Ministry of Agriculture & Rural Development Milestone 7 Classical Swine Fever (CSF): Development of a new classical swine fever vaccine Research Reports / Technical Notes Chris Morrissy 1 Table of Contents 1. Institute Information _____________________________________________2 2. Project Abstract _________________________________________________4 3. Executive Summary______________________________________________4 4. Progress to Date ________________________________________________6 4.1 Technical reports on the development of an improved CSF vaccine __ 6 5. Conclusions ____________________________________________________8 2 1. Institute Information Project Name: Classical Swine Fever (CSF): Development of a new classical swine fever vaccine [Project Code: 014/07 VIE] Vietnamese Institution: Veterinary Research Centre (subordinated to the National Veterinary Company – NAVETCO) Vietnamese Project Team Leader: Dr Tran Xuan Hanh Australian Organisation: Australian Animal Health Laboratory (AAHL), PMB 24, Geelong, VIC 3220, Australia Australian Personnel: Mr Chris Morrissy Date commenced: 01/03/2008 Completion date (original): 01/03/2010 Completion date (revised): Reporting period: Contact Officer(s) In Australia: Team Leader Name: Mr Chris Morrissy Telephone: +61 3 5227 5000 Position: Diagnostic Virologist, Supervisor Mammalian Virology, AAHL Fax: +61 3 5227 5555 Organisation: AAHL, PMB 24, Geelong, VIC 3220, Australia Email: chris.morrissy@csiro.au In Australia: Administrative Contact Name: Mr Christopher Morrissy Telephone: +61 3 5227 5000 Position: Patents Contracts Officer Fax: +61 3 5227 5555 Organisation: AAHL, PMB 24, Geelong, VIC 3220, Australia Email: christopher.morrissy@csiro.au 3 In Vietnam: Name: Dr Tran Xuan Hanh Telephone: +84 8 8225955 Position: Deputy Director of National Veterinary Company [NAVETCO] Fax: +84 8 8225060 Organisation: NAVETCO 29 Nguyen Dinh Chieu street, Dist.1. Ho Chi Minh City, Vietnam Email: tranxuananh2002@yahoo.com 4 2. Project Abstract Vaccinating pigs to prevent CSF is a common measure among swine producers in Vietnam, but nevertheless, CSF still causes significant losses to both commercial and smallholder agriculture. Current locally produced Vietnamese CSF vaccines are reliant on vaccine production in experimentally infected animals leading to inherent variation in routine vaccine production capacity and related quality control considerations in Vietnam. This project focuses on the development of an improved cell culture propagated CSF vaccine with the aim to greatly enhance both the supply and quality assurance of locally produced vaccines. In addition, for greatest economic and societal impact, it will also be necessary to tailor vaccination protocols to Vietnamese-specific field conditions thereby facilitating the control of CSF in Vietnam. The major objectives of this project are: 1. To develop an improved cell culture propagated vaccine CSF vaccine to allow the production of a cheap and high quality vaccine. 2. To enhance the diagnostic capability of Vietnamese laboratories to facilitate the control of CSF. 3. To contribute to the education and training of veterinarians and small farm holders on how to use the improved CSF vaccine effectively. Successful realisation these project objectives will improve the capacity of Vietnam to control CSF with resultant benefits to the pig industry and to smallholder agricultural producers in particular. 3. Executive Summary The attenuated Chinese “c-strain” of CSF is used for the control and prevention of CSF worldwide. In Vietnam, current CSF vaccines are based on the Chinese “c-strain” of CSF propagated in experimental animals [rabbits and calves], which can be in short supply or in poor health. This leads to inherent variation in vaccine quality, limited production capacity and is compounded by the logistics associated with conventional vaccine virus production. The successful implementation of a cell culture-based approach would obviate current requirements for the in vivo titration of vaccine virus and related quality assurance considerations with regard to freedom from adventitious agents. In this Milestone Report we document achievement of central project objectives in relation to the successful adaptation, growth and characterisation of a c-strain CSF vaccine in a quality assured cell culture system. Specifically, the attenuated c-strain of CSF currently used for vaccine production in experimental animals was successfully adapted to growth following serial passage in the continuous porcine kidney cell line, PK-15A. 5 The successful adaptation of the vaccine virus to cell culture was associated with a concomitant increase in the level of vaccine virus production ranging from 10 2.23 TCID 50 /ml at passage 1, rising to a plateau of around 10 6.33 TCID 50 /ml at passage 12. The optimal conditions for improved vaccine virus production in cell culture was determined to be at a multiplicity of infection of 0.02 with virus pools harvested at between 72 and 96 hrs post-infection having average titres of approximately 10 6.0 TCID 50 /ml. Following adaptation of the virus to PK-15A virus titres remained stable at approximately 10 6.0 TCID 50 /ml and successful adaptation to PK-15A was associated with genotypic changes between the cell culture adapted and the parental in vivo propagated vaccine virus. Specifically, successful adaptation to PK-15A was associated with a decrease in E2 gene nucleotide sequence identity to 99.5% as compared to the parental vaccine virus. As observed following quantitative virus titre measurement, this genetic change was stable from passage 9 over the course of subsequent serial passage in PK-15A. The effect of genetic changes following successful adaptation to PK-15A was further investigated in relation to possible changes in the parental virus with regard to vaccine safety and efficacy. A direct comparison of the virulence of the parental in vivo propagated and improved cell culture adapted vaccine in experimentally infected rabbits revealed no differences in virulence as determined following the monitoring of clinical signs, viremia and the elicitation of an immune response. In addition, trials conducted by NAVETO in relation to vaccine potency demonstrated solid protection in pigs vaccinated with the improved cell culture adapted CSF vaccine with no evidence of the replication or shedding of virulent virus following challenge. Furthermore, 100% of vaccinated pigs elicited an antibody response that persisted for at least 24 weeks. NAVETCO have successfully adapted the conventional CSF c-strain vaccine virus to PK-15A cell culture and have produced seed lots of the improved vaccine for subsequent scale-up production. The improved PK-15A cell culture propagated c-strain CSF vaccine exhibited excellent growth characteristics, was stable following adaptation to cell culture and was shown in animal trials to be both safe and highly efficacious. The future availability of this improved CSF vaccine in the field will greatly facilitate the control of CSF in Vietnam. 6 4. Progress to Date [in relation to Milestone 7 Deliverables] 4.1 Technical reports on the development of an improved CSF vaccine • Cell culture techniques for CSF c-strain adaptation Candidate cell lines, comprising the continuous cell cultures: Pig Kidney [PK- 15A], Swine Testes [ST] and Lamb Testes [LT]) were assayed in relation to their potential utility for the in vitro propagation of the CSF c-strain vaccine. These cell cultures had been subject to quality control testing at AAHL and were shown to be free of possible adventitious agents such as BVDV and PCV. Preliminary testing at AAHL demonstrated that the continuous cell line PK-15A was more permissive to infection with CSF virus and accordingly this cell line was selected for future use. The quality assured PK-15A cell line was transferred from AAHL to NAVETCO and a cell bank of PK-15A established to facilitate subsequent studies in relation to vaccine production in vitro. Consequently, all resultant data in relation to the adaptation and growth characteristics of the c-strain CSF vaccine in vitro refers to the passage number post-transfer of the PK-15A cell line and establishment of the cell bank at NAVETCO. In addition, this cell line and all subsequent seed lots of virus pools generated during the adaptation of the c-strain CSF vaccine to PK-15A cell culture were subject to quality assurance by NAVETCO under their accredited “in-house” quality assurance system. This was facilitated by earlier training activities undertaken at AAHL and enabled following subsequent successful capacity development in the field of cell culture and enhanced CSF diagnostic capabilities at NAVETCO. • Adaptation of c-strain CSF virus on a range of cell lines The c-strain CSF virus currently used for vaccine production in experimental animals at NAVETCO was used as the parental virus for subsequent adaptation and growth in cell culture. As related earlier, preliminary research at AAHL had demonstrated that PK-15A was more permissive to CSF virus infection than either the ST or LT cell lines. Accordingly, subsequent efforts at NAVETCO focused on the adaptation and growth characteristics of the c- strain CSF virus in PK-15A continuous cell culture. The successful adaptation of the c-strain of CSF virus following serial passage in PK-15A cell culture was readily apparent following virus-specific labelling as shown in Attachment 1. Of particular significance, the successful adaptation following serial passage in PK-15 at NAVETCO was accompanied by a marked and progressive increase in infectious virus production as determined following virus titration. Specifically, ranging from 10 2.23 TCID 50 /ml following 1 passage to a plateau of around 10 6.33 TCID 50 /ml at passage 12 as shown in Attachment 2. 7 • Characterisation of c-strain CSF vaccine virus Following successful adaptation and subsequent highly productive propagation in PK-15A, the growth characteristics, genotype and biological properties of the adapted c-strain CSF vaccine virus were further investigated. The results of a NAVETCO study to investigate the optimum multiplicity of infection [moi] to achieve highest yields are given in Attachment 3 and demonstrate that an moi of 0.02 and harvesting time between 72-96 hrs post- infection was optimal for vaccine production. Resultant virus pools with a titre of approximately 10 6.0 TCID 50 /ml were used for all subsequent work. The genotype of the adapted c-strain CSF vaccine was also monitored during the course of serial passage in PK-15A at NAVETCO. Using the parental reference virus propagated in rabbits as a control, results [detailed in Attachment 4] demonstrated initial genetic change in the E2 gene [99.8-99.6% nucleotide sequence identity] following successful adaptation to PK-15A. The observed adaptation was associated with a concomitant increase in infectious virus titre [as detailed earlier in Attachment 2] and the adapted virus exhibited subsequent genetic stability after passage 9 [99.5% nucleotide sequence identity]. Central to the utility of the PK-15A adapted c-strain as a vaccine is proof with regard to both its safety and also its efficacy as determined by challenge with virulent CSF virus post-vaccination. Data obtained as part of a NAVETCO study detailed the absence of virus shedding in pigs inoculated with the improved cell culture propagated c-strain CSF vaccine. In addition, the improved cell culture propagated c-strain CSF vaccine exhibited identical virulence of following direct comparison with the conventional experimental animal propagated vaccine in inoculated rabbits [Attachment 5]. The potency and antibody response elicited by the improved cell culture adapted c-strain CSF vaccine was further investigated. The results of trials conducted by NAVETO in relation to vaccine potency as determined by measurement of clinical signs, viremia, virus shedding and quantification of the antibody response of pigs challenged 21 days post-vaccination are shown in Attachment 6. Of particular significance, solid protection following challenge with reference virulent CSF virus was observed in vaccinated pigs with no evidence of replication or shedding of virulent virus following challenge. In addition, 100% of vaccinated pigs elicited an antibody response with immunity observed initially at day 2 post-vaccination and subsequently increased significantly and persisted throughout the course of the 24-week timeframe of the experiment [Attachment 6]. 8 Taken together, this data represents proof of the successful adaptation and productive growth of the c-strain CSF vaccine in PK-15A cell culture. Of particular significance, safety and efficacy profiling has confirmed the utility of the improved CSF vaccine. • Laboratory protocols An earlier report has detailed the successful and sustainable capacity development in relation to the establishment of cell culture techniques and required quality assurance compliance at NAVETCO. During the course of this project enhancement of CSF technical competencies at NAVETCO were attained and applied in conjunction with pre-existing CSF vaccine efficacy testing capabilities under their “in-house” quality assurance system to facilitate the testing of the improved c-strain adapted CSF vaccine. Specifically, pre-existing quality assured protocols for vaccine efficacy and safety determination were used. For example, in relation to efficacy and safety investigations of the improved vaccine, the challenge virus used was the national CSF virus isolate used in the quality control of the conventional experimental animal propagated vaccine currently produced by NAVETCO. In experimental studies at NAVETCO, 3 week-old pigs from vaccinated sows were used. Piglets were housed in the NAVETCO animal facility until maternal antibody levels dropped and were both seronegative and negative for CSF antigen prior to experimental usage at 8-10 weeks of age. All vaccine virulence, efficacy and safety assays were conducted at NAVETCO and performed in accordance with “in-house” quality assured standard operating procedures. The successful implementation of additional capacity development in relation to the propagation of the improved c-strain adapted CSF vaccine in cell culture under the “in-house” quality assurance system at NAVETCO is given in Attachment 7. This document is the title page of the improved cell culture vaccine production SOP that is currently implemented at NAVETCO. If required, addition details in relation to available SOPs and quality assurance documentation in support of the fulfilment of specific project milestones and deliverables can be obtained from NAVETCO. 9 5. Conclusions Earlier successful capacity developments in the field of virus propagation by cell culture and the enhancement of CSF diagnostic capabilities at NAVETCO have facilitated the achievement of project milestones. Specifically, NAVETCO have successfully adapted the c-strain CSF vaccine virus to growth in PK-15A cell culture under quality assured conditions and the resultant candidate improved vaccine has been further characterised both in vitro and in vivo. Vaccine production in cell culture achieved levels in the order of approximately 10 6.0 TCID 50 /ml and quality assured seed lots of virus pools were subject to further characterisation. Genotyping of the improved vaccine highlighted that a small number of nucleotide sequence differences had arisen during the course of adaptation and serial passage in PK-15A. However, direct virulence comparison of the improved vaccine with the parental vaccine virus in vivo in rabbits clearly demonstrated no differences in virulence. In addition, experimental trials conducted by NAVETO in relation to vaccine potency demonstrated solid protection in pigs vaccinated with the improved cell culture adapted CSF vaccine with no evidence of the replication or shedding of virulent virus following challenge. Furthermore, 100% of vaccinated pigs elicited an antibody response. The improved cell culture adapted vaccine has been shown to be both safe and highly efficacious. Quality assured seed lots of the improved vaccine have been established with an aim to perform additional safety, efficacy and field trials of the improved vaccine in due course. . Centre (subordinated to the National Veterinary Company – NAVETCO) Vietnamese Project Team Leader: Dr Tran Xuan Hanh Australian Organisation: Australian Animal Health Laboratory (AAHL), PMB 24,. Ministry of Agriculture & Rural Development Milestone 7 Classical Swine Fever (CSF): Development of a new classical swine fever vaccine Research Reports / Technical Notes. all resultant data in relation to the adaptation and growth characteristics of the c-strain CSF vaccine in vitro refers to the passage number post-transfer of the PK-1 5A cell line and establishment