Meningococcal infections*

Bull. Org. mond. Sante 1971, 45, 287-290Bull. Wid Hlth Org. Meningococcal Infections* 4. Stability of Group A and Group C Polysaccharide Vaccines MALCOLM S. ARTENSTEIN Polysaccharide vaccines preparedfrom meningococci ofserogroups A and C were shown to be stable for long periods when stored in the cold-for 14-26 months or more when stored as lyophilized powder and for 18 days to 9 months or longer when stored frozen in the fluid state. Lyophilized group A vaccine was unaffected by being kept at room temperature (20-25C) for 12 days or at 35°Cfor 3 days. Determination of storage conditions for vaccines and other biological preparations is an important practical problem. In the absence of an animal model to assess the immunogenicity or potency of meningo- coccal polysaccharide vaccines, it has been necessary to test these materials in man (Gotschlich, Gold- schneider & Artenstein, 1969). The present report describes the stability of the products in the lyo- philized state and under various conditions of stor- age after hydration, based upon their ability to produce antibodies in human volunteers. MATERIALS AND METHODS Vaccine Purified meningococcal polysaccharides were pro- duced by the method of Gotschlich, Liu & Arten- stein (1969). Group C vaccine lot C-6 and group A vaccine lots A-5 and A-6 were prepared at the Walter Reed Army Institute of Research, Washington, D.C. Vaccine lots A-7, C-7, and C-8 were produced by the Squibb Institute for Medical Research under a contract with the US Army Medical Research and Development Command. Subjects Informed voluntary consent was obtained from army recruits in accordance with US Army regu- lations. A single 50-,ug dose of vaccine was admi- nistered to each subject subcutaneously in the deltoid area. Some lots were given by intradermal injection * From the Department of Bacterial Diseases, Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington, D.C., USA. or by jet injector apparatus, routes that give com- parable antibody titres (Artenstein et al., 1970). Serum specimens were obtained prior to, and 2 weeks after administration of the vaccine. Antibody assay Purified polysaccharide antigens were prepared as described by Gotschlich, Liu & Artenstein (1969). Indirect haemagglutination tests were performed according to methods previously described (Arten- stein et al., 1970). Except where specifically indi- cated otherwise, groups of sera being compared were tested on the same day with the same reagents. Sera from subjects who were nasopharyngeal carriers of group A or C meningococci were not included in the calculations. RESULTS Tables 1 and 2 show the results of storage of lyophilized vaccines at -20°C (deep-freeze unit) or at 4-50C (refrigerator). Group A polysaccharides showed no loss of immunogenicity when stored for 26 months in a deep-freeze unit or for 18 months in a refrigerator. Group C vaccines retained their po- tency for at least 14 months when kept refrigerated. The minor fluctuations in geometric mean titre rises probably represent variations in sensitivity of the assays, which in these experiments were not per- formed on the same day. Following hydration with sterile distilled water, vaccines of groups A and C were stored frozen or at 4-50C prior to being used to immunize volunteers. For control purposes, freshly hydrated vaccine was injected into comparable groups of volunteers. The 2726 - 287- 2 M. S. ARTENSTEIN Table 1. Immunogenicity of lyophilized group A polysaccharine vaccines after storage Serological response LtN. Temperature Duration- ______ -_______Lot No. of storage of storage No. of subjects Percentage Mean antibod ,(months) positive/no. pstv rie1g2tested ie(o A-5 -20°C 8a 4/4 100 5.5 26 31/33 94 3.8 A-6 4°C 1 a 4/5 80 3.8 6 31/37 84 3.1 18 28/32 88 3.7 A-7 4°C 6 7/7 100 3.4 7 34/36 95 5.3 14 30/34 88 3.5 a Intradermal injection. results (Table 3) indicate that the group C polysaccha- ride is stable for 20 days under refrigeration and for at least 9 months when frozen. Group A poly- saccharide retained its potency when frozen for 18 days. Lot A-7 vaccine was also tested after being warmed under various conditions. Lyophilized vaccine, stored Table 2. Immunogenicity of lyophilized Group C vaccines after storage at 4°C