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Titration method for an ointment type smallpox vaccine in embryonated chicken eggs
Journal of Biological Standardization 1974 2, 65-68
Titration method for an ointment type smallpox vaccine in embryonated chicken eggs*
B. Mettier,t Ingrid Haenzelt and M. Majer$
The titration in embryonated eggs of a new ointment type smallpox vaccine is described. The vaccine consists of silicone-incorporated freeze-dried vaccinia virus. The vaccine is treated with low-viscosity silicone fluid in order to facilitate the subsequent extraction of the virus with a buffered saline solution. The extracted virus is titrated in chorioallantoie membranes of chick embryos.
INTRODUCTION An ointment type smallpox vaccine was developed by dispersing freeze-dried vaccinia virus in high-viscosity silicone oil (Mettier & Majer, 1972). T h e silicone-incorporated virus has been shown to be well protected from atmospheric influences, thus maintaining its stability. The ointment type vaccine can be used with all vaccination techniques based on skin scarification without prior rehydration. I n a clinical trial satisfactory take rates were achieved in primary and revaccinations (Stransky, 1973). Potency and stability of the ointment type vaccine can be determined in the rabbit skin (W.H.O., 1959) using vaccine dilutions prepared with low-viscosity silicone fluid. These vaccine dilutions, however, cannot be assayed in embryonated eggs because the silicone fluid used is harmful to the chorioallantoic membranes. Because the egg titration method is more sensitive than the rabbit skin test (Krag & Weis Bentzon, 1963) we developed a procedure which allows for the titration in embryonated eggs of the vaccinia virus in the ointment type vaccine. * Received for publication 9 April 1973. t Swiss Serum and Vaccine Institute Berne, Berne, Switzerland. Present address : Behringwerke Abr, Marburg, Lahn, Germany. 5
65
B. M E T T I E R , I. H A E N Z E L AND M. MAJER MATERIALS
AND METHODS
Freeze-dried smallpox vacdne 'Lancy-Vaxina' was used. This vaccine contains the Lister strain of vaccinia virus and was prepared in our Institute in the skin of sheep according to standard techniques (W.H.O., 1968). The vaccine was stored at - 2 0 °C.
Ointment type smallpox vaccine 'Unguantigen Berna' (Lots Nos SD5 and SD6a) was prepared by the method of Mettier & Majer (1972). Amounts of lg of the vaccine were stored at - 2 0 °C.
Titration method The freeze-dried vaccine was reconstituted and diluted with 0.004 V-McIlvaine's buffer (pH 7.2) using amounts of 0.5 ml virus suspension and 4-5 ml diluent for each log10 dilution step. Intermediate dilutions were prepared when deemed necessary. The viscosity of the ointment type vaccine was decreased by adding a low-viscosity silicone fluid: to 0.5 g of the vaccine 4.5 ml dimethylpolysiloxane DC 200 0.65 centistokes (Dow Corning Corporation, Midland, Michigan) were added to a test tube (15 x 100 ram). The mixture was shaken until homogenized. Thereafter 5"0 ml of McIlvaine's buffer were added and the mixture was vigorously shaken by hand for five minutes. The silicone-in-water emulsion was then centrifuged for ten minutes at 600 g. The lower aqueous phase containing the extracted 10-fold filnted virus was collected with a Pasteur pipette. For the pock count assay the identical dilution procedure to that used for the assay of the freeze-dried vaccine was applied.
Pock count assay Pock counts were estimated by the conventional method (W.H.O. 1966). The dropped chorioallantoic membranes (CAM) of each of four to five embryonated chicken eggs per group were inoculated with a 0.1-ml amount of the diluted virus suspension. The pocks were counted after a 48-h incubation period at 37 °C. For calculation of vaccine titres virus dilutions were used which yielded approximately 25 pocks per membrane.
Stability test The stability test was performed according to the World Health Organization Requirements for Smallpox Vaccine (W.H.O., 1966).
