Vol. 3, No. 4 Printed in U.S.A.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1976, p. 460-462 Copyright (© 1976 American Society for Microbiology
Improved Method for Production of Attenuated Venezuelan Equine Encephalomyelitis (TC-83 Strain) Vaccine FRANCIS E. COLE, JR.,* CARL E. PEDERSEN, JR., DAVID M. ROBINSON,' AND GERALD A. EDDY U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701 Received for publication 17 December 1975
Primary chicken embryo cell cultures were evaluated as an alternate cell system for the production of attenuated Venezuelan equine encephalomyelitis (TC-83 strain) vaccine. The TC-83 strain virus was shown to remain stable during 10 serial passages in chicken embryo cell culture with regard to plaque size and morphology, virus yield, potency, and virulence for mice and hamsters. Venezuelan equine encephalomyelitis (VEE)
vaccine, TC-83 strain, is currently propagated in second-passage, fetal guinea pig heart (GPH) cell cultures (1, 5). Inherent disadvantages with these cultures include: (i) difficulty in obtaining sufficient numbers of "clean" pregnant guinea pigs; (ii) tedious and time-consuming production procedures; (iii) small yields of cells obtained per guinea pig from fetal hearts; and (iv) long incubation period (ca. 10 days) from original planting through outgrowth of secondpassage cultures. This study was made to determine whether chicken embryo cell (CEC) cultures could be substituted for GPH cell cultures to produce a vaccine with acceptable potency and the same characteristics as the original product. Attenuated VEE virus was received as 82ndpassage fetal GPH cell culture fluid from Merrell-National Laboratories (5). Trinidad strain VEE virus was used as challenge virus in potency assays (7). Plaque assays were performed in duck embryo cell cultures as described by Pedersen et al. (6). Virus titers were expressed as plaqueforming units (PFU) per ml. In vivo titrations were performed in 1- to 3-day-old CD-1 strain white mice (Charles River Mouse Farms, Wilmington, Mass.), using groups of six mice each inoculated intracerebrally (i.c.) with 0.03 ml of log,), dilutions of virus. End points were determined by the method of Reed and Muench (8) and expressed as median lethal doses (LD5() per ml. CEC cultures in 75-cm2 flasks were prepared as described previously (2) using pathogen-free eggs (SPAFAS Inc., Norwich, Conn.) and incubated at 35 C until confluent cell sheets were Present address: U.S. Army Medical Research Unit, Kuala Lumpur, Department of State, Washington, D.C. 20521.
obtained (18 to 24 h). Cultures were then inoculated with TC-82 strain VEE virus at a multiplicity of 0.005 PFU/cell. Virus was adsorbed for 1 h at 35 C, the infecting fluid was drained, and 20 ml of medium 199 containing 0.25% human serum albumin and antibiotics was added. After incubation for 22 to 24 h culture fluids were harvested, pooled, and clarified by centrifugation and membrane filtration (3). Samples of clarified virus were then used as inoculum for a fresh set of CEC cultures as described above, using 1.0 ml of an empirical dilution of 10-5. Since vaccine production requires continued availability of master and working seed virus stocks, 10 serial passages were made to determine the effect of such passage on viral characteristics. Samples were taken at selected passage levels and stored at -70 C for subsequent testing. The original VEE vaccine seed (TC-82) and the empirically chosen third (TC-82-3) and tenth (TC-82-10) CEC-passaged viruses were titrated for potency in 350-g Hartley strain guinea pigs (West Jersey Biological Supply Farms, Wenonah, N.J.). Groups of 10 guinea pigs were inoculated intraperitoneally (i.p.) with 0.5 ml of log1,, dilutions of virus. After 21 days guinea pigs were challegened i.p. with 10: guinea pig LD5, of Trinidad strain VEE virus; those surviving for 10 days were considered to have received an immunizing dose of virus. Guinea pig i.p. median immunizing doses (IPID50,) were determined by the method of Reed and Muench (8). Mice and hamsters were used to determine the stability of certain known characteristics of the virus after CEC culture passage. Each of 40 weanling mice (12 to 14 g, CD-1 strain) was inoculated i.p. with 0.3 ml and i.c. with 0.03 ml of either undiluted TC-82-3 or TC-82-10. Mice given TC-82 served as controls. The same vi-
