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Considerations Involved in the Standardization of Polyvalent Respiratory Vaccines
Considerations Involved in the Standardization of Polyvalent Respiratory Vaccines* By F. J. MURRAY, KATHERINE A. LUDWIG, and MILTON J. POTER Certain aspeas of standardization of polyvalent respiratory vaccines are discussed. A mouse-protection index may be used satisfactorily for the standardization of pneumococci or other mouse-virulent organisms. Of prime importance is the determination, under standardized conditions, that each lot is as good as those preceding.
group was subtracted from the logarithm of the LDSo of each test group, the difference being multiplied by an arbitrary factor of 10. This method has been very satisfactory for comparing various lots of a respiratory vaccine’ with regard to the individual pneumococcal types. I n our studies, 18-20 Gm. albino mice were given a single intraperitoneal injection of 5 X lo9 formalin-killed cells, and seven days later were challenged with a range of dilutions permitting calculation of the LDsoby the method of Reed and Muench (2). Some idea of the type of range encountered and the reproducibility to be expected may be obtained from the data on various batches in Table I.
of biological products is generally highly desirable from the standpoint of the manufacturer, since it gives assurance that each lot is as good as those preceding. It is obvious that any standard selected should be one that could be achieved in the great majority of cases, and yet it should be rigid enough to be of real value. Consideration of the possibilities for standardizing polyvalent respiratory TABLEI.-ANTIGENICITY OF LOTSOF PNEUMOCOCvaccines reveals a multiplicity of complications. cus VACCINES Since respiratory vaccines generally contain VPCsome organisms which are unable in the living cine, D. 10 x Log state to infect animals, an animal-protection test Protection pneuNo. LDa Index monaac Lot Mice Control is not possible for all of the constituents of such I A 48 10-9.20 62 vaccines. The pneumococci, on the other hand, B 48 59 do lend themselves to a mouse-protection test so 53 C 48 10-8.89 D 60 10-8.60 55 that a standard might be based on the antigenicity E 48 10-9.‘o 71 of these organisms. One must decide whether F 48 1 0 - 9 . 6 0 64 to test the individual suspensions of killed pneuI1 A 48 36 mococci before pooling, or the pooled material, B 48 10-9.00 35 or the finished product. Although an evaluation 31 I11 A 48 10-8.60 B 48 10-8.62 35 of the finished product would appear most meanC 48 37 ingful, an evaluation of the individual vaccine D 48 10-9.00 37 suspensions is more practical and provides assur30 VII A 48 10-9.00 ance that each lot is as antigenic as the last. B 48 10-9.00 32 If the individual pneumococcus types contained 34 VIII A 48 1 0 - 9 . 2 4 B 48 <30 in a given polyvalent vaccine are found to have 21 C 48 10-*.76 the same antigenic activity, it is possible to establish one numerical index. If there is a variation between types, however, one must choose beObviously, types 11, 111, VII, and VIII could tween using more than one index or using the be expected to attain a protection index of 30 lowest of the indexes. When the aim is merely (protection against 1,000 LD5O doses) in every to prove a lot “as good as” rather than to indi- lot. Type I apparently is much more antigenic, cate “how good,” this problem is of little import- and thus every lot of vaccine could be expected ance. to reach an index of 50 (100,000 LDso doses). We have previously described a method for In this case, if one index is desired, 30 would have comparing vaccines based on an LDw calculation to be the choice, but obviously 30 is much too and expressed as a logarithm protection index X lenient a requirement for our type I. 10 (1). The logarithm of the LD50 of the control Our results have been uniform with mice from different sources of supply and with either sex. HE STANDARDIZATION
*
Received September 27 1950 from the Department of Bacteriology, Research Ladoratokies, The Wm. S. Merrell CO.,Cincinnati, Ohio.
1
197
Oravax. registered trade mark of the Wm. S. Merrell Co.
198
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
Obviously, these factors should be checked and if it is found that a particular strain or sex gives an irregular or unusual response, this must be taken into consideration in the evaluation of results. The variables of animal tests are numerous, but fortunately more and more of them are being revealed. Results niay be influenced by varying the close of vaccine, or the waiting period before challenge, or even by varying the species of test animal. By the proper combination of factors a wide range of results may be obtained and this should tend to minimize the importance of a high protection index. It appears far more important to establish that under a standardized set of conditions one can repeatedly protect against a given number of challenging doses, rather than to strive for protection against higher and higher doses with the identical vaccine.