Serological response Duration Lot No. of storage No. of sub- Percenta Mean(months) jects positive/ p gsitive antibody no. tested p rise (1g2) C-6 3a 52/53 98 5.5 11 21/22 96 5.1 13 23/25 92 4.1 C-7 1 a 25/29 86 5.3 3 26/26 100 5.1 14 49/49 100 6.9 C-8 3 29/29 100 6.0 9 35/35 100 5.3 a Jet injection. for 18 months in the refrigerator, was held at rc onil temperature for 12 days prior to hydration and injected into 26 volunteers. Altogether, 24 subjects received vaccine that was incubated for 3 days at 35°C prior to hydration. For the control group of 23 subjects, vaccine was stored cold until it was hydrated just before use. The results of antibody tests on these subjects (Table 4) showed no significant differences in antibody response. DISCUSSION The purified meningococcal polysaccharide vaccines appear to be very stable when stored in the cold as a lyophilized powder or, after hydration, when frozen at - 20°C. Since the vaccines as at present con- stituted contain no preservative, simple refrigeration of the fluid vaccine appears undesirable even though potency is retained for 20 days and more. The data presented also show that group A vaccine when lyophilized is stable for periods of 3-12 days at warmer temperatures, a characteristic of considerable importance for vaccines that may be used in tropical areas. Further study is necessary, however, to make certain that all production lots of vaccine will show similar stability upon warming. Such studies would be considerably facilitated by the development of physicochemical methods for char- acterizating these polysaccharides. 288 MENINGOCOCCAL INFECTIONS. 4 Table 3. Stability of groups A and C meningococcal polysaccharide vaccines following hydration Geometric mean No. of subjects with Vaccine Conditions Serum haemagglutination titre (log2) no response a Control b] Experimental Control b [Experimental C-6 refrigerated for pre- 1.6 1.7 20 days post- 5.7 5.9 2/25 1/23 C-8 frozen at pre- 1.2 1.4 - 20'C for 3 months post- 6.5 6.4 0/35 2/35 C-6 frozen at pre- 2.0 2.5 - 20'C for 9 months post- 7.1 7.0 1/22 0/21 A-7 frozen at pre- 1.8 1.7 - 20'C for 18 days post- 5.9 5.8 3/27 4/45 a Less than 4-fold increase. b Freshly hydrated vaccine. Table 4. Effect of warm storage on immunogenicity of lyophilized group A polysaccharide vaccine (Lot A-7) Storage conditions Geometric meanStorage ~~~~~antibody rise (log2) 1. control 4.3 2. 35°C for 3 days 3.9 3. room temperature (20-250C) for 12 days 3.7 ACKNOWLEDGEMENTS The studies were made possible by the assistance of the following Preventive Medicine Officers of the US Army Medical Corps: Lt Col. R. L. Coultrip, Lt Col. W. P. Morgan, Lt Col. J. M. Sowell, Maj. J. G. Zimmerly, Capt. R. L. Cohen, and Capt. C. E. Weidmer, who super- vised the immunizations and collection of specimens. Col. A. Leibovitz and Lt Col. C. D. Smith of the Sixth Army Medical Laboratory performed many of the carrier surveys. W. C. Branche, Jr, Ph.D., H. D. Fleet, and C. Harkins provided excellent technical assistance. RESUME INFECTIONS MJNINGOCOCCIQUES: 4. STABILITP- (DE VACCINS POLYSACCHARIDIQUES DU GROUPE A ET DU GROUPE C On a evalue la stabilite de vaccins antimeningococciques (groupes A et C) conserves sous des formes et pendant des durees variables en prenant comme critere l'aptitude a susciter une reponse immunitaire chez des volontaires. Apres lyophilisation, les vaccins du groupe C gardent leur pouvoir immunogene pendant 14 mois au moins de conservation a 4°C. L'activite des vaccins lyophilises du groupe A n'est pas alteree par un stockage de 26 mois 289 290 M. S. ARTENSTEIN a - 20°C et de 18 mois a 4'C; leur pouvoir immunogene est intact apres 12 jours A la temperature ambiante et apres 3 jours a 35°C. Apres rehydratation, les vaccins du groupe C restent actifs pendant 3 semaines s'ils sont conserves a 4°C et pendant 9 mois au moins a - 20°C, tandis que les vaccins du groupe A conservent leur pouvoir immunog6ne pen- dant 18 jours a -20°C. REFERENCES Artenstein, M. S., Gold, R., Zimmerly, J. G., Wyle, F. A., Branche, W. C., Jr & Harkins, C. (1970) J. infect. Dis., 121, 372-377 Gotschlich, E. C., Goldschneider, I. & Artenstein, M. S. (1969) J. Exp. Med., 129, 1367-1384 Gotschlich, E. C., Liu, T. Y. & Artenstein, M. S. (1969) J. exp. Med., 129, 1349-1365