Immunological response of Moroccan chi!dren and newborns to oral pollovlrus vaccine prepared on Vero cells M.T. Lahrech *~ and P. Caudrelier t Immunogenicity of the oral poliovirus vaccine prepared on Vero cells ( Veto OP V) has been assessed in a two-stage study conducted in Rabat, Morocco. During the first stage, the vaccine produced seroconversion in most of the 200 children vaccinated m a three dose primary immunization, and in most of the 56 children boostered. During the second stage, four doses were given at O, 6, 10 and 14 weeks of age to 55 newborns. At the same time, 50 control newborns were given reference O P V prepared on primary monkey kidney cells ( P M K OPV). No thfference could be shown between the two vaccines in terms of immunogenicity. Therefore, permitting a more standardized production, continuous cell lines should be used for the production of OP V. .J
.
Keywords:Poliovirus; Vero cells; children; oral vaccine
Poliomyelitis remains a public health problem in developing countries x'2. For over 30 years, primary African green monkey kidney cells have been used in the production of safe and effective live poliovirus vaccines. However, it is becoming increasingly difficult to acquire the appropriate monkeys (Cercopithecus aethiops), which more frequently may be infected by unknown or unexpected viral contaminants. The Vero cell line which was established from an African green monkey kidney possesses the beneficial characteristics of most continuous cell lines (CCLs). It meets the current World Health Organization 3 and US Food and Drug Administration 4 guidelines on the use of continuous cell lines for the production of biologicals, and is used for the production of rabies and inactivated poliovirus vaccines. The new attenuated oral poliovirus vaccine was prepared on Vero cells cultivated on microcarriers in 5001 tanks 5 and controlled according to traditional oral poliovirus (OPV) regulations 6. Its immunogenicity and safety were assessed and compared with those of a reference P M K cell vaccine. The trial was conducted between June 1987 and July 1988 at Mother and Child Welfare Centres responsible for vaccinating the children of Rabat (Morocco). Fully informed consent of parents or guardians was obtained for all children involved in the study. In a first stage, 200 children (mean age 3.2 months) were given three doses of Vero OPV at one-month intervals. Additionally, 56 children (mean age 20.7 months) were given a booster dose of OPV. Adsorbed diphtheria-pertussis-tetanus (DPT) vaccine was injected to all children on the same day as the OPV. In a second stage, 105 newborns received *Service de P6diatrie 4, H6pital d'Enfants, Rabat, Morocco. tlnsUtut M6rieux, Lyon, France. tTo whom correspondence should be addressed. (Received 24 October 1989; revised '7 December 1989; accepted 27 March 1990) 0264-410X/90/040306-02 O 1990 Butterworth-HeinernannLtd
306 Vaccine, Vol. 8, August 1990
four doses of OPV prepared on either P M K (n = 50) or Vero (n = 55) cells. Vaccines were given at birth and at 6, 10 and 14 weeks of age. Adsorbed D P T vaccine was injected to all children on the same day as the last three doses of OPV. Monodose vials (0.5 ml) of magnesium chloride stabilized OPV, containing at least 6, 5 and 5.5 log median tissue culture infectious dose (TCIDso) per dose for poliovirus type 1, 2 and 3, respectively, were prepared by the Institut M6rieux, France. Titration controls of samples returned at the end of the study confirmed the good vaccine storage condition. Blood was draXvn in all children on enrolment in the study, on the day of the last dose of vaccine and one month after that. Serum was separated by centrifugation and stored at - 20°C until tested. Neutralizing antibodies against three reference wild poliovirus strains (Mahoney, Mefl and Saukett) were measured at Institut M6rieux and Laboratoire National de la Sant6, Lyon. Positivity threshold used was 1:5 (Ref. 7). Seroconversion was defined by an at least fourfold increase in antibody titre when compared to the theoretical titre expected if no vaccination had been performed. This titre was calculated individually, assuming that the half-life of maternal antibodies is 4 weeks 8. After the three dose primary immunization, 88%, 95% and 74% of the children evidenced antibodies for polio type 1, 2 and 3, respectively. Regardless of the serotype, there was a statistically significant increase in antibody titres, as early as the second dose. The vaccine also induced a booster response in the 56 18-month-old children (Table 1). Digestive-type adverse reactions (diarrhoea and vomiting) were reported in less than 2% of cases. The few febrile reactions (2.7%) can be attributed to the simultaneous injection of D P T vaccine 9. Follow-up of the children for three months following primary vaccination showed no medium-term safety
