 strain has been widely used internationally as a commercial vaccine for chickens. There are no reports of human infection from the parental strain. Based on this and the wide use of serotype 1 Rispens vaccines, the chimeric virus is unlikely to be virulent in humans.
5.2 Probability of human exposure
Human exposure to the Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera is to be limited to employees in the manufacturing facility, veterinarians, animal technicians, and poultry farm operators. The probability of human exposure or risk from the chimeric virus is equal to or less than the risk from the parental serotype 1 strain.
5.3 Possible outcomes of human exposure
This vaccine strain has not been inoculated into humans, thus the actual outcome is unknown. As the parental strain has not contributed to human infections, human exposure to Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera is not expected to be a health concern.
5.4 Pathogenicity of parent microorganisms in humans
The parental strain has not been associated with pathogenicity in humans.
5.5 Effect of gene manipulation on pathogenicity in humans
This vaccine strain has not been inoculated into humans, thus the actual outcome is unknown. As the parental strain has not contributed to human infections, human exposure to Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera is not expected to contribute to pathogenicity.
5.6 Risk associated with widespread use of the vaccine
No risks to human safety associated with the widespread use of the vaccine have been identified.
6 Animal safety
6.1 Previous safe use
Study Report No. 11-069 confirmed a lack of in vivo recombination events in day old birds co-administered both the chimeric vaccine strain and either a virulent Marek's disease virus strain or a Marek's serotype 3 strain. This was based on examination for gross MDV lesions and attempts to isolate virus by PCR from different tissues and organs.
The field safety study (Report No. 14-058) monitored approximately 57,400 birds, with half vaccinated by the in ovo route and half by subcutaneous vaccination of day old chicks. The hatchability was higher in the test vaccine groups compared to the in house treated birds. There was no evidence of leucosis, including Marek's disease, in any of the condemned birds.
6.2 Fate of the vaccine in target and non-target species
The manufacturer demonstrated in Report No. 10-066 that the vaccine virus showed significantly decreased tissue tropism in SPF day old chickens when compared to the commercial strain. This was further supported by a larger study (No. 10-156) with 50 vaccinates compared to 50 each of positive control birds (commercial Serotype 2 vaccine), sham vaccinates and negative controls, followed for 49 days.
Safety in non-target species was demonstrated in quail, turkeys, ducks, pheasants and pigeons with a 10X dose of the vaccine (Report No. 11-036). No lesions were observed, and the vaccine did not induce a humoral response, thereby confirming its restricted host range.
6.3 Potential of shed and/or spread from vaccinate to contact target and non-target animals
Report No. 10-065 demonstrated that the virus was not shed and spread from subcutaneously vaccinated day old chickens to non-vaccinated in-contact chickens.
6.4 Reversion to virulence resulting from back passage in animals
The manufacturer performed a back passage study with five passages in chickens (Report No. 10-130). No increase in morbidity or mortality was observed with in vivo passage, demonstrating that the recombinant virus does not acquire pathogenicity when subjected to a series of in vivo passages. Shed /spread to in-contact negative control birds was not detected. Virus recovery was assessed by PCR. A further study (PHN3257) corroborated these results for in ovo safety.
6.5 Effect of overdose in target and potential non-target species
The vaccine has been administered subcutaneously to SPF chickens at a 10X dose without any observed complications (clinical signs, weight, gross lesions). Birds were monitored for 120 days (Report No. 11-059). A similar study was done to confirm safety by in ovo vaccination (Report No. 16-131)
6.6 The extent of the host range and the degree of mobility of the vector
The Marek's Disease Vaccine, serotype 1, Live Virus is restricted in its host range to avian species, including turkeys, chickens, ducks, and quail. Safety in non-target species was demonstrated in quail, turkeys, ducks, pheasants and pigeons with a 10X dose of the vaccine (Report No. 11-036). No lesions were observed and the vaccine did not induce a humoral response, thereby confirming its restricted host range.
7 Affected environment
7.1 Extent of release into the environment
The vast majority of vaccinated chickens will be housed indoors in biosecure facilities, and thus will have little direct exposure to the environment. However, limited release of the vaccine organism may occur when poultry houses are cleaned out, or through the vented air.