460
ruses were
461
NOTES
VOL. 3, 1976
titrated i.c. in weanling mice using
(TC-82) and the Merrell-National Laboratories
log,0 dilutions. Lakeview strain golden Syrian TC-83 vaccine (Table 1). Thus the virus plaque
hamsters (85 to 95 g; Lakeview Hamster ColNewfield, N.J.) were inoculated i.p. with 103 hamster IPID50 of TC-82, TC-82-3, or TC-8210 using 100 hamsters for each virus preparation. All mice and hamsters were observed for 21 days for deaths. No substantial changes were apparent when the titers and plaque size and morphology obtained with CEC-passaged virus were compared with those of the starting seed material
ony,
TABLE 1. Effect ofserial passage in CEC cultures on titer and plaque size of strain TC-82 VEE virus Titer: CEC passage no.
SMIC
LD50/ ml
(log10)
PFU/ml
Plaque sizea (mm)
1.5-3.5 9.2 5.2 x 109 TC-82 seed 1.5-3.5 9.4 x 109 1 10.0 1.5-3.5 2.1 x 1010 2 10.4 1.5-3.5 1.2 x 1010 3 10.0 1.5-3.0 2.0 x 1010 4 10.9 4.1 x 10'0 1.5-3.0 10.7 5 4.9 x 1010 1.5-3.0 10.7 6 1.5-3.0 7 11.0 2.8 x 1010 10.0 1.4 x 1010 1.5-3.0 8 4.4 x 1010 1.5-3.0 10.5 9 1.5-3.0 8.6 x 1010 11.0 10 1.5-3.5 1.6 x 105 5.2 TC-83 vaccine0 a Range in size of 100 plaques examined at each passage level. b Merrell-National Laboratories commercial vaccine, as diluted before freeze-drying for human use.
TABLE 2. Reponse of weanling mice to combined i.p. and i.c. inoculation with TC-82 strain VEE virus after serial passage in CEC cultureSa Virus
markers were stable, and high yields of virus were obtained in the CEC culture system. Inoculation of weanling mice by the combined i.c. and i.p. routes resulted in no more than 10% deaths (Table 2). Challenge of the surviving mice on day 21 postinoculation with 103 LD50 of Trinidad strain VEE virus resulted in 100% survival, indicating that they had received a protective dose of the vaccine viruses. Mouse neurovirulence was also unaffected by serial CEC passage (Table 3), with a few deaths occurring through the range of virus dilutions tested. Both of these findings are in agreement with those of Berge et al. (1) and R. W. McKinney (personal communication) in studies on the original TC-83 vaccine showing that only few mouse deaths occurred and were not dose related. Hamsters inoculated with 103 IPID50 of the TC-82 starting seed virus or CEC-passaged viruses (TC-83-3 or TC-82-10; Table 4) exhibited a range of vaccine mortality (16 to 22%) similar to that observed by Jahrling et al. (4) and F. E. Cole, Jr. (unpublished observations) with TC83 vaccine administered to more than 500 hamsters. Potency tests in guinea pigs indicated that effective vaccines were obtained with CEC-passaged virus (Table 5). CEC culture is in excellent cell system for the production of attenuated VEE vaccine. Vaccine virus comparable in all established characteristics to the original vaccine virus may be prepared in this system more easily than in the TABLE 4. Response of hamsters to i.p. inoculation with TC-82 strain VEE virus after serial passage in CEC cultures Percent surviving (no./total)b Virus' 84 (84/100) TC-82 79 (79/100) TC-82-3 78 (78/100) TC-82-10 a Each animal received ca. 103 IPIDs. b Differences between groups are not statistically
Percent surviving (no./total) inoculation with undiluted virus
90 (36/40) TC-82b TC-82-3c 90 (36/40) 100 (40/40) TC-82-10c a Differences between groups are not statistically significant. b Inoculum titered ca. 5 x 109 PFU/ml. c Inoculum titered ca. 1 x 1010 PFU/ml.
significant.