Vol. XL, No. 4
SUMMARY 1. Some aspects of standardization of polyvalent respiratory vaccines have been considered in this paper. 2. Pneumococci or other mouse-virulent organisms may be standardized according to a mouse-protection index. 3. Attainment of higher protective indexes by variations in test conditions is meaningless. It is far more important to determine, under standardized conditions, that each lot is as good as those preceding. REFERENCES (1) Ludwig, K. A,, Murray, F. J., and Stauhach, E. J., J . Zmmunol.. 62,257(1949). (2) Reed, I,. J., aud Mueuch, H.. A m . J . H y g . , 27, 493 (1938).
A Study of the Repeated Administration of Tinnevelly and Alexandria Senna to Mice* By MARIBELLE WOODS and I. W. GROTE To determine if the same mice could be used repeatedly over long periods of time without change in sensitivity, two sets of experiments were conducted. In the first set of experiments no tolerance to either Tinnevelly or Alexandria senna developed. In the second set of experiments, 3 1 o u t of 40 mice survived the twenty-three-week period. The average percentage laxative response for the period was 64 per cent (range 38-92 er cent). The responses of individual subjects were roughly random. It woulef therefore seem that mice may be used once a week for quantitative assay of laxative activity of senna.
PROCEDURE for determining the laxative activity in mice of senna preparations has been previously reported (1). The finely ground drug was suspended in boiling distilled water and 0.5-cc. doses were given by stomach tube. The results were considered positive if the feces were softened sufficiently to stain the underside of newsprint paper, and negative if no stain was visible. T o determine if the same mice could be given a weekly dose of senna over long periods of time without changing their sensitivity t o the drug, two sets of experiments were conducted.
A
STANDARDIZED
* Received May 5, 1950, from the Research Laboratories, The Chattanooga Medicine Company, Chattanooga. Tenn. Presented to the Scientific Section, A. PH. A,, Atlantic City meeting, May, 1050.
EXPERIMENTAL In the first set of experiments, two groups of 25 male mice each were individually numbered and a record was kept, week by week, of each animal’s response to senna and his weight. Tinnevelly and Alexandria senna were given on alternate weeks at three dosage levels, namely 8.3, 6.3, or 5.0 mg. That is, one week group I would receive 8.3 mg. ot Tinnevelly senna, while group I1 would receive the same dosage of Alexandria. The following week the two groups would receive the same dosage as the first week, but the kinds of senna were reversed for the groups. This procedure was followed in succeeding weeks until each group had received three different doses of two kinds of senna. Then this identical procedure was repeated so that each group again received the same dosage and kind of senna which had been received six weeks before. This w a s to determine whether the five weekly doses which had intervened had changed the mouse’s
group was subtracted from the logarithm of the LDSo of each test group, the difference being multiplied by an arbitrary factor of 10. This method has been very satisfactory for comparing various lots of a respiratory vaccine’ with regard to the individual pneumococcal types. I n our studies, 18-20 Gm. albino mice were given a single intraperitoneal injection of 5 X lo9 formalin-killed cells, and seven days later were challenged with a range of dilutions permitting calculation of the LDsoby the method of Reed and Muench (2). Some idea of the type of range encountered and the reproducibility to be expected may be obtained from the data on various batches in Table I.
of biological products is generally highly desirable from the standpoint of the manufacturer, since it gives assurance that each lot is as good as those preceding. It is obvious that any standard selected should be one that could be achieved in the great majority of cases, and yet it should be rigid enough to be of real value. Consideration of the possibilities for standardizing polyvalent respiratory TABLEI.-ANTIGENICITY OF LOTSOF PNEUMOCOCvaccines reveals a multiplicity of complications. cus VACCINES Since respiratory vaccines generally contain VPCsome organisms which are unable in the living cine, D. 10 x Log state to infect animals, an animal-protection test Protection pneuNo. LDa Index monaac Lot Mice Control is not possible for all of the constituents of such I A 48 10-9.20 62 vaccines. The pneumococci, on the other hand, B 48 59 do lend themselves to a mouse-protection test so 53 C 48 10-8.89 D 60 10-8.60 55 that a standard might be based on the antigenicity E 48 10-9.‘o 71 of these organisms. One must decide whether F 48 1 0 - 9 . 6 0 64 to test the individual suspensions of killed pneuI1 A 48 36 mococci before pooling, or the pooled material, B 48 10-9.00 35 or the finished product. Although an evaluation 31 I11 A 48 10-8.60 B 48 10-8.62 35 of the finished product would appear most meanC 48 37 ingful, an evaluation of the individual vaccine D 48 10-9.00 37 suspensions is more practical and provides assur30 VII A 48 10-9.00 ance that each lot is as antigenic as the last. B 48 10-9.00 32 If the individual pneumococcus types contained 34 VIII A 48 1 0 - 9 . 2 4 B 48 <30 in a given polyvalent vaccine are found to have 21 C 48 10-*.76 the same antigenic activity, it is possible to establish one numerical index. If there is a variation between types, however, one must choose beObviously, types 11, 111, VII, and VIII could tween using more than one index or using the be expected to attain a protection index of 30 lowest of the indexes. When the aim is merely (protection against 1,000 LD5O doses) in every to prove a lot “as good as” rather than to indi- lot. Type I apparently is much more antigenic, cate “how good,” this problem is of little import- and thus every lot of vaccine could be expected ance. to reach an index of 50 (100,000 LDso doses). We have previously described a method for In this case, if one index is desired, 30 would have comparing vaccines based on an LDw calculation to be the choice, but obviously 30 is much too and expressed as a logarithm protection index X lenient a requirement for our type I. 10 (1). The logarithm of the LD50 of the control Our results have been uniform with mice from different sources of supply and with either sex. HE STANDARDIZATION
*
Received September 27 1950 from the Department of Bacteriology, Research Ladoratokies, The Wm. S. Merrell CO.,Cincinnati, Ohio.
1
197
Oravax. registered trade mark of the Wm. S. Merrell Co.
198
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
Obviously, these factors should be checked and if it is found that a particular strain or sex gives an irregular or unusual response, this must be taken into consideration in the evaluation of results. The variables of animal tests are numerous, but fortunately more and more of them are being revealed. Results niay be influenced by varying the close of vaccine, or the waiting period before challenge, or even by varying the species of test animal. By the proper combination of factors a wide range of results may be obtained and this should tend to minimize the importance of a high protection index. It appears far more important to establish that under a standardized set of conditions one can repeatedly protect against a given number of challenging doses, rather than to strive for protection against higher and higher doses with the identical vaccine.
Vol. XL, No. 4
SUMMARY 1. Some aspects of standardization of polyvalent respiratory vaccines have been considered in this paper. 2. Pneumococci or other mouse-virulent organisms may be standardized according to a mouse-protection index. 3. Attainment of higher protective indexes by variations in test conditions is meaningless. It is far more important to determine, under standardized conditions, that each lot is as good as those preceding. REFERENCES (1) Ludwig, K. A,, Murray, F. J., and Stauhach, E. J., J . Zmmunol.. 62,257(1949). (2) Reed, I,. J., aud Mueuch, H.. A m . J . H y g . , 27, 493 (1938).
A Study of the Repeated Administration of Tinnevelly and Alexandria Senna to Mice* By MARIBELLE WOODS and I. W. GROTE To determine if the same mice could be used repeatedly over long periods of time without change in sensitivity, two sets of experiments were conducted. In the first set of experiments no tolerance to either Tinnevelly or Alexandria senna developed. In the second set of experiments, 3 1 o u t of 40 mice survived the twenty-three-week period. The average percentage laxative response for the period was 64 per cent (range 38-92 er cent). The responses of individual subjects were roughly random. It woulef therefore seem that mice may be used once a week for quantitative assay of laxative activity of senna.
PROCEDURE for determining the laxative activity in mice of senna preparations has been previously reported (1). The finely ground drug was suspended in boiling distilled water and 0.5-cc. doses were given by stomach tube. The results were considered positive if the feces were softened sufficiently to stain the underside of newsprint paper, and negative if no stain was visible. T o determine if the same mice could be given a weekly dose of senna over long periods of time without changing their sensitivity t o the drug, two sets of experiments were conducted.
A
STANDARDIZED
* Received May 5, 1950, from the Research Laboratories, The Chattanooga Medicine Company, Chattanooga. Tenn. Presented to the Scientific Section, A. PH. A,, Atlantic City meeting, May, 1050.
EXPERIMENTAL In the first set of experiments, two groups of 25 male mice each were individually numbered and a record was kept, week by week, of each animal’s response to senna and his weight. Tinnevelly and Alexandria senna were given on alternate weeks at three dosage levels, namely 8.3, 6.3, or 5.0 mg. That is, one week group I would receive 8.3 mg. ot Tinnevelly senna, while group I1 would receive the same dosage of Alexandria. The following week the two groups would receive the same dosage as the first week, but the kinds of senna were reversed for the groups. This procedure was followed in succeeding weeks until each group had received three different doses of two kinds of senna. Then this identical procedure was repeated so that each group again received the same dosage and kind of senna which had been received six weeks before. This w a s to determine whether the five weekly doses which had intervened had changed the mouse’s