7.2 Persistence of the vector in the environment and cumulative impacts
The manufacturer demonstrated that the vaccine virus was only detected on day 0 on 'infected' sterile pine shavings.
7.3 Extent of exposure of non-target species
The extent of exposure of non-target species is expected to be curbed by the fact that vaccine administration predominantly occurs in housed domestic poultry without access to the outside.
7.4 Behaviour of parent microorganisms and vector in non-target species
The parental HVT virus is not known to be pathogenic to other species.
8 Environmental consequences
8.1 Risks and Benefits
The benefits of this genetically modified vaccine are (1) that the vaccine is suitable for use in embryonated chicken eggs and young chicks, (2) that the vaccine is restricted in its host range and will not affect co-reared turkeys, and (3) that the chimeric virus shows decreased tissue tropism and no residual pathogenicity. The immune response in young chicks in the presence of maternal antibodies has not been assessed.
8.2 Relative safety compared to other vaccines
Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera is expected to be similar or slightly safer to the currently licensed, parent strain CVI988-based vaccine.
9 Mitigative measures
9.1 Worker safety
The vaccine will be manufactured at Merial, Inc. (Athens, Georgia), a veterinary biologics establishment licensed by the United States Department of Agriculture (USDA). Individuals working with the vaccine such as employees in the production facility, as well as veterinarians, animal technicians, and poultry operators, can be exposed to the live genetically modified organism. Since the recombinant vaccine virus is based on a naturally non-pathogenic virus backbone that is unable to replicate in mammalian cells, human exposure is not anticipated to be a human health concern. The use of the in ovo route of vaccine administration proposed for Prevexxion Rn should help reduce the incidence of accidental vaccinator self-injection compared to live bird vaccination. Moreover, since the vaccine does not contain any adjuvant, the risk of clinical problems due to accidental self-injection of oil adjuvant is removed.
9.2 Handling vaccinated or exposed animals
Since chicks reared in a biosecure facility are not often handled directly by humans, and poultry workers typically employ precautionary biosafety measures, exposure through handling vaccinated chicks is not expected to be great. However, poultry workers could become exposed to the vaccine virus through dust and air inside barns that might be contaminated with virus shed through feather dander. Again, the recombinant virus is not believed to be pathogenic to humans.
The vaccine licensing regulations in Canada require manufacturers to report all significant suspected adverse reactions to the CFIA within 15 days of receiving notice from an owner or a veterinarian. Veterinarians may also report suspected adverse reactions directly to the CFIA. If an adverse reaction complaint is received by the CCVB, the manufacturer is asked to investigate and prepare a report for the owner's veterinarian and the CFIA. If the problem is resolved to the satisfaction of the veterinarian or client, usually, no further action is requested by the CCVB. However, if the outcome of the investigation is unsatisfactory, the CCVB may initiate regulatory action, depending on the case, which may include further safety testing, temporary stoppage of product sales, or product withdrawal from the market.
No special monitoring of the human safety of the product will be carried out.
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.
Manufacturer: Merial, Inc.
1730 Olympic Drive
Athens, GA 30601
Importer: Merial Canada, Inc.
5180 South Service Road
Burlington, ON L7L 5H4
12 Conclusions and actions
Based on our assessment of the available information, the CCVB has concluded that the importation and use of Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera (USDA Code 16L1.R0; CCVB file number 800VV/M1.4/R2.1) in Canada would not be expected to have any significant adverse effect on the target species, non-target species, humans and 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, the Permit to Import Veterinary Biologics held by Merial Canada, Inc. may be amended to allow the importation and distribution of the following product in Canada:
Marek's Disease Vaccine, Serotype 1, Live Herpesvirus Chimera (Trade Name: Prevexxion Rn), CCVB File 800VV/M1.4/R2.1, USDA Product Code 16L1.R0.
All serials of this product must be released by the USDA prior to importation into Canada. All conditions described in the Permit to Import Veterinary Biologics must be followed with respect to the importation and sale of this product.
- Date modified: