NEW FOWL POX VACCINE EVALUATION Evaluation of fowl pox (FPV) strains free of reticuloendotheliosis virus as vaccines for use in Australian poultry flocks A report for the Rural Industries Research and Development Corporation by Dr. David B. Boyle September 2003 RIRDC Publication No 03/086 RIRDC Project No CSA-16A
2003 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 58653 5 ISSN 1440-6845 New Fowl Pox Vaccine Evaluation Evaluation of fowl pox (FPV) strains free of reticuloendotheliosis virus (REV) as vaccines for use in Australian poultry flocks Publication No. 03/086 Project No. CSA-16A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Dr. David Boyle CSIRO Livestock Industries Australian Animal Health Laboratory Private Bag 24 GEELONG VIC 3220 Phone: 03 52275018 Fax: 03 52275555 Email: david.boyle@csiro.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 Email: rirdc@rirdc.gov.au. Website: http://www.rirdc.gov.au Published in September 2003 Printed on environmentally friendly paper by Canprint ii
Foreword Ongoing problems with recurrent fowl pox disease have been experienced in some sectors of the meat chicken breeder industry over the past half decade. The only vaccine strain available in Australia (FPV M strain) has proven ineffective in controlling the problem on certain breeder farms. Although suitable for day old vaccination, this strain has not proven effective for revaccination of broiler breeder flocks coming into production. No other fowl pox virus (FPV) vaccine strains are available in Australia. Earlier studies have shown that the previously available FPV S vaccine is contaminated with reticuloendotheliosis virus REV), with the REV provirus integrated into the FPV genome. A small RIRDC-funded project was previously conducted by this CSIRO research team to remove the REV provirus from the FPV S vaccine and two field strains. The integrated REV genome was successfully removed from FPV S vaccine strain and two field strains, FPV 59vac and 62vac, in the course of that project (RIRDC Project CSA-8A Removal of reticuloendotheliosis (REV) contamination from fowl pox (FPV) S and FPV field strains for use as enhanced fowl pox vaccines in Australia ). Preliminary in vivo testing of these strains has shown them to be free of REV contamination. The project reported on herein was aimed at undertaking vaccine efficacy, safety and adventitious agent testing prior to the conduct of a field trial for the registration of one of these candidate vaccine strains for use in Australian poultry flocks. The benefits to the industry to accrue from this work will be the availability of a new FPV vaccine strain that is more immunogenic than the currently available vaccine strains. The new vaccine strain should be suitable to boost immunity to FPV in mature age birds and thus reduce the losses in broiler breeder flocks associated with fowl pox disease. This project was funded from industry revenue, which is matched, by funds provided by the Federal Government. This research project was funded from industry revenue, which is matched by funds provided by the Federal Government. This report is a new addition to RIRDC s diverse range of over 900 research publications and forms part of our Chicken Meat R&D program, which aims to support increased sustainability and profitability in the chicken meat industry by focusing research and development on those areas that will enable the industry to become more efficient and globally competitive and that will assist in the development of good industry and product images. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at www.rirdc.gov.au/reports/index.htm purchases at www.rirdc.gov.au/eshop Simon Hearn Managing Director Rural Industries Research and Development Corporation iii
Acknowledgements CSIRO wishes to acknowledge the technical support provided by various staff members during the conduct of this project, including Mrs. Mary Ann Anderson, Mr. Tony Pye, Ms. Maria Cardoso and Mr. Matthew Rudd. Abbreviations FPV = REV = NSW = mm = SPF = GMP = CIT = fowl pox virus reticuloendotheliosis virus New South Wales millimetres specific pathogen free good manufacturing practice chick inoculation test iv
Contents Foreword... iii Acknowledgements...iv Abbreviations...iv List of Figures...v Executive Summary...vi 1. Introduction...1 2. Objectives...1 3. Methodology...2 Strain Selection for GMP Manufacture... 2 Vaccination... 2 Vaccine take... 2 Vaccine efficacy... 2 REV freedom... 2 Safety, Potency and Chick Inoculation Testing (CIT)... 2 Vaccine production... 2 Safety, potency and CIT tests... 2 4. Detailed Results...3 Strain Selection for GMP Manufacture... 3 Vaccine take... 3 Vaccine efficacy... 4 REV Freedom... 5 Safety, Potency and Chick Inoculation Testing... 6 Vaccine production... 6 Safety, Potency and CIT tests... 6 5. Discussion...7 6. Implications...7 7. Recommendations...7 8. References...7 List of Figures Figure 1. Vaccine take as assessed by wing web thickness post vaccination... 3 Figure 2. Vaccine efficacy assessed by wing web thickness post challenge... 4 Figure 3. Antibodies to REV in vaccinated chickens at 21 days post vaccination... 5 v