S h o r t Paper: M.T. L a h r e c h a n d P. C a u d r e l i e r Table 1
Geometric mean antibody titre and seroconversion rates of Veto OPV (first stage of the study) Primary immunization (n=200) Type 1
Type 2
Booster (n=56) Type 3
Type 1
Type 2
Type 3
Preimmunization Children with antibodies (%) Geometric mean titre
74 6
84 11
42 3
96 125
98 237
87 60
Postimmunization Children with antibodies (%) Fourfold increase in titre (%) Geometric mean titre
88 66 58
95 74 211
74 62 22
98 33 298
100 37 896
94 35 124
Table 2
Geometric mean antibody titre and seroconversion rates in Vero and PMK cell OPV groups (second stage of the study) Vero OPV Type 1
Type 2
PMK OPV Type 3
Type 1
81 14
90 33
Type 2
Type 3
Before immunization Newborns with antibodies (%) Geometric mean titre
93 31
After three doses Newborns with antibodies (%) Seroconversion rate Geometric mean titre
95 74 42
(n=42) 100 98 212
86 86 33
95 81 39
(n=42) 100 93 147
79 81 41
After four doses Newborns with antibodies (%) Seroconversion rate Geometric mean titre
95 92 45
(n=39) 100 100 267
90 95 39
86 86 62
(n=35) 100 97 263
89 94 43
88 28
problems. After the four dose primary immunization, 95%, 100% and 90% of the newborns who were given Vero OPV showed antibodies for the respective polio serotypes. Figures are comparable after P M K OPV: 86%, 100% and 89% . Regardless of the serotype, primary immunization produced a statistically significant increase in antibody titres. Results after three or four doses did not differ. Finally, no difference could be seen between the two vaccines for seropositivity and seroconversion rates, as well "as geometric mean titres
(Table 2). In this study, immunogenicity of Vero OPV was good, and comparable to that of a reference P M K OPV. Most children and newborns showed antibodies at the end of the primary immunization series. For polio type 3 after the three dose series, results are weaker but are in line with reports of moderate seroprotection rates in specific developing countries, in the international literature in particular for types 1 and 31° . Vero OPV was also well tolerated. One question about the use of CCLs concerned the risk of reversion to neurovirulence, but a comparative study showed no difference in terms of poliovirus excretion and reversion patterns between Vero and P M K OPV 11. Therefore, because CCLs permit a more standardized production, and do not change the safety and immunogenicity of oral poliovirus vaccine, they should be advantageously used for the production of this vaccine.
83 32
64 14
References 1 Anon. Poliomyelitis in 1985. Week. Epidemiol. Record 1987, 62, 273-288 2 Assad, F. and LjungarsoEsteves, K. World overview of poliomyelitis: Regional patterns and trends. Rev. Infect. Dis. 1984, 6(2), $302-307 3 WHO Expert Committee on Biological Standardization. Requirements for continuous cell lines use for biological products. WHO Tech. Rep. 1987, 745 4 Points to consider in the characterization of cell lines used to produce Biologicals. Office of Biologics Research and Review, Centers for Drugs and Biologics, FDA (USA), 1984 5 Montagnon, B.J. Polio and rabies vaccines produced in continuous cell lines: a reality for Vero line. Dev. Biol. Stand. 1989, 70, 27-47 6 WHO Expert Committee on Biological Standardization. Requirements for poliomyelitis vaccine (oral). WHO Tech. Rep. 1983, 687 7 Grenier, B., Hamza, B., Biron, G., Xueref, C., Viarme, F. and Roumiantzeff, M. Seroimmunity following vaccination in infants by an inactivated poliovirus vaccine prepared on Vero cells. Rev. Infect. Dis. 1984, 6(2), $545-547 8 Dong, D.X., Hu, X.M., Liu, W.J., Li, J.S., Jin, Y.C., Tan, S.G. et al. Immunization of neonates with trivalent oral poliomyelitis vaccine (Sabin). Bull. WHO 1986, 64, 853-860 9 Diphtheria, tetanus and pertussis: Guidelines for vaccine prophylaxis and other preventive measures. Recommendation of the ACIP. Morbid. Mortal. Week. Rep. 1985, 27, 405-428 10 Wright, P. Current worldwide polio situation and policies. In: Poliomyelitis Vaccines: Re-evaluating Policy Options. Institute of Medicine meeting, Washington, 21-22 January 1988 11 Brigaud, M., Aymard, M. and Caudrelier, P. Comparative study of poliovirus excretion after vaccination of infants with two OPV prepared on Vero cells or monkey kidney cells. In: Proceedings of Vl International Conference on Comparative and Applied Virology, Banff, Alberta, 15-21 October 1989
Vaccine, Vol. 8, A u g u s t 1990
307
.