RESULTS
The accuracy of the titration method for the ointment type vaccine was examined by the analysis of day-to-day variations. The ointment type vaccine Lot No. SD5 and our freeze-dried laboratory standard vaccine were tested simultaneously as stated under 'Materials and Methods' twice a week for six weeks. The average log10 titres, the range of titre values and the standard deviations were calculated. The average titre of the ointment type vaccine was 8.44. Based on the range of log10 titre values (0.26 and 0"19) and the standard deviations (0-086 and 0-055) for the freeze-dried and for the ointment type vaccine, respectively, it becomes evident that the results of the testing method for 66
TITRATION
OF O I N T M E N T
SMALLPOX VACCINE
the ointment type vaccine were at least as reproducible as the results of the standard egg titration method for the freeze-dried vaccine. The results are summarized in Table 1. TABLE 1. Evaluation of the pock counts in eggs
Number of tests Range of the average pock count/membrane Average log 10 p.f.u./ml Range of titre values Standard deviation
Freeze-dried vaccine*
Ointment vaccine Lot No. SD5
i2
12
21-38"~
21-32:~
8"74 0"26 0.086
8"44 0"19 0"055
* Laboratory standard vaccine. t At vaccine dilution I0 -s'a. At vaccine dilution 10J ' ° . The ointment type vaccine Lot No. SD6a and the freeze-dried virus suspension used for its preparation were tested for stability. Log~0 titre values of samples incubated for 7, 14 and 28 days at 37 °C were compared with the titres of control samples stored at - 2 0 °C. Differences between titres of samples stored at - 2 0 °C and of those incubated at 37 °C for 28 days were 0.25 and 0.20 for the ointment type vaccine and the freezedried vaccine, respectively, indicating good stability of both preparations. It is interesting to note (see Table 2) that the extraction process used for recovering the vaccinia virus from the ointment was efficient in that almost all the virus was recovered. TABLE 2. Stability test Days 7 14 28
Freeze-dried vaccine - 20 °C 37 °C 8-80 8-83 8-79
8"76 8-79 8"59
Ointment type vaccine -20 °C 37 °C 8"66 8"67 8"73
8"48 8"54 8"48
Results of the stability test in logt0 titre values of the ointment vaccine (Lot No. SD6a) and the freeze-dried vaccinia virus suspension used for the preparation of the ointment vaccine.
DISCUSSION Freeze-dried smallpox vaccines on account of their stability are preferentially used. However, a freeze-dried vaccine has the disadvantage that it must be rehydrated for use, which by many physicians is considered a tedious procedure particularly when the vaccine is used in single doses. Furthermore, after reconstitution the vaccine may deteriorate rapidly. To overcome these disadvantages we have developed an ointment type smallpox vaccine by dispersing freeze-dried vaccinia virus in high-viscosity silicone oil (Mettier & Majer, 1972). It could be shown that this vaccine has the stability characteristics of the 5*
67
B. M E T T I E R , I. H A E N Z E L AND M. MAJER freeze-dried vaccine. Whereas the potency of the ointment type vaccine was shown in clinical trials (Stransky, 1973), its laboratory testing was hampered by the water-repellent properties of silicones. Although the vaccine can be diluted with low-viscosity silicone fluid for testing in the skin of rabbits, it cannot be assayed in embryonated eggs because the silicone fluid used is harmful to the chorioallantoic membrane. The presently described method which consists of first lowering the viscosity of the ointment type vaccine and second the extraction of the virus with a buffered saline solution now also permits its testing for potency in embryonated eggs. The extracted virus can be titrated in the standard pock count assay. With this method approximately threequarters of the calculated number of pock forming units were recovered from the silicone ointment vaccine. The reproducibility of the titre values for the ointment type vaccine was comparable to that for the freeze-dried vaccine. The results of the CAM titration assay confirmed the previous results obtained with the rabbit skin test as well as those obtained in the vaccination studies. The described extraction method permits the accurate assay of the ointment type vaccine with regard to stability. Both the ointment type vaccine and the freeze-dried vaccinia virus suspension used for its preparation do fulfil the World Health Organization Requirements for Smallpox Vaccine (W.H.O., 1966).
Acknowledgement
We wish to thank Mrs Marilyn Rutz for her valuable technical assistance.
REFERENCES Krag, P. & Weis Bentzon, M. (1963). The international reference preparation of smallpox vaccine. An international collaborative assay. Bulletin of the Worm Health Organization 29, 299-309. Mettier, B. & Majer, M. (1972). A new stable smallpox vaccine. Smallpox Vaccine Conference, Bilthoven 11-13 October. Stransky, M. (1973). Salbenimpfstoff fiir pockenimpfungen. Schweiz. Medizinische Wochenschrift 103, 20-21. World Health Organization (1959). Requirements for biological substances. World Health Organization No. 180. World Health Organization (1966). Requirements for biological substances. World Health Organization Technical Report Series No. 323. World Health Organization (1968). Methodology of freeze-dried smallpox vaccine production. World Health Organization SE/68.3 Rev. 1.