Executive Summary This project aimed to complete the development and laboratory evaluation of a new FPV vaccine strain that is more immunogenic than the currently available vaccine strain and which should be suitable to boost immunity to FPV in mature age birds and thus reduce the losses in broiler breeder flocks associated with fowl pox disease. Vaccine efficacy, safety and adventitious agent testing was undertaken in the course of this project on reticuloendotheliosis free fowl pox strains derived from the fowl pox standard vaccine strain and two field strains. The reticuloendotheliosis free status of these strains was confirmed in this project. One strain was selected for manufacture by the commercialising partner in the project, Intervet Pty. Ltd., on the basis of vaccine take, freedom from reticuloendotheliosis virus and protection provided in laboratory based vaccination and challenge experiments. This strain meets the European Pharmacopoeia requirements for safety, potency and freedom from adventitious poultry pathogens. Intervet Pty. Ltd., in collaboration with industry, will conduct a field trial to determine suitability for registration of this strain for use by the Australian poultry industry. vi
1. Introduction The unique association of fowl pox virus with reticuloendothelosis virus which we have described (Hertig et.al., 1997) has not previously been documented. Our studies have shown that the FPV S strain vaccine, withdrawn from use in Australia because of suspected REV contamination, does not contain free REV but has REV provirus integrated into its genome. Unpublished studies show also that most if not all Australian fowl pox field strains similarly carry the REV provirus integrated into the FPV genome. Upon infection of chickens with the FPV S vaccine, infectious REV is produced in the chicken. The emergence of variant strains of fowl pox not protected against by existing vaccines has been documented in the United States of America (Fatunmbi and Reed, 1996). Our own studies on field isolates of fowl pox from New South Wales have documented a wide range of genotypes and phenotypes present in the field (Boyle et. al., 1996). We have no data on the efficacy of existing vaccines against these field isolates. Our previous studies on the use of fowl pox virus as a vector for vaccine antigens have established the necessary techniques for the specific deletion of the REV provirus genome from FPV S and field strains. Approximately 20 peer reviewed publications have been prepared by the author s research group in this area in the past decade. Using these techniques we now have derived strains of FPV free of REV provirus. Upon infection of chickens these strains do not lead to REV infection. One large broiler breeder operation, in particular, has experienced ongoing problems with recurrent fowl pox diseases over the past half decade. The only vaccine stain available in Australia has proven ineffective in controlling this problem. Although suitable for day old vaccination, this strain has not proven effective for revaccination of broiler breeder flocks coming into production. No other FPV vaccine strains are available in Australia. Our studies have shown that the previously available FPV S vaccine is contaminated with REV, with the REV provirus being integrated into the FPV genome. RIRDC previously funded the author s research team to conduct a small project to remove the REV provirus from the FPV S vaccine and two field strains. The integrated REV genome was successfully removed from FPV S vaccine and two field strains, FPV 59vac and 62vac, in the course of this earlier project. Preliminary in vivo testing of these strains has shown them to be free of REV contamination (RIRDC Project CSA-8A Removal of reticuloendotheliosis (REV) contamination from fowl pox (FPV) S and FPV field strains for use as enhanced fowl pox vaccines in Australia). The current project was aimed at undertaking vaccine efficacy, safety and adventitious agent testing prior to the conduct of a field trial for the registration of one of these strains for use in Australian poultry flocks. 2. Objectives The project objectives were to undertake vaccine efficacy, safety and adventitious agent testing on reticuloendotheliosis (REV) free fowl pox virus (FPV) strains derived from FPV S (Standard vaccine strain) and two field strains, FPV 59vac and FPV 62vac.These studies were conducted in collaboration with a commercial partner (Intervet Pty. Ltd.) to ensure that the results would be suitable for registration purposes and manufacture of selected strain(s) for delivery to the Australian poultry industry as new FPV vaccine(s). 1