Keywords:Poliovirus; Vero cells; children; oral vaccine
Poliomyelitis remains a public health problem in developing countries x'2. For over 30 years, primary African green monkey kidney cells have been used in the production of safe and effective live poliovirus vaccines. However, it is becoming increasingly difficult to acquire the appropriate monkeys (Cercopithecus aethiops), which more frequently may be infected by unknown or unexpected viral contaminants. The Vero cell line which was established from an African green monkey kidney possesses the beneficial characteristics of most continuous cell lines (CCLs). It meets the current World Health Organization 3 and US Food and Drug Administration 4 guidelines on the use of continuous cell lines for the production of biologicals, and is used for the production of rabies and inactivated poliovirus vaccines. The new attenuated oral poliovirus vaccine was prepared on Vero cells cultivated on microcarriers in 5001 tanks 5 and controlled according to traditional oral poliovirus (OPV) regulations 6. Its immunogenicity and safety were assessed and compared with those of a reference P M K cell vaccine. The trial was conducted between June 1987 and July 1988 at Mother and Child Welfare Centres responsible for vaccinating the children of Rabat (Morocco). Fully informed consent of parents or guardians was obtained for all children involved in the study. In a first stage, 200 children (mean age 3.2 months) were given three doses of Vero OPV at one-month intervals. Additionally, 56 children (mean age 20.7 months) were given a booster dose of OPV. Adsorbed diphtheria-pertussis-tetanus (DPT) vaccine was injected to all children on the same day as the OPV. In a second stage, 105 newborns received *Service de P6diatrie 4, H6pital d'Enfants, Rabat, Morocco. tlnsUtut M6rieux, Lyon, France. tTo whom correspondence should be addressed. (Received 24 October 1989; revised '7 December 1989; accepted 27 March 1990) 0264-410X/90/040306-02 O 1990 Butterworth-HeinernannLtd
306 Vaccine, Vol. 8, August 1990
four doses of OPV prepared on either P M K (n = 50) or Vero (n = 55) cells. Vaccines were given at birth and at 6, 10 and 14 weeks of age. Adsorbed D P T vaccine was injected to all children on the same day as the last three doses of OPV. Monodose vials (0.5 ml) of magnesium chloride stabilized OPV, containing at least 6, 5 and 5.5 log median tissue culture infectious dose (TCIDso) per dose for poliovirus type 1, 2 and 3, respectively, were prepared by the Institut M6rieux, France. Titration controls of samples returned at the end of the study confirmed the good vaccine storage condition. Blood was draXvn in all children on enrolment in the study, on the day of the last dose of vaccine and one month after that. Serum was separated by centrifugation and stored at - 20°C until tested. Neutralizing antibodies against three reference wild poliovirus strains (Mahoney, Mefl and Saukett) were measured at Institut M6rieux and Laboratoire National de la Sant6, Lyon. Positivity threshold used was 1:5 (Ref. 7). Seroconversion was defined by an at least fourfold increase in antibody titre when compared to the theoretical titre expected if no vaccination had been performed. This titre was calculated individually, assuming that the half-life of maternal antibodies is 4 weeks 8. After the three dose primary immunization, 88%, 95% and 74% of the children evidenced antibodies for polio type 1, 2 and 3, respectively. Regardless of the serotype, there was a statistically significant increase in antibody titres, as early as the second dose. The vaccine also induced a booster response in the 56 18-month-old children (Table 1). Digestive-type adverse reactions (diarrhoea and vomiting) were reported in less than 2% of cases. The few febrile reactions (2.7%) can be attributed to the simultaneous injection of D P T vaccine 9. Follow-up of the children for three months following primary vaccination showed no medium-term safety
S h o r t Paper: M.T. L a h r e c h a n d P. C a u d r e l i e r Table 1
Geometric mean antibody titre and seroconversion rates of Veto OPV (first stage of the study) Primary immunization (n=200) Type 1
Type 2
Booster (n=56) Type 3
Type 1