68
Titration method for an ointment type smallpox vaccine in embryonated chicken eggs*
B. Mettier,t Ingrid Haenzelt and M. Majer$
The titration in embryonated eggs of a new ointment type smallpox vaccine is described. The vaccine consists of silicone-incorporated freeze-dried vaccinia virus. The vaccine is treated with low-viscosity silicone fluid in order to facilitate the subsequent extraction of the virus with a buffered saline solution. The extracted virus is titrated in chorioallantoie membranes of chick embryos.
INTRODUCTION An ointment type smallpox vaccine was developed by dispersing freeze-dried vaccinia virus in high-viscosity silicone oil (Mettier & Majer, 1972). T h e silicone-incorporated virus has been shown to be well protected from atmospheric influences, thus maintaining its stability. The ointment type vaccine can be used with all vaccination techniques based on skin scarification without prior rehydration. I n a clinical trial satisfactory take rates were achieved in primary and revaccinations (Stransky, 1973). Potency and stability of the ointment type vaccine can be determined in the rabbit skin (W.H.O., 1959) using vaccine dilutions prepared with low-viscosity silicone fluid. These vaccine dilutions, however, cannot be assayed in embryonated eggs because the silicone fluid used is harmful to the chorioallantoic membranes. Because the egg titration method is more sensitive than the rabbit skin test (Krag & Weis Bentzon, 1963) we developed a procedure which allows for the titration in embryonated eggs of the vaccinia virus in the ointment type vaccine. * Received for publication 9 April 1973. t Swiss Serum and Vaccine Institute Berne, Berne, Switzerland. Present address : Behringwerke Abr, Marburg, Lahn, Germany. 5
65
B. M E T T I E R , I. H A E N Z E L AND M. MAJER MATERIALS
AND METHODS
Freeze-dried smallpox vacdne 'Lancy-Vaxina' was used. This vaccine contains the Lister strain of vaccinia virus and was prepared in our Institute in the skin of sheep according to standard techniques (W.H.O., 1968). The vaccine was stored at - 2 0 °C.
Ointment type smallpox vaccine 'Unguantigen Berna' (Lots Nos SD5 and SD6a) was prepared by the method of Mettier & Majer (1972). Amounts of lg of the vaccine were stored at - 2 0 °C.
Titration method The freeze-dried vaccine was reconstituted and diluted with 0.004 V-McIlvaine's buffer (pH 7.2) using amounts of 0.5 ml virus suspension and 4-5 ml diluent for each log10 dilution step. Intermediate dilutions were prepared when deemed necessary. The viscosity of the ointment type vaccine was decreased by adding a low-viscosity silicone fluid: to 0.5 g of the vaccine 4.5 ml dimethylpolysiloxane DC 200 0.65 centistokes (Dow Corning Corporation, Midland, Michigan) were added to a test tube (15 x 100 ram). The mixture was shaken until homogenized. Thereafter 5"0 ml of McIlvaine's buffer were added and the mixture was vigorously shaken by hand for five minutes. The silicone-in-water emulsion was then centrifuged for ten minutes at 600 g. The lower aqueous phase containing the extracted 10-fold filnted virus was collected with a Pasteur pipette. For the pock count assay the identical dilution procedure to that used for the assay of the freeze-dried vaccine was applied.
Pock count assay Pock counts were estimated by the conventional method (W.H.O. 1966). The dropped chorioallantoic membranes (CAM) of each of four to five embryonated chicken eggs per group were inoculated with a 0.1-ml amount of the diluted virus suspension. The pocks were counted after a 48-h incubation period at 37 °C. For calculation of vaccine titres virus dilutions were used which yielded approximately 25 pocks per membrane.
Stability test The stability test was performed according to the World Health Organization Requirements for Smallpox Vaccine (W.H.O., 1966).