3. Methodology Strain Selection for GMP Manufacture Vaccination Three REV free FPV strains derived from FPV S (FPV 79b), FPV 59vac (FPV 81b) and FPV 62vac (FPV 85a) strains were evaluated in vaccination and challenge experiments in three-week-old specific pathogen free poultry (SPF). Chickens (five to six per group) were vaccinated by wing web inoculation using industry standard bifurcated needle fowl pox vaccine applicators. Vaccination with the FPV Mild vaccine strain (Fort Dodge) (FPV M) was used for comparison with the strains for assessment of vaccine take and protective efficacy. Vaccine take Vaccine take was monitored by measuring, every two to three days, wing web thickness at the inoculation site using callipers. Vaccine efficacy The efficacy of the vaccine was determined by challenging the chickens with the NSW field isolate FPV 62vac by wing web inoculation in the wing opposite to that used for vaccination. Wing web thickness was monitored for ten to 14 days post challenge. The chickens were also examined for spread of the challenge virus from the primary challenge site. REV freedom Freedom from REV contamination was determined in the vaccination and challenge experiment. Serum samples collected at 21 days post vaccination were tested for antibodies to REV using a commercial ELISA antibody test. Safety, Potency and Chick Inoculation Testing (CIT) Vaccine production Intervet Pty. Ltd. manufactured the chosen strain under GMP conditions at their Bendigo plant. A master seed, working seed and three pilot batches of vaccine were prepared using their standard FPV vaccine production methods. FPV titre in the seeds and pilot vaccine batches was determined at CSIRO by plaque assay on chicken embryo skin cells. Safety, potency and CIT tests Safety, potency and chick inoculation test (CIT) tests were conducted as per European Pharmacopoeia, Fourth Edition September 2001 on master and working seeds and pilot vaccine lots of the fowl pox vaccine. The only significant protocol modification was that potency test chickens were challenged by wing stab inoculation rather than by feather follicle inoculation. Wing web stab inoculation allowed the measurement of responses by monitoring wing web thickness following vaccination and challenge. The safety test was conducted with the pilot batch with the highest titre. The CIT test was conducted on a pool of all five batches (two seeds and three pilots). The potency test was conduced on the pilot batch with the lowest titre of virus. 2
4. Detailed Results Strain Selection for GMP Manufacture Vaccine take Vaccine take as assessed by wing web thickness measurements showed that FPV 79b and FPV 85a gave a larger and more prolonged local lesion in comparison with the FPV M Vaccine and FPV 81b (Figure 1). For FPV M and FPV 81b the wing web lesion peaked by five to seven days post vaccination and was substantially resolved by ten days post vaccination. In comparison for FPV 79b and FPV 85a maximum lesion thickness occurred at seven days post vaccination and the lesion was not fully resolved until 13 to17 days post vaccination. None of the vaccinated chickens developed FPV lesions at sites other than the vaccination site. Mean Wing Web Thickness Post Vaccination 7 6 5 mm +/- SD 4 3 2 Unvacc FPV M FPV 79b FPV 81b FPV 85a 1 0 0 3 5 7 10 13 17 Days Post Vaccination Figure 1. Vaccine take as assessed by wing web thickness post vaccination. 3
Vaccine efficacy Vaccine efficacy was assessed by challenge with a virulent NSW field strain, FPV 62vac. Wing web thickness was measured for ten days following challenge (Figure 2) and visual assessment of challenge virus take was also undertaken. All chickens vaccinated with FPV 85a were fully protected no change occurred in wing web thickness and no visible lesion developed at the challenge inoculation site. For FPV M, FPV 79b and FPV 81b, a visible lesion developed at the challenge inoculation site for three or four of the five chickens challenged. This was accompanied by a small increase in wing web thickness measured at days three to seven post challenge. Mean Wing Web Thickness Post Challenge 6 5 mm +/- SD 4 3 2 Unvacc FPV M FPV 79b FPV 81b FPV 85a 1 0 0 3 5 7 10 Days Post Challenge Figure 2. Vaccine efficacy assessed by wing web thickness post challenge. 4
REV Freedom Freedom from REV contamination of the FPV vaccine strains was confirmed, since none of the chickens vaccinated developed antibodies to REV by 21 days post vaccination. In comparison, all chickens vaccinated at the same time with the parent strains from which the vaccine strains were derived developed antibodies to REV (Figure 3). REV ELISA Antibody Response Post Vaccination 12000 10000 Mean Antibody Titre 8000 6000 4000 2000 Day 21 Post Vaccination 0 Unvacc FPV M FPV 79b FPV 81b FPV 85a FPV S FPV 59vac FPV 62vac Virus Strain Figure 3. Antibodies to REV in vaccinated chickens at 21 days post vaccination. For comparison the parent strains used for generation of the REV free FPV strains were also tested (FPV S, 59vac and 62vac). Strain FPV 85a was selected for further vaccine development on the basis of freedom from REV contamination, effective vaccine take in all chickens, satisfactory growth in cell culture and provision of the best protection when challenged with the field strain from which this vaccine was derived. 5