TABLE 3. Response of weanling mice to i.c. inoculation with decimal dilutions of TC-83 strain VEE virus after serial passage in CEC culturesa Percent surviving (no./total) i.c. inoculation with virus dilution of: Virus 10- I
10-2
90 (9/10) TC-82b 90 (9/10) 80 (24/30) TC-82-3c 97 (29/30) 97 (29/30) 100 (30/30) TC-82-10r a Differences between control group (TC-82) b Inoculum titered ca. 5 x 109 PFU/ml. ' Inoculum titered ca. 1010 PFU/ml.
10-3
10-4
10-5
10-6
100 (10/10)
100 (10/10)
90 (9/10)
100 (10/10)
97 (29/30) 73 (22/30) 80 (24/30) 83 (25/30) 93 (28/30) 100 (30/30) 100 (30/30) 100 (30/30) and other groups are not statistically significant.
462
J. CLIN. MICROBIOL.
NOTES
TABLE 5. Potency of TC-82 strain VEE virus after serial passage in CEC cultures Titer/0.5 ml in: Virus
Suckling ml (log1O) mice0 0.5GPIPID50a/ (log10
LD5O)
DECcl
DECr culPU
tr
8.9 2.6 x 109 TC-82 8.1 8.6 9.7 6.0 x 109 TC-82-3 TC-82-10 8.9 10.7 4.3 x 1010 a Guinea pig intraperitoneal median immunizing dose. b Titrated via i.c. route; value corrected to 0.5 ml. c Duck embryo cell.
fetal GPH cell system currently used. The availability of embryonated eggs free of adventitious agents and the acceptability of this cell system for licensed vaccines (9) are additional advantages. LITERATURE CITED 1. Berge, T. 0., I. S. Banks, and W. D. Tigertt. 1961. Attenuation of Venezuelan equine encephalomyelitits virus by in vitro cultivation in guinea pig heart cells. Am. J. Hyg. 73:209-218.
2. Cole, F. E., Jr. 1971. Inactivated Eastern equine encephalomyelitis vaccine propagated in rolling-bottle cultures of chick embryo cells. Appl. Microbiol.
22:842-845. 3. Cole, F. E., Jr., S. W. May, and D. M. Robinson. 1973. Formalin-inactivated Venezuelan equine encephalomyelitits (Trinidad strain) vaccine produced in rolling-bottle cultures of chicken embryo cells. Appl. Microbiol. 25:262-265. 4. Jahrling, P. B., E. Dendy, and G. A. Eddy. 1974. Correlates to increased lethality of attenuated Venezuelan encephalitis virus vaccine for immunosuppressed hamsters. Infect. Immun. 9:924-930. 5. McKinney, R. W., T. 0. Berge, W. D. Sawyer, W. D, Tigertt, and D. Crozier. 1963. Use of an attenuated strain of Venezuelan equine encephalomyelitis virus for immunization in man. Am. J. Trop. Med. Hyg. 12:597-603. 6. Pedersen, C. E. Jr., D. R. Slocum, and N. H. Levitt. 1972. Chromatography of Venezuelan equine encephalomyelitis virus strains on calcium phosphate. Appl. Microbiol. 24:91-95. 7. Randall, R., and J. W. Mills. 1944. Fatal encephalitis in man due to the Venezuelan virus of equine encephalitis in Trinidad. Science 99:225-226. 8. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27:493-497. 9. U.S. Code of Federal Regulations, Title 21-Food and Drugs, Chapter 1, subchapter F-Biologics. 1975.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1976, p. 460-462 Copyright (© 1976 American Society for Microbiology
Improved Method for Production of Attenuated Venezuelan Equine Encephalomyelitis (TC-83 Strain) Vaccine FRANCIS E. COLE, JR.,* CARL E. PEDERSEN, JR., DAVID M. ROBINSON,' AND GERALD A. EDDY U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701 Received for publication 17 December 1975
Primary chicken embryo cell cultures were evaluated as an alternate cell system for the production of attenuated Venezuelan equine encephalomyelitis (TC-83 strain) vaccine. The TC-83 strain virus was shown to remain stable during 10 serial passages in chicken embryo cell culture with regard to plaque size and morphology, virus yield, potency, and virulence for mice and hamsters. Venezuelan equine encephalomyelitis (VEE)