RESULTS
The accuracy of the titration method for the ointment type vaccine was examined by the analysis of day-to-day variations. The ointment type vaccine Lot No. SD5 and our freeze-dried laboratory standard vaccine were tested simultaneously as stated under 'Materials and Methods' twice a week for six weeks. The average log10 titres, the range of titre values and the standard deviations were calculated. The average titre of the ointment type vaccine was 8.44. Based on the range of log10 titre values (0.26 and 0"19) and the standard deviations (0-086 and 0-055) for the freeze-dried and for the ointment type vaccine, respectively, it becomes evident that the results of the testing method for 66
TITRATION
OF O I N T M E N T
SMALLPOX VACCINE
the ointment type vaccine were at least as reproducible as the results of the standard egg titration method for the freeze-dried vaccine. The results are summarized in Table 1. TABLE 1. Evaluation of the pock counts in eggs
Number of tests Range of the average pock count/membrane Average log 10 p.f.u./ml Range of titre values Standard deviation
Freeze-dried vaccine*
Ointment vaccine Lot No. SD5
i2
12
21-38"~
21-32:~
8"74 0"26 0.086
8"44 0"19 0"055
* Laboratory standard vaccine. t At vaccine dilution I0 -s'a. At vaccine dilution 10J ' ° . The ointment type vaccine Lot No. SD6a and the freeze-dried virus suspension used for its preparation were tested for stability. Log~0 titre values of samples incubated for 7, 14 and 28 days at 37 °C were compared with the titres of control samples stored at - 2 0 °C. Differences between titres of samples stored at - 2 0 °C and of those incubated at 37 °C for 28 days were 0.25 and 0.20 for the ointment type vaccine and the freezedried vaccine, respectively, indicating good stability of both preparations. It is interesting to note (see Table 2) that the extraction process used for recovering the vaccinia virus from the ointment was efficient in that almost all the virus was recovered. TABLE 2. Stability test Days 7 14 28
Freeze-dried vaccine - 20 °C 37 °C 8-80 8-83 8-79
8"76 8-79 8"59
Ointment type vaccine -20 °C 37 °C 8"66 8"67 8"73
8"48 8"54 8"48
Results of the stability test in logt0 titre values of the ointment vaccine (Lot No. SD6a) and the freeze-dried vaccinia virus suspension used for the preparation of the ointment vaccine.
DISCUSSION Freeze-dried smallpox vaccines on account of their stability are preferentially used. However, a freeze-dried vaccine has the disadvantage that it must be rehydrated for use, which by many physicians is considered a tedious procedure particularly when the vaccine is used in single doses. Furthermore, after reconstitution the vaccine may deteriorate rapidly. To overcome these disadvantages we have developed an ointment type smallpox vaccine by dispersing freeze-dried vaccinia virus in high-viscosity silicone oil (Mettier & Majer, 1972). It could be shown that this vaccine has the stability characteristics of the 5*
67
B. M E T T I E R , I. H A E N Z E L AND M. MAJER freeze-dried vaccine. Whereas the potency of the ointment type vaccine was shown in clinical trials (Stransky, 1973), its laboratory testing was hampered by the water-repellent properties of silicones. Although the vaccine can be diluted with low-viscosity silicone fluid for testing in the skin of rabbits, it cannot be assayed in embryonated eggs because the silicone fluid used is harmful to the chorioallantoic membrane. The presently described method which consists of first lowering the viscosity of the ointment type vaccine and second the extraction of the virus with a buffered saline solution now also permits its testing for potency in embryonated eggs. The extracted virus can be titrated in the standard pock count assay. With this method approximately threequarters of the calculated number of pock forming units were recovered from the silicone ointment vaccine. The reproducibility of the titre values for the ointment type vaccine was comparable to that for the freeze-dried vaccine. The results of the CAM titration assay confirmed the previous results obtained with the rabbit skin test as well as those obtained in the vaccination studies. The described extraction method permits the accurate assay of the ointment type vaccine with regard to stability. Both the ointment type vaccine and the freeze-dried vaccinia virus suspension used for its preparation do fulfil the World Health Organization Requirements for Smallpox Vaccine (W.H.O., 1966).
Acknowledgement
We wish to thank Mrs Marilyn Rutz for her valuable technical assistance.
REFERENCES Krag, P. & Weis Bentzon, M. (1963). The international reference preparation of smallpox vaccine. An international collaborative assay. Bulletin of the Worm Health Organization 29, 299-309. Mettier, B. & Majer, M. (1972). A new stable smallpox vaccine. Smallpox Vaccine Conference, Bilthoven 11-13 October. Stransky, M. (1973). Salbenimpfstoff fiir pockenimpfungen. Schweiz. Medizinische Wochenschrift 103, 20-21. World Health Organization (1959). Requirements for biological substances. World Health Organization No. 180. World Health Organization (1966). Requirements for biological substances. World Health Organization Technical Report Series No. 323. World Health Organization (1968). Methodology of freeze-dried smallpox vaccine production. World Health Organization SE/68.3 Rev. 1.
68