Safety, Potency and Chick Inoculation Testing Vaccine production The FPV 85a strain derived from NSW field isolate FPV 62vac was provided to Intervet Pty. Ltd. on 31/7/2001 for GMP manufacture using their standard FPV vaccine production protocols. CSIRO Microstores identification of the virus provided to Intervet is 9910-05-2000. A full passage history from the time of receipt of FPV 62vac by CSIRO AAHL (21/10/1997), including the source of biological materials used during the derivation of FPV 85a, was provided to Intervet. Intervet prepared master and working seeds and three pilot vaccine lots under GMP conditions at their Bendigo plant. Samples of these seeds and pilot vaccine lots were provided to CSIRO for titration and for safety, potency and CIT testing. Safety, Potency and CIT tests Safety, potency and CIT tests were conducted at CSIRO AAHL in three to four week old SPF chickens as per European Pharmacopoeia, Fourth Edition September 2001. The only significant protocol modification was that potency test chickens were challenged by wing stab inoculation rather than by feather follicle inoculation. Wing web stab inoculation allowed the measurement of responses by monitoring wing web thickness following vaccination and challenge. Safety Test. 11/11 chickens inoculated at three to four weeks of age via wing web stab inoculation with 10 x the recommended vaccine dose of a pilot vaccine batch survived for 21 days. None of the chickens showed any signs of disease other than the localised fowl pox lesion at the primary inoculation site. Potency Test. 24/24 chickens inoculated at three to four weeks of age via wing web stab inoculation with the recommended vaccine dose of a pilot vaccine batch developed a localised fowl pox lesion at the primary inoculation site. 24/24 non-vaccinate controls showed no signs of infection. 24/24 vaccinated chickens challenged by wing web stab inoculation at 21 days post vaccination with a field strain of FPV (FPV 62vac the strain from which the vaccine was developed) were completely protected from the development of a fowl pox lesion at the inoculation site. 24/24 non-vaccinated controls similarly challenged developed a fowl pox lesion at the challenge inoculation site. CIT Test. 12 chickens were inoculated and re-inoculated with a pool of two seed stocks and three pilot vaccine lots. All chickens survived for six weeks post primary vaccination. Serum samples collected three weeks post the secondary inoculation were negative for antibodies to all poultry pathogens tested. The FPV vaccine strain based on FPV 85a (a REV free derivative of field strain FPV 62vac) was therefore shown to meet the European Pharmacopoeia, Fourth Edition September 2001 requirements for safety, potency and freedom from adventitious poultry pathogens (CIT test). 6
5. Discussion We have been able to identify a suitable Australian FPV candidate vaccine strain from which the REV provirus has been removed. This strain provides good protection in vaccination and challenge experiments in SPF chickens. Freedom from REV contamination has been confirmed. Master and working seeds for vaccine production have been prepared by Intervet Pty. Ltd. Three pilot vaccine batches were prepared under GMP manufacturing conditions. Safety, potency and CIT tests conducted on the master and working seeds and the pilot vaccine batches confirm that the FPV vaccine based on FPV 85a (a REV free derivative of field strain FPV 62vac) meets the European Pharmacopoeia, Fourth Edition September 2001 requirements for safety, potency and freedom from adventitious poultry pathogens (CIT test). 6. Implications The FPV candidate vaccine strain that has been developed will need to be evaluated in field trials to be conducted by Intervet Pty. Ltd. to ascertain if it is suitable for use by the Australian poultry industry for the control of fowl pox disease. Subject to the suitability of the candidate vaccine for use in control of fowl pox in Australia being demonstrated in these field trials and the vaccine subsequently being registered for use for this purpose, then the Australian poultry industries will have available an alternative, more immunogenic vaccine for the control of fowl pox in the future. 7. Recommendations That Intervet Pty. Ltd. Conduct, with the collaboration of the industry, suitable field trials to ascertain the suitability of the developed strain for use by the Australian poultry industry for the control of fowl pox disease and for national registration. 8. References Boyle, D.B. RIRDC Project CSA-8A Removal of reticuloendotheliosis (REV) contamination from fowlpox (FPV) S and FPV field strains for use as enhanced fowlpox vaccines in Australia. Final Report August 2000. Boyle, D.B., Pye, A.D. and Coupar, B.E.H. (1996) Comparison of field and vaccine strains of Australian fowlpox viruses. Archives of Virology 142 : 737-748 Fatunmbi, O.O. and Reed, V.M. (1996) Evaluation of a commercial modified live virus fowl pox vaccine for the control of variant fowlpox virus infections. Avian Diseases 40: 582-587. Hertig, C.H., Coupar, B.E.H., Gould, A.R. and Boyle, D.B. (1997) Field and vaccine strains of fowlpox virus carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus. Virology 1997 Sep 1;235(2):367-376 7