vaccine, TC-83 strain, is currently propagated in second-passage, fetal guinea pig heart (GPH) cell cultures (1, 5). Inherent disadvantages with these cultures include: (i) difficulty in obtaining sufficient numbers of "clean" pregnant guinea pigs; (ii) tedious and time-consuming production procedures; (iii) small yields of cells obtained per guinea pig from fetal hearts; and (iv) long incubation period (ca. 10 days) from original planting through outgrowth of secondpassage cultures. This study was made to determine whether chicken embryo cell (CEC) cultures could be substituted for GPH cell cultures to produce a vaccine with acceptable potency and the same characteristics as the original product. Attenuated VEE virus was received as 82ndpassage fetal GPH cell culture fluid from Merrell-National Laboratories (5). Trinidad strain VEE virus was used as challenge virus in potency assays (7). Plaque assays were performed in duck embryo cell cultures as described by Pedersen et al. (6). Virus titers were expressed as plaqueforming units (PFU) per ml. In vivo titrations were performed in 1- to 3-day-old CD-1 strain white mice (Charles River Mouse Farms, Wilmington, Mass.), using groups of six mice each inoculated intracerebrally (i.c.) with 0.03 ml of log,), dilutions of virus. End points were determined by the method of Reed and Muench (8) and expressed as median lethal doses (LD5() per ml. CEC cultures in 75-cm2 flasks were prepared as described previously (2) using pathogen-free eggs (SPAFAS Inc., Norwich, Conn.) and incubated at 35 C until confluent cell sheets were Present address: U.S. Army Medical Research Unit, Kuala Lumpur, Department of State, Washington, D.C. 20521.
obtained (18 to 24 h). Cultures were then inoculated with TC-82 strain VEE virus at a multiplicity of 0.005 PFU/cell. Virus was adsorbed for 1 h at 35 C, the infecting fluid was drained, and 20 ml of medium 199 containing 0.25% human serum albumin and antibiotics was added. After incubation for 22 to 24 h culture fluids were harvested, pooled, and clarified by centrifugation and membrane filtration (3). Samples of clarified virus were then used as inoculum for a fresh set of CEC cultures as described above, using 1.0 ml of an empirical dilution of 10-5. Since vaccine production requires continued availability of master and working seed virus stocks, 10 serial passages were made to determine the effect of such passage on viral characteristics. Samples were taken at selected passage levels and stored at -70 C for subsequent testing. The original VEE vaccine seed (TC-82) and the empirically chosen third (TC-82-3) and tenth (TC-82-10) CEC-passaged viruses were titrated for potency in 350-g Hartley strain guinea pigs (West Jersey Biological Supply Farms, Wenonah, N.J.). Groups of 10 guinea pigs were inoculated intraperitoneally (i.p.) with 0.5 ml of log1,, dilutions of virus. After 21 days guinea pigs were challegened i.p. with 10: guinea pig LD5, of Trinidad strain VEE virus; those surviving for 10 days were considered to have received an immunizing dose of virus. Guinea pig i.p. median immunizing doses (IPID50,) were determined by the method of Reed and Muench (8). Mice and hamsters were used to determine the stability of certain known characteristics of the virus after CEC culture passage. Each of 40 weanling mice (12 to 14 g, CD-1 strain) was inoculated i.p. with 0.3 ml and i.c. with 0.03 ml of either undiluted TC-82-3 or TC-82-10. Mice given TC-82 served as controls. The same vi-
460
ruses were
461
NOTES
VOL. 3, 1976
titrated i.c. in weanling mice using
(TC-82) and the Merrell-National Laboratories
log,0 dilutions. Lakeview strain golden Syrian TC-83 vaccine (Table 1). Thus the virus plaque
hamsters (85 to 95 g; Lakeview Hamster ColNewfield, N.J.) were inoculated i.p. with 103 hamster IPID50 of TC-82, TC-82-3, or TC-8210 using 100 hamsters for each virus preparation. All mice and hamsters were observed for 21 days for deaths. No substantial changes were apparent when the titers and plaque size and morphology obtained with CEC-passaged virus were compared with those of the starting seed material
ony,
TABLE 1. Effect ofserial passage in CEC cultures on titer and plaque size of strain TC-82 VEE virus Titer: CEC passage no.