Type 2
Type 3
Preimmunization Children with antibodies (%) Geometric mean titre
74 6
84 11
42 3
96 125
98 237
87 60
Postimmunization Children with antibodies (%) Fourfold increase in titre (%) Geometric mean titre
88 66 58
95 74 211
74 62 22
98 33 298
100 37 896
94 35 124
Table 2
Geometric mean antibody titre and seroconversion rates in Vero and PMK cell OPV groups (second stage of the study) Vero OPV Type 1
Type 2
PMK OPV Type 3
Type 1
81 14
90 33
Type 2
Type 3
Before immunization Newborns with antibodies (%) Geometric mean titre
93 31
After three doses Newborns with antibodies (%) Seroconversion rate Geometric mean titre
95 74 42
(n=42) 100 98 212
86 86 33
95 81 39
(n=42) 100 93 147
79 81 41
After four doses Newborns with antibodies (%) Seroconversion rate Geometric mean titre
95 92 45
(n=39) 100 100 267
90 95 39
86 86 62
(n=35) 100 97 263
89 94 43
88 28
problems. After the four dose primary immunization, 95%, 100% and 90% of the newborns who were given Vero OPV showed antibodies for the respective polio serotypes. Figures are comparable after P M K OPV: 86%, 100% and 89% . Regardless of the serotype, primary immunization produced a statistically significant increase in antibody titres. Results after three or four doses did not differ. Finally, no difference could be seen between the two vaccines for seropositivity and seroconversion rates, as well "as geometric mean titres
(Table 2). In this study, immunogenicity of Vero OPV was good, and comparable to that of a reference P M K OPV. Most children and newborns showed antibodies at the end of the primary immunization series. For polio type 3 after the three dose series, results are weaker but are in line with reports of moderate seroprotection rates in specific developing countries, in the international literature in particular for types 1 and 31° . Vero OPV was also well tolerated. One question about the use of CCLs concerned the risk of reversion to neurovirulence, but a comparative study showed no difference in terms of poliovirus excretion and reversion patterns between Vero and P M K OPV 11. Therefore, because CCLs permit a more standardized production, and do not change the safety and immunogenicity of oral poliovirus vaccine, they should be advantageously used for the production of this vaccine.
83 32
64 14
References 1 Anon. Poliomyelitis in 1985. Week. Epidemiol. Record 1987, 62, 273-288 2 Assad, F. and LjungarsoEsteves, K. World overview of poliomyelitis: Regional patterns and trends. Rev. Infect. Dis. 1984, 6(2), $302-307 3 WHO Expert Committee on Biological Standardization. Requirements for continuous cell lines use for biological products. WHO Tech. Rep. 1987, 745 4 Points to consider in the characterization of cell lines used to produce Biologicals. Office of Biologics Research and Review, Centers for Drugs and Biologics, FDA (USA), 1984 5 Montagnon, B.J. Polio and rabies vaccines produced in continuous cell lines: a reality for Vero line. Dev. Biol. Stand. 1989, 70, 27-47 6 WHO Expert Committee on Biological Standardization. Requirements for poliomyelitis vaccine (oral). WHO Tech. Rep. 1983, 687 7 Grenier, B., Hamza, B., Biron, G., Xueref, C., Viarme, F. and Roumiantzeff, M. Seroimmunity following vaccination in infants by an inactivated poliovirus vaccine prepared on Vero cells. Rev. Infect. Dis. 1984, 6(2), $545-547 8 Dong, D.X., Hu, X.M., Liu, W.J., Li, J.S., Jin, Y.C., Tan, S.G. et al. Immunization of neonates with trivalent oral poliomyelitis vaccine (Sabin). Bull. WHO 1986, 64, 853-860 9 Diphtheria, tetanus and pertussis: Guidelines for vaccine prophylaxis and other preventive measures. Recommendation of the ACIP. Morbid. Mortal. Week. Rep. 1985, 27, 405-428 10 Wright, P. Current worldwide polio situation and policies. In: Poliomyelitis Vaccines: Re-evaluating Policy Options. Institute of Medicine meeting, Washington, 21-22 January 1988 11 Brigaud, M., Aymard, M. and Caudrelier, P. Comparative study of poliovirus excretion after vaccination of infants with two OPV prepared on Vero cells or monkey kidney cells. In: Proceedings of Vl International Conference on Comparative and Applied Virology, Banff, Alberta, 15-21 October 1989
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307