SMIC
LD50/ ml
(log10)
PFU/ml
Plaque sizea (mm)
1.5-3.5 9.2 5.2 x 109 TC-82 seed 1.5-3.5 9.4 x 109 1 10.0 1.5-3.5 2.1 x 1010 2 10.4 1.5-3.5 1.2 x 1010 3 10.0 1.5-3.0 2.0 x 1010 4 10.9 4.1 x 10'0 1.5-3.0 10.7 5 4.9 x 1010 1.5-3.0 10.7 6 1.5-3.0 7 11.0 2.8 x 1010 10.0 1.4 x 1010 1.5-3.0 8 4.4 x 1010 1.5-3.0 10.5 9 1.5-3.0 8.6 x 1010 11.0 10 1.5-3.5 1.6 x 105 5.2 TC-83 vaccine0 a Range in size of 100 plaques examined at each passage level. b Merrell-National Laboratories commercial vaccine, as diluted before freeze-drying for human use.
TABLE 2. Reponse of weanling mice to combined i.p. and i.c. inoculation with TC-82 strain VEE virus after serial passage in CEC cultureSa Virus
markers were stable, and high yields of virus were obtained in the CEC culture system. Inoculation of weanling mice by the combined i.c. and i.p. routes resulted in no more than 10% deaths (Table 2). Challenge of the surviving mice on day 21 postinoculation with 103 LD50 of Trinidad strain VEE virus resulted in 100% survival, indicating that they had received a protective dose of the vaccine viruses. Mouse neurovirulence was also unaffected by serial CEC passage (Table 3), with a few deaths occurring through the range of virus dilutions tested. Both of these findings are in agreement with those of Berge et al. (1) and R. W. McKinney (personal communication) in studies on the original TC-83 vaccine showing that only few mouse deaths occurred and were not dose related. Hamsters inoculated with 103 IPID50 of the TC-82 starting seed virus or CEC-passaged viruses (TC-83-3 or TC-82-10; Table 4) exhibited a range of vaccine mortality (16 to 22%) similar to that observed by Jahrling et al. (4) and F. E. Cole, Jr. (unpublished observations) with TC83 vaccine administered to more than 500 hamsters. Potency tests in guinea pigs indicated that effective vaccines were obtained with CEC-passaged virus (Table 5). CEC culture is in excellent cell system for the production of attenuated VEE vaccine. Vaccine virus comparable in all established characteristics to the original vaccine virus may be prepared in this system more easily than in the TABLE 4. Response of hamsters to i.p. inoculation with TC-82 strain VEE virus after serial passage in CEC cultures Percent surviving (no./total)b Virus' 84 (84/100) TC-82 79 (79/100) TC-82-3 78 (78/100) TC-82-10 a Each animal received ca. 103 IPIDs. b Differences between groups are not statistically
Percent surviving (no./total) inoculation with undiluted virus
90 (36/40) TC-82b TC-82-3c 90 (36/40) 100 (40/40) TC-82-10c a Differences between groups are not statistically significant. b Inoculum titered ca. 5 x 109 PFU/ml. c Inoculum titered ca. 1 x 1010 PFU/ml.
significant.
TABLE 3. Response of weanling mice to i.c. inoculation with decimal dilutions of TC-83 strain VEE virus after serial passage in CEC culturesa Percent surviving (no./total) i.c. inoculation with virus dilution of: Virus 10- I
10-2
90 (9/10) TC-82b 90 (9/10) 80 (24/30) TC-82-3c 97 (29/30) 97 (29/30) 100 (30/30) TC-82-10r a Differences between control group (TC-82) b Inoculum titered ca. 5 x 109 PFU/ml. ' Inoculum titered ca. 1010 PFU/ml.
10-3
10-4
10-5
10-6
100 (10/10)
100 (10/10)
90 (9/10)
100 (10/10)
97 (29/30) 73 (22/30) 80 (24/30) 83 (25/30) 93 (28/30) 100 (30/30) 100 (30/30) 100 (30/30) and other groups are not statistically significant.
462
J. CLIN. MICROBIOL.
NOTES
TABLE 5. Potency of TC-82 strain VEE virus after serial passage in CEC cultures Titer/0.5 ml in: Virus
Suckling ml (log1O) mice0 0.5GPIPID50a/ (log10
LD5O)
DECcl
DECr culPU
tr
8.9 2.6 x 109 TC-82 8.1 8.6 9.7 6.0 x 109 TC-82-3 TC-82-10 8.9 10.7 4.3 x 1010 a Guinea pig intraperitoneal median immunizing dose. b Titrated via i.c. route; value corrected to 0.5 ml. c Duck embryo cell.
fetal GPH cell system currently used. The availability of embryonated eggs free of adventitious agents and the acceptability of this cell system for licensed vaccines (9) are additional advantages. LITERATURE CITED 1. Berge, T. 0., I. S. Banks, and W. D. Tigertt. 1961. Attenuation of Venezuelan equine encephalomyelitits virus by in vitro cultivation in guinea pig heart cells. Am. J. Hyg. 73:209-218.
2. Cole, F. E., Jr. 1971. Inactivated Eastern equine encephalomyelitis vaccine propagated in rolling-bottle cultures of chick embryo cells. Appl. Microbiol.
22:842-845. 3. Cole, F. E., Jr., S. W. May, and D. M. Robinson. 1973. Formalin-inactivated Venezuelan equine encephalomyelitits (Trinidad strain) vaccine produced in rolling-bottle cultures of chicken embryo cells. Appl. Microbiol. 25:262-265. 4. Jahrling, P. B., E. Dendy, and G. A. Eddy. 1974. Correlates to increased lethality of attenuated Venezuelan encephalitis virus vaccine for immunosuppressed hamsters. Infect. Immun. 9:924-930. 5. McKinney, R. W., T. 0. Berge, W. D. Sawyer, W. D, Tigertt, and D. Crozier. 1963. Use of an attenuated strain of Venezuelan equine encephalomyelitis virus for immunization in man. Am. J. Trop. Med. Hyg. 12:597-603. 6. Pedersen, C. E. Jr., D. R. Slocum, and N. H. Levitt. 1972. Chromatography of Venezuelan equine encephalomyelitis virus strains on calcium phosphate. Appl. Microbiol. 24:91-95. 7. Randall, R., and J. W. Mills. 1944. Fatal encephalitis in man due to the Venezuelan virus of equine encephalitis in Trinidad. Science 99:225-226. 8. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27:493-497. 9. U.S. Code of Federal Regulations, Title 21-Food and Drugs, Chapter 1, subchapter F-Biologics. 1